Bacteriophage Ecology Group
Reference Abstracts (All)
Dedicated to the ecology and evolutionary biology of the parasites of unicellular organisms (UOPs)
© Stephen T. Abedon
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© Phage et al. last updated on Monday, December 31, 2007
  1. Phenotypic transformation including host-range transition through superinfection of T-even phages. Abe,M., Izumoji,Y., Tanji,Y. (2007). FEMS Microbiol. Lett. 269:145-152. Mosaic genome design, considered evidence of horizontal gene transfer, is prominent in T-even phage tail fiber genes involved in host recognition. The possibility of direct gene transfer was assessed through superinfection with two virulent phages T2 and PP01, which caused host recognition shift. Two recombinant phages designated as TPr03 and TPr04 were isolated. PCR-restriction fragment length polymorphism analysis and sequence analysis suggested that 18% of the TPr03 and 38% of the TPr04 genome derived from PP01. Both isolates showed host ranges identical to PP01. The results suggested the possibility of generating various recombinant phages by intentional dual infections and of the occasional occurrence in nature of generation of phage showing new characteristics through superinfection, followed by the genomic recombination. [TOP OF PAGE]

  2. Bacteriophage evolution given spatial constraint. Abedon,S.T., Culler,R.R. (2007). J. Theor. Biol. 248:111-119. Spatial structure can impede mixing, diffusion, and motility. In microbiology laboratories, spatial structure is commonly achieved via formation of agar gels, within which bacteriophage (phage) replication results in localized clearings called plaques. Developing a better understanding of phage plaque formation is relevant because of the ubiquity of phage plaquing in the laboratory; because plaque size has been employed as a measure of phage fitness; because many bacteria exist within environments that display significant spatial structure (e.g., biofilms, soils, sediments, and in or on plant or animal tissues); and because spatial structure could impede phage exploitation of bacterial communities. There is, however, a relative dearth of experimentation and analysis considering phage plaque formation from the perspective of selection acting on individual phage growth parameters—latent period, burst size, and adsorption rate. Here we consider the impact of these parameters on rates of plaque wavefront velocity (rates of radial plaque enlargement), especially as functions of existing phage and environmental properties. We do so based on analyses of published equations which predict plaque enlargement rates. These indicate that greater wavefront velocities should be associated with (i) latent period reductions, (ii) larger burst sizes, or (iii) faster virion binding to bacteria. We suggest, however, that deviations could occur, respectively, (i) if virion adsorption is ''slow'' or if burst sizes are large, (ii) if burst sizes are already large, or (iii) if virion binding rates are already fast, bacterial densities are especially high, or burst sizes are large. Higher initial lawn bacterial densities could also contribute to faster plaque expansion, but only if adsorption is otherwise slow or burst sizes are large. By contrast, faster virion diffusion is always expected to result in greater plaque wavefront velocities. Overall, we provide a snapshot of how phage populations may respond evolutionarily to selection for more-rapid propagation during spatially constrained growth. [TOP OF PAGE]

  3. Optimizing bacteriophage plaque fecundity. Abedon,S.T., Culler,R.R. (2007). J. Theor. Biol. 249:582-592. Bacteriophages (phages), the viruses of bacteria, form visible lesions within bacterial lawns (called plaques), which are employed ubiquitously in phage isolation and characterization. Plaques also can serve as models for phage population growth within environments that display significant spatial structure, e.g. soils, sediments, animal mucosal tissue, etc. Furthermore, phages growing within plaques, in experimental evolution studies, may become adapted to novel conditions, may be selected for faster expansion, or may evolve toward producing more virions per plaque. Here, we examine the evolution of the latter, greater plaque fecundity, considering especially tradeoffs between phage latent period and phage burst size. This evolution is interesting because genetically lengthening latent periods, as seen with phage lysis-timing mutants, should increase phage burst sizes, as more time is available for phage-progeny maturation during infection. Genetically shortening latent periods, however, is a means toward producing larger phage plaques since phage virions then can spend more time diffusing rather than infecting. With these larger plaques more bacteria become phage infected, resulting in more phage bursts. Given this conflict between latent period's impact on per-plaque burst number versus per-infection burst size, and based on analysis of existing models of plaque expansion, we provide two assertions. First, latent periods that optimize plaque fecundity are longer (e.g. at least two-fold longer) than latent periods that optimize plaque size (or that optimize phage population growth within broth). Second, if increases in burst size can contribute to plaque size (i.e. larger plaques with larger bursts), then latent-period optima that maximize plaque fecundity should be longer still. As a part of our analysis, we provide a means for predicting latent-period optima-for maximizing either plaque size or plaque fecundity-which is based on knowledge of only phage eclipse period and the relative contribution of phage burst size versus latent period toward plaque size. [TOP OF PAGE]

  4. 5500 phages examined in the electron microscope. Ackermann,H.-W. (2007). Arch. Virol. 152:227-243. "Phages" include viruses of eubacteria and archaea. At least 5568 phages have been examined in the electron microscope since the introduction of negative staining in 1959. Most virions (96%) are tailed. Only 208 phages (3.7%) are polyhedral, filamentous, or pleomorphic. Phages belong to one order, 17 families, and three "floating" groups. Phages are found in 11 eubacterial and archaeal phyla and infect 154 host genera, mostly of the phyla Actinobacteria, Firmicutes, and Proteobacteria. Of the tailed phages, 61% have long, noncontractile tails and belong to the family Siphoviridae. Convergent evolution is visible in the morphology of certain phage groups. [TOP OF PAGE]

  5. Bacteriophage-encoded toxins: the l-holin protein causes caspase-independent non-apoptotic cell death of eukaryotic cells. Agu,C.A., Klein,R., Lengler,J., Schilcher,F., Gregor,W., Peterbauer,T., Blasi,U., Salmons,B., Gunzburg,W.H., Hohenadl,C. (2007). Cellular microbiology 9:1753-1765. The bacteriophage-encoded holin proteins are known to promote bacterial cell lysis by forming lesions within the cytoplasmic membrane. Recently, we have shown that the bacteriophage l-holin protein exerts cytotoxic activity also in eukaryotic cells accounting for a reduced tumour growth in vivo. In order to elucidate the mechanisms of l-holin-induced mammalian cell death, detailed biochemical and morphological analyses were performed. Colocalization analyses by subcellular fractionation and organelle-specific fluorescence immunocytochemistry indicated the presence of the l-holin protein in the endoplasmic reticulum and in mitochondria. Functional studies using the mitochondria-specific fluorochrome JC-1 demonstrated a loss of mitochondrial transmembrane potential in response to l-holin expression. Morphologically, these cells exhibited unfragmented nuclei but severe cytoplasmic vacuolization representing signs of oncosis/necrosis rather than apoptosis. Consistently, Western blot analyses indicated neither an activation of effector caspases 3 and 7 nor cleavage of the respective substrate poly(ADP-ribose) polymerase (PARP) in an apoptosis-specific manner. These findings suggest that the l-holin protein mediates a caspase-independent non-apoptotic mode of cell death. [TOP OF PAGE]

  6. UV light inactivation of Mycobacterium avium subsp. paratuberculosis in milk as assessed by FASTPlaqueTB phage assay and culture. Altic,L.C., Rowe,M.T., Grant,I.R. (2007). Appl. Environ. Microbiol. 73:3728-3733. UV light inactivation of Mycobacterium avium subsp. paratuberculosis in Middlebrook 7H9 broth and whole and semiskim milk was investigated using a laboratory-scale UV machine that incorporated static mixers within UV-penetrable pipes. UV treatment proved to be less effective in killing M. avium subsp. paratuberculosis suspended in milk (0.5- to 1.0-log(10) reduction per 1,000 mJ/ml) than that suspended in Middlebrook 7H9 broth (2.5- to 3.3-log(10) reduction per 1,000 mJ/ml). The FASTPlaqueTB phage assay provided more rapid enumeration of surviving M. avium subsp. paratuberculosis (within 24 h) than culture on Herrold's egg yolk medium (6 to 8 weeks). Despite the fact that plaque counts were consistently 1 to 2 log(10) lower than colony counts throughout the study, UV inactivation rates for M. avium subsp. paratuberculosis derived using the phage assay and culture results were not significantly different (P = 0.077). [TOP OF PAGE]

  7. Propagation of fluorescent viruses in growing plaques. Alvarez,L.J., Thomen,P., Makushok,T., Chatenay,D. (2007). Biotech. Bioeng. 96:615-621. To study virus propagation, we have developed a method by which the propagation of the Lambda bacteriophage can be observed and quantified. This is done by creating a fusion protein of the capsid protein gpD and the enhanced yellow fluorescent protein (EYFP). We show that this fusion allows capsid formation and that the modified viruses propagate on a surface covered with host bacteria thus forming fluorescent plaques. The intensity of fluorescence in a growing plaque determines the distribution of phages. This provides a new tool to study the propagation of infection at the microscopic level. [TOP OF PAGE]

  8. Lytic phage as a specific and selective probe for detection of Staphylococcus aureus--A surface plasmon resonance spectroscopic study. Balasubramanian,S., Sorokulova,I.B., Vodyanoy,V.J., Simonian,A.L. (2007). Biosensors & bioelectronics 22:948-955. Rapid and reliable detection of harmful pathogens at low levels are vital due to the related environmental and economical impact. While antibodies (monoclonal or polyclonal) are successfully employed in many immunoanalysis procedures as a biorecognition element, many of them remain costly with a comparatively short shelf life and uncertain manufacturability. Additionally, they suffer from several limitations, such as susceptibility to hostile environmental stresses such as temperature, pH, ionic strength, and cross-reactivity. The development of easy available, sensitive, and robust alternative molecular recognition elements, capable of providing a very high level of selectivity are very attractive to industry and may benefit in multiple areas. Several attempts have been made to utilize fluorescent-tagged bacteriophages and phage-displayed peptides for bacterial detection. However, involvement of complex labeling and detecting procedures make these approaches time-consuming and complicated. Here, we are reporting for the first time, the label-free detection of Staphylococcus aureus using lytic phage as highly specific and selective biorecognition element and surface plasmon resonance-based SPREETA sensor as a detection platform. Lytic phage was immobilized on the gold surface of SPREETA sensor via trouble-free direct physical adsorption. The detection limit was found to be 10(4) cfu/ml. Detection specificity was investigated by an inhibition assay while selectivity was examined with Salmonella typhimurium. The preliminary results using lytic phage as a probe for bacterial detection, in combination with SPR platform are promising and hence can be employed for rapid and label-free detection of different bacterial pathogens. [TOP OF PAGE]

  9. CRISPR provides acquired resistance against viruses in prokaryotes. Barrangou,R., Fremaux,C., Deveau,H., Richards,M., Boyaval,P., Moineau,S., Romero,D.A., Horvath,P. (2007). Science (New York, N. Y. ) 315:1709-1712. Clustered regularly interspaced short palindromic repeats (CRISPR) are a distinctive feature of the genomes of most Bacteria and Archaea and are thought to be involved in resistance to bacteriophages. We found that, after viral challenge, bacteria integrated new spacers derived from phage genomic sequences. Removal or addition of particular spacers modified the phage-resistance phenotype of the cell. Thus, CRISPR, together with associated cas genes, provided resistance against phages, and resistance specificity is determined by spacer-phage sequence similarity. [TOP OF PAGE]

  10. Temperate bacteriophages affect pulsed-field gel electrophoresis patterns of Campylobacter jejuni. Barton,C., Ng,L.K., Tyler,S.D., Clark,C.G. (2007). J. Clin. Microbiol. 45:386-391. The recently sequenced genome of Campylobacter jejuni RM1221 revealed the presence of three integrated bacteriophage-like elements. In this study, genes from the first element, a Mu-like bacteriophage, were amplified by PCR and used to probe pulsed-field gels of clinical C. jejuni strains obtained from a waterborne outbreak (Ontario, Canada, 2000). These highly similar strains differed only by their pulsed-field gel electrophoresis (PFGE) patterns due to an apparent insertion or deletion of a 40-kb fragment. Bacteriophage probes hybridized to these different bands in Southern blot analysis, indicating that homologues of bacteriophage genes were present in the outbreak strains. Investigation of the bacteriophage insertion sites in these isolates suggested that bacteriophage acquisition, loss, or transposition was responsible for the PFGE pattern variation. The bacteriophage gene sequences were similar, but not identical, in the outbreak strains and RM1221, indicating that differences may exist between the bacteriophages. [TOP OF PAGE]

  11. Development of a bacteriophage model system to investigate virus inactivation methods used in the treatment of bone allografts. Bienek,C., MacKay,L., Scott,G., Jones,A., Lomas,R., Kearney,J.N., Galea,G. (2007). Cell and tissue banking 8:115-124. Bone allografts are commonly used in a variety of surgical procedures, to reconstruct lost bone stock and to provide mechanical support during the healing process. Due to concerns regarding the possibility of disease transmission from donor to recipient, and of contamination of grafts during retrieval and processing procedures, it is common practice to sterilise bone allografts prior to issue for clinical use. It is vital that the sterilisation processes applied to allografts are validated to demonstrate that they achieve the required level of bioburden reduction, and by extension that validated models are used for these studies. Two common sterilisation protocols applied to bone allografts are gamma irradiation and ethylene oxide gas sterilisation, and there are currently no validated models available for measuring the anti-viral efficacy of ethylene oxide treatment with regard to bone allografts or readily useable models for assessing the anti-viral efficiency of gamma irradiation treatment. We have developed and validated models for both these sterilisation processes, using the bacteriophage jx174, and utilised the models to measure the antiviral activity of the standard ethylene oxide and gamma irradiation sterilisation processes applied to bone allografts by the National Blood Service. For the irradiation model, we also utilised bacterial spores (Bacillus pumilus). Our results show that ethylene oxide sterilisation (which can only be applied to lyophilised grafts) inactivated > 6.1 log(10) of the model virus, and gamma irradiation (at 25 -40 kGy and applied to frozen allografts) inactivated 3.6 - 4.0 log(10) of the model virus and > 4 log(10) of the bacterial spores. Gamma irradiation at this dosage is therefore not in itself a sterilisation process with respect to viruses. [TOP OF PAGE]

  12. Effects of wastewater disinfection on waterborne bacteria and viruses. Blatchley,E.R., Gong,W.L., Alleman,J.E., Rose,J.B., Huffman,D.E., Otaki,M., Lisle,J.T. (2007). Water Environ. Res. 79:81-92. Wastewater disinfection is practiced with the goal of reducing risks of human exposure to pathogenic microorganisms. In most circumstances, the efficacy of a wastewater disinfection process is regulated and monitored based on measurements of the responses of indicator bacteria. However, inactivation of indicator bacteria does not guarantee an acceptable degree of inactivation among other waterborne microorganisms (e.g., microbial pathogens). Undisinfected effluent samples from several municipal wastewater treatment facilities were collected for analysis. Facilities were selected to provide a broad spectrum of effluent quality, particularly as related to nitrogenous compounds. Samples were subjected to bench-scale chlorination and dechlorination and UV irradiation under conditions that allowed compliance with relevant discharge regulations and such that disinfectant exposures could be accurately quantified. Disinfected samples were subjected to a battery of assays to assess the immediate and long-term effects of wastewater disinfection on waterborne bacteria and viruses. In general, (viable) bacterial populations showed an immediate decline as a result of disinfectant exposure; however, incubation of disinfected samples under conditions that were designed to mimic the conditions in a receiving stream resulted in substantial recovery of the total bacterial community. The bacterial groups that are commonly used as indicators do not provide an accurate representation of the response of the bacterial community to disinfectant exposure and subsequent recovery in the environment. UV irradiation and chlorination/dechlorination both accomplished measurable inactivation of indigenous phage; however, the extent of inactivation was fairly modest under the conditions of disinfection used in this study. UV irradiation was consistently more effective as a virucide than chlorination/dechlorination under the conditions of application, based on measurements of virus (phage) diversity and concentration. Taken together, and when considered in conjunction with previously published research, the results of these experiments illustrate several important limitations of common disinfection processes as applied in the treatment of municipal wastewaters. In general, it is not clear that conventional disinfection processes, as commonly implemented, are effective for control of the risks of disease transmission, particularly those associated with viral pathogens. Microbial quality in receiving streams may not be substantially improved by the application of these disinfection processes; under some circumstances, an argument can be made that disinfection may actually yield a decrease in effluent and receiving water quality. Decisions regarding the need for effluent disinfection must account for site-specific characteristics, but it is not clear that disinfection of municipal wastewater effluents is necessary or beneficial for all facilities. When direct human contact or ingestion of municipal wastewater effluents is likely, disinfection may be necessary. Under these circumstances, UV irradiation appears to be superior to chlorination in terms of microbial quality and chemistry and toxicology. This advantage is particularly evident in effluents that contain appreciable quantities of ammonia-nitrogen or organic nitrogen. [TOP OF PAGE]

  13. Clonal interference is alleviated by high mutation rates in large populations. Bollback,J.P., Huelsenbeck,J.P. (2007). Mol. Biol. Evol. 24:1397-1406. When a beneficial mutation is fixed in a population that lacks recombination, the genetic background linked to that mutation is fixed. As a result, beneficial mutations on different backgrounds experience competition, or "clonal interference," that can cause asexual populations to evolve more slowly than their sexual counterparts. Factors such as a large population size (N) and high mutation rates (mu) increase the number of competing beneficial mutations, and hence are expected to increase the intensity of clonal interference. However, recent theory suggests that, with very large values of Nmu, the severity of clonal interference may instead decline. The reason is that, with large Nmu, genomes including both beneficial mutations are rapidly created by recurrent mutation, obviating the need for recombination. Here, we analyze data from experimentally evolved asexual populations of a bacteriophage and find that, in these nonrecombining populations with very large Nmu, recurrent mutation does appear to ameliorate this cost of asexuality. [TOP OF PAGE]

  14. Local interactions select for lower pathogen infectivity. Boots,M., Mealor,M. (2007). Science 315:1284-1286. Theory suggests that the current rapid increase in connectivity and consequential changes in the structure of human, agricultural, and wildlife populations may select for parasite strains with higher infectivity. We carried out a test of this spatial theory by experimentally altering individual host movement rates in a model host/pathogen system by altering the viscosity of their environment. In our microevolutionary selection experiments, the infectivity of the virus was, as predicted by the theory, reduced in the most viscous populations. We therefore provide empirical support for the theory that population structure affects the evolution of infectious organisms. [TOP OF PAGE]

  15. Bacteria-eating virus approved as food additive. Bren,L. (2007). FDA consumer 41:20-22. Not all viruses harm people. The Food and Drug Administration has approved a mixture of viruses as a food additive to protect people. The additive can be used in processing plants for spraying onto ready-to-eat meat and poultry products to protect consumers from the potentially life-threatening bacterium Listeria monocytogenes (L. monocytogenes). [TOP OF PAGE]

  16. Bacteriophage-based bioluminescent bioreporter for the detection of Escherichia coli 0157:H7. Brigati,J.R., Ripp,S.A., Johnson,C., Iakova,P.A., Jegier,P., Sayler,G.S. (2007). J. Food Prot. 70:1386-1392. The rapid detection of pathogenic bacteria in food and water is vital for the prevention of foodborne illness. In this study, the lux reporter genes were used in a new bioassay that allows pathogen monitoring without multiple sample manipulations or the addition of exogenous substrate. A recombinant phage specific for Escherichia coli 0157:H7 was constructed that, upon infection, catalyzes the synthesis of N-(3-oxohexanoyl)-L-homoserine lactone (OHHL). This phage PP01 derivative carries the luxI gene from Vibrio fischeri under the control of the phage promoter PL. OHHL produced by infected E. coli 0157:H7 induces bioluminescence in bioreporter cells carrying the V. fischeri lux operon. The ability of phage PP0-luxl to detect several strains of E. coli 0157:H7 was confirmed in a 96-well plate assay. In this assay, luxCDABE bioreporter cells capable of detecting OHHL were mixed with phage PPOI-luxl and E. coli 0157:H7, and luminescence was monitored. Reporter phages induced light in bioreporter cells within I h when exposed to 10(4) CFU/ml of E. coli 0157:H7 and were able to detect 10 CFU/ml in pure culture with a preincubation step (total detection time, 4 h). The detection method was also applied to contaminated apple juice and was able to detect 10(4) CFU/ml of E. coli 0157:H7 in 2 h after a 6-h preincubation. [TOP OF PAGE]

  17. The impact of migration from parasite-free patches on antagonistic host-parasite coevolution. Brockhurst,M.A., Buckling,A., Poullain,V., Hochberg,M.E. (2007). Evolution 61:1238-1243. Natural populations of hosts and parasites are often subdivided and patchily distributed such that some regions of a host species' range will be free from a given parasite. Host migration from parasite-free to parasite-containing patches is expected to alter coevolutionary dynamics by changing the evolutionary potential of antagonists. Specifically, host immigration can favor parasites by increasing transmission opportunities, or hosts by introducing genetic variation. We tested these predictions in coevolving populations of Pseudomonas fluorescens and phage Phi2 that received immigrants from phage-free populations. We observed a negative quadratic relationship between sympatric resistance to phage and host immigration rate (highest at intermediate immigration) but a positive quadratic relationship between coevolution rate and host immigration rate (lowest at intermediate immigration). These results indicate that for a wide range of rates, host immigration from parasite-free patches can increase the evolutionary potential of parasites, and increase the coevolutionary rate if parasite adaptation is limiting in the absence of immigration. [TOP OF PAGE]

  18. Experimental coevolution with bacteria and phage. The Pseudomonas fluorescens--F2 model system. Brockhurst,M.A., Morgan,A.D., Fenton,A., Buckling,A. (2007). Infec. Genet. Evol. 7:547-552. Parasites are ubiquitous in biological systems and antagonistic coevolution between hosts and parasites is thought be a major ecological and evolutionary force. Recent experiments using laboratory populations of bacteria and their parasitic viruses, phage, have provided the first direct empirical evidence of antagonistic coevolution in action. In this article we describe this model system and synthesise recent findings that address the causes and consequences of antagonistic coevolution. [TOP OF PAGE]

  19. Phage metagenomics. Casas,V., Rohwer,F. (2007). Meth. Enzymol. 421:259-268. The vast majority of novel DNA sequences deposited in the databases now comes from environmental phage DNA sequences. Methods are presented for the cloning and sequencing of phage DNA that might otherwise be lethal to bacterial host vectors or contain modified DNA bases that prevent standard cloning of such sequences. In addition, methods are presented for the isolation of viral particles directly from soil and sediment environmental samples or from large volumes of environmental water samples. The viral particles are then purified by cesium-chloride density centrifugation followed by DNA extraction. This purified viral metagenomic DNA is then used for cloning and sequencing. [TOP OF PAGE]

  20. A possible heterodimeric prophage-like element in the genome of the insect endosymbiont Sodalis glossinidius. Clark,A.J., Pontes,M., Jones,T., Dale,C. (2007). J. Bacteriol. 189:2949-2951. Extrachromosomal element pSOG3 (52,162 nucleotides) in the genome of Sodalis glossinidius contains redundant phage-related gene pairs, indicating that it may have been formed by the fusion of two ancestral phage genomes followed by gene degradation. We suggest that pSOG3 is a prophage that has undergone genome degeneration accompanying host adaptation to symbiosis. [TOP OF PAGE]

  21. Microbiological performance of common water treatment devices for household use in India. Clasen,T., Menon,S. (2007). International Journal of Environmental Health Research 17:83-93. Diarrhoea and other diseases associated with unsafe drinking water are a leading cause of mortality and morbidity worldwide and in India. Household-based water treatment has been shown to be an effective means of reducing this disease burden. Numerous such devices are manufactured and sold all over the world. We tested the microbiological performance of a leading brand of each of three common types of water treatment devices designed for household use in India: a ceramic candle gravity filter, an iodine resin gravity filter and an iodine resin faucet mounted filter. The ceramic candle filter and the iodine resin faucet filter reduced bacteria by more than 4 logs. However, the reduction of the MS2 phage (surrogate for viruses) and 3 micron microspheres (surrogate for protozoan cysts) in these devices was lower than log 3.4 and log 2.6, respectively. There were also high levels of residual iodide (and in some cases, iodine) in treated water from the iodine-based devices. While household water treatment could play an important role in India, standards are necessary so that consumers can ensure that the devices they purchase and use in the home are effective and safe. [TOP OF PAGE]

  22. Phage-antibiotic synergy (PAS): b-lactam and quinolone antibiotics stimulate virulent phage growth. Comeau,A., Tétart,F., Trojet,S.A., Prère,M.-F., Krisch,H.M. (2007). PLoS One 2:e799 Although the multiplication of bacteriophages (phages) has a substantial impact on the biosphere, comparatively little is known about how the external environment affects phage production. Here we report that sub-lethal concentrations of certain antibiotics can substantially stimulate the host bacterial cell's production of some virulent phage. For example, a low dosage of cefotaxime, a cephalosporin, increased an uropathogenic Escherichia coli strain's production of the phage FMFP by more than 7-fold. We name this phenomenon Phage-Antibiotic Synergy (PAS). A related effect was observed in diverse host-phage systems, including the T4-like phages, with b-lactam and quinolone antibiotics, as well as mitomycin C. A common characteristic of these antibiotics is that they inhibit bacterial cell division and trigger the SOS system. We therefore examined the PAS effect within the context of the bacterial SOS and filamentation responses. We found that the PAS effect appears SOS-independent and is primarily a consequence of cellular filamentation; it is mimicked by cells that constitutively filament. The fact that completely unrelated phages manifest this phenomenon suggests that it confers an important and general advantage to the phages. [TOP OF PAGE]

  23. Shiga toxin and Shiga toxin-encoding phage do not facilitate Escherichia coli O157:H7 colonization in sheep. Cornick,N.A., Helgerson,A.F., Sharma,V. (2007). Appl. Environ. Microbiol. 73:344-346. Isogenic strains of Escherichia coli O157:H7, missing either stx(2) or the entire Stx2-encoding phage, were compared with the parent strain for their abilities to colonize sheep. The absence of the phage or of the Shiga toxin did not significantly impact the magnitude or duration of shedding of E. coli O157:H7. [TOP OF PAGE]

  24. Conservation and diversity in the immunity regions of wild phages with the immunity specificity of phage lambda. Degnan,P.H., Michalowski,C.B., Babic,A.C., Cordes,M.H.J., Little,J.W. (2007). Mol. Microbiol. 64:232-244. The gene regulatory circuitry of phage lambda is among the best-understood circuits. Much of the circuitry centres around the immunity region, which includes genes for two repressors, CI and Cro, and their cis-acting sites. Related phages, termed lambdoid phages, have different immunity regions, but similar regulatory circuitry and genome organization to that of lambda, and show a mosaic organization, arising by recombination between lambdoid phages. We sequenced the immunity regions of several wild phages with the immunity specificity of lambda, both to determine whether natural variation exists in regulation, and to analyse conservation and variability in a region rich in well-studied regulatory elements. CI, Cro and their cis-acting sites are almost identical to those in lambda, implying that regulatory mechanisms controlled by the immunity region are conserved. A segment adjacent to one of the operator regions is also conserved, and may be a novel regulatory element. In most isolates, different alleles of two regulatory proteins (N and CII) flank the immunity region; possibly the lysis-lysogeny decision is more variable among isolates. Extensive mosaicism was observed for several elements flanking the immunity region. Very short sequence elements or microhomologies were also identified. Our findings suggest mechanisms by which fine-scale mosaicism arises. [TOP OF PAGE]

  25. Host density impacts relative fitness of bacteriophage f6 genotypes in structured habitats. Dennehy,J.J., Abedon,S.T., Turner,P.E. (2007). Evolution 61:2516-2527. Spatially structured environments may impact evolution by restricting population sizes, limiting opportunities for genetic mixis, or weakening selection against deleterious genotypes. When habitat structure impedes dispersal, low-productivity (less virulent) infectious parasites may benefit from their prudent exploitation of local hosts. Here we explored the combined ability for habitat structure and host density to dictate the relative reproductive success of differentially productive parasites. To do so, we allowed two RNA bacteriophage ?6 genotypes to compete in structured and unstructured (semi-solid versus liquid) habitats while manipulating the density of Pseudomonas hosts. In the unstructured habitats, the more-productive phage strain experienced a relatively constant fitness advantage regardless of starting host density. By contrast, in structured habitats, restricted phage dispersal may have magnified the importance of local productivity, thus allowing the relative fitness of the less-productive virus to improve as host density increased. Further data suggested that latent period (duration of cellular infection) and especially burst size (viral progeny produced per cell) were the phage "life-history" traits most responsible for our results. We discuss the relevance of our findings for selection occurring in natural phage populations and for the general evolutionary epidemiology of infectious parasites. [TOP OF PAGE]

  26. Virus population extinction via ecological traps. Dennehy,J.J., Friedenberg,N.A., Yang,Y.W., Turner,P.E. (2007). Ecol. Lett. 10:230-240. Populations are at risk of extinction when unsuitable or when sink habitat exceeds a threshold frequency in the environment. Sinks that present cues associated with high-quality habitats, termed ecological traps, have especially detrimental effects on net population growth at metapopulation scales. Ecological traps for viruses arise naturally, or can be engineered, via the expression of viral-binding sites on cells that preclude viral reproduction. We present a model for virus population growth in a heterogeneous host community, parameterized with data from populations of the RNA bacteriophage fi6 presented with mixtures of suitable host bacteria and either neutral or trap cells. We demonstrate that viruses can sustain high rates of population growth in the presence of neutral non-hosts as long as some host cells are present, whereas trap cells dramatically reduce viral fitness. In addition, we demonstrate that the efficacy of traps for viral elimination is frequency dependent in spatially structured environments such that population viability is a nonlinear function of habitat loss in dispersal-limited virus populations. We conclude that the ecological concepts applied to species conservation in altered landscapes can also contribute to the development of trap cell therapies for infectious human viruses. [TOP OF PAGE]

  27. Abortive phage resistance mechanism AbiZ speeds the lysis clock to cause premature lysis of phage-infected Lactococcus lactis. Durmaz,E., Klaenhammer,T.R. (2007). J. Bacteriol. 189:1417-1425. The conjugative plasmid pTR2030 has been used extensively to confer phage resistance in commercial Lactococcus starter cultures. The plasmid harbors a 16-kb region, flanked by insertion sequence (IS) elements, that encodes the restriction/modification system LlaI and carries an abortive infection gene, abiA. The AbiA system inhibits both prolate and small isometric phages by interfering with the early stages of phage DNA replication. However, abiA alone does not account for the full abortive activity reported for pTR2030. In this study, a 7.5-kb region positioned within the IS elements and downstream of abiA was sequenced to reveal seven additional open reading frames (ORFs). A single ORF, designated abiZ, was found to be responsible for a significant reduction in plaque size and an efficiency of plaquing (EOP) of 10(-6), without affecting phage adsorption. AbiZ causes phage phi31-infected Lactococcus lactis NCK203 to lyse 15 min early, reducing the burst size of phi31 100-fold. Thirteen of 14 phages of the P335 group were sensitive to AbiZ, through reduction in either plaque size, EOP, or both. The predicted AbiZ protein contains two predicted transmembrane helices but shows no significant DNA homologies. When the phage phi31 lysin and holin genes were cloned into the nisin-inducible shuttle vector pMSP3545, nisin induction of holin and lysin caused partial lysis of NCK203. In the presence of AbiZ, lysis occurred 30 min earlier. In holin-induced cells, membrane permeability as measured using propidium iodide was greater in the presence of AbiZ. These results suggest that AbiZ may interact cooperatively with holin to cause premature lysis. [TOP OF PAGE]

  28. Gene flow reverses an adaptive cline in a coevolving host-parasitoid interaction. Forde,S.E., Thompson,J.N., Bohannan,B.J.M. (2007). Am. Nat. 169:794-801. Many natural populations are characterized by clinal patterns of adaptation, but it is unclear how gene flow and environmental gradients interact to drive such clines. We addressed this question by directly manipulating dispersal and productivity in an experimental landscape containing a microbial parasitoid, the bacteriophage T7, and its host, the bacterium Escherichia coli. We observed that the adaptation of parasitoids increased on hosts originating from lower-productivity communities in the absence of gene flow. However, adaptation decreased along the same productivity gradient with experimentally imposed gene flow of the host and parasitoid. This occurred despite relatively low rates of gene flow. [TOP OF PAGE]

  29. A Survey of the bacteriophage WO in the endosymbiotic bacteria Wolbachia. Gavotte,L., Henri,H., Stouthamer,R., Charif,D., Charlat,S., Bouletreau,M., Vavre,F. (2007). Mol. Biol. Evol. 24:427-435. Bacteriophages are common viruses infecting prokaryotes. In addition to their deadly effect, phages are also involved in several evolutionary processes of bacteria, such as coding functional proteins potentially beneficial to them, or favoring horizontal gene transfer through transduction. The particular lifestyle of obligatory intracellular bacteria usually protects them from phage infection. However, Wolbachia, an intracellular alpha-proteobacterium, infecting diverse arthropod and nematode species and best known for the reproductive alterations it induces, harbors a phage named WO, which has recently been proven to be lytic. Here, phage infection was checked in 31 Wolbachia strains, which induce 5 different effects in their hosts and infect 25 insect species and 3 nematodes. Only the Wolbachia infecting nematodes and Trichogramma were found devoid of phage infection. All the 25 detected phages were characterized by the DNA sequence of a minor capsid protein gene. Based on all data currently available, phylogenetic analyses show a lack of congruency between Wolbachia or insect and phage WO phylogenies, indicating numerous horizontal transfers of phage among the different Wolbachia strains. The absence of relation between phage phylogeny and the effects induced by Wolbachia suggests that WO is not directly involved in these effects. Implications on phage WO evolution are discussed. [TOP OF PAGE]

  30. Characterization of a Leuconostoc gelidum bacteriophage from pork. Greer,G.G., Dilts,B.D., Ackermann,H.W. (2007). Int. J. Food Microbiol. 114:370-375. A new bacteriophage (phage ggg) and its host, Leuconostoc gelidum LRC-BD, were isolated from vacuum-packaged pork loins. Homogenates of pork loin tissue were enriched with L. gelidum LRC-BD to isolate phages. Cultural, biochemical and genetic methods were used to compare L. gelidum LRC-BD and the type strain, L. gelidum ATCC 49366. The phages were characterized by host range, morphology and phage-bacterial interaction in All Purpose Tween (APT) broth and on pork adipose tissue. With the exception of its inability to produce dextran from sucrose and the fermentation of l-arabinose, L. gelidum LRC-BD was culturally and biochemically similar to L. gelidum ATCC 49366. DNA-relatedness of the strains was confirmed by sequencing of the 16s rRNA gene. Electron microscopic observation revealed that phage ggg was a member of the Siphoviridae. The host range was limited to L. gelidum isolates from meats. Phages were able to replicate and limit the growth of L. gelidum LRC-BD in APT broth incubated aerobically and anaerobically at 4 degrees C, with a multiplicity of infection (MOI) of 0.001. When inoculated pork adipose tissue was stored at 4 degrees C in air or vacuum, phages could multiply but a higher MOI (0.01 to 1000) was necessary to limit the growth of L. gelidum LRC-BD. Naturally occurring phages may affect the numbers of L. gelidum and other lactic acid bacteria residing in meats and thereby alter the storage quality or the preservative potential of competitive strains. [TOP OF PAGE]

  31. Bacteriophages: an appraisal of their role in the treatment of bacterial infections. Hanlon,G.W. (2007). International Journal of Antimicrobial Agents 30:118-128. Bacteriophages were first used successfully to treat bacterial infections a decade before penicillin was discovered. However, the excitement that greeted those initial successes was short-lived, as a lack of understanding of basic phage biology subsequently led to a catalogue of clinical failures. As a consequence, bacteriophage therapy was largely abandoned in the West in favour of the newly emerging antibiotics. Now, as the problem of antibiotic resistance becomes ever more acute, a number of scientists and clinicians are looking again at bacteriophages as a therapeutic option in the treatment of bacterial infections. The chances of success second time round would appear to be much better given our current extensive knowledge of bacteriophage biology following their important role in underpinning the advances in molecular biology. We also have available to us the experience of nearly 80 years of clinical usage in the countries of the former Soviet Union and Eastern Europe as well as a political climate that encourages sharing of that knowledge. This review outlines those features of bacteriophages that contribute to their utility in therapy and explores the potential for their re-introduction into Western medicine. An abundance of clinical evidence is available in the Soviet literature but much of this is technically flawed and a more realistic appraisal of the clinical value of phages can be obtained from animal studies conducted in the West. As interest in bacteriophages increases, a number of companies throughout the world have begun investing in phage technology and this has led to novel approaches to therapy, some of which will be discussed. [TOP OF PAGE]

  32. Isolation and characterization of the Serratia entomophila antifeeding prophage. Hurst,M.R.H., Beard,S.S., Jackson,T.A., Jones,S.M. (2007). FEMS Microbiol. Lett. 270:42-48. The Serratia entomophila antifeeding prophage (Afp) is thought to form a virus-like structure that has activity towards the New Zealand grass grub, Costelytra zealandica. Through the trans based expression of AnfA1, an RfaH - like transcriptional antiterminator, the Afp, was able to be induced. The expressed Afp was purified and visualized by electron microscopy. The Afp resembled a phage tail-like bacteriocin, exhibiting two distinct morphologies: an extended and a contracted form. The purified Afp conferred rapid activity towards C. zealandica larvae, causing cessation of feeding and a change to an amber colouration within 48 h postinoculation, with increased dose rates causing larval mortality. [TOP OF PAGE]

  33. Identification of structural genes for Clostridium botulinum type C neurotoxin-converting phage particles. Hwang,H.J., Lee,J.C., Yamamoto,Y., Sarker,M.R., Tsuchiya,T., Oguma,K. (2007). FEMS Microbiol. Lett. 270:82-89. The structural genes for strain C-Stockholm (c-st) phage particles, a representative type C toxin-converting phage of Clostridium botulinum, have been determined. First, by determining the N-terminal amino acid sequences of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) bands of c-st phage particles, it became clear that four proteins, 14, 25, 32 and 42 kDa, are the products of the ORFs, cst166, cst165, cst160 and cst164, respectively, of the c-st phage genome. The Western blot analyses reacting these phage bands with an antiphage serum prepared previously indicated that the products of cst165 and cst160 are the main proteins of the phage particles. Then, six candidates for the phage structural proteins, including cst165 and cst160 gene products, were prepared as recombinant proteins. Also, the protein corresponding to the cst164 gene product was excised from SDS-PAGE gels. The antibodies against these seven proteins were prepared in rabbits, and finally, the reaction of these antibodies to the c-st phage particles was analyzed by electron microscopy. It was concluded that a sheath protein and a head protein of the c-st phage are the products of genes cst160 and cst165, respectively, and that these two proteins are conserved in the other three converting phages, but not in the nonconverting phage. [TOP OF PAGE]

  34. Evaluation of a rapid microbial detection method via phage lytic amplification assay coupled with Live/Dead fluorochromic stains. Jassim,S.A.A., Griffiths,M.W. (2007). Lett. Appl. Microbiol. 44:673-678. AIMS: To develop a method for rapid detection of bacteria via bacteriophage amplification coupled with exogenous fluorochromic stains. METHODS AND RESULTS: A method for the rapid detection of bacteria was developed which consisted of exposing the sample suspected to contain target cells to host-specific phage. After at least one infection cycle, bacteria known to be infected by the phage (helper cells) were added and the number of nascent phage particles was estimated using the Live/Dead BacLight Bacterial Viability kit. Using Pseudomonas aeruginosa, it was shown that the dead helper cell population following phage infection was proportional to the initial number of target cells present in the original sample. Approximately 1 x 10(1) CFU per ml of P. aeruginosa could be detected within 4 h without the need for enrichment. CONCLUSIONS: The phage lytic amplification assay coupled with exogenous fluorochromic stains was able to detect approx. 1 x 10(1) CFU per ml of the target bacterium within 4 h. SIGNIFICANCE AND IMPACT OF THE STUDY: A method to detect low number of bacterial cells in a sample within 4 h without the need for enrichment was developed. [TOP OF PAGE]

  35. Molecular characterization of T4-type bacteriophages in a rice field. Jia,Z., Ishihara,R., Nakajima,Y., Asakawa,S., Kimura,M. (2007). Environ. Microbiol. 9:1091-1096. Bacteriophages, the viruses that infect bacteria, are the most abundant biological entities in the biosphere and play a key role in global biogeochemical cycling. All T4-type bacteriophage isolates tested so far have a conserved genetic module that encodes the virion components including gene 23 (g23), the major capsid protein. Molecular analysis of the g23 sequence revealed a remarkable level of diversity of T4-type bacteriophages isolated from rice straw and surface soil in a Japanese rice field. It was found that g23 sequences obtained from the rice field were quite distinctive from those obtained in marine environments. Phylogenetic analysis showed that most of these g23 sequences belonged to two novel subgroups of T4-type bacteriophages, although some of them were related to well-studied subgroups of T4-type bacteriophages, such as marine cyanophage isolates of exoT-evens. [TOP OF PAGE]

  36. Enterobacter sakazakii bacteriophages can prevent bacterial growth in reconstituted infant formula. Kim,K.P., Klumpp,J., Loessner,M.J. (2007). Int. J. Food Microbiol. 115:195-203. Reconstituted infant formula has been implicated in outbreaks of Enterobacter sakazakii infections, causing high mortality and serious sequelae. Current prevention methods appear to be insufficient to ensure that such foods are free of E. sakazakii. In this study, the usefulness of bacteriophages for biocontrol of E. sakazakii was investigated. Of a total of six new E. sakazakii phages isolated from sewage and UV irradiated cultures, two were selected for further study by electron microscopy, DNA restriction analysis and SDS-PAGE of structural proteins. Purified phages were used to control bacterial growth in broth medium and reconstituted infant formula. Both phages effectively prevented development of E. sakazakii in formula at various temperatures (12, 24 and 37 degrees C), the efficiency of which was dependent upon intrinsic lysis properties and the applied phage concentration. We conclude that application of specific bacteriophages may provide a means for efficient prevention of E. sakazakii infection through reconstituted infant formula. [TOP OF PAGE]

  37. Going for baroque at the Escherichia coli K1 cell surface. King,M.R., Steenbergen,S.M., Vimr,E.R. (2007). Trends Microbiol. 15:196-202. Phase variation is usually thought of as the stochastic switching between alternatively expressed ('on') and unexpressed ('off') phenotypic states. However, coupling synthesis of a monotonous homopolysaccharide to a mechanism of random but incomplete chemical modification produces almost infinite structural variation. Potentially limitless variability implies that evolution can produce highly ornate or extravagant flourishes reminiscent of the baroque style. Here, we describe an analysis of capsular polysialic acid form variation in Escherichia coli K1, demonstrating that the large number of variant structures is controlled by a single contingency locus. The mechanism for generating maximum structural diversity from maximal genetic parsimony is conferred by a simple translational switch carried on a K1-specific prophage. [TOP OF PAGE]

  38. Sunlight-mediated inactivation of MS2 coliphage via exogenous singlet oxygen produced by sensitizers in natural waters. Kohn,T., Nelson,K.L. (2007). Environ. Sci. Technol. 41:192-197. Pathogens in sunlit surface waters can be damaged directly by UVB light. Indirect inactivation by reactive oxygen species (ROS) generated by sunlight interacting with external sensitizer molecules may also be important, but this mechanism has not been conclusively demonstrated. To better understand the role of ROS, we investigated the inactivation of MS2 coliphage, a commonly used surrogate for human enteric viruses, in water samples irradiated with a solar simulator and containing different types of sensitizers: waste stabilization pond (WSP) constituents, Fluka humic acid (FHA), and Suwannee River humic acid (SRHA). Inactivation of MS2 by the indirect mechanism was significant for all three sensitizers, and the efficiency of the sensitizers at inactivating MS2 was FHA > SRHA > WSP. Both dissolved and particulate fractions in the WSP water contributed to inactivation. In the WSP water, the indirect process was quantitatively more importantthan direct damage by UVB light, due to the rapid attenuation of UVB compared to the longer wavelengths that may initiate the indirect mechanism. Singlet oxygen (1O2) was the most important ROS involved in the inactivation of MS2. The addition of histidine, a 1O2 quencher, decreased inactivation, whereas inactivation rate constants increased in solutions of D20. Selective quenchers for other ROS showed little or no protective effect. Inactivation in WSP water was a function of the steady-state 102 concentration and could be described by a second-order rate expression. [TOP OF PAGE]

  39. [Diversity and dynamics of bacteriophages in horse feces]. Kulikova,E.E., Isaeva,A.S., Rotkina,A.S., Manykin,A.A., Letarov,A.V. (2007). Mikrobiologiia 76:271-278. The complex cellulolytic microbial community of the horse intestines is a convenient model for studying the ecology of bacteriophages in natural habitats. Unlike the rumen of the ruminants, this community of the equine large intestine is not subjected to digestion. The inner conditions of the horse gut are much more stable in comparison to other mammals, due to the fact that the horse diet remains almost unchanged and the intervals between food consumption and defecation are much shorter than the whole digestive cycle. The results of preliminary analysis of the structure and dynamics of the viral community of horse feces, which combines direct and culture methods, are presented. In horse fecal samples, we detected more than 60 morphologically distinct phage types, the majority of which were present as a single phage particle. This indicates that the community includes no less than several hundreds of phage types. Some phage types dominated and constituted 5-11% of the total particle count each. The most numerous phage type had an unusual morphology: the tails of its members were extremely long (about 700 nm), flexible, and irretractable, while their heads were 100 nm in diameter. Several other phage types with similar but not identical properties were detected. The total coliphage plaque count of the samples taken from three animals revealed significant fluctuations in the phage titers. During the observation time, the maximum titer ranged within four orders of magnitude (10(3)-10(7) plaque forming units (PFU)/g); the minimum titer ranged within two orders of magnitude. The samples contained two to five morphologically distinct and potentially competitive coliphage types, specific to a single Escherichia coli strain. [TOP OF PAGE]

  40. Phage cocktail to control the exponential growth of normal flora in processed sputum specimens grown overnight in liquid medium for rapid TB diagnosis. Kumar,V., Balaji,S., Gomathi,N.S., Venkatesan,P., Sekar,G., Jayasankar,K., Narayanan,P.R. (2007). J. Microbiol. Meth. 68:536-542. The mechanical pressure exerted during centrifugation and the chemical pressure experienced when sputum specimens are processed, leave the tubercle bacilli in the sputum unsuitable for rapid detection especially in phage based assays. Thus, growing Mycobacterium tuberculosis in broth, at least overnight, is mandatory for allowing the tubercle bacilli to recoup. During this time the surviving colonizing flora grow faster and overgrow tubercle bacilli interfering with TB diagnosis. In the present study normal flora surviving the action of 4% NaOH was isolated and characterized. Phages capable of killing 14 different species representing this normal flora were isolated from soil and sewage samples and characterized. A novel and bio-friendly approach to treat sputum samples with a cocktail of three phages capable of killing most of the 14 representative organisms and not infecting mycobacteria is explored to control the overgrowth of colonizing bacteria in broth culture. While 26 of the 100 sputum samples processed by modified Petroff's procedure showed growth of colonizing flora on blood agar, all of them when grown in broth overnight showed mixed, confluent growth. The addition of phagebiotics controlled them all, showing a significant reduction in colony forming units but resulting in few discrete colonies in 54 samples. Isolation of phages capable of controlling these surviving organisms and including them in the phagebiotics mixture should lead to the control of colonizing bacteria effectively. [TOP OF PAGE]

  41. Abortive infection mechanisms and prophage sequences significantly influence the genetic makeup of emerging lytic lactococcal phages. Labrie,S.J., Moineau,S. (2007). J. Bacteriol. 189:1482-1487. In this study, we demonstrated the remarkable genome plasticity of lytic lactococcal phages that allows them to rapidly adapt to the dynamic dairy environment. The lytic double-stranded DNA phage ul36 was used to sequentially infect a wild-type strain of Lactococcus lactis and two isogenic derivatives with genes encoding two phage resistance mechanisms, AbiK and AbiT. Four phage mutants resistant to one or both Abi mechanisms were isolated. Comparative analysis of their complete genomes, as well as morphological observations, revealed that phage ul36 extensively evolved by large-scale homologous and nonhomologous recombination events with the inducible prophage present in the host strain. One phage mutant exchanged as much as 79% of its genome compared to the core genome of ul36. Thus, natural phage defense mechanisms and prophage elements found in bacterial chromosomes contribute significantly to the evolution of the lytic phage population. [TOP OF PAGE]

  42. Importance of widespread gene transfer agent genes in a-proteobacteria. Lang,A.S., Beatty,J.T. (2007). Trends Microbiol. 15:54-62. The gene transfer agent produced by Rhodobacter capsulatus (RcGTA) is a model for several virus-like elements that seem to function solely for mediating gene exchange. Several genes that encode RcGTA are clearly related to bacteriophage genes but the cellular regulatory mechanisms that control RcGTA production indicate that RcGTA is more than just a defective prophage. Genome sequencing projects show that seemingly functional RcGTA-like structural gene clusters are present in many other species of alpha-proteobacteria, which might also produce RcGTA-like particles. Here, we use the genomic sequence data that are currently available to identify candidate GTA-producing species and propose an evolutionary scheme for RcGTA-like elements in the alpha-proteobacteria. [TOP OF PAGE]

  43. Preharvest control of Escherichia coli O157 in cattle. LeJeune,J.T., Wetzel,A.N. (2007). Journal of Animal Science 85:E73-E80 Bovine manure is an important source of Escherichia coli O157 contamination of the environment and foods; therefore, effective interventions targeted at reducing the prevalence and magnitude of fecal E. coli O157 excretion by live cattle (preharvest) are desirable. Preharvest intervention methods can be grouped into 3 categories: 1) exposure reduction strategies, 2) exclusion strategies, and 3) direct antipathogen strategies. Exposure reduction involves environmental management targeted at reducing bovine exposure to E. coli O157 through biosecurity and environmental niche management such as feed and drinking water hygiene, reduced exposure to insects or wildlife, and improved cleanliness of the bedding or pen floor. In the category of exclusion, we group vaccination and dietary modifications such as selection of specific feed components; feeding of prebiotics, probiotics, or both; and supplementation with competitive exclusion cultures to limit proliferation of E. coli O157 in or on exposed animals. Direct antipathogen strategies include treatment with sodium chlorate, antibiotics, bacteriophages, in addition to washing of animals before slaughter. Presently, only 1 preharvest control for E. coli O157 in cattle has been effective and has gained widespread adoption-the feeding probiotic Lactobacillus acidophilus. More research into the effectiveness of parallel and simultaneous application of 1 or more preharvest control strategies, as well as the identification of new pre-harvest control methods, may provide practical means to substantially reduce the incidence of human E. coli O157-related illness by intervening at the farm level. [TOP OF PAGE]

  44. Antiviral effects on bacteriophages and rotavirus by cranberry juice. Lipson,S.M., Sethi,L., Cohen,P., Gordon,R.E., Tan,I.P., Burdowski,A., Stotzky,G. (2007). Phytomedicine : international journal of phytotherapy and phytopharmacology 14:23-30. Studies were undertaken to investigate the antiviral effects of comestible juices, especially cranberry juice, on non-related viral species. After exposure of bacteriophage T2 to a commercially available cranberry (Vaccinium macrocarpon) juice cocktail (CJ), virus infectivity titer was no longer detectible. After a 60-min exposure to orange (OJ) and grapefruit juices (GJ), phage infectivity was reduced to 25-35% of control, respectively. Similar data were observed for the bacteriophage T4. CJ inactivation of phage T4 was rapid, dose-dependent, and occurred at either 4 or 23 degrees C. Neither pH nor differences in sugar/carbohydrate levels among the juices may be ascribed to the recognized antiviral effects. Further studies were performed to identify the occurrence of antiviral activity by CJ to a mammalian enteric virus. The treatment of the simian rotavirus SA-11 with a 20% CJ suspension was sufficient to inhibit hemagglutination. Under scanning and transmission electron microscopy, CJ was observed to inhibit the adsorption of phage T4 to its bacterial host cells and prevented the replication of rotavirus in its monkey kidney (MA-104) host cells, respectively. The data suggest, for the first time, a non-specific antiviral effect towards unrelated viral species (viz., bacteriophages T2 and T4 and the simian rotavirus SA-11) by a commercially available cranberry fruit juice drink. [TOP OF PAGE]

  45. Effective inhibition of lytic development of bacteriophages lambda, P1 and T4 by starvation of their host, Escherichia coli. Los,M., Golec,P., Los,J.M., Weglewska-Jurkiewicz,A., Czyz,A., Wegrzyn,A., Wegrzyn,G., Neubauer,P. (2007). BMC Biotechnol. 7:13 BACKGROUND: Bacteriophage infections of bacterial cultures cause serious problems in genetic engineering and biotechnology. They are dangerous not only because of direct effects on the currently infected cultures, i.e. their devastation, but also due to a high probability of spreading the phage progeny throughout a whole laboratory or plant, which causes a real danger for further cultivations. Therefore, a simple method for quick inhibition of phage development after detection of bacterial culture infection should be very useful. RESULTS: Here, we demonstrate that depletion of a carbon source from the culture medium, which provokes starvation of bacterial cells, results in rapid inhibition of lytic development of three Escherichia coli phages, lambda, P1 and T4. Since the effect was similar for three different phages, it seems that it may be a general phenomenon. Moreover, similar effects were observed in flask cultures and in chemostats. CONCLUSION: Bacteriophage lytic development can be inhibited efficiently by carbon source limitation in bacterial cultures. Thus, if bacteriophage contamination is detected, starvation procedures may be recommended to alleviate deleterious effects of phage infection on the culture. We believe that this strategy, in combination with the use of automated and sensitive bacteriophage biosensors, may be employed in the fermentation laboratory practice to control phage outbreaks in bioprocesses more effectively. [TOP OF PAGE]

  46. Phage passage after extended processing in small-virus-retentive filters. Lute,S., Bailey,M., Combs,J., Sukumar,M., Brorson,K. (2007). Biotechnology and applied biochemistry 47:141-151. Retention of a two small phages (PhiX-174 and pp7) by direct-flow small-virus-retentive filters [Viresolve NFP (normal-flow parvovirus), Virosart CPV (canine parvovirus), Ultipor DV20 and Planova 20N] was studied using a commercial-process fluid. Phage passage occurred in each filter type, particularly when overloaded with phage. Clearances of pp7 and PhiX-174 were similar for any given filter brand, arguing that the two phages are equivalent for testing small-virus-retentive filters. The patterns of flux under constant pressure and instantaneous LRV (log reduction value) in relationship to cumulative phage load differed between brands, consistent with the current industry understanding that each brand possesses specific performance attributes. Phages are a powerful and universal tool for evaluating filter performance. Validation of filter performance with phages such as pp7 or PhiX-174 as models for small mammalian viruses represents an attractive alternative to the current practice. [TOP OF PAGE]

  47. Bacteriophage biopanning in human tumour biopsies to identify cancer-specific targeting ligands. Maruta,F., Akita,N., Nakayama,J., Miyagawa,S., Ismail,T., Rowlands,D.C., Kerr,D.J., Fisher,K.D., Seymour,L.W., Parker,A.L. (2007). Journal of drug targeting 15:311-319. Intravenous targeting of anticancer agents should improve both efficacy and therapeutic index. However, rational design of targeting constructs requires detailed definition of receptor targets and must take account of polarised tissue architecture that may restrict access to chosen receptors from the bloodstream. Bacteriophage biopanning provides a solution to this problem, identifying targeting sequences by functional selection rather than design, although reiterative panning in polarized human tumours has not previously been attempted. Here, we report an ex vivo, intra-arterial method for biopanning in freshly-resected human tumours, enabling reiterative selection of oligopeptide sequences capable of intravascular targeting to human colorectal tumours. Significant consensus was observed after two rounds of panning in tumours from different patients, and lead sequences demonstrated tumour targeting in samples from unrelated patients. This novel approach may be applicable to a wide range of settings, thus enabling iteration of consensus targeting sequences for tumour imaging and selective delivery of anticancer agents. [TOP OF PAGE]

  48. Microbiology. New bacterial defense against phage invaders identified. Marx,J. (2007). Science (New York, N. Y. ) 315:1650-1651. [first two paragraphs] Humans are not alone in having to fend off pathogens; even the simplest organisms are under a constant threat of invasion. Bacteria, for example, are awash in a sea of viruses known as bacteriophages. "Every 2 days, half the bacteria on Earth are killed [by bacteriophages]," says phage expert Vincent Fischetti of Rockefeller University in New York City. "It's a constant battle." Researchers have now identified a new defense mechanism that helps bacteria hold their own in this battle. ¶ On page 1709, a team led by Philippe Horvath and Rodolphe Barrangou of Danisco, a Danish company that produces bacterial cultures and other materials for the food-processing industry, reports that bacteria use a system, apparently akin to the RNA interference (RNAi) system of higher organisms, to block phage reproduction, thus making them resistant to infection. [TOP OF PAGE]

  49. Crash of a population of the marine heterotrophic flagellate Cafeteria roenbergensis by viral infection. Massana,R., del Campo,J., Dinter,C., Sommaruga,R. (2007). Environ. Microbiol. 9:2660-2669. Viruses are known as important mortality agents of marine microorganisms. Most studies focus on bacterial and algal viruses, and few reports exist on viruses infecting marine heterotrophic protists. Here we show results from several incubations initiated with a microbial assemblage from the central Indian Ocean and amended with different amounts of organic matter. Heterotrophic flagellates developed up to 30 000 cells ml-1 in the most enriched incubation. A 18S rDNA clone library and fluorescent in situ hybridization counts with newly designed probes indicated that the peak was formed by Cafeteria roenbergensis and Caecitellus paraparvulus (90% and 10% of the cells respectively). Both taxa were below detection in the original sample, indicating a strong positive selective bias during the enrichment. During the peak, C. roenbergensis cells were observed with virus-like particles in the cytoplasm, and 4 days later this taxa could not be detected. Transmission electron microscopy confirmed the viral nature of these particles, which were large (280 nm), had double-stranded DNA, and were produced with a burst size of ~70. This virus was specific of C. roenbergensis as neither C. paraparvulus that was never seen infected, nor other flagellate taxa that developed in later stages of the incubation, appeared attacked. This is one of the few reports on a heterotrophic flagellate virus and the implications of this finding in the Indian Ocean are discussed. [TOP OF PAGE]

  50. Viral proteomics. Maxwell,K.L., Frappier,L. (2007). Microbiol. Mol. Biol. Rev. 71:398-411. Viruses have long been studied not only for their pathology and associated disease but also as model systems for molecular processes and as tools for identifying important cellular regulatory proteins and pathways. Recent advances in mass spectrometry methods coupled with the development of proteomic approaches have greatly facilitated the detection of virion components, protein interactions in infected cells, and virally induced changes in the cellular proteome, resulting in a more comprehensive understanding of viral infection. In addition, a rapidly increasing number of high-resolution structures for viral proteins have provided valuable information on the mechanism of action of these proteins as well as aided in the design and understanding of specific inhibitors that could be used in antiviral therapies. In this paper, we discuss proteomic studies conducted on all eukaryotic viruses and bacteriophages, covering virion composition, viral protein structures, virus-virus and virus-host protein interactions, and changes in the cellular proteome upon viral infection. [TOP OF PAGE]

  51. Simple colorimetric microplate test of phage lysis in Salmonella enterica. McLaughlin,M.R. (2007). J. Microbiol. Meth. 69:394-398. A simple microplate method, based on conversion of tetrazolium to formazan, was devised for rapidly assessing Salmonella survival after phage treatment. Results were easily interpretable. Monitoring with a microplate reader was useful, but not required. The method was used in defining phage-Salmonella interactions for selection of phage biocontrol cocktails. [TOP OF PAGE]

  52. Phage therapy of Pseudomonas aeruginosa infection in a mouse burn wound model. McVay,C.S., Velasquez,M., Fralick,J.A. (2007). Antimicrob. Agents Chemother. 51:1934-1938. Mice compromised by a burn wound injury and subjected to a fatal infection with Pseudomonas aeruginosa were administered a single dose of a Pseudomonas aeruginosa phage cocktail consisting of three different P. aeruginosa phages by three different routes: the intramuscular (i.m.), subcutaneous (s.c.), or intraperitoneal (i.p.) route. The results of these studies indicated that a single dose of the P. aeruginosa phage cocktail could significantly decrease the mortality of thermally injured, P. aeruginosa-infected mice (from 6% survival without treatment to 22 to 87% survival with treatment) and that the route of administration was particularly important to the efficacy of the treatment, with the i.p. route providing the most significant (87%) protection. The pharmacokinetics of phage delivery to the blood, spleen, and liver suggested that the phages administered by the i.p. route were delivered at a higher dose, were delivered earlier, and were delivered for a more sustained period of time than the phages administered by the i.m. or s.c. route, which may explain the differences in the efficacies of these three different routes of administration. [TOP OF PAGE]

  53. On kinetics of phage adsorption. Moldovan,R., Chapman-McQuiston,E., Wu,X.L. (2007). Biophys. J. 93:303-315. Adsorption of l-phage on sensitive bacteria Escherichia coli is a classical problem but not all issues have been resolved. One of the outstanding problems is the rate of adsorption, which in some cases appears to exceed the theoretical limit imposed by the law of random diffusion. We revisit this problem by conducting experiments along with new theoretical analyses. Our measurements show that upon incubating l-phage with bacteria Ymel, the population of unbound phage in a salt buffer decreases with time and in general obeys a double-exponential function characterized by a fast (t1) and a slow (t2) decay time. We found that both the fast and the slow processes are specific to interactions between l-phage and its receptor LamB. Such specificity motivates a kinetic model that describes the interaction between the phage and the receptor as an on-and-off process followed by an irreversible binding. The latter may be a signature of the initiation of DNA translocation. The kinetic model successfully predicts the double exponential behavior seen in the experiment and allows the corresponding rate constants to be extracted from single measurements. The weak temperature dependence of the reversible and the irreversible binding rate suggests that phage retention by the receptor is entropic in nature and that a molecular key-lock interaction may be an appropriate description of the interaction between the phage tail and the receptor. [TOP OF PAGE]

  54. Highly sensitive phage-based biosensor for the detection of b-galactosidase. Nanduri,V., Balasubramanian,S., Sista,S., Vodyanoy,V.J., Simonian,A.L. (2007). Analytica chimica acta 589:166-172. Development of real-time sensor based on the target-specific probe that make possible sensitive, rapid and selective detection and monitoring of the particular antigen molecules could be of substantial importance to the many applications. Because of its high specificity to the target molecules, excellent temperature stability, and easy production, bacterial phage might serve as a powerful biorecognition probe in biosensor applications. Here, we report extremely sensitive and specific label-free direct detection of model antigen, beta-galactosidase (beta-gal), based on surface plasmon resonance (SPR) spectroscopy. The beta-gal specific landscape phage 1G40 has been immobilized on the gold surface of SPR SPREETA sensor chip through physical adsorption [V. Nanduri, A.M. Samoylova, V.Petrenko, V. Vodyanoy and A.L.Simonian, Comparison of optical and acoustic wave phage biosensors, 206th Meeting of The Electrochemical Society, Honolulu, Hawaii, October 3-8, (2004)]. Another non-specific to the beta-gal phage, a wild-type phage F8-5, was used in the reference channel. The concentration-dependent binding of beta-gal in both channels were assessed by monitoring the sensor optical response as a function of time under different experimental conditions, and the concentration of beta-gal was computed in differential mode. Concentrations of beta-gal between 10(-12) M and 10(-7) M could be readily detected, with linear part of calibration curve between 10(-9) M and 10(-6) M. When beta-gal was pre-incubated with different concentrations of free 1G40 phage prior to exposure to the biosensor, concentration-dependent inhibition was observed, indicating on biosensor high specificity toward beta-gal. Apart from a flow through mode used to deliver the samples to the surface for the SPR sensor, batch mode sensing was also employed to study the binding of beta-gal to immobilized phage on the SPR sensor surface. Experiments using a flow through mode provided more consistent results in the full dose range and showed higher sensitivity as opposed to the batch mode studies. The mean K(d) and binding valences for the flow through mode studies was 1.3+/-0.001 nM and 1.5+/-0.03, in comparison to 26+/-0.003 nM and 2.4+/-0.01 for the batch mode studies. The average thickness of phage 1G40 adlayer deposited through flow through and batch mode was 3+/-0.002 and 0.66+/-0.001 nm, respectively. [TOP OF PAGE]

  55. The recombinant phage lysin LysK has a broad spectrum of lytic activity against clinically relevant staphylococci, including methicillin resistant Staphylococcus aureus. O'Flaherty,S., Coffey,A., Meaney,W.J., Fitzgeral,G.F., Ross,R.P. (2007). J. Bacteriol. 197:7161-7164. This study concerns the cloning, characterization, and expression of the lysin (LysK) from staphylococcal phage K in Lactococcus lactis. Lactococcal lysates containing recombinant LysK were found to inhibit a range of different species of staphylococci isolated from bovine and human infection sources, including methicillin-resistant Staphylococcus aureus. LysK thus has potential as an antimicrobial for applications in the prevention and/or treatment of infections caused by staphylococci. [TOP OF PAGE]

  56. Phage diversity in a methanogenic digester. Park,M.O., Ikenaga,H., Watanabe,K. (2007). Microb. Ecol. 53:98-103. It has been shown that phages are present in natural and engineered ecosystems and influence the structure and performance of prokaryotic communities. However, little has been known about phages occurring in anaerobic ecosystems, including those in methanogenic digesters for waste treatment. This study investigated phages produced in an upflow anaerobic sludge blanket methanogenic digester treating brewery wastes. Phage-like particles (PLPs) in the influent and effluent of the digester were concentrated and purified by sequential filtration and quantified and characterized by transmission electron microscopy (TEM), fluorescence assay, and field inversion gel electrophoresis (FIGE). Results indicate that numbers of PLPs in the effluent of the digester exceeded 1 x 10(9) L-1 and at least 10 times greater than those in the influent, suggesting that substantial amounts of PLPs were produced in the digester. A production rate of the PLPs was estimated at least 5.2 x 10(7) PLPs day-1 L-1. TEM and FIGE showed that a variety of phages were produced in the digester, including those affiliated with Siphoviridae, Myoviridae, and Cystoviridae. [TOP OF PAGE]

  57. High viral infection rates in Antarctic and Arctic bacterioplankton. Sawstrom,C., Graneli,W., Laybourn-Parry,J., Anesio,A.M. (2007). Environ. Microbiol. 9:250-255. The frequency of visibly phage-infected bacterial cells (FVIB) and the average number of phages per cell [i.e. burst size (BS)] were determined in Antarctic and Arctic ultra-oligotrophic freshwater environments. Water samples were collected from two Antarctic freshwater lakes and cryoconite holes from a glacier in the Arctic. Data from this bipolar study show the highest FVIB (average 26.1%, range 5.1% to 66.7%) and the lowest BS (average 4, range 2-15) ever reported in the literature. The bacterial density is low in these ultra-oligotrophic freshwater environments but a large proportion of the bacteria are visibly infected. Our results suggest that a constant virioplankton population can be maintained in these extreme environments even though host density is low and often slow growing. [TOP OF PAGE]

  58. Isolation and characterization of a bacteriophage with an unusually large genome from the Great Salt Plains National Wildlife Refuge, Oklahoma, USA. Seaman,P.F., Day,M.J. (2007). FEMS Microbiol. Ecol. 60:1-13. In this study we present a bacteriophage isolated from the Great Salt Plains National Wildlife Refuge (GSP) that is shown to have a genome size of 340 kb, unusually large for a bacterial virus. Transmission electron microscopy analysis of the virion showed this to be a Myoviridae, the first reported to infect the genus Halomonas. This temperate phage, PhigspC, exhibits a broad host range, displaying the ability to infect two different Halomonas spp. also isolated from the GSP. The phage infection process demonstrates a high level of tolerance towards temperature, pH and salinity; however, free virions are rapidly inactivated in water unless supplemented with salt. We show that susceptibility to osmotic shock is correlated with the density of the packaged DNA (rho(pack)). Lysogens of Halomonas salina GSP21 were detrimental to host fitness at 10% salinity, but the lysogen was able to grow faster than the wild type at 20% salinity. From these results we propose that the extensive genome of PhigspC may encode environmentally relevant genes (ERGs); genes that are perhaps not essential for the phage life cycle but increase host and phage fitness in some environmental conditions. [TOP OF PAGE]

  59. Evolution and the complexity of bacteriophages. Serwer,P. (2007). Virol. J. 4:30 BACKGROUND: The genomes of both long-genome (> 200 Kb) bacteriophages and long-genome eukaryotic viruses have cellular gene homologs whose selective advantage is not explained. These homologs add genomic and possibly biochemical complexity. Understanding their significance requires a definition of complexity that is more biochemically oriented than past empirically based definitions. HYPOTHESIS: Initially, I propose two biochemistry-oriented definitions of complexity: either decreased randomness or increased encoded information that does not serve immediate needs. Then, I make the assumption that these two definitions are equivalent. This assumption and recent data lead to the following four-part hypothesis that explains the presence of cellular gene homologs in long bacteriophage genomes and also provides a pathway for complexity increases in prokaryotic cells: (1) Prokaryotes underwent evolutionary increases in biochemical complexity after the eukaryote/prokaryote splits. (2) Some of the complexity increases occurred via multi-step, weak selection that was both protected from strong selection and accelerated by embedding evolving cellular genes in the genomes of bacteriophages and, presumably, also archaeal viruses (first tier selection). (3) The mechanisms for retaining cellular genes in viral genomes evolved under additional, longer-term selection that was stronger (second tier selection). (4) The second tier selection was based on increased access by prokaryotic cells to improved biochemical systems. This access was achieved when DNA transfer moved to prokaryotic cells both the more evolved genes and their more competitive and complex biochemical systems. TESTING THE HYPOTHESIS: I propose testing this hypothesis by controlled evolution in microbial communities to (1) determine the effects of deleting individual cellular gene homologs on the growth and evolution of long genome bacteriophages and hosts, (2) find the environmental conditions that select for the presence of cellular gene homologs, (3) determine which, if any, bacteriophage genes were selected for maintaining the homologs and (4) determine the dynamics of homolog evolution. IMPLICATIONS OF THE HYPOTHESIS: This hypothesis is an explanation of evolutionary leaps in general. If accurate, it will assist both understanding and influencing the evolution of microbes and their communities. Analysis of evolutionary complexity increase for at least prokaryotes should include analysis of genomes of long-genome bacteriophages. [TOP OF PAGE]

  60. Propagating the missing bacteriophages: a large bacteriophage in a new class. Serwer,P., Hayes,S.J., Thomas,J.A., Hardies,S.C. (2007). Virol. J. 4:21 The number of successful propagations/isolations of soil-borne bacteriophages is small in comparison to the number of bacteriophages observed by microscopy (great plaque count anomaly). As one resolution of the great plaque count anomaly, we use propagation in ultra-dilute agarose gels to isolate a Bacillus thuringiensis bacteriophage with a large head (95 nm in diameter), tail (486 x 26 nm), corkscrew-like tail fibers (187 x 10 nm) and genome (221 Kb) that cannot be detected by the usual procedures of microbiology. This new bacteriophage, called 0305phi8-36 (first number is month/year of isolation; remaining two numbers identify the host and bacteriophage), has a high dependence of plaque size on the concentration of a supporting agarose gel. Bacteriophage 0305phi8-36 does not propagate in the traditional gels used for bacteriophage plaque formation and also does not produce visible lysis of liquid cultures. Bacteriophage 0305phi8-36 aggregates and, during de novo isolation from the environment, is likely to be invisible to procedures of physical detection that use either filtration or centrifugal pelleting to remove bacteria. Bacteriophage 0305phi8-36 is in a new genomic class, based on genes for both structural components and DNA packaging ATPase. Thus, knowledge of environmental virus diversity is expanded with prospect of greater future expansion. [TOP OF PAGE]

  61. Grazer and virus-induced mortality of bacterioplankton accelerates development of Flectobacillus populations in a freshwater community. Simek,K., Weinbauer,M.G., Hornak,K., Jezbera,J., Nedoma,J., Dolan,J.R. (2007). Environ. Microbiol. 9:789-800. We present a detailed analysis of the effects of distinct bacterial mortality factors, viral lysis and heterotrophic nanoflagellates (HNF) bacterivory, associated with the development of filamentous Flectobacillus populations. Reservoir bacterioplankton communities were subjected to additions of both HNF and viruses together, or HNF alone, and then incubated in situ in dialyses bags. For distinct bacterial groups, mortality or growth stimulation was analysed by examining bacterial prey ingested in HNF food vacuoles with fluorescence in situ hybridization (FISH) and via FISH with microautoradiography (MAR-FISH). We also developed a semi-quantitative MAR-FISH-based estimation of relative activities of Flectobacillus populations (targeted by the R-FL615 probe). Bacterial groups vulnerable to HNF predation (mainly clusters of Betaproteobacteria), or discriminated against (Actinobacteria), were detected. Bacterial lineages most vulnerable to virus-lysis (mainly the Betaproteobacteria not targeted by the R-BT065 probe, of the Polynucleobacter cluster) were identified by comparing treatments with HNF alone to HNF and viruses together. Filaments affiliated with the Flectobacillus cluster appeared in both treatments, but were about twice as abundant, long and active as in incubations with viruses and HNF as compared with HNF alone. Viruses appeared to selectively suppress several bacterial groups, perhaps enhancing substrate availability thus stimulating growth and activity of filamentous Flectobacillus. [TOP OF PAGE]

  62. Occurrence and persistence of bacterial and viral faecal indicators in wastewater biofilms. Skraber,S., Helmi,K., Willame,R., Ferreol,M., Gantzer,C., Hoffmann,L., Cauchie,H.M. (2007). Water Sci. Technol. 55:377-385. Biofilms within wastewater treatment plants can capture enteric microorganisms initially present in the water phase immobilising them either definitively or temporarily. Consequently, fates of microorganisms may totally change depending on whether they interact or not with biofilms. In this study, we assessed the stability of wastewater biofilms comparing the evolution of the concentrations of bacteria (heterotrophic plate count [HPC], thermotolerant coliforms [TC]) and viral (somatic coliphages [SC] and F-specific phages [F +]) indicators in the biofilms and in the corresponding wastewaters at 4 and 20 dgrees C. Additionally, we assessed the monthly occurrence of these bacterial and viral indicators as well as of pathogenic protozoa (Cryptosporidium oocysts and Giardia cysts) in three native wastewater biofilms for four months. Our results show that viral indicators (SC and F + ) persist longer in biofilms than in the corresponding wastewaters at 4 degrees C as well as at 20 degrees C. In contrast, persistence of bacterial indicators (TC and HPC) depends on both the temperature and the matrix. Differences between viral and bacterial persistence are discussed. Monthly analysis of native wastewater biofilms shows that bacterial and viral indicators, as well as Cryptosporidium oocysts and Giardia cysts, attach to wastewater biofilms to a concentration that remains stable in time, probably as a result of a dynamic equilibrium between attachment and detachment processes. [TOP OF PAGE]

  63. Microbial source tracking: a forensic technique for microbial source identification? Stapleton,C.M., Wyer,M.D., Kay,D., Crowther,J., McDonald,A.T., Walters,M., Gawler,A., Hindle,T. (2007). Journal of environmental monitoring : JEM 9:427-439. As the requirements of the Water Framework Directive (WFD) and the US Clean Water Act (USCWA) for the maintenance of microbiological water quality in 'protected areas' highlight, there is a growing recognition that integrated management of point and diffuse sources of microbial pollution is essential. New information on catchment microbial dynamics and, in particular, the sources of faecal indicator bacteria found in bathing and shellfish harvesting waters is a pre-requisite for the design of any 'programme of measures' at the drainage basin scale to secure and maintain compliance with existing and new health-based microbiological standards. This paper reports on a catchment-scale microbial source tracking (MST) study in the Leven Estuary drainage basin, northwest England, an area for which quantitative faecal indicator source apportionment empirical data and land use information were also collected. Since previous MST studies have been based on laboratory trials using 'manufactured' samples or analyses of spot environmental samples without the contextual microbial flux data (under high and low flow conditions) and source information, such background data are needed to evaluate the utility of MST in USCWA total maximum daily load (TMDL) assessments or WFD 'Programmes of Measures'. Thus, the operational utility of MST remains in some doubt. The results of this investigation, using genotyping of Bacteroidetes using polymerase chain reaction (PCR) and male-specific ribonucleic acid coliphage (F + RNA coliphage) using hybridisation, suggest some discrimination is possible between livestock- and human-derived faecal indicator concentrations but, in inter-grade areas, the degree to which the tracer picture reflected the land use pattern and probable faecal indicator loading were less distinct. Interestingly, the MST data was more reliable on high flow samples when much of the faecal indicator flux from catchment systems occurs. Whilst a useful supplementary tool, the MST information did not provide quantitative source apportionment for the study catchment. Thus, it could not replace detailed empirical measurement of microbial flux at key catchment outlets to underpin faecal indicator source apportionment. Therefore, the MST techniques reported herein currently may not meet the standards required to be a useful forensic tool, although continued development of the methods and further catchment scale studies could increase confidence in such methods for future application. [TOP OF PAGE]

  64. Inactivation of calcium-dependent lactic acid bacteria phages by phosphates. Suarez,V.B., Capra,M.L., Rivera,M., Reinheimer,J.A. (2007). J. Food Prot. 70:1518-1522. The capacity of three phosphates to interrupt the lytic cycle of four specific autochthonal bacteriophages of lactic acid bacteria used as starters was assayed. The phosphates used (polyphosphates A and B and sodium tripolyphosphate-high solubility [TAS]) were selected on the basis of their capacity to sequester divalent cations, which are involved in the lytic cycle of certain bacteriophages. The assays were performed in culture media (deMan Rogosa Sharpe and Elliker broths) and reconstituted (10%, wt/vol) commercial skim milk to which phosphates had been added at concentrations of 0.1, 0.3, and 0.5% (wt/vol). Phosphate TAS was the most inhibitory one, since it was able to inhibit the lytic cycle of all bacteriophages studied, in both broths and milk. In broth, polyphosphates A and B inhibited the lytic cycle of only two bacteriophages at the maximal concentration used (0.5%), whereas in milk, they were not capable of maintaining the same inhibitory effect. [TOP OF PAGE]

  65. Removal of particle-associated bacteriophages by dual-media filtration at different filter cycle stages and impacts on subsequent UV disinfection. Templeton,M.R., Andrews,R.C., Hofmann,R. (2007). Water Res. 41:2393-2406. This bench-scale study investigated the passage of particle-associated bacteriophage through a dual-media (anthracite-sand) filter over a complete filter cycle and the effect on subsequent ultraviolet (UV) disinfection. Two model viruses, bacteriophages MS2 and T4, were considered. The water matrix was de-chlorinated tap water with either kaolin or Aldrich humic acid (AHA) added and coagulated with alum to form floc before filtration. The turbidity of the influent flocculated water was 6.4+/-1.5 NTU. Influent and filter effluent turbidity and particle counts were measured as well as headloss across the filter media. Filter effluent samples were collected for phage enumeration during three filter cycle stages: (i) filter ripening; (ii) stable operation; and (iii) end of filter cycle. Stable filter operation was defined according to a filter effluent turbidity goal of <0.3 NTU. Influent and filter effluent samples were subsequently exposed to UV light (254 nm) at 40 mJ/cm(2) using a low pressure UV collimated beam. The study found statistically significant differences (alpha=0.05) in the quantity of particle-associated phage present in the filter effluent during the three stages of filtration. There was reduced UV disinfection efficiency due to the presence of particle-associated phage in the filter effluent in trials with bacteriophage MS2 and humic acid floc. Unfiltered influent water samples also resulted in reduced UV inactivation of phage relative to particle-free control conditions for both phages. Trends in filter effluent turbidity corresponded with breakthrough of particle-associated phage in the filter effluent. The results therefore suggest that maintenance of optimum filtration conditions upstream of UV disinfection is a critical barrier to particle-associated viruses. [TOP OF PAGE]

  66. Low-cost rapid detection of rifampicin resistant tuberculosis using bacteriophage in Kampala, Uganda. Traore,H., Ogwang,S., Mallard,K., Joloba,M.L., Mumbowa,F., Narayan,K., Kayes,S., Jones-Lopez,E.C., Smith,P.G., Ellner,J.J., Mugerwa,R.D., Eisenach,K.D., McNerney,R. (2007). Annals of clinical microbiology and antimicrobials 6:1 BACKGROUND: Resistance to anti-tuberculosis drugs is a serious public health problem. Multi-drug resistant tuberculosis (MDR-TB), defined as resistance to at least rifampicin and isoniazid, has been reported in all regions of the world. Current phenotypic methods of assessing drug susceptibility of M. tuberculosis are slow. Rapid molecular methods to detect resistance to rifampicin have been developed but they are not affordable in some high prevalence countries such as those in sub Saharan Africa. A simple multi-well plate assay using mycobacteriophage D29 has been developed to test M. tuberculosis isolates for resistance to rifampicin. The purpose of this study was to investigate the performance of this technology in Kampala, Uganda. METHODS: In a blinded study 149 M. tuberculosis isolates were tested for resistance to rifampicin by the phage assay and results compared to those from routine phenotypic testing in BACTEC 460. Three concentrations of drug were used 2, 4 and 10 microg/ml. Isolates found resistant by either assay were subjected to sequence analysis of a 81 bp fragment of the rpoB gene to identify mutations predictive of resistance. Four isolates with discrepant phage and BACTEC results were tested in a second phenotypic assay to determine minimal inhibitory concentrations. RESULTS: Initial analysis suggested a sensitivity and specificity of 100% and 96.5% respectively for the phage assay used at 4 and 10 microg/ml when compared to the BACTEC 460. However, further analysis revealed 4 false negative results from the BACTEC 460 and the phage assay proved the more sensitive and specific of the two tests. Of the 39 isolates found resistant by the phage assay 38 (97.4%) were found to have mutations predictive of resistance in the 81 bp region of the rpoB gene. When used at 2 mug/ml false resistant results were observed from the phage assay. The cost of reagents for testing each isolate was estimated to be 1.3 US dollars when testing a batch of 20 isolates on a single 96 well plate. Results were obtained in 48 hours. CONCLUSION: The phage assay can be used for screening of isolates for resistance to rifampicin, with high sensitivity and specificity in Uganda. The test may be useful in poorly resourced laboratories as a rapid screen to differentiate between rifampicin susceptible and potential MDR-TB cases. [TOP OF PAGE]

  67. Prevalence and numbers of coliphages and Campylobacter jejuni bacteriophages in New Zealand foods. Tsuei,A.C., Carey-Smith,G.V., Hudson,J.A., Billington,C., Heinemann,J.A. (2007). Int. J. Food Microbiol. 116:121-125. Vegetable samples were tested for the presence of coliphages. None of the 55 samples contained these phages at concentrations greater than 10 g(-1) (the limit of detection). Spiking and recovery experiments indicated that the method was efficient at detecting coliphage T4 added to the food, and so it was concluded that phage titres were not being falsely underestimated. In addition 51 samples of chicken skin from retail portions were tested for the presence and numbers of coliphages and for presence only of Campylobacter jejuni phages. Coliphages were isolated from 46 samples (90.2% positive), at up to 2570 PFU 10 g sample(-1) but no C. jejuni phages were isolated. Several other methods were used to isolate C. jejuni phages from retail chicken but none was successful. However, when pooled whole chicken rinses from 39 flocks were tested for the presence of C. jejuni phages, 11 (28.2%) of the flocks were positive. It is possible that phages present on birds at the start of processing were either inactivated or simply diluted out during spin chilling. These data add to the body of information indicating that phages can readily be isolated from certain foods and indicate that consumers are exposed to them on a regular basis. [TOP OF PAGE]

  68. Phage-resistant mutants of Lactobacillus delbrueckii may have functional properties that differ from those of parent strains. Vinderola,G., Marco,M.B., Guglielmotti,D.M., Perdigon,G., Giraffa,G., Reinheimer,J., Quiberoni,A. (2007). Int. J. Food Microbiol. 116:96-102. Three commercial phage sensitive Lactobacillus delbrueckii strains (identified as Ab(1), YSD V and Ib(3)), and four spontaneous phage-resistant mutants isolated from them were tested for their capacity to activate the gut mucosal immune response in mice, as indicated by the numbers of IgA-producing cells. Random Amplified Polymorphic DNA (RAPD) analysis revealed a strong genetic homology between the sensitive strains and their respective derivatives. The phage-resistant mutants exhibited high levels of phage resistance, elevated stability of this phenotype and technological properties comparable to those of their respective parent strains. The tolerance to acidic conditions, bile salts and lysozyme was strain dependent and total cell viability losses as a result of exposure to all three stresses ranged from 2.0 to 3.7 log units. All the strains were highly resistant to a simulated gastric solution of pH 3, while significant additional losses in cell viability were observed when acid treated cells were exposed to bile salts and lysozyme. BALB/c mice received pure cultures of Lb. delbrueckii sensitive and phage-resistant strains for 2, 5 or 7 consecutive days. The ability of the parent strains to activate the small intestine immune response was preserved or enhanced in phage-resistant mutants. The maximal proliferation of IgA(+) cells was observed at day 5 or 7, depending on the strain. Mutants isolated in this study using natural selection strategies had improved phage resistance, adequate technological properties and satisfactory gut mucosal immunostimulation ability, and so would be good candidates for industrial applications in functional foods. [TOP OF PAGE]

  69. Efficacy of bacteriophage therapy against gut-derived sepsis caused by Pseudomonas aeruginosa in mice. Watanabe,R., Matsumoto,T., Sano,G., Ishii,Y., Tateda,K., Sumiyama,Y., Uchiyama,J., Sakurai,S., Matsuzaki,S., Imai,S., Yamaguchi,K. (2007). Antimicrob. Agents Chemother. 51:446-452. We evaluated the efficacy of bacteriophage (phage) therapy by using a murine model of gut-derived sepsis caused by Pseudomonas aeruginosa that closely resembles the clinical pathophysiology of septicemia in humans. Oral administration of a newly isolated lytic phage strain (KPP10) significantly protected mice against mortality (survival rates, 66.7% for the phage-treated group versus 0% for the saline-treated control group; P<0.01). Mice treated with phage also had lower numbers of viable P. aeruginosa cells in their blood, liver, and spleen. The levels of inflammatory cytokines (tumor necrosis factor alpha TNF-alpha, interleukin-1beta [IL-1beta], and IL-6) in blood and liver were significantly lower in phage-treated mice than in phage-untreated mice. The number of viable P. aeruginosa cells in fecal matter in the gastrointestinal tract was significantly lower in phage-treated mice than in the saline-treated control mice. We also studied the efficacy of phage treatment for intraperitoneal infection caused by P. aeruginosa and found that phage treatment significantly improved the survival of mice, but only under limited experimental conditions. In conclusion, our findings suggest that oral administration of phage may be effective against gut-derived sepsis caused by P. aeruginosa. [TOP OF PAGE]

  70. Bacteriophage in the Environment. Weinbauer,M.G., Agis,M., Bonilla-Findji,O., Malits,A., Winter,C. (2007). pp. 61-92. In In McGrath,S. and van Sinderen,D. (eds.), Bacteriophage: Genetics and Molecular Biology. Caister Academic Press, Norfolk, UK. One and a half decades ago, it was detected that phages are much more abundant in the water column of freshwater and marine habitats than previously thought and that they can cause significant mortality of bacterioplankton. Methods in phage community ecology have been developed to assess phage-induced mortality of bacterioplankton and its role for food web process and biogeochemical cycles, to genetically fingerprint phage communities or populations and estimate viral biodiversity by metagenomics. The release of lysis products by phages converts organic carbon from particulate (cells) to dissolved forms (lysis products), which makes organic carbon more bio-available and thus acts as a catalyst of geochemical nutrient cycles. Phages are not only the most abundant biological entities but probably also the most diverse ones. The majority of the sequence data obtained from phage communities has no equivalent in data bases. These data and other detailed analyses indicate that phage-specific genes and ecological traits are much more frequent than previously thought. In order to reveal the meaning of this genetic and ecological versatility, studies have to be performed with communities and at spatiotemporal scales relevant for microorganisms. [TOP OF PAGE]

  71. Targeted drug-carrying bacteriophages as antibacterial nanomedicines. Yacoby,I., Bar,H., Benhar,I. (2007). Antimicrob. Agents Chemother. 51:2156-2163. While the resistance of bacteria to traditional antibiotics is a major public health concern, the use of extremely potent antibacterial agents is limited by their lack of selectivity. As in cancer therapy, antibacterial targeted therapy could provide an opportunity to reintroduce toxic substances to the antibacterial arsenal. A desirable targeted antibacterial agent should combine binding specificity, a large drug payload per binding event, and a programmed drug release mechanism. Recently, we presented a novel application of filamentous bacteriophages as targeted drug carriers that could partially inhibit the growth of Staphylococcus aureus bacteria. This partial success was due to limitations of drug-loading capacity that resulted from the hydrophobicity of the drug. Here we present a novel drug conjugation chemistry which is based on connecting hydrophobic drugs to the phage via aminoglycoside antibiotics that serve as solubility-enhancing branched linkers. This new formulation allowed a significantly larger drug-carrying capacity of the phages, resulting in a drastic improvement in their performance as targeted drug-carrying nanoparticles. As an example for a potential systemic use for potent agents that are limited for topical use, we present antibody-targeted phage nanoparticles that carry a large payload of the hemolytic antibiotic chloramphenicol connected through the aminoglycoside neomycin. We demonstrate complete growth inhibition toward the pathogens Staphylococcus aureus, Streptococcus pyogenes, and Escherichia coli with an improvement in potency by a factor of approximately 20,000 compared to the free drug. [TOP OF PAGE]

  72. Specific electrochemical phage sensing for Bacillus cereus and Mycobacterium smegmatis. Yemini,M., Levi,Y., Yagil,E., Rishpon,J. (2007). Bioelectrochemistry (Amsterdam, Netherlands) 70:180-184. The rapid and reliable detection of pathogenic microorganisms is an important issue for the safety and security of our society. Here we describe the use of a sensitive, inexpensive, amperometric, phage-based biosensor for the detection of extremely low concentrations of Bacillus cereus and Mycobacterium smegmatis as models for Bacillus anthracis (the causative agent of anthrax) and for Mycobacterium tuberculosis (the causative agent of tuberculosis), respectively. The detection procedure developed here enabled the determination of bacteria at a low concentration of 10 viable cells/mL within 8 h. This experimental setup allows the simultaneous analysis of up to eight independent samples, using disposable screen-printed electrodes. [TOP OF PAGE]

  73. A quantitative comet assay: Imaging and analysis of virus plaques formed with a liquid overlay. Zhu,Y., Yin,J. (2007). J. Virol. Meth. 139:100-102. Although the plaque assay defines a "gold-standard" for measuring virus infectivity, its reliance on plaque counting limits its sensitivity. When the assay is performed with a liquid overlay, instead of agar overlay, spontaneous flows can promote a uni-directional spread of infection, creating elongated regions of cytopathology that resemble comets. As a model system comet and plaque cultures of vesicular stomatitis virus (VSV) on baby hamster kidney (BHK-21) cells were compared. Host-cell monolayers were infected with VSV particles, incubated 15 h in the presence of liquid or agar overlays and stained. VSV formed significantly larger comets than plaques, consistent with a mechanism of flow-enhanced spread. When antiviral drug (5-fluorouracil) was incorporated into the liquid overlay, comet sizes were reduced in a dose-dependent manner. Images of infected monolayers, acquired using a simple digital scanner, enabled a quantification of the inhibitory effect of the drug on infectivity. The resulting measure of drug susceptibility was found to be 18-fold more sensitive than the IC50 measure attained by the traditional plaque-reduction assay. This quantitative comet assay has the potential to similarly enhance the sensitivity of infection measures for other plaque-forming viruses. [TOP OF PAGE]

  74. Phage ecology. Abedon,S.T. (2006). pp. 37-46. In In Calendar,R. and Abedon,S.T. (eds.), The Bacteriophages. Oxford University Press, Oxford. Phages are found nearly everywhere bacteria are found and phage ecology is the study of the interactions between phages and their environments. These interactions are consequential, particularly to the extent that they affect bacteria. During the molecular characterization of phages, however, traditionally only minimal consideration of their ecology is made. Bucking these trends, here I consider, from a phage's perspective, organismal, population, community, and ecosystem ecology (Table 1). For additional approaches to the review of phage ecology as well as the related field of phage environmental microbiology see [REFS] plus various recent reviews of aquatic and ecosystem phage ecology [REFS]. Visit www.phage.org for additional phage-ecology resources. [TOP OF PAGE]

  75. Classification of Bacteriophages. Ackermann,H.-W. (2006). pp. 8-16. In In Calendar,R. and Abedon,S.T. (eds.), The Bacteriophages. Oxford University Press, Oxford. [first three paragraphs] Bacteriophages or "phages" were discovered twice in a short time. The British pathologist Frederick William Twort described in 1915 the glassy transformation of "Micrococcus" colonies by an unknown agent. Félix Hubert d'Hérelle, a French Canadian then working at the Pasteur Institute of Paris, observed the destruction of Shigella bacteria in broth (5). Contrary to Twort, he clearly recognized the viral nature of this phenomenon and devoted the rest of his scientific career to it. He coined the term "bacteriophage," devised several techniques still in use, and introduced the phage treatment of infectious diseases. For him, there was only one bacteriophage species with many races, the Bacteriophagum intestinale (6). ¶ The viral nature of bacteriophages was recognized in 1940 with the advent of the electron microscope. In 1962, Lwoff, Horne, and Tournier published a system of viruses based on morphology and nucleic acid type. They proposed the order Urovirales for tailed phages and the families Inoviridae and Microviridae for filamentous and fX-type phages, respectively (9). A further milestone was the recognition of six basic phage types: tailed phages, filamentous phages, and icosahedral phages with single-stranded DNA or single-stranded RNA. This simple scheme, proposed by Bradley in 1967 (4), is still the basis of the present edifice of phage classification. ¶ In 1971, the International Committee on Taxonomy of Viruses (ICTV) classified phages into six genera corresponding to five of Bradley's basic types, namely the T4, l, fX174, MS2, and fd phage groups, and the newly described type PM2 (16). New phage groups were added over time. The most recent development is the establishment of the order Caudavirales for tailed phages and of 15 tailed phage genera (1, 10, 14). At present, at least 5136 bacterial viruses have been examined in the electron microscope (3). This makes bacteriophages the largest viral group in nature. [TOP OF PAGE]

  76. Evaluation of the factors controlling the time-dependent inactivation rate coefficients of bacteriophage MS2 and PRD1. Anders,R., Chrysikopoulos,C.V. (2006). Environ. Sci. Technol. 40:3237-3242. Static and dynamic batch experiments were conducted to study the effects of temperature and the presence of sand on the inactivation of bacteriophage MS2 and PRD1. The experimental data suggested that the inactivation process can be satisfactorily represented by a pseudo-first-order expression with time-dependent rate coefficients. The time-dependent rate coefficients were used to determine pertinent thermodynamic properties required for the analysis of the molecular processes involved in the inactivation of each bacteriophage. A combination of high temperature and the presence of sand appears to produce the greatest disruption to the surrounding protein coat of MS2. However, the lower activation energies for PRD1 indicate a weaker dependence of the inactivation rate on temperature. Instead, the presence of air-liquid and air-solid interfaces appears to produce the greatest damage to specific viral components that are related to infection. These results indicate the importance of using thermodynamic parameters based on the time-dependent inactivation model to better predict the inactivation of viruses in groundwater. [TOP OF PAGE]

  77. The marine viromes of four oceanic regions. Angly,F.E., Felts,B., Breitbart,M., Salamon,P., Edwards,R.A., Carlson,C., Chan,A.M., Haynes,M., Kelley,S., Liu,H., Mahaffy,J.M., Mueller,J.E., Nulton,J., Olson,R., Parsons,R., Rayhawk,S., Suttle,C.A., Rohwer,F. (2006). PLoS Biol. 4:e368 Viruses are the most common biological entities in the marine environment. There has not been a global survey of these viruses, and consequently, it is not known what types of viruses are in Earth's oceans or how they are distributed. Metagenomic analyses of 184 viral assemblages collected over a decade and representing 68 sites in four major oceanic regions showed that most of the viral sequences were not similar to those in the current databases. There was a distinct "marine-ness" quality to the viral assemblages. Global diversity was very high, presumably several hundred thousand of species, and regional richness varied on a North-South latitudinal gradient. The marine regions had different assemblages of viruses. Cyanophages and a newly discovered clade of single-stranded DNA phages dominated the Sargasso Sea sample, whereas prophage-like sequences were most common in the Arctic. However most viral species were found to be widespread. With a majority of shared species between oceanic regions, most of the differences between viral assemblages seemed to be explained by variation in the occurrence of the most common viral species and not by exclusion of different viral genomes. These results support the idea that viruses are widely dispersed and that local environmental conditions enrich for certain viral types through selective pressure. [TOP OF PAGE]

  78. Micromonas pusilla reovirus: a new member of the family Reoviridae assigned to a novel proposed genus (Mimoreovirus). Attoui,H., Jaafar,F.M., Belhouchet,M., de Micco,P., de Lamballerie,X., Brussaard,C.P. (2006). J. Gen. Virol. 87:1375-1383. Micromonas pusilla reovirus (MpRV) is an 11-segmented, double-stranded RNA virus isolated from the marine protist Micromonas pusilla. Sequence analysis (including conserved termini and presence of core motifs of reovirus polymerase), morphology and physicochemical properties confirmed the status of MpRV as a member of the family Reoviridae. Electron microscopy showed that intact virus particles are unusually larger (90-95 nm) than the known size of particles of viruses belonging to the family Reoviridae. Particles that were purified on caesium chloride gradients had a mean size of 75 nm (a size similar to the size of intact particles of members of the family Reoviridae), indicating that they lost outer-coat components. The subcore particles had a mean size of 50 nm and a smooth surface, indicating that MpRV belongs to the non-turreted Reoviridae. The maximum amino acid identity with other reovirus proteins was 21 %, which is compatible with values existing between distinct genera. Based on morphological and sequence findings, this virus should be classified as the representative of a novel genus within the family Reoviridae, designated Mimoreovirus (from Micromonas pusilla reovirus). The topology of the phylogenetic tree built with putative polymerase sequences of the family Reoviridae suggested that the branch of MpRV could be ancestral. Further analysis showed that segment 1 of MpRV was much longer (5792 bp) than any other reovirus segment and encoded a protein of 200 kDa (VP1). This protein exhibited significant similarities to O-glycosylated proteins, including viral envelope proteins, and is likely to represent the additional outer coat of MpRV. [TOP OF PAGE]

  79. Use of the Weibull model for lactococcal bacteriophage inactivation by high hydrostatic pressure. Avsaroglu,M.D., Buzrul,S., Alpas,H., Akcelik,M., Bozoglu,F. (2006). Int. J. Food Microbiol. 108:78-83. Four lactococcal bacteriophages (fLl6-2, fLl35-6, fLd66-36 and fLd67-42) in M17 broth were pressurized at 300 and 350 MPa at room temperature and their survival curves were determined at various time intervals. Tailing (monotonic upward concavity) was observed in all survival curves. The resulting non-linear semi-logarithmic survival curves were described by the Weibull model and goodness of fit of this model was investigated. Regression coefficients (R2), root mean square error (RMSE), residual and correlation plots strongly suggested that Weibull model produced a better fit to the data than the traditional linear model. Hazard plots suggested that the Weibull model was fully appropriate for the data being analyzed. These results have confirmed that the Weibull model, which is mostly utilized to describe the inactivation of bacterial cells or spores by heat and pressure, could be successfully used in describing the lactococcal bacteriophage inactivation by high hydrostatic pressure. [TOP OF PAGE]

  80. A recombinant bacteriophage-based assay for the discriminative detection of culturable and viable but nonculturable Escherichia coli O157:H7. Awais,R., Fukudomi,H., Miyanaga,K., Unno,H., Tanji,Y. (2006). Biotechnol. Prog. 22:853-859. A previously green fluorescent protein (GFP)-labeled PP01 virulent bacteriophage, specific to Escherichia coli O157:H7, was used to construct lysozyme-inactivated GFP-labeled PP01 phage (PP01e-/GFP). The new recombinant phage lacked lytic activity because of the inactivation of gene e, which produces the lysozyme responsible for cell lysis. Gene e was inactivated by inserting an amber stop codon. Prolonged incubation of E. coli O157:H7 cells with PP01e-/GFP did not lead to cell lysis, while the propagation of PP01e-/GFP in host cells increased the intensity of green fluorescence. Retention of cell morphology and increase in fluorescence enabled the direct visualization and enumeration of E. coli O157:H7 cells within an hour. The PP01e-/GFP system, when combined with nutrient uptake analysis, further allowed the discriminative detection of culturable, viable but nonculturable (VBNC), and dead cells in the stress-induced aquatic environment. Stress-induced cells, which retained culturability, allowed phage propagation and produced bright green florescence. Nonculturable cells (VBNC and dead) allowed only phage adsorption but no proliferation and remained low fluorescent. The low-fluorescent nonculturable cells were further differentiated into VBNC and dead cells on the basis of nutrient uptake analysis. The low-fluorescent cells, which grew in size by nutrient incorporation during prolonged incubation in nutrient medium, were defined as metabolically active and in the VBNC state. The elongated VBNC cells were then easily recognizable from dead cells. The proposed assay enabled the detection and quantification of VBNC cells. Additionally, it revealed the proportion of culturable to VBNC cells within the population, as opposed to conventional techniques, which demonstrate VBNC cells as a differential value of the total viable count and the culturable cell count. [TOP OF PAGE]

  81. Prophages of Staphylococcus aureus Newman and their contribution to virulence. Bae,T., Baba,T., Hiramatsu,K., Schneewind,O. (2006). Mol. Microbiol. 62:1035-1047. Four prophages (phiNM1-4) were identified in the genome of Staphylococcus aureus Newman, a human clinical isolate. phiNM1, phiNM2 and phiNM4, members of the siphoviridae family, insert at different sites (poiA, downstream of isdB and geh) in the staphylococcal chromosome. phiNM3, a beta-haemolysin (hlb) converting phage, encodes modulators of innate immune responses (sea, sak, chp and scn) in addition to other virulence genes. Replication of phiNM1, phiNM2 and phiNM4 occurs in culture and during animal infection, whereas phiNM3 prophage replication was not observed. Prophages were excised from the chromosome and S. aureus variants lacking phiNM3 or phiNM1, phiNM2 and phiNM4 displayed organ specific virulence defects in a murine model of abscess formation. S. aureus Newman lacking all four prophages was unable to cause disease, thereby revealing essential contributions of prophages to the pathogenesis of staphylococcal infections. [TOP OF PAGE]

  82. Identification of a diagnostic marker to detect freshwater cyanophages of filamentous cyanobacteria. Baker,A.C., Goddard,V.J., Davy,J., Schroeder,D.C., Adams,D.G., Wilson,W.H. (2006). Appl. Environ. Microbiol. 72:5713-5719. Cyanophages are viruses that infect the cyanobacteria, globally important photosynthetic microorganisms. Cyanophages are considered significant components of microbial communities, playing major roles in influencing host community diversity and primary productivity, terminating cyanobacterial water blooms, and influencing biogeochemical cycles. Cyanophages are ubiquitous in both marine and freshwater systems; however, the majority of molecular research has been biased toward the study of marine cyanophages. In this study, a diagnostic probe was developed to detect freshwater cyanophages in natural waters. Oligonucleotide PCR-based primers were designed to specifically amplify the major capsid protein gene from previously characterized freshwater cyanomyoviruses that are infectious to the filamentous, nitrogen-fixing cyanobacterial genera Anabaena and Nostoc. The primers were also successful in yielding PCR products from mixed virus communities concentrated from water samples collected from freshwater lakes in the United Kingdom. The probes are thought to provide a useful tool for the investigation of cyanophage diversity in freshwater environments. [TOP OF PAGE]

  83. Bacteriophages of freshwater Brevundimonas vesicularis isolates. Beilstein,F., Dreiseikelmann,B. (2006). Res. Microbiol. 157:213-219. Nine strains of Brevundimonas vesicularis were isolated from surface water of three ponds in Bielefeld, Germany. With those strains as indicators seven bacteriophages with different host ranges were isolated. Molecular characterization showed that all phages contained linear double-stranded DNA with a similar genome size of about 37 kb. Restriction analysis and hybridization of phage DNAs revealed that three of these phages are closely related to each other. These phages had morphologies typical of the family Siphoviridae. Their genomes contained cohesive ends. Four phages were classified into the family of Podoviridae. Restriction analysis of the DNAs of these phages did not reveal any similarities. The DNA of these phages were terminally redundant. All phages were unable to transduce plasmids or marker genes. [TOP OF PAGE]

  84. Bacteroides spp. as reliable marker of sewage contamination in Hawaii's environmental waters using molecular techniques. Betancourt,W.Q., Fujioka,R.S. (2006). Water Sci. Technol. 54:101-107. Standard PCR (SPCR) and quantitative PCR (QPCR) assays using primers for general and for human-specific Bacteroides 16S rRNA markers were selected as the molecular tests to assess sewage contamination in recreational waters of Hawaii and these same water samples were assayed for culturable concentrations of selected faecal microbial indicators. The results of this study showed that the general primer for Bacteroides was not useful because ambient and polluted water samples were positive for this marker. However, use of human-specific primers reliably detected sewage contamination. The human-specific Bacteroides detection data supported previously reported conclusions that concentrations of alternative faecal indicators (C. perfringens, FRNA coliphages) but not traditional faecal indicators (faecal coliform, E. coli, enterococci) are reliable indicators of faecal contamination in Hawaii's environmental waters. The QPCR assay for the human-specific Bacteroides 16S rRNA marker was faster, more sensitive and more reliable than comparable SPCR assay because OPCR assay provided additional information such as melting temperatures, which confirmed that the right amplicons were being measured and Ct values, which indicated the relative level of faecal contamination. [TOP OF PAGE]

  85. Virus-bacterium interactions in water and sediment of West African inland water system. Bettarel,Y., Bouvy,M., Dumont,C., Sime-Ngando,T. (2006). Appl. Environ. Microbiol. 72:5274-5282. The ecology of virioplankton in tropical aquatic ecosystems is poorly documented, and in particular, there are no references concerning African continental waters in the literature. In this study, we examined virusbacterium interactions in the pelagic and benthic zones of seven contrasting shallow inland waters in Senegal, including one hypersaline lake. SYBR Gold-stained samples revealed that in the surface layers of the sites, the numbers of viruses were in the same range as the numbers of viruses reported previously for productive temperate systems. Despite high bacterial production rates, the percentages of visibly infected cells (as determined by transmission electron microscopy) were similar to the lowest percentages (range, 0.3 to 1.1%; mean, 0.5%) found previously at pelagic freshwater or marine sites, presumably because of the local environmental and climatic conditions. Since the percentages of lysogenic bacteria were consistently less than 8% for pelagic and benthic samples, lysogeny did not appear to be a dominant strategy for virus propagation at these sites. In the benthic samples, viruses were highly concentrated, but paradoxically, no bacteria were visibly infected. This suggests that sediment provides good conditions for virus preservation but ironically is an unfavorable environment for proliferation. In addition, given the comparable size distributions of viruses in the water and sediment samples, our results support the paradigm that aquatic viruses are ubiquitous and may have moved between the two compartments of the shallow systems examined. Overall, this study provides additional information about the relevance of viruses in tropical areas and indicates that the intensity of virus-bacterium interactions in benthic habitats may lower than the intensity in the adjacent bodies of water. [TOP OF PAGE]

  86. Control of bacteriophage contamination in commercial microbiology and fermentation facilities. Bogosian,G. (2006). pp. 667-673. In In Calendar,R. and Abedon,S.T. (eds.), The Bacteriophages. Oxford University Press, Oxford. [first two paragraph] There are an estimated 5 x 10(30) bacterial cells on Earth (29), suggesting a global population of about 1 x 10(31) bacteriophages. Roger Hendrix, in his characteristic whimsical fashion, has calculated that if bacteriophages were the size of cockroaches, they would cover the surface of the Earth in a layer 50,000 km deep. This type of thinking sends chills through those who work in commercial fermentation facilities, where bacteriophages really are considered cockroaches. ¶ Experienced members of the commercial fermentation industry live in constant fear of bacteriophage contamination. Products obtained from the large-scale fermentation of bacteria include a wide variety of foods, pharmaceuticals, vitamins, solvents, enzymes, and more. The loss of an entire fermentation batch to bacteriophage contamination is a dramatic and unsettling event, often leading to such heavy bacteriophage contamination of the fermentation facility that further fermentation runs are not possible for an extended period of time. In addition, raw materials, isolation and purification equipment, and finished product may be contaminated by bacteriophage. The economic setbacks from product loss, raw material spoilage, and nonproductive operation costs can be substantial. Bacteriophage contamination can become an extremely distressing and frustrating problem for fermentation-based industries. There have been instances when bacteriophage outbreaks have taken months to bring under control. This chapter explores the possible causes of bacteriophage outbreaks, and the most effective approaches to handle the problem. [TOP OF PAGE]

  87. The tripartite associations between bacteriophage, Wolbachia, and arthropods. Bordenstein,S.R., Marshall,M.L., Fry,A.J., Kim,U., Wernegreen,J.J. (2006). PLoS Pathog. 2:e43 By manipulating arthropod reproduction worldwide, the heritable endosymbiont Wolbachia has spread to pandemic levels. Little is known about the microbial basis of cytoplasmic incompatibility (CI) except that bacterial densities and percentages of infected sperm cysts associate with incompatibility strength. The recent discovery of a temperate bacteriophage (WO-B) of Wolbachia containing ankyrin-encoding genes and virulence factors has led to intensifying debate that bacteriophage WO-B induces CI. However, current hypotheses have not considered the separate roles that lytic and lysogenic phage might have on bacterial fitness and phenotype. Here we describe a set of quantitative approaches to characterize phage densities and its associations with bacterial densities and CI. We enumerated genome copy number of phage WO-B and Wolbachia and CI penetrance in supergroup A- and B-infected males of the parasitoid wasp Nasonia vitripennis. We report several findings: (1) variability in CI strength for A-infected males is positively associated with bacterial densities, as expected under the bacterial density model of CI, (2) phage and bacterial densities have a significant inverse association, as expected for an active lytic infection, and (3) CI strength and phage densities are inversely related in A-infected males; similarly, males expressing incomplete CI have significantly higher phage densities than males expressing complete CI. Ultrastructural analyses indicate that approximately 12% of the A Wolbachia have phage particles, and aggregations of these particles can putatively occur outside the Wolbachia cell. Physical interactions were observed between approximately 16% of the Wolbachia cells and spermatid tails. The results support a low to moderate frequency of lytic development in Wolbachia and an overall negative density relationship between bacteriophage and Wolbachia. The findings motivate a novel phage density model of CI in which lytic phage repress Wolbachia densities and therefore reproductive parasitism. We conclude that phage, Wolbachia, and arthropods form a tripartite symbiotic association in which all three are integral to understanding the biology of this widespread endosymbiosis. Clarifying the roles of lytic and lysogenic phage development in Wolbachia biology will effectively structure inquiries into this research topic. [TOP OF PAGE]

  88. Prophage Finder: a prophage loci prediction tool for prokaryotic genome sequences. Bose,M., Barber,R.D. (2006). In silico biology 6:223-227. Prophage loci often remain under-annotated or even unrecognized in prokaryotic genome sequencing projects. A PHP application, Prophage Finder, has been developed and implemented to predict prophage loci, based upon clusters of phage-related gene products encoded within DNA sequences. This application provides results detailing several facets of these clusters to facilitate rapid prediction and analysis of prophage sequences. Prophage Finder was tested using previously annotated prokaryotic genomic sequences with manually curated prophage loci as benchmarks. Additional analyses from Prophage Finder searches of several draft prokaryotic genome sequences are available through the Web site (http://bioinformatics.uwp.edu/~phage/DOEResults.php) to illustrate the potential of this application. [TOP OF PAGE]

  89. Transport of coliphage in the presence and absence of manure suspension. Bradford,S.A., Tadassa,Y.F., Jin,Y. (2006). J. Environ. Qual. 35:1692-1701. Mechanisms of coliphage transport and fate in the presence and absence of manure suspension were studied in saturated column experiments. In the presence of manure suspension, little inactivation of indigenous somatic coliphage occurred and the transport was controlled by deposition. The deposition followed a power law distribution with depth, and the magnitude increased with decreasing sand size. Comparison of the cumulative size distribution of manure components in the suspension initially and after passage through sand, suggested that particles retained by mechanical filtration and/or straining decreased the effective pore size and potentially induced straining of the somatic coliphage. A 2-site kinetic deposition model was used to estimate the magnitudes of attachment and straining in the presence of manure suspension, and provided a good description of the data. Modeling results indicated that straining accounted for 16 to 42% of the deposited somatic coliphage, and that both straining and attachment increased with decreasing sand size due to smaller pores and higher surface area, respectively. In the absence of manure suspension, fX174 (a representative somatic coliphage) and MS2 (a male-specific RNA coliphage) transport was controlled by inactivation induced by the solid phase. This conclusion was based on comparison of coliphage transport behavior at 5 and 20 degrees C, mass balance information, and numerical modeling. Comparison of somatic coliphage transport data in the presence and absence of manure suspension revealed much higher effluent concentrations in the presence of manure. This difference was attributed to lower inactivation and higher detachment rates. The observed coliphage transport behavior suggests that survival of viruses may be extended in the presence of manure suspensions, and that transport studies conducted in the absence of manure suspension may not accurately characterize the transport potential of viruses in manure-contaminated environments. [TOP OF PAGE]

  90. Spatial heterogeneity and the stability of host-parasite coexistence. Brockhurst,M.A., Buckling,A., Rainey,P.B. (2006). J. Evol. Biol. 19:374-379. Spatially heterogeneous environments can theoretically promote more stable coexistence of hosts and parasites by reducing the risk of parasite attack either through providing permanent spatial refuges or through providing ephemeral refuges by reducing dispersal. In experimental populations of Pseudomonas aeruginosa and the bacteriophage PP7, spatial heterogeneity promoted stable coexistence of host and parasite, while coexistence was significantly less stable in the homogeneous environment. Phage populations were found to be persisting on subpopulations of sensitive bacteria. Transferring populations to fresh microcosms every 24 h prevented the development of permanent spatial refuges. However, the lower dispersal rates in the heterogeneous environment were found to reduce parasite transmission thereby creating ephemeral refuges from phage attack. These results suggest that spatial heterogeneity can stabilize an otherwise unstable host-parasite interaction even in the absence of permanent spatial refuges. [TOP OF PAGE]

  91. Are phytoplankton population density maxima predictable through analysis of host and viral genomic DNA content? Brown,C.M., Lawrence,J.E., Campbell,D.A. (2006). J. Mar. Bio. Assoc. UK 86:491-498. Phytoplankton:virus interactions are important factors in aquatic nutrient cycling and community succession. The number of viral progeny resulting from an infection of a cell critically influences the propagation of infection and concomitantly the dynamics of phytoplankton populations. Host nucleotide content may be the resource limiting viral particle assembly.We present evidence for a strong linear correlation between measured viral burst sizes and viral burst sizes predicted from the host DNA content divided by the viral genome size, across a diversity of phytoplankton:viral pairs. An analysis of genome sizes therefore supports predictions of taxon-specifc phytoplankton population density thresholds beyond which viral proliferation can trim populations or terminate phytoplankton blooms. We present corollaries showing that host:virus interactions may place evolutionary pressure towards genome reduction of both phytoplankton hosts and their viruses. [TOP OF PAGE]

  92. Ecology of microbial invasions: amplification allows virus carriers to invade more rapidly when rare. Brown,S.P., Le Chat,L., De Paepe,M., Taddei,F. (2006). Curr. Biol. 16:2048-2052. Locally adapted residents present a formidable barrier to invasion . One solution for invaders is to kill residents . Here, we explore the comparative ecological dynamics of two distinct microbial mechanisms of killing competitors, via the release of chemicals (e.g., bacteriocins ) and via the release of parasites (e.g., temperate phage ). We compared the short-term population dynamics of susceptible E. coli K12 and isogenic carriers of phage varphi80 in experimental cultures to that anticipated by mathematical models using independently derived experimental parameters. Whereas phages are a direct burden to their carriers because of probabilistic host lysis, by killing competitor bacteria they can indirectly benefit bacterial kin made immune by carrying isogenic phage. This is similar to previously described bacteriocin-mediated effects. However, unlike chemical killing, viable phage trigger an epidemic among susceptible competitors, which become factories producing more phage. Amplification makes phage carriers able to invade well-mixed susceptibles even faster when rare, whereas chemical killers can only win in a well-mixed environment when sufficiently abundant. We demonstrate that for plausible parameters, the release of chemical toxins is superior as a resident strategy to repel invasions, whereas the release of temperate phage is superior as a strategy of invasion. [TOP OF PAGE]

  93. Prophage genomics. Brüssow,H. (2006). pp. 17-25. In In Calendar,R. and Abedon,S.T. (eds.), The Bacteriophages. Oxford University Press, Oxford. [first paragraph] At the time of this writing, the Genbank phage database comprises 200 complete phage genome sequences. An equivalent amount of phage sequences were passively acquired as prophages in bacterial genome sequencing projects. The scientific value of these prophage sequences was only recently recognized (13, 14). It goes beyond their potential to double the content of the current phage database and to correct a bias of the database towards selected phage systems (coliphages, dairy phages, mycobacteriophages) (9). These prophage sequences allowed a first insight into the evolution of phage-host genome interactions at a molecular level. This analysis turned out to be especially fruitful for bacterial pathogens (1, 5, 49). [TOP OF PAGE]

  94. Lactobacillus phages. Brüssow,H., Suárez,J.E. (2006). pp. 653-663. In In Calendar,R. and Abedon,S.T. (eds.), The Bacteriophages. Oxford University Press, Oxford. [first paragraph] The studies conducted with Lactobacillus bacteriophages reflect the economic and medical importance of their hosts. Due to the variety of food fermentation lactobacilli-based processes that can become disrupted by phage development, and the diversity of mucosal surfaces colonized by lactobacilli, phage research has not concentrated on a single Lactobacillus species or phage. Lactobacillus phage research also is frequently directed by practical needs. For example, the industrially relevant properties of lactic acid bacteria are often plasmid-encoded. These plasmids are intrinsically unstable, which frequently leads to strain degeneration. The DNA-integration mechanism used by temperate Lactobacillus phages therefore has been studied to develop tools for chromosomal stabilization of economically important traits (57, 73). In addition, many fermented products undergo a ripening period that may last several months. Early lysis of the starter bacteria might accelerate ripening through release of the intracellular enzymes into the food matrix. To this aim, the expression of cloned phage-lysis cassettes, controlled by inducible promoters, has been studied (19). Furthermore, the need of phage-insensitive Lactobacillus-based fermentation starters has prompted the generation of strains harbouring a cI-like repressor gene or a phage replication origin (3, 59). [TOP OF PAGE]

  95. Evolution of tailed phages-insights from comparative phage genomics. Brüssow,H., Desiere,F. (2006). pp. 26-36. In In Calendar,R. and Abedon,S.T. (eds.), The Bacteriophages. Oxford University Press, Oxford. [first paragraph] Many phage researchers believe that phages are as old as their bacterial hosts. If this hypothesis is true, then we have to postulate elements of vertical evolution for phages. In view of the postulated antiquity of these relationships we might not expect sequence similarities between more distantly related phages. Comparative phage genomics can reveal DNA sequence, protein sequence or gene map similarities in the absence of sequence similarities between increasingly diverged phages. For even more distant relationships, data from structural biology can be informative. Recent genomics-based ideas on phage evolution are dominated by two interpretations. In one view all double-stranded DNA tailed phage genomes are mosaics with access, by horizontal exchange, to a large common genetic pool, but in which access to the gene pool is not uniform for all phages (22). In this hypothesis horizontal gene transfer dominates over vertical evolution. In fact, it is in some way an updated version of the classical modular theory of phage evolution developed 30 years ago on the basis of heteroduplex mapping with lambdoid coliphages (2, 7). Other investigators studying phages from dairy bacteria observed strong elements of vertical evolution in the structural gene cluster of phages that were not erased by horizontal gene transfer events (5). The two hypotheses are not mutually exclusive since they only set a different balance for horizontal and vertical elements in phage evolution. In the following we will present the basic observations leading to the two hypotheses, search for a synthesis of both concepts and challenge this unifying view with recent phage sequence data. [TOP OF PAGE]

  96. Antagonistic coevolution with parasites increases the cost of host deleterious mutations. Buckling,A., Wei,Y., Massey,R.C., Brockhurst,M.A., Hochberg,M.E. (2006). Proc. R. Soc. Lond. B Biol. Sci. 273:45-49. The fitness consequences of deleterious mutations are sometimes greater when individuals are parasitized, hence parasites may result in the more rapid purging of deleterious mutations from host populations. The significance of host deleterious mutations when hosts and parasites antagonistically coevolve (reciprocal evolution of host resistance and parasite infectivity) has not previously been experimentally investigated. We addressed this by coevolving the bacterium Pseudomonas fluorescens and a parasitic bacteriophage in laboratory microcosms, using bacteria with high and low mutation loads. Directional coevolution between bacterial resistance and phage infectivity occurred in all populations. Bacterial population fitness, as measured by competition experiments with ancestral genotypes in the absence of phage, declined with time spent coevolving. However, this decline was significantly more rapid in bacteria with high mutation loads, suggesting the cost of bacterial resistance to phage was greater in the presence of deleterious mutations (synergistic epistasis). As such, resistance to phage was more costly to evolve in the presence of a high mutation load. Consistent with these data, bacteria with high mutation loads underwent less rapid directional coevolution with their phage populations, and showed lower levels of resistance to their coevolving phage populations. These data suggest that coevolution with parasites increases the rate at which deleterious mutations are purged from host populations. [TOP OF PAGE]

  97. Optimality models of phage life history and parallels in disease evolution. Bull,J.J. (2006). J. Theor. Biol. 241:928-938. Optimality models constitute one of the simplest approaches to understanding phenotypic evolution. Yet they have shortcomings that are not easily evaluated in most organisms. Most importantly, the genetic basis of phenotype evolution is almost never understood, and phenotypic selection experiments are rarely possible. Both limitations can be overcome with bacteriophages. However, phages have such elementary life histories that few phenotypes seem appropriate for optimality approaches. Here we develop optimality models of two phage life history traits, lysis time and host range. The lysis time models show that the optimum is less sensitive to differences in host density than suggested by earlier analytical work. Host range evolution is approached from the perspective of whether the virus should avoid particular hosts, and the results match optimal foraging theory: there is an optimal ''diet'' in which host types are either strictly included or excluded, depending on their infection qualities. Experimental tests of both models are feasible, and phages provide concrete illustrations of many ways that optimality models can guide understanding and explanation. Phage genetic systems already support the perspective that lysis time and host range can evolve readily and evolve without greatly affecting other traits, one of the main tenets of optimality theory. The models can be extended to more general properties of infection, such as the evolution of virulence and tissue tropism. [TOP OF PAGE]

  98. Evolutionary feedback mediated through population density, illustrated with viruses in chemostats. Bull,J.J., Millstein,J., Orcutt,J., Wichman,H.A. (2006). Am. Nat. 167:E39-E51 A cornerstone of evolutionary ecology is that population density affects adaptation: r and K selection is the obvious example. The reverse is also appreciated: adaptation impacts population density. Yet, empirically demonstrating a direct connection between population density and adaptation is challenging. Here, we address both evolution and ecology of population density in models of viral (bacteriophage) chemostats. Chemostats supply nutrients for host cell growth, and the hosts are prey for viral reproduction. Two different chemostat designs have profoundly different consequences for viral evolution. If host and virus are confined to the same chamber, as in a predator-prey system, viral regulation of hosts feeds back to maintain low viral density (measured as infections per cell). Viral adaptation impacts host density but has a small effect on equilibrium viral density. More interesting are chemostats that supply the viral population with hosts from a virus-free refuge. Here, a type of evolutionary succession operates: adaptation at low viral density leads to higher density, but high density then favors competitive ability. Experiments support these models with both phenotypic and molecular data. Parallels to these designs exist in many natural systems, so these experimental systems may yield insights to the evolution and regulation of natural populations. [TOP OF PAGE]

  99. Pharmacodynamics of non-replicating viruses, bacteriocins and lysins. Bull,J.J., Regoes,R.R. (2006). Proc. R. Soc. Lond. B Biol. Sci. 273:2703-2712. The pharmacodynamics of antibiotics and many other chemotherapeutic agents is often governed by a 'multi-hit' kinetics, which requires the binding of several molecules of the therapeutic agent for the killing of their targets. In contrast, the pharmacodynamics of novel alternative therapeutic agents, such as phages and bacteriocins against bacterial infections or viruses engineered to target tumour cells, is governed by a 'single-hit' kinetics according to which the agent will kill once it is bound to its target. In addition to requiring only a single molecule for killing, these agents bind irreversibly to their targets. Here, we explore the pharmacodynamics of such 'irreversible, single-hit inhibitors' using mathematical models. We focus on agents that do not replicate, i.e. in the case of phage therapy, we deal only with non-lytic phages and in the case of cancer treatment, we restrict our analysis to replication of incompetent viruses. We study the impact of adsorption on dead cells, heterogeneity in adsorption rates and spatial compartmentalization. [TOP OF PAGE]

  100. The Bacteriophages. Calendar,R., Abedon,S.T. (2006). Oxford University Press, Oxford.[TOP OF PAGE]

  101. General aspects of lysogeny. Campbell,A.M. (2006). pp. 66-73. In In Calendar,R. and Abedon,S.T. (eds.), The Bacteriophages. Oxford University Press, Oxford. [first paragraph] Cell lines harboring latent viruses are common both in eukaryotes and in prokaryotes. Prokaryotes harboring latent phages are lysogenic, and the latent form of the phage is called a prophage. Phages that can enter such a latent state are called temperate, although some authors erroneously refer to them as lysogenic. [TOP OF PAGE]

  102. Isolation and sequencing of a temperate transducing phage for Pasteurella multocida. Campoy,S., Aranda,J., Alvarez,G., Barbe,J., Llagostera,M. (2006). Appl. Environ. Microbiol. 72:3154-3160. A temperate bacteriophage (F108) has been isolated through mitomycin C induction of a Pasteurella multocida serogroup A strain. F108 has a typical morphology of the family Myoviridae, presenting a hexagonal head and a long contractile tail. F108 is able to infect all P. multocida serogroup A strains tested but not those belonging to other serotypes. Bacteriophage F108, the first P. multocida phage sequenced so far, presents a 30,505-bp double-stranded DNA genome with cohesive ends (CTTCCTCCCC cos site). The F108 genome shows the highest homology with those of Haemophilus influenzae HP1 and HP2 phages. Furthermore, an F108 prophage attachment site in the P. multocida chromosome has been established to be inside a gene encoding tRNA(Leu). By using several chromosomal markers that are spread along the P. multocida chromosome, it has been demonstrated that F108 is able to perform generalized transduction. This fact, together with the absence of pathogenic genes in the F108 genome, makes this bacteriophage a valuable tool for P. multocida genetic manipulation. [TOP OF PAGE]

  103. Selection of an Escherichia coli O157:H7 bacteriophage for persistence in the circulatory system of mice infected experimentally. Capparelli,R., Ventimiglia,I., Roperto,S., Fenizia,D., Iannelli,D. (2006). Clin. Microbiol. Infect. 12:248-253. A bacteriophage lytic for Escherichia coli O157:H7 was isolated from bovine manure. Following in-vivo selection, the phage acquired the capacity to persist in the circulatory system of mice for at least 38 days. When mice were infected experimentally with E. coli O157:H7 (10(7) CFU/mouse), simultaneous injection of the mice with phage (10(8) PFU/mouse) cleared E. coli O157:H7 from the mice within 48 h. [TOP OF PAGE]

  104. Characterization of a new virulent phage (MLC-A) of Lactobacillus paracasei. Capra,M.L., Del,L.Q., Ackermann,H.W., Moineau,S., Reinheimer,J.A. (2006). J. Dairy Sci. 89:2414-2423. A new virulent bacteriophage (MLC-A) was recently isolated in Argentina from a probiotic dairy product containing a strain of Lactobacillus paracasei. Observation of the lysate with an electron microscope revealed bacteriophage particles with an icosahedral capsid of 57 +/- 2 nm; with a collar and a noncontractile tail of 156 +/- 3 nm terminating with a baseplate to which a tail fiber was attached. Therefore, phage MLC-A belongs to the Siphoviridae family. This phage was able to survive the pasteurization process and was resistant to alcohols and sodium hypochlorite (400 mg/kg). Only peracetic acid could inactivate high-titer suspensions of phages in a short time. The maximum rates of phage adsorption to its host cells were obtained at 30 degrees C with a pH between 5 and 7, and in the presence of calcium or magnesium ions. The host range of phage MLC-A encompassed L. paracasei and Lactobacillus casei strains, but it was not able to infect Lactobacillus rhamnosus or Lactobacillus gasseri strains. One-step growth kinetics of its lytic development revealed latent and burst periods of 30 and 135 min, respectively, with a burst size of about 69 +/- 4 plaque-forming units per infected cell. Phage MLC-A had a distinctive restriction profile when compared with the 2 well-studied Lactobacillus phages, PL-1 and J-1. The genome size of the MLC-A phage was estimated to be approximately 37 kb. This study presents the description of the first phage specific for L. paracasei isolated in Argentina. The isolation of phage MLC-A indicates that, beside lactic acid bacteria starters, probiotic cultures can also be sensitive to virulent phages in industrial processes. [TOP OF PAGE]

  105. Isolation and characterization of bacteriophages infecting Salmonella spp. Carey-Smith,G.V., Billington,C., Cornelius,A.J., Hudson,J.A., Heinemann,J.A. (2006). FEMS Microbiol. Lett. 258:182-186. Bacteriophages infecting Salmonella spp. were isolated from sewage using soft agar overlays containing three Salmonella serovars and assessed with regard to their potential to control food-borne salmonellae. Two distinct phages, as defined by plaque morphology, structure and host range, were obtained from a single sample of screened sewage. Phage FGCSSa1 had the broadest host range infecting six of eight Salmonella isolates and neither of two Escherichia coli isolates. Under optimal growth conditions for S. Enteritidis PT160, phage infection resulted in a burst size of 139 PFU but was apparently inactive at a temperature typical of stored foods (5 degrees C), even at multiplicity of infection values in excess of 10 000. While neither isolate had characteristics that would make them candidates for biocontrol of Salmonella spp. in foods, phage FGCSSa1 behaved unusually when grown on two Salmonella serotypes at 37 degrees C in that the addition of phages appeared to retard growth of the host, presumably by the lysis of a fraction of the host cell population. [TOP OF PAGE]

  106. Widespread occurrence of phage-encoded exotoxin genes in terrestrial and aquatic environments in Southern California. Casas,V., Miyake,J., Balsley,H., Roark,J., Telles,S., Leeds,S., Zurita,I., Breitbart,M., Bartlett,D., Azam,F., Rohwer,F. (2006). FEMS Microbiol. Lett. 261:141-149. Many human diseases are caused by pathogens that produce exotoxins. The genes that encode these exotoxins are frequently encoded by mobile DNA elements such as plasmids or phage. Mobile DNA elements can move exotoxin genes among microbial hosts, converting avirulent bacteria into pathogens. Phage and bacteria from water, soil, and sediment environments represent a potential reservoir of phage- and plasmid-encoded exotoxin genes. The genes encoding exotoxins that are the causes of cholera, diphtheria, enterohemorrhagic diarrhea, and Staphylococcus aureus food poisoning were found in soil, sediment, and water samples by standard PCR assays from locations where the human diseases are uncommon or nonexistent. On average, at least one of the target exotoxin genes was detected in approximately 15% of the more than 300 environmental samples tested. The results of standard PCR assays were confirmed by quantitative PCR (QPCR) and Southern dot blot analyses. Agreement between the results of the standard PCR and QPCR ranged from 63% to 84%; and the agreement between standard PCR and Southern dot blots ranged from 50% to 66%. Both the cholera and shiga exotoxin genes were also found in the free phage DNA fraction. The results indicate that phage-encoded exotoxin genes are widespread and mobile in terrestrial and aquatic environments. [TOP OF PAGE]

  107. Fecal contamination of agricultural soils before and after hurricane-associated flooding in North Carolina. Casteel,M.J., Sobsey,M.D., Mueller,J.P. (2006). Journal of environmental science and health. Part A, Toxic/hazardous substances & environmental engineering 41:173-184. Hurricane Floyd and other storms in 1999 caused widespread and extensive flooding of eastern North Carolina and environmental contamination with fecal wastes from municipal wastewater and livestock operations. Because wastewater contains high levels of pathogenic micro-organisms, principal health risks to humans from flooding are consumption of crops grown in fecally contaminated soil and ingestion of contaminated water. Flood waters polluted with microbial and other contaminants also may be detrimental to the health of livestock and plant crops. In the present study, agricultural soils impacted by flood waters were analyzed for bacterial and viral indicators of fecal contamination. Total coliforms, fecal coliforms, Escherichia coli, spores of Clostridium perfringens, and both male specific (F+) and somatic coliphages were recovered from soil and assayed in liquid culture media. A number of samples were positive for the presence of fecal coliforms, E. coli, and coliphages, indicating the presence of human or animal feces. Most samples were positive for total coliforms, and almost all samples contained high levels of Cl. perfringens spores. The levels of Cl. perfringens spores were significantly (P < 0.001) higher in flooded soil (post-Hurricane Floyd) compared to pre-flood soil. Persistent fecal contamination of soil, as demonstrated by the high levels of Cl. perfringens spores, suggests the need for additional or alternative measures to protect crop-growing areas, including prospective microbiological monitoring and improved protection of watersheds from incidents capable of releasing fecal material. [TOP OF PAGE]

  108. Isolation of lytic Pseudomonas aeruginosa bacteriophages from worldwide collected water samples. Ceyssens,P.J., Lavigne,R., Hertveldt,K., Volckaert,G. (2006). Communications in agricultural and applied biological sciences 71:95-98. [TOP OF PAGE]

  109. Bacteriophage WO-B and Wolbachia in natural mosquito hosts: infection incidence, transmission mode and relative density. Chauvatcharin,N., Ahantarig,A., Baimai,V., Kittayapong,P. (2006). Molecular Ecology 15:2451-2461. Bacteriophages of Wolbachia bacteria have been proposed as a potential transformation tool for genetically modifying mosquito vectors. In this study, we report the presence of the WO-B class of Wolbachia-associated phages among natural populations of several mosquito hosts. Eighty-eight percent (22/25) of Wolbachia-infected mosquito species surveyed were found to contain WO-B phages. WO-B phage orf7 sequence analysis suggested that a single strain of WO-B phage was found in most singly (23/24) or doubly (1/1) Wolbachia-infected mosquitoes. However, the single Wolbachia strain infecting Aedes perplexus was found to harbour at least two different WO-B phages. Phylogenetic analysis suggested that horizontal transmission of WO-B phages has occurred on an evolutionary scale between the Wolbachia residing in mosquitoes. On an ecological scale, a low trend of co-transmission occurred among specific WO-B phages within Wolbachia of each mosquito species. Assessment of the density of WO-B phage by real-time quantitative polymerase chain reaction (RTQ-PCR) revealed an average relative density of 7.76 x 10(5)+/- 1.61 x 10(5) orf7 copies per individual mosquito for a single Wolbachia strain infecting mosquitoes, but a threefold higher density in the doubly Wolbachia-infected Aedes albopictus. However, the average combined density of WO-B phage(s) did not correlate with that of their Wolbachia hosts, which varied in different mosquito species. We also confirmed the presence of WO-B-like virus particles in the laboratory colony of Ae. albopictus (KLPP) morphologically, by transmission electron microscopy (TEM). The viral-like particles were detected after purification and filtration of Ae. albopictus ovary extract, suggesting that at least one WO-B-like phage is active (temperate) within the Wolbachia of this mosquito vector. Nevertheless, the idea of utilizing these bacteriophages as transformation vectors still needs more investigation and is likely to be unfeasible. [TOP OF PAGE]

  110. Induction of multiple prophages from a marine bacterium: a genomic approach. Chen,F., Wang,K., Stewart,J., Belas,R. (2006). Appl. Environ. Microbiol. 72:4995-5001. Approximately 70% of sequenced bacterial genomes contain prophage-like structures, yet little effort has been made to use this information to determine the functions of these elements. The recent genomic sequencing of the marine bacterium Silicibacter sp. strain TM1040 revealed five prophage-like elements in its genome. The genomes of these prophages (named prophages 1 to 5) are approximately 74, 30, 39, 36, and 15 kb long, respectively. To understand the function of these prophages, cultures of TM1040 were treated with mitomycin C to induce the production of viral particles. A significant increase in viral counts and a decrease in bacterial counts when treated with mitomycin C suggested that prophages were induced from TM1040. Transmission electron microscopy revealed one dominant type of siphovirus, while pulsed-field gel electrophoresis demonstrated two major DNA bands, equivalent to 35 and 75 kb, in the lysate. PCR amplification with primer sets specific to each prophage detected the presence of prophages 1, 3, and 4 in the viral lysate, suggesting that these prophages are inducible, but not necessarily to the same level, while prophages 2 and 5 are likely defective or non-mitomycin C-inducible phages. The combination of traditional phage assays and modern microbial genomics provides a quick and efficient way to investigate the functions and inducibility of prophages, particularly for a host harboring multiple prophages with similar sizes and morphological features. [TOP OF PAGE]

  111. Comparative analysis of tandem T7-like promoter containing regions in enterobacterial genomes reveals a novel group of genetic islands. Chen,Z., Schneider,T.D. (2006). Nucleic Acids Research 34:1133-1147. Based on molecular information theory, 10 T7-like promoter models were built for the T7 group of phages and used to scan their host genomes and closely related genomes. 38 genomes were scanned and 12 clusters of tandem promoters were identified in nine enteropathogens. Comparative analysis of these tandem promoter-bearing regions reveals that they are similar to each other, forming prophage-like islands of 4-13 kb. Each island appears to contain two or three tandem T7-like promoters within a stretch of 150-620 bases, but there are no corresponding RNA polymerase (RNAP) genes. The promoters would transcribe two to five putative phage-related proteins, but none of these resemble known phage structural proteins. An integrase belonging to the Int family of site-specific recombinases is encoded upstream of the tandem promoters. A direct repeat of 17-24 bases was found on the ends of all 12 islands. Comparative analysis of the islands shows that these islands appear to have recombined with each other. These results suggest that the islands could encode a group of satellite phages. Activation and function of the islands may depend on transcription by a T7-like RNAP after infection by a T7-like phage or foreign DNA that encodes a T7-like RNAP. [TOP OF PAGE]

  112. Leaching of phage from Class B biosolids and potential transport through soil. Chetochine,A.S., Brusseau,M.L., Gerba,C.P., Pepper,I.L. (2006). Appl. Environ. Microbiol. 72:665-671. The objective of this study was to investigate leaching and transport of viruses, specifically those of an indigenous coliphage host specific to Escherichia coli ATTC 15597 (i.e., MS-2), from a biosolid-soil matrix. Serial extractions of 2% and 7% (solids) class B biosolid matrices were performed to determine the number of phage present in the biosolids and to evaluate their general leaching potential. Significant concentrations of coliphage were removed from the biosolids for each sequential extraction, indicating that many phage remained associated with the solid phase. The fact that phage was associated with or attached to solid particles appeared to influence the potential for release and subsequent transport of phage under saturated-flow conditions, which was examined in a series of column experiments. The results indicated that less than 8% of the indigenous coliphage initially present in the biosolids leached out of the biosolid-soil matrix. A fraction of this was subsequently transported through the sandy porous medium with minimal retention. The minimal retention observed for the indigenous phage, once released from the biosolids, was consistent with the results of control experiments conducted to examine MS-2 transport through the porous medium. [TOP OF PAGE]

  113. Bacteriophages and biotechnology: vaccines, gene therapy and antibacterials. Clark,J.R., March,J.B. (2006). Trends Biotechnol. 24:212-218. In recent years it has been recognized that bacteriophages have several potential applications in the modern biotechnology industry: they have been proposed as delivery vehicles for protein and DNA vaccines; as gene therapy delivery vehicles; as alternatives to antibiotics; for the detection of pathogenic bacteria; and as tools for screening libraries of proteins, peptides or antibodies. This diversity, and the ease of their manipulation and production, means that they have potential uses in research, therapeutics and manufacturing in both the biotechnology and medical fields. It is hoped that the wide range of scientists, clinicians and biotechnologists currently researching or putting phages to practical use are able to pool their knowledge and expertise and thereby accelerate progress towards further development in this exciting field of biotechnology. [TOP OF PAGE]

  114. Mimivirus and the emerging concept of "giant" virus. Claverie,J.-M., Ogata,H., Audic,S., Abergel,C., Suhre,K., Fournier,P.-E. (2006). Virus Res. 17:133-144. The recently discovered Acanthamoeba polyphaga Mimivirus is the largest known DNA virus. Its particle size (750 nm), genome length (1.2 million bp) and large gene repertoire (911 protein coding genes) blur the established boundaries between viruses and parasitic cellular organisms. In addition, the analysis of its genome sequence identified many types of genes never before encountered in a virus, including aminoacyl-tRNA synthetases and other central components of the translation machinery previously thought to be the signature of cellular organisms. In this article, we examine how the finding of such a giant virus might durably influence the way we look at microbial biodiversity, and lead us to revise the classification of microbial domains and life forms. We propose to introduce the word "girus" to recognize the intermediate status of these giant DNA viruses, the genome complexity of which makes them closer to small parasitic prokaryotes than to regular viruses. [TOP OF PAGE]

  115. Virus isolation studies suggest short-term variations in abundance in natural cyanophage populations of the Indian Ocean. Clokie,M., Millard,A.D., Mehta,J.Y., Mann,N.H. (2006). J. Mar. Bio. Assoc. UK 86:499-505. Cyanophage abundance has been shown to fluctuate over long timescales and with depth, but little is known about how it varies over short timescales. Previous short-term studies have relied on counting total virus numbers and therefore the phages which infect cyanobacteria cannot be distinguished from the total count. ¶ In this study, an isolation-based approach was used to determine cyanophage abundance from water samples collected over a depth profile for a 24h period from the Indian Ocean. Samples were used to infect Synechococcus sp. WH7803 and the number of plaque forming units (pfu) at each time point and depth were counted. At 10m phage numbers were similar for most time-points, but there was a distinct peak in abundance at 0100 hours. Phage numbers were lower at 25m and 50m and did not show such strong temporal variation. No phages were found below this depth. Therefore, we conclude that only the abundance of phages in surface waters showed a clear temporal pattern over a short timescale. Fifty phages from a range of depths and time points were isolated and purified. The molecular diversity of these phages was estimated using a section of the phage-encoded psbD gene and the results from a phylogenetic analysis do not suggest that phages from the deeper waters form a distinct subgroup. [TOP OF PAGE]

  116. Transcription of a 'photosynthetic' T4-type phage during infection of a marine cyanobacterium. Clokie,M.R.J., Shan,J., Bailey,S., Jia,Y., Krisch,H.M., West,S., Mann,N.H. (2006). Environ. Microbiol. 8:827-835. The transcription of S-PM2 phage following infection of Synechococcus sp. WH7803, a marine cyanobacterium, was analysed by quantitative real-time PCR. Unlike the distantly related coliphage T4, there were only two (early and late) instead of three (early, middle and late) classes of transcripts during the developmental cycle of the phage. This difference is consistent with the absence from the S-PM2 genome of T4-like middle mode promoter sequences and the transcription factors associated with their recognition. Phage S-PM2 carries the 'photosynthetic' genes psbA and psbD that encode homologues of the host photosystem II proteins D1 and D2. Transcripts of the phage psbA gene appeared soon after infection and remained at high levels until lysis. Throughout the course of infection, the photosynthetic capacity of the cells remained constant. A considerable transient increase in the abundance of the host psbA transcripts occurred shortly after infection, suggesting that the host responds to the trauma of phage infection in a similar way as it does to a variety of other environmental stresses. The very substantial transcription of the phage psbA gene during the latter phase of phage infection suggests that S-PM2 has acquired this cellular gene to ensure that D1 levels and thus photosynthesis are fully maintained until the infected cell finally lyses. Unexpectedly, transcripts of a phage-encoded S-layer protein gene were among the earliest and most abundant detected, suggesting that this partial homologue of a host protein plays an important role in the S-PM2 infection process. [TOP OF PAGE]

  117. Marine cyanophages and light. Clokie,M.R.J., Mann,N.H. (2006). Environ. Microbiol. 8:2074-2082. In contrast to the phages of heterotrophic hosts, light can play a key role in all aspects of the life cycle of phages infecting ecologically important marine unicellular cyanobacteria of the genera Synechococcus and Prochlorococcus. Phage adsorption, replication, modulation of the host cell metabolism, and survival in the environment following lysis, all exhibit light-dependent components. The analysis of cyanophage genomes has revealed the acquisition of key photosynthetic genes during the course of evolution, such as those encoding central components of the light harvesting apparatus. These discoveries are beginning to reveal novel features of the interactions between parasite and host that shape the biology of both. [TOP OF PAGE]

  118. Genetic richness of vibriophages isolated in a coastal environment. Comeau,A.M., Chan,A.M., Suttle,C.A. (2006). Environ. Microbiol. 8:1164-1176. The purpose of this study was to characterize Vibrio parahaemolyticus viruses (VpVs) isolated from different environments within and adjacent to the Strait of Georgia, and to examine the relative influences of distance and environment on host-range and genetic richness. Nearly all seawater enrichment cultures (29/31) generated isolates, implying that VpVs were widespread in the virioplankton, yet at low abundances (< 1 l(-1)). Viruses were not detected in sediments (n = 99). Fourteen of the 16 viruses characterized were siphoviruses, with genome sizes ranging from approximately 45-106 kb, and half were capable of infecting other Vibrio species. The VpVs infected bacteria isolated from oysters and sediments fairly well (55% and 46% of the host-virus combinations, respectively), but were unable to infect many of the bacteria isolated from the water column (< 13% of 112 combinations). When compared with VpVs from oysters, it was clear that the major determinant of phenotypic (host-range) and genetic richness (by the DP-RAPD assay) was not geography, but the source environment from which the VpVs originated. Therefore, the VpV population within the Strait of Georgia is a highly diverse mixture of phenotypes and genotypes. [TOP OF PAGE]

  119. In vivo transduction of an Stx-encoding phage in ruminants. Cornick,N.A., Helgerson,A.F., Mai,V., Ritchie,J.M., Acheson,D.W.K. (2006). Appl. Environ. Microbiol. 72:5086-5088. We assessed the ability of a kanamycin-marked Stx phage to move into a commensal, ovine Escherichia coli strain in the ruminant gastrointestinal tract. Transduction was detected in 19/24 sheep tested, resulting in the recovery of 47 transductants. Subtherapeutic doses of the quinolone antibiotic enrofloxacin did not increase the rate of transduction. [TOP OF PAGE]

  120. Phase-variable surface structures are required for infection of Campylobacter jejuni by bacteriophages. Coward,C., Grant,A.J., Swift,C., Philp,J., Towler,R., Heydarian,M., Frost,J.A., Maskell,D.J. (2006). Appl. Environ. Microbiol. 72:4638-4647. This study characterizes the interaction between Campylobacter jejuni and the 16 phages used in the United Kingdom typing scheme by screening spontaneous mutants of the phage-type strains and transposon mutants of the sequenced strain NCTC 11168. We show that the 16 typing phages fall into four groups based on their patterns of activity against spontaneous mutants. Screens of transposon and defined mutants indicate that the phage-bacterium interaction for one of these groups appears to involve the capsular polysaccharide (CPS), while two of the other three groups consist of flagellatropic phages. The expression of CPS and flagella is potentially phase variable in C. jejuni, and the implications of these findings for typing and intervention strategies are discussed. [TOP OF PAGE]

  121. Using bacteriophages to reduce formation of catheter-associated biofilms by Staphylococcus epidermidis. Curtin,J.J., Donlan,R.M. (2006). Antimicrob. Agents Chemother. 50:1268-1275. Use of indwelling catheters is often compromised as a result of biofilm formation. This study investigated if hydrogel-coated catheters pretreated with a coagulase-negative bacteriophage would reduce Staphylococcus epidermidis biofilm formation. Biofilms were developed on hydrogel-coated silicone catheters installed in a modified drip flow reactor. Catheter segments were pretreated with the lytic S. epidermidis bacteriophage 456 by exposing the catheter lumen to a 10-log-PFU/ml culture of the bacteriophage for 1 h at 37 degrees C prior to biofilm formation. The untreated mean biofilm cell count was 7.01+/-0.47 log CFU/cm2 of catheter. Bacteriophage treatment with and without supplemental divalent cations resulted in log-CFU/cm2 reductions of 4.47 (P<0.0001) and 2.34 (P=0.001), respectively. Divalent cation supplementation without bacteriophage treatment provided a 0.67-log-CFU/cm2 reduction (P=0.053). Treatment of hydrogel-coated silicone catheters with an S. epidermidis bacteriophage in an in vitro model system significantly reduced viable biofilm formation by S. epidermidis over a 24-h exposure period, suggesting the potential of bacteriophage for mitigating biofilm formation on indwelling catheters and reducing the incidence of catheter-related infections. [TOP OF PAGE]

  122. Possible association between phages, Hoc protein, and the immune system. Dabrowska,K., Switala-Jelen,K., Opolski,A., Gorski,A. (2006). Arch. Virol. 151:209-215. Mammals have become "an environment" for enterobacterial phage life cycles. Therefore it could be expected that bacteriophages adapt to them. This adaptation must comprise bacteriophage proteins. Gp Hoc seems to have significance neither for phage particle structure nor for phage antibacterial activity. It is evidently not necessary for the "typical" antibacterial actions of bacteriophages. But the rules of evolution make it improbable that gp Hoc really has no function, and non-essential genes of T4-type phages are probably important for phages' adaptation to their particular lifestyle. More interesting is the eukaryotic origin of gp Hoc: a resemblance to immunoglobulin-like proteins that reflects their evolutionary relation. Substantial differences in biological activity between T4 and a mutant that lacks gp Hoc were observed in a mammalian system. Hoc protein seems to be one of the molecules predicted to interact with mammalian organisms and/or modulate these interactions. [TOP OF PAGE]

  123. In vivo efficacy of phage therapy for Mycobacterium avium infection as delivered by a nonvirulent mycobacterium. Danelishvili,L., Young,L.S., Bermudez,L.E. (2006). Microb. Drug Res. 12:1-6. The emergence of mycobacteria resistant to currently available antimicrobial agents has become an important problem in modern medicine. Mycobacterium avium and M. tuberculosis are intracellular pathogens that replicate and survive within the mononuclear phagocytes. TM4 is a lytic mycobacteriophage that kills both extracellular M. avium and M. tuberculosis. When delivered by M. smegmatis transiently infected with TM4, it kills both M. avium and M. tuberculosis within RAW 264.7 macrophages. To evaluate the treatment of M. avium infection with phage in vivo, C57 BL/6 mice were infected with M. avium 109 and, 7 days later, treated either once or twice with TM4 phage (7.9 x 10(10) PFU/ml), M. smegmatis (4 x 10(8) cFU/ml), or M. smegmatis with TM4 phage delivered intravenously (i.v.). Treatment with TM4 phage alone or M. smegmatis without TM4 did not show a significant decrease in number of intracellular bacteria in the spleen compared with untreated control. In contrast, administration of M. smegmatis-TM4 resulted in a significant decrease in the number of M. avium in the spleen. However, 23% of bacteria recovered from treated mice were resistant to TM4. These in vivo studies confirmed the in vitro findings that an avirulent mycobacterium can be used as a carrier to deliver antimycobacterial phage intracellularly. [TOP OF PAGE]

  124. Soil inactivation of DNA viruses in septic seepage. Davies,C.M., Logan,M.R., Rothwell,V.J., Krogh,M., Ferguson,C.M., Charles,K., Deere,D.A., Ashbolt,N.J. (2006). J. Appl. Microbiol. 100:365-374. AIMS: To generate field-relevant inactivation data for incorporation into models to predict the likelihood of viral contamination of surface waters by septic seepage. METHODS AND RESULTS: Inactivation rates were determined for PRD1 bacteriophage and Adenovirus 2 in two catchment soils under a range of temperature, moisture and biotic status regimes. Inactivation rates presented for both viruses were significantly different at different temperatures and in different soil types (alpha = 0.05). Soil moisture generally did not significantly affect virus inactivation rate. Biotic status significantly affected inactivation rates of PRD1 in the loam soil but not the clay-loam soil. Adenovirus 2 was inactivated more rapidly in the loam soil than PRD1 bacteriophage. CONCLUSIONS: Virus inactivation rates incorporated into models should be appropriate for the climate/catchment in question with particular regard to soil type and temperature. Given that PRD1 is similar in size to adenoviruses, yet more conservative with regard to inactivation in soil, it may be a useful surrogate in studies of Adenovirus fate and transport. SIGNIFICANCE AND IMPACT OF THE STUDY: A better understanding of the factors that govern virus fate and transport in catchments would facilitate the design of barrier measures to prevent viral contamination of surface waters by septic seepage. [TOP OF PAGE]

  125. Adsorption of viruses to soil: impact of anaerobic treatment. Davis,J.A., Farrah,S.R., Wilkie,A.C. (2006). Water Sci. Technol. 54:161-167. The adsorption of viruses in untreated flushed dairy manure wastewater (FDMW), anaerobically digested flushed dairy manure wastewater (ADFDMW) and groundwater to sandy soil was investigated. Batch adsorption studies showed differential adsorption of viruses in groundwater to soil. Less than 75% of PRD1 and MS2 added to groundwater adsorbed after 1 h, but greater than 95% of FX174 and poliovirus 1 adsorbed to the soil. Adsorption differences in groundwater were related to the isoelectric points of the viruses. Suspending phages in untreated and treated wastewater reduced adsorption compared with groundwater. For MS2, more phages were adsorbed using ADFDMW than with FDMW. Adsorption of poliovirus 1 was not affected by FDMW and ADFDMW. Small column studies (6 x 2.5 cm) produced a similar trend in that adsorption was observed with groundwater and both FDMW and ADFDMW reduced virus adsorption. Groundwater, FDMW or ADFDMW did not affect the adsorption of poliovirus 1 in column studies. The major difference between FDMW and ADFDMW was in mobilisation of adsorbed viruses. The application of FDMW to soil columns with adsorbed viruses caused significantly more viruses to be mobilised than did the application of rainwater or ADFDMW. These results showed that treating FDMW by anaerobic digestion increased the adsorption of viruses to soil and decreased detachment of adsorbed viruses. As the potential for new zoonotic pathogens becomes known, the treatment of animal wastes may become mandatory. The assessment and management of viruses in manure for addressing possible risk to animal and human health is of interest. [TOP OF PAGE]

  126. Viruses' life history: Towards a mechanistic basis of a trade-off between survival and reproduction among phages. de Paepe,M., Taddei,F. (2006). PLoS Biol. 4:e193 Life history theory accounts for variations in many traits involved in the reproduction and survival of living organisms, by determining the constraints leading to trade-offs among these different traits. The main life history traits of phages—viruses that infect bacteria—are the multiplication rate in the host, the survivorship of virions in the external environment, and their mode of transmission. By comparing life history traits of 16 phages infecting the bacteria Escherichia coli, we show that their mortality rate is constant with time and negatively correlated to their multiplication rate in the bacterial host. Even though these viruses do not age, this result is in line with the trade-off between survival and reproduction previously observed in numerous aging organisms. Furthermore, a multiple regression shows that the combined effects of two physical parameters, namely, the capsid thickness and the density of the packaged genome, account for 82% of the variation in the mortality rate. The correlations between life history traits and physical characteristics of virions may provide a mechanistic explanation of this trade-off. The fact that this trade-off is present in this very simple biological situation suggests that it might be a fundamental property of evolving entities produced under constraints. Moreover, such a positive correlation between mortality and multiplication reveals an underexplored trade-off in host–parasite interactions. [TOP OF PAGE]

  127. Evaluation of the natural virucidal activity of teas for use in the phage amplification assay. de Siqueira,R.S., Dodd,C.E.R., Rees,C.E.D. (2006). Int. J. Food Microbiol. 111:259-262. Many natural products have intrinsic antimicrobial activity. In this study we have examined infusions from nine types of loose-leaf tea for their ability to inactivate bacteriophage, for use as an alternative to plant extract in a phage-based Salmonella detection assay. The results demonstrated that tea infusions, either freshly prepared or stored at 4 degrees C had virucidal action against two phages, Felix 01 and P22. Crucially, for use in the detection assay, there was no antibacterial effect of the virucide on the target bacteria. Therefore, tea was a good candidate to replace pomegranate as the virucidal agent in the phage amplification assay. [TOP OF PAGE]

  128. Viral ecology and the maintenance of novel host use. Dennehy,J.J., Friedenberg,N.A., Holt,R.D., Turner,P.E. (2006). Am. Nat. 167:429-439. Viruses can occasionally emerge by infecting new host species. However, the early phases of emergence can hinge upon ecological sustainability of the virus population, which is a product of both within-host population growth and between-host transmission. Insufficient growth or transmission can force virus extinction before the latter phases of emergence, where genetic adaptations that improve host use may occur. We examined the early phase of emergence by studying the population dynamics of RNA phages in replicated laboratory environments containing native and novel host bacteria. To predict the breadth of transmission rates allowing viral persistence on each species, we developed a simple model based on in vitro data for phage growth rate over a range of initial population densities on both hosts. Validation of these predictions using serial passage experiments revealed a range of transmission rates for which the native host was a source and the novel host was a sink. In this critical range of transmission rates, periodic exposure to the native host was sufficient for the maintenance of the viral population on the novel host. We argue that this effect should facilitate adaptation by the virus to utilize the novel host--often crucial in subsequent phases of emergence. [TOP OF PAGE]

  129. Bacteriophage migration via nematode vectors: host-parasite-consumer interactions in laboratory microcosms. Dennehy,J.J., Friedenberg,N.A., Yang,Y.W., Turner,P.E. (2006). Appl. Environ. Microbiol. 72:1974-1979. Pathogens vectored by nematodes pose serious agricultural, economic, and health threats; however, little is known of the ecological and evolutionary aspects of pathogen transmission by nematodes. Here we describe a novel model system with two trophic levels, bacteriophages and nematodes, each of which competes for bacteria. We demonstrate for the first time that nematodes are capable of transmitting phages between spatially distinct patches of bacteria. This model system has considerable advantages, including the ease of maintenance and manipulation at the laboratory bench, the ability to observe many generations in short periods, and the capacity to freeze evolved strains for later comparison to their ancestors. More generally, experimental studies of complex multispecies interactions, host-pathogen coevolution, disease dynamics, and the evolution of virulence may benefit from this model system because current models (e.g., chickens, mosquitoes, and malaria parasites) are costly to maintain, are difficult to manipulate, and require considerable space. Our initial explorations centered on independently assessing the impacts of nematode, bacterium, and phage population densities on virus migration between host patches. Our results indicated that virus transmission increases with worm density and host bacterial abundance; however, transmission decreases with initial phage abundance, perhaps because viruses eliminate available hosts before migration can occur. We discuss the microbial growth dynamics that underlie these results, suggest mechanistic explanations for nematode transmission of phages, and propose intriguing possibilities for future research. [TOP OF PAGE]

  130. Study of the morphological diversity of bacteriophages in Lake Baikal. Drucker,V.V., Dutova,N.V. (2006). Doklady biological sciences : proceedings of the Academy of Sciences of the USSR, Biological sciences sections / translated from Russian 410:421-423. [TOP OF PAGE]

  131. Shared architecture of bacteriophage SPO1 and herpesvirus capsids. Duda,R.L., Hendrix,R.W., Huang,W.M., Conway,J.F. (2006). Curr. Biol. 16:R11-R13 [first paragraph] Viruses have probably existed for as long as cells. Recent structural studies of viral capsids have revealed similarities that span the domains of life and point to distant evolutionary connections between viruses that pre-date the division of their host organisms into domains [1-3]. Comparisons of adenovirus and phage PRD1 demonstrate this emerging theme: these viruses share a unique T=25 capsid geometry with unusual 'trimeric hexons' and a common core fold for the major capsid proteins [4]. We describe a novel structural link between herpesviruses and the bacteriophage SPO1 revealed by cryo-electron microscopy (cryoEM) data showing that the SPO1 capsid has icosahedral geometry with triangulation number T=16, a value previously associated uniquely with herpesviruses, as well as an asymmetric capsid surface molecule reminiscent of the 'triplex' molecule of HSV-1. We propose that the similarities go deeper, to a common capsid protein core fold of the phage HK97 class [5]. The shared architecture suggests a common ancestor for herpesviruses and phage SPO1 and supports a distinct lineage for herpesviruses and the tailed phages. [TOP OF PAGE]

  132. Pleiotropic costs of niche expansion in the RNA bacteriophage f6. Duffy,S., Turner,P.E., Burch,C.L. (2006). Genetics 172:751-757. Natural and experimental systems have failed to universally demonstrate a trade-off between generalism and specialism. When a trade-off does occur it is difficult to attribute its cause to antagonistic pleiotropy without dissecting the genetic basis of adaptation, and few previous experiments provide these genetic data. Here we investigate the evolution of expanded host range (generalism) in the RNA virus f6, an experimental model system allowing adaptive mutations to be readily identified. We isolated 10 spontaneous host range mutants on each of three novel Pseudomonas hosts and determined whether these mutations imposed fitness costs on the standard laboratory host. Sequencing revealed that each mutant had one of nine nonsynonymous mutations in the f6 gene P3, important in host attachment. Seven of these nine mutations were costly on the original host, confirming the existence of antagonistic pleiotropy. In addition to this genetically imposed cost, we identified an epigenetic cost of generalism that occurs when phage transition between host types. Our results confirm the existence in f6 of two costs of generalism, genetic and environmental, but they also indicate that the cost is not always large. The possibility for cost-free niche expansion implies that varied ecological conditions may favor host shifts in RNA viruses. [TOP OF PAGE]

  133. Characterization of Streptococcus thermophilus host range phage mutants. Duplessis,M., Levesque,C.M., Moineau,S. (2006). Appl. Environ. Microbiol. 72:3036-3041. To investigate phage-host interactions in Streptococcus thermophilus, a phage-resistant derivative (SMQ-301R) was obtained by challenging a Tn917 library of phage-sensitive strain S. thermophilus SMQ-301 with virulent phage DT1. Mutants of phages DT1 and MD2 capable of infecting SMQ-301 and SMQ-301R were isolated at a frequency of 10(-6). Four host range phage mutants were analyzed further and compared to the two wild-type phages. Altogether, three genes (orf15, orf17, and orf18) contained point mutations leading to amino acid substitutions and were responsible for the expanded host range. These three proteins were also identified in both phages by N-terminal sequencing and/or matrix-assisted laser desorption ionization-time-of-flight mass spectrometry. The results suggest that at least three phage structural proteins may be involved in phage-host interactions in S. thermophilus. [TOP OF PAGE]

  134. Bacteriophage T5 structure reveals similarities with HK97 and T4 suggesting evolutionary relationships. Effantin,G., Boulanger,P., Neumann,E., Letellier,L., Conway,J.F. (2006). J. Mol. Biol. 361:993-1002. Evolutionary relationships between viruses may be obscure by protein sequence but unmasked by structure. Analysis of bacteriophage T5 by cryo-electron microscopy and protein sequence analysis reveals analogies with HK97 and T4 that suggest a mosaic of such connections. The T5 capsid is consistent with the HK97 capsid protein fold but has a different geometry, incorporating three additional hexamers on each icosahedral facet. Similarly to HK97, the T5 major capsid protein has an N-terminal extension, or Delta-domain that is missing in the mature capsid, and by analogy with HK97, may function as an assembly or scaffold domain. This Delta-domain is predicted to be largely coiled-coil, as for that of HK97, but is approximately 70% longer correlating with the larger capsid. Thus, capsid architecture appears likely to be specified by the Delta-domain. Unlike HK97, the T5 capsid binds a decoration protein in the center of each hexamer similarly to the "hoc" protein of phage T4, suggesting a common role for these molecules. The tail-tube has unusual trimeric symmetry that may aid in the unique two-stage DNA-ejection process, and joins the tail-tip at a disk where tail fibers attach. This intriguing mix of characteristics embodied by phage T5 offers insights into virus assembly, subunit function, and the evolutionary connections between related viruses. [TOP OF PAGE]

  135. [T4-type bacteriophages: ubiquitous components of the "dark matter" of the biosphere]. Filee,J., Comeau,A.M., Suttle,C.A., Krisch,H.M. (2006). Med. Sci. 22:111-112. [TOP OF PAGE]

  136. Infection paradox: high abundance but low impact of freshwater benthic viruses. Filippini,M., Buesing,N., Bettarel,Y., Sime-Ngando,T., Gessner,M.O. (2006). Appl. Environ. Microbiol. 72:4893-4898. The discovery of an abundant and diverse virus community in oceans and lakes has profoundly reshaped ideas about global carbon and nutrient fluxes, food web dynamics, and maintenance of microbial biodiversity. These roles are exerted through massive viral impact on the population dynamics of heterotrophic bacterioplankton and primary producers. We took advantage of a shallow wetland system with contrasting microhabitats in close proximity to demonstrate that in marked contrast to pelagic systems, viral infection, determined directly by transmission electron microscopy, and consequently mortality of prokaryotes were surprisingly low in benthic habitats in all seasons. This was true even though free viruses were abundant throughout the year and bacterial infection and mortality rates were high in surrounding water. The habitats in which we found this pattern include sediment, decomposing plant litter, and biofilms on aquatic vegetation. Overall, we detected viruses in only 4 of a total of approximately 15,000 bacterial cells inspected in these three habitats; for comparison, nearly 300 of approximately 5,000 cells suspended in the water column were infected. The strikingly low incidence of impact of phages in the benthos may have important implications, since a major portion of microbial biodiversity and global carbon and nutrient turnover are associated with surfaces. Therefore, if failure to infect benthic bacteria is a widespread phenomenon, then the global role of viruses in controlling microbial diversity, food web dynamics, and biogeochemical cycles would be greatly diminished compared to predictions based on data from planktonic environments. [TOP OF PAGE]

  137. Reinventing phage therapy: are the parts greater than the sum? Fischetti,V.A., Nelson,D., Schuch,R. (2006). Nat. Biotech. 24:1508-1511. Although whole phage continue to generate interest as an alternative to antibiotics, focus is shifting to the use of purified phage components as antibacterial agents. [TOP OF PAGE]

  138. Effect of goethite coating and humic acid on the transport of bacteriophage PRD1 in columns of saturated sand. Foppen,J.W.A., Okletey,S., Schijven,J.F. (2006). J. Contam. Hydrol. 85:287-301. The transport of bacteriophage PRD1, a model virus, was studied in columns containing sediment mixtures of quartz sand with goethite-coated sand and using various solutions consisting of monovalent and divalent salts and humic acid (HA). Without HA and in the absence of sand, the inactivation rate of PRD1 was found to be as low as 0.014 day(-1) (at 5+/-3 degrees C), but in the presence of HA it was much lower (0.0009 day(-1)), indicating that HA helps PRD1 to survive. When the fraction of goethite in the sediment was increased, the removal of PRD1 also increased. However, in the presence of HA, C/C0 values of PRD1 increased by as much as 5 log units, thereby almost completely eliminating the effect of addition of goethite. The sticking efficiency was not linearly dependent on the amount of goethite added to the quartz sand; this is apparently due to surface charge heterogeneity of PRD1. Our results imply that, in the presence of dissolved organic matter (DOM), viruses can be transported for long distances thanks to two effects: attachment is poor because DOM has occupied favourable sites for attachment and inactivation of virus may have decreased. This conclusion justifies making conservative assumptions about the attachment of viruses when calculating protection zones for groundwater wells. [TOP OF PAGE]

  139. The origin of viruses and their possible roles in major evolutionary transitions. Forterre,P. (2006). Virus Res. 117:5-16. Viruses infecting cells from the three domains of life, Archaea, Bacteria and Eukarya, share homologous features, suggesting that viruses originated very early in the evolution of life. The three current hypotheses for virus origin, e.g. the virus first, the escape and the reduction hypotheses are revisited in this new framework. Theoretical considerations suggest that RNA viruses may have originated in the nucleoprotein world by escape or reduction from RNA-cells, whereas DNA viruses (at least some of them) might have evolved directly from RNA viruses. The antiquity of viruses can explain why most viral proteins have no cellular homologues or only distantly related ones. Viral proteins have replaced the ancestral bacterial RNA/DNA polymerases and primase during mitochondrial evolution. It has been suggested that replacement of cellular proteins by viral ones also occurred in early evolution of the DNA replication apparatus and/or that some DNA replication proteins originated directly in the virosphere and were later on transferred to cellular organisms. According to these new hypotheses, viruses played a critical role in major evolutionary transitions, such as the invention of DNA and DNA replication mechanisms, the formation of the three domains of life, or else, the origin of the eukaryotic nucleus. [TOP OF PAGE]

  140. Sequencing Bacillus anthracis typing phages gamma and cherry reveals a common ancestry. Fouts,D.E., Rasko,D.A., Cer,R.Z., Jiang,L., Fedorova,N.B., Shvartsbeyn,A., Vamathevan,J.J., Tallon,L., Althoff,R., Arbogast,T.S., Fadrosh,D.W., Read,T.D., Gill,S.R. (2006). J. Bacteriol. 188:3402-3408. The genetic relatedness of the Bacillus anthracis typing phages Gamma and Cherry was determined by nucleotide sequencing and comparative analysis. The genomes of these two phages were identical except at three variable loci, which showed heterogeneity within individual lysates and among Cherry, Wbeta, Fah, and four Gamma bacteriophage sequences. [TOP OF PAGE]

  141. Phage_Finder: automated identification and classification of prophage regions in complete bacterial genome sequences. Fouts,D.E. (2006). Nucleic Acids Research 34:5839-5851. Phage_Finder, a heuristic computer program, was created to identify prophage regions in completed bacterial genomes. Using a test dataset of 42 bacterial genomes whose prophages have been manually identified, Phage_Finder found 91% of the regions, resulting in 7% false positive and 9% false negative prophages. A search of 302 complete bacterial genomes predicted 403 putative prophage regions, accounting for 2.7% of the total bacterial DNA. Analysis of the 285 putative attachment sites revealed tRNAs are targets for integration slightly more frequently (33%) than intergenic (31%) or intragenic (28%) regions, while tmRNAs were targeted in 8% of the regions. The most popular tRNA targets were Arg, Leu, Ser and Thr. Mapping of the insertion point on a consensus tRNA molecule revealed novel insertion points on the 5' side of the D loop, the 3' side of the anticodon loop and the anticodon. A novel method of constructing phylogenetic trees of phages and prophages was developed based on the mean of the BLAST score ratio (BSR) of the phage/prophage proteomes. This method verified many known bacteriophage groups, making this a useful tool for predicting the relationships of prophages from bacterial genomes. [TOP OF PAGE]

  142. Ig-like domains on bacteriophages: a tale of promiscuity and deceit. Fraser,J.S., Yu,Z., Maxwell,K.L., Davidson,A.R. (2006). J. Mol. Biol. 359:496-507. The immunoglobulin (Ig) fold is one of the most important structures in biology, playing essential roles in the vertebrate immune response, cell adhesion, and many other processes. Through bioinformatic analysis, we have discovered that Ig-like domains are often found in the constituent proteins of tailed double-stranded (ds) DNA bacteriophage particles, and are likely displayed on the surface of these viruses. These phage Ig-like domains fall into three distinct sequence families, which are similar to the classic immunoglobulin domain (I-Set), the fibronectin type 3 repeat (FN3), and the bacterial Ig-like domain (Big2). The phage Ig-like domains are very promiscuous. They are attached to more than ten different functional classes of proteins, and found in all three morphogenetic classes of tailed dsDNA phages. In addition, they reside in phages that infect a diverse set of gram negative and gram positive bacteria. These domains are deceptive because many are added to larger proteins through programmed ribosomal frameshifting, so that they are not always detected by standard protein sequence searching procedures. In addition, the presence of unrecognized Ig-like domains in a variety of phage proteins with different functions has led to gene misannotation. Our results demonstrate that horizontal gene transfer involving Ig-like domain encoding DNA has occurred commonly between diverse classes of both lytic and temperate phages, which otherwise display very limited sequence similarities to one another. We suggest that phage may have been an important vector in the spread of Ig-like domains through diverse species of bacteria. While the function of the phage Ig-like domains is unknown, several lines of evidence suggest that they may play an accessory role in phage infection by weakly interacting with carbohydrates on the bacterial cell surface. [TOP OF PAGE]

  143. Commensal bacteria influence Escherichia coli O157:H7 persistence and Shiga toxin production in the mouse intestine. Gamage,S.D., Patton,A.K., Strasser,J.E., Chalk,C.L., Weiss,A.A. (2006). Infect. Immun. 74:1977-1983. The presence of commensal flora reduced colonization of Escherichia coli O157:H7 and production of Shiga toxin (Stx) in the murine intestine. Stx production was not detected in mice colonized with E. coli that were resistant to the Shiga toxin phage, but it was detected in mice colonized with phage-susceptible E. coli. [TOP OF PAGE]

  144. Bacteriophage as pollution indicators. Gerba,C. (2006). pp. 695-701. In In Calendar,R. and Abedon,S.T. (eds.), The Bacteriophages. Oxford University Press, Oxford. [first three paragraphs] Because of the difficulty and cost of detecting waterborne enteric pathogens, indicator organisms have been used since the beginning of the twentieth century. Much of the work in this area has focused on bacterial indicators, such as coliform and fecal coliform bacteria. However, it has been recognized in the last 30 years that these traditional indicators do not always reflect the waterborne occurrence of human pathogenic viruses and protozoa. Thus, bacteriophages have been investigated as a better indicator of these groups of pathogens in water, as models of enteric virus removal by treatment processes, and to examine the fate and transport of enteric viruses in the environment (12). ¶ The term "indicator organism" is often not clearly defined. By contrast, an "index organism" is usually defined as one related to the occurrence of a selected surrogate microorganism or microorganisms (Table 45-1). The relationship may be direct, such as an index of human viruses, or indirect, such as an index of fecal pollution or types of fecal pollution (i.e., human or animal) (27). The criteria for an index organism are very similar to those commonly used for bacterial fecal indicators. An indicator organism, on the other hand, is measured to check the performance of a treatment process against previously set standards. For example, an indicator is used to evaluate the performance of drinking-water disinfection for the inactivation of enteric viruses. To serve as an effective indicator, the resistance of both the indicator organism and the pathogen to the disinfectant should be similar. ¶ Three main groups of bacteriophages infecting enteric bacteria have received the greatest amount of study in the assessment of water quality. These are the somatic coliphages, the F-specific RNA coliphages, and the bacteriophages infecting Bacteroides fragilis (Table 45-2). This chapter largely concerns the use of these bacteriophages as indicators of fecal pollution and as index organisms of pathogenic human enteric viruses. [TOP OF PAGE]

  145. Ciprofloxacin and trimethoprim cause phage induction and virulence modulation in Staphylococcus aureus. Goerke,C., Koller,J., Wolz,C. (2006). Antimicrob. Agents Chemother. 50:171-177. In Staphylococcus aureus strains of human origin, phages which integrate into the chromosomal gene coding for b-hemolysin (hlb) are widely distributed. Most of them encode accessory virulence determinants such as staphylokinase (sak) or enterotoxins. Here, we analyzed the effects of ciprofloxacin and trimethoprim on phage induction and expression of phage-encoded virulence factors by using isolates from patients with cystic fibrosis for which the induction of hlb-converting phages was demonstrated in vivo (C. Goerke, S. Matias y Papenberg, S. Dasbach, K. Dietz, R. Ziebach, B. C. Kahl, and C. Wolz, J. Infect. Dis. 189:724-734, 2004) as well as a f13 lysogen of phage-cured strain 8325-4. Treatment of lysogens with subinhibitory concentrations of either antibiotic resulted in (i) delysogenization of strains resembling the isolates picked up after chronic lung infection and (ii) replication of phages in the bacterial host in a dose-dependent manner. Ciprofloxacin treatment resulted in enhanced recA transcription, indicating involvement of the SOS response in phage mobilization. Induction of f13 was linked to elevated expression of the phage-encoded virulence gene sak, chiefly due to the activation of latent phage promoters. In summary, we could show the induction of hlb-converting phages and a subsequent virulence modulation of the host bacterium by ciprofloxacin and trimethoprim. [TOP OF PAGE]

  146. Dynamic modelling of viral impact on cyanobacterial populations in shallow lakes: implications of burst size. Gons,H.J., Hoogveld,H.L., Simis,S.G.H., Tijdens,M. (2006). J. Mar. Bio. Assoc. UK 86:537-542. Laboratory experiments with whole water-columns from shallow, eutrophic lakes repeatedly showed collapse of the predominant filamentous cyanobacteria. The collapse could be due to viral activity, from the evidence of electron microscopy of infected cyanobacterial cells and observed dynamics of virus-like particles. Burst-size effects on single-host single-virus dynamics was modelled for nutrient-replete growth of the cyanobacteria and fixed viral decay rate in the water column. The model combined previously published equations for nutrient-replete cyanobacterial growth and virus–host relationship. According to the model results, burst sizes greater than 200 to 400 virions per cell would result in host extinction, whereas lower numbers would allow coexistence, and even stable population densities of host and virus. High-nutrient status of the host cells might accommodate a large burst size. The ecological implication could be that burst-size increase accompanying a transition from phosphorus to light-limited cyanobacterial growth might destabilize the virus–host interaction and result in the population collapse observed in the experiments. [TOP OF PAGE]

  147. Bacteriophages and transplantation tolerance. Gorski,A., Kniotek,M., Perkowska-Ptasinska,A., Mroz,A., Przerwa,A., Gorczyca,W., Dabrowska,K., Weber-Dabrowska,B., Nowaczyk,M. (2006). Transplant. Proc. 38:331-333. Our recent findings suggest that bacteriophages (phages) may not only eliminate bacteria, but also modulate immune functions. In this communication, we demonstrate that phages may strongly inhibit human T-cell activation and proliferation as well as activation of the nuclear transcription factor NF-kappaB in response to a viral pathogen. Phage administration in vivo can diminish cellular infiltration of allogeneic skin allografts. Thus, phage treatment should be considered in antibiotic-resistant posttransplantation infections. Furthermore, phages could find a broader application in clinical transplantation. [TOP OF PAGE]

  148. Response of four types of coliphages to high hydrostatic pressure. Guan,D., Kniel,K., Calci,K.R., Hicks,D.T., Pivarnik,L.F., Hoover,D.G. (2006). Food Microbiol. 23:546-551. Pressure inactivation of four types of coliphages, jiX 174 (ssDNA virus), MS2 (ssRNA virus), l imm434 (dsDNA virus) and T4 (dsDNA virus), was studied to evaluate their potential as human enteric viral surrogates for use in validation of commercial pressure processing treatments. Phage var jX 174 demonstrated an unexpected high resistance to pressure with no more than 1-log(10) reduction observed following exposures to 350-600 MPa. There was no greater than 1-log(10) reduction below 500 MPa for MS2 in modified phosphate-buffered saline, but a 3.3-log(10) reduction was observed for MS2 pressurized at 600 MPa. Coliphages l imm434 and T4 were relatively sensitive to pressure in demonstrating inactivation at 350 MPa. At 21 degrees C, l imm434 was inactivated in modified phosphate-buffered saline or Dulbecco's Modified Eagle's Medium plus 5% fetal bovine sera by at least 7.5-log(10) when exposed to 400 MPa for 5 min. Treatment at 450 MPa for 5 min was necessary to obtain a log(10) reduction of 6-7 for T4. [TOP OF PAGE]

  149. Detection of multiple antibiotic–resistant Salmonella enterica Serovar Typhimurium DT104 by phage replication–competitive enzyme-linked immunosorbent assay. Guan,J., Chan,M., Allain,B., Mandeville,R., Brooks,B.W. (2006). J. Food Prot. 739-742. A phage replication-competitive enzyme-linked immunosorbent assay (PR-cELISA) was developed for the detection of multiple antibiotic-resistant Salmonella Typhimurium DT104. In the PR-cELISA procedure, a phage, BP1, was inoculated into a log-phase bacterial culture at a ratio of 1:100. After a 3-h incubation of the mixture, BP1 replication was measured by cELISA based on the competitive binding between BP1 and biotinylated BP1 to Salmonella Typhimurium smooth lipopolysaccharide. Among the 84 Salmonella strains and 9 non-Salmonella strains that were tested by PR-cELISA, BP1 detected 39 of 40 Salmonella Typhimurium strains, 2 of 10 Salmonella non-Typhimurium somatic group B strains, and 5 of 18 Salmonella somatic group D1 strains. With the addition of chloramphenicol to the culture medium, PR-cELISA detected all 27 multiple antibiotic-resistant Salmonella Typhimurium DT104 and none of the other Salmonella strains or non-Salmonella strains tested. The results demonstrated that PR-cELISA has potential applications for the detection of multiple antibiotic-resistant Salmonella Typhimurium DT104. [TOP OF PAGE]

  150. Characterization of spontaneous phage-resistant derivatives of Lactobacillus delbrueckii commercial strains. Guglielmotti,D.M., Reinheimer,J.A., Binetti,A.G., Giraffa,G., Carminati,D., Quiberoni,A. (2006). Int. J. Food Microbiol. 111:126-133. A total of 44 spontaneous phage-resistant mutants were isolated from three commercial Lactobacillus delbrueckii strains by secondary culture and agar plate methods. Phenotypic characteristics related to their phage-resistance capacities, i.e. plaquing efficiency, phage-resistance stability, lysogeny and adsorption rates were determined. The morphological, biochemical (sugar fermentation patterns) and technological (acidifying and proteolytic activities and acidification kinetics) properties of mutants were also studied. Amplification and restriction analysis of the 16S rRNA gene (PCR-ARDRA) was applied to confirm strain identity at the subspecies level. Random amplification of polymorphic DNA (RAPD-PCR) was used to determine genetic diversity among the isolates and their respective parent strains. The secondary culture method was the most useful for obtaining phage-resistant mutants. Phage resistance stability was a variable property among the isolates, but a high level of resistance was exhibited as quantified by the efficiency of plaquing. Furthermore, a total absence of spontaneous lysogeny was demonstrated. Adsorption rates were heterogeneously distributed among the three groups of mutants. All mutants isolated from two sensitive strains were similar to them with respect to technological properties. Two groups of mutants with distinctive technological properties were isolated from the other sensitive strain. PCR-ARDRA revealed that two out of three sensitive strains identified commercially as Lb. delbrueckii subsp. bulgaricus were actually Lb. delbrueckii subsp. lactis. Some of the phage-resistant mutants that were obtained might be used in culture rotation programs without regulatory restrictions when commercial strains become sensitive to phages present in industrial environments. [TOP OF PAGE]

  151. Augmentation of the antimicrobial efficacy of antibiotics by filamentous phage. Hagens,S., Habel,A., Blasi,U. (2006). Microb. Drug Resist. 12:164-168. A significant increase in sensitivity to several antibiotics was observed in vitro after infection of the two Pseudomonas aeruginosa strains O1 and K with the filamentous phage Pf3 and Pf1, respectively. Moreover, upon infection with phage Pf1 a P. aeruginosa K strain harboring a plasmid-borne gentamicin resistance gene could be resensitized to the antibiotic. We further show that BALB/c mice were rescued from lethal infections with P. aeruginosa K by concomitant treatment with phage Pf1 and low concentrations of gentamicin, neither of which was able to cure the infection when administered alone. [TOP OF PAGE]

  152. Phages of Lactococcus lactis. Hammer,K., Brønsted,L. (2006). pp. 572-592. In In Calendar,R. and Abedon,S.T. (eds.), The Bacteriophages. Oxford University Press, Oxford. [third paragarph] The industrial use of L. lactis in vast amounts for milk fermentations is providing a gigantic large-scale environment for phage reproduction and evolution. Phage infections are difficult to avoid, since pasteurized milk is not sterile and may contain phages that potentially destroys the fermentation and hence the product. Furthermore, the use of defined strains as starter cultures has greatly limited the number of industrial strains used and hence made it easier for phage contaminants to proliferate. After the dairy production failures in the mid 1930s were recognized to be caused by phage infections (117) research into phages and phage resistance has been a major issue in the lactococcal field. Phage resistance systems are outside the scope of this chapter, for reviews see (2, 40). [TOP OF PAGE]

  153. Virus retention and transport in chemically heterogeneous porous media under saturated and unsaturated flow conditions. Han,J., Jin,Y., Willson,C.S. (2006). Environ. Sci. Technol. 40:1547-1555. Retention and transport of colloids and microorganisms are complex processes, especially in the vadose zone due to the more complicated water flow regime and additional interfacial reactions involved. In this study, we examined the retention and transport behavior of two bacteriophages, MS-2 and fX174, in homogeneous and chemically heterogeneous media under variably saturated conditions. Column experiments with glass beads (treated to have either hydrophilic or hydrophobic surface properties) were conducted using a phosphate-buffered saline solution at different pore water ionic strengths ranging from 0.025 to 0.163 M. In columns packed with 100% hydrophilic glass beads, retention of the viruses increased with decreasing water content and increasing ionic strength, a result similar to those reported in the literature. However, greater retention of both MS-2 and fX174 was observed in saturated columns than in unsaturated columns packed with a 1:1 mixture of hydrophilic and hydrophobic glass beads, especially at high ionic strengths. This result contradicts the common belief that viruses (and colloids in general) are subject to greater removal in unsaturated media. Our study suggests that while the mechanisms controlling colloid interfacial interactions (i.e., attachment on solid-water and air-water interfaces and film straining) on the pore scale are relevant, nonuniform wetting conditions due to heterogeneous grain surface hydrophobicity can strongly influence water flow and phase interconnection. Under these conditions, hydrodynamic effects on the mesopore scale will dominate pore-scale interfacial reactions in controlling the extent of colloid retention and movement in unsaturated media. [TOP OF PAGE]

  154. Seasonal profiles of human noroviruses and indicator bacteria in a wastewater treatment plant in Tokyo, Japan. Haramoto,E., Katayama,H., Oguma,K., Yamashita,H., Tajima,A., Nakajima,H., Ohgaki,S. (2006). Water Sci. Technol. 54:301-308. The seasonal profiles of microorganisms in raw sewage, secondary-treated sewage, and final effluent at a wastewater treatment plant in Tokyo, Japan, were quantitatively determined each month for one year, from July 2003 to June 2004. Human noroviruses, which were determined by real-time PCR, in raw sewage varied from 0.17-260 copies/mL for genotype 1 and from 2.4-1900 copies/mL for genotype 2, showing much higher values in winter, the epidemic season. The concentration of total coliforms, Escherichia coli, or F-specific phages in raw sewage was almost constant throughout the year. Human noroviruses of genotype 2 were removed most effectively (3.69 log10 on average) at the wastewater treatment plant, followed by E. coli (3.37 log10), total coliforms (3.05 loglo), F-specific phages (2.81 log10), and human noroviruses of genotype 1 (2.27 log10). The removal ratio of human noroviruses was almost constant, independent of the initial concentration of the viruses in raw sewage, which led to the increasing concentration of human noroviruses in final effluent in winter. None of the tested bacteria was judged to be a reliable indicator of human noroviruses in final effluent. [TOP OF PAGE]

  155. Mycobacteriophages. Hatfull,G.F. (2006). pp. 602-620. In In Calendar,R. and Abedon,S.T. (eds.), The Bacteriophages. Oxford University Press, Oxford. [first paragraph] Mycobacteriophages are viruses of the Mycobacteria. The interest in these phages derives in large part from the medical significance and biological idiosyncrasies of their hosts. Mycobacteria are acid-fast staining bacteria with characteristic waxy cell walls that can be readily divided into two groups based on their growth rate; slow-growers such as Mycobacterium tuberculosis that have a doubling time of 24 hrs and fast-growers such as Mycobacterium smegmatis with 3-4 hr doubling times [for reviews see (8, 32)]. Several mycobacterial species are important human and animal pathogens with the most notorious being M. tuberculosis and Mycobacterium leprae, the causative agents of tuberculosis and leprosy respectively (8). The extent of these diseases is alarming - M. tuberculosis is the leading cause of human mortality from a single infectious disease and the increased prevalence of multiple drug resistant M. tuberculosis strains greatly complicates its treatment. A study of the mycobacteriophages offers potential for the development of novel methodologies for the diagnosis, prevention and treatment of these diseases as well as revealing interesting biological features of their unusual bacterial hosts (30, 32, 33). [TOP OF PAGE]

  156. Exploring the mycobacteriophage metaproteome: phage genomics as an educational platform. Hatfull,G.F., Pedulla,M.L., Jacobs-Sera,D., Cichon,P.M., Foley,A., Ford,M.E., Gonda,R.M., Houtz,J.M., Hryckowian,A.J., Kelchner,V.A., Namburi,S., Pajcini,K.V., Popovich,M.G., Schleicher,D.T., Simanek,B.Z., Smith,A.L., Zdanowicz,G.M., Kumar,V., Peebles,C.L., Jacobs,W.R.J., Lawrence,J.G., Hendrix,R.W. (2006). PLoS Genetics 2:e92 Bacteriophages are the most abundant forms of life in the biosphere and carry genomes characterized by high genetic diversity and mosaic architectures. The complete sequences of 30 mycobacteriophage genomes show them collectively to encode 101 tRNAs, three tmRNAs, and 3,357 proteins belonging to 1,536 "phamilies" of related sequences, and a statistical analysis predicts that these represent approximately 50% of the total number of phamilies in the mycobacteriophage population. These phamilies contain 2.19 proteins on average; more than half (774) of them contain just a single protein sequence. Only six phamilies have representatives in more than half of the 30 genomes, and only three-encoding tape-measure proteins, lysins, and minor tail proteins-are present in all 30 phages, although these phamilies are themselves highly modular, such that no single amino acid sequence element is present in all 30 mycobacteriophage genomes. Of the 1,536 phamilies, only 230 (15%) have amino acid sequence similarity to previously reported proteins, reflecting the enormous genetic diversity of the entire phage population. The abundance and diversity of phages, the simplicity of phage isolation, and the relatively small size of phage genomes support bacteriophage isolation and comparative genomic analysis as a highly suitable platform for discovery-based education. [TOP OF PAGE]

  157. Bug killers. Hausler,T. (2006). Nat. Med. 12:600-601. Viruses that can kill bacteria were once wildly popular. Will the rising problem of antibiotic resistance bring them back? Thomas Häusler reports. [TOP OF PAGE]

  158. Viruses vs. Superbugs: A Solution to the Antibiotic Crisis. Häusler,T. (2006). Macmillan, [TOP OF PAGE]

  159. Viral and bacterial assemblage covariance in oligotrophic waters of the West Florida Shelf (Gulf of Mexico). Hewson,I., Winget,D.M., Williamson,K.E., Fuhrman,J.A., Wommack,K.E. (2006). J. Mar. Bio. Assoc. UK 86:591-603. Viruses are hypothesized to cause enhanced diversity in bacterial communities by regulating the outcome of intertaxon competition. However, concomitant documentation of viral and bacterial assemblage composition in oligotrophic waters are rare, particularly in situ over time, and there is almost no information on the temporal variability in virioplankton assemblage composition in oligotrophic water masses. Assemblage composition of viruses (via pulsed-field gel electrophoresis, PFGE) and bacteria (via automated rRNA intergenic spacer analysis, ARISA) was compared during surface lagrangian drifter deployments in the oligotrophic Gulf of Mexico during summer 2001, 2002, and 2003. In vertical profile, viruses and bacteria both had maximum abundances in surface waters, which decreased with depth; however, the richness of their assemblages was not significantly different between depths, suggesting independence of biomass and diversity. Viral assemblages changed rapidly (0.17-0.32 Jaccard index d-1), which was similar to the rate of change in bacterial assemblages reported in surface waters. Patterns of viral and bacterial assemblage composition were significantly related (P<0.001, r=0.58 between node ranks), and both assemblages clustered primarily by year and then by depth. These cultivation-independent observations demonstrate relationships between viral and bacterial assemblages, which are dynamic in patches of open ocean water. Even at the relatively low phylogenetic resolution of the ARISA and PFGE methods, the results support the idea that viruses may influence the species composition of host assemblages. [TOP OF PAGE]

  160. Viral impacts upon marine bacterioplankton assemblage structure. Hewson,I., Fuhrman,J.A. (2006). J. Mar. Bio. Assoc. UK 86:577-589. This study examined the relationship between viral infection and the richness, diversity and composition of bacterial assemblages in the water column. Viruses were enriched by ultrafiltration, added to water column incubation experiments at 15 locations in the North Atlantic, North Pacific, Gulf of Mexico and Southern California. In a separate experiment, viruses were removed from bacterioplankton by diafiltration at the San Pedro Ocean Time Series Station. Bacterial assemblage composition was observed using a high throughput and sensitive molecular fingerprinting analysis, automated rRNA intergenic spacer analysis (ARISA). Diazotrophs were used as a model functional group to represent rare organisms hypothesized to benefit from viral activity, and their richness and diversity was determined by terminal restriction fragment length polymorphism of a nitrogenase gene fragment (nifH). The enrichment and removal experiments demonstrated mixed impacts of viral pressure upon bacterial communities, and we observed significant effects of viruses on several microbial parameters in all but two experiments. However, there was no consistent response of viral enrichment on total bacterial and diazotroph assemblages at stations with similar environmental conditions, suggesting that untested variables, small spatial scale factors, or stochastic processes influence the outcome of viral activities. Across all experiments, the relative abundance of the more common operational taxonomic units (OTUs) in fingerprints were not significantly impacted compared to the abundance of rare OTUs. These data indicate that viruses may have significant influence upon community structure of bacterioplankton; however, effects were not consistent between sampling locations nor water masses. [TOP OF PAGE]

  161. The cyanophage molecular mixing bowl of photosynthesis genes. Hill,E. (2006). PLoS Biol. 4:e264 [first paragraph] Among the wealth of microbial organisms inhabiting marine environments, cyanobacteria (blue-green algae) are the most abundant photosynthetic cells. Prochlorococcus and Synechococcus, the two most common cyanobacteria, account for 30% of global carbon fixation (through the photosynthetic process in which sugars are manufactured from carbon dioxide and water). By drawing on natural resources, these microbes use photosystems (PS) I and II (the two reaction centers in photosynthesis) to harness energy. [TOP OF PAGE]

  162. Babies and bacteria: phage typing, bacteriologists, and the birth of infection control. Hillier,K. (2006). Bull. Hist. Med. 80:733-761. During the 1950s, Staphylococcus aureus became a major source of hospital infections and death, particularly in neonates. This situation was further complicated by the fact that Staphylococcus quickly gained resistance to most antibiotics. Controlling these infections was a pressing concern for hospital workers, especially bacteriologists who tackled it through the use of a new epidemiologic tool: phage typing. This article argues that during the mid- to late 1950s a series of staphylococcal hospital and nursery epidemics united phage typers, brought international recognition to the usefulness of their technique, and, in the process, contributed to the establishment of the new field of infection control. Through the use of this new tool, phage typers established themselves as experts in infection control and, in some places, became essential members of newly formed infection-control committees. The nursery epidemics represent a particularly important test for phage typing and infection control, for this staphylococcal strain (80/81) was especially virulent and spread rapidly beyond the hospital to the wider community. The epidemiologic information provided by phage typers was vital for devising practical advice on how to control this deadly strain of Staphylococcus and also for transforming the role of the hospital bacteriologist from mere technician into infection-control expert. [TOP OF PAGE]

  163. Evaluation of the influence of bacteriophage titer on the treatment of colibacillosis in broiler chickens. Huff,W.E., Huff,G.R., Rath,N.C., Donoghue,A.M. (2006). Poult. Sci. 85:1373-1377. Two studies were conducted to determine the efficacy of bacteriophage SPR02 and DAF6 at varying titers to treat colibacillosis in chickens. In Study 1, the treatments consisted of a control, i.m. injection of bacteriophage SPR02 or DAF6, Escherichia coli airsac challenge, and E. coli challenge followed by treatment at different titers with bacteriophage SPR02 or DAF6. The E. coli- challenged birds were injected with 6 x 10(4) cfu into the left thoracic airsac at 7 d of age. Immediately after the birds were challenged with E. coli, they were treated by administration of bacteriophage SPR02 or DAF6 by i.m. injection into the left thigh with 4 x 10(8), 10(6), 10(4), or 10(2) pfu. Study 2 was identical to Study 1, with the exception that the E. coli challenge was increased to 9 x 10(4) cfu, and the titers of SPR02 and DAF6 were slightly less at 3 x 10(8), 10(6), 10(4), and 10(2) pfu. Both studies were concluded when the birds were 3 wk of age. Mortality in the birds challenged with E. coli in Studies 1 and 2 was 48 and 47%, respectively. The only consistently effective bacteriophage treatment was the highest titer (10(8) pfu) of bacteriophage SPR02, which significantly reduced mortality from 48 and 47% in the birds only challenged with E. coli (positive control) to 7% in both studies, which was not significantly different from the unchallenged negative control treatments. These studies indicate that an effective multiplicity of infection for i.m. treatment with SPR02 was 10(4) in this experimental model of colibacillosis. Bacteriophage administered at sufficient titers can be effective therapeutic agents and provide an alternative to antibiotics in the treatment of bacterial diseases. [TOP OF PAGE]

  164. Shiga toxin-producing Escherichia coli, faecal coliforms and coliphage in animal feeds. Hutchison,M.L., Thomas,D.J.I., Walters,L.D., Avery,S.M. (2006). Lett. Appl. Microbiol. 43:205-210. AIMS: Animal feeds (n = 226), collected from pastures or feeding troughs on UK farms and from feed manufacturers' bulk stores, were analysed for Escherichia coli harbouring shiga-toxin genes (stx), faecal coliforms, coliphages and stx-harbouring bacteriophages. METHODS AND RESULTS: Samples comprised of 79 fresh grasses, 26 silages and 121 dried or heat-processed feeds (DPF). Five of the 79 (6.3%) fresh grass samples contained stx(2)-E. coli. stx-E. coli were not detected in the silages or DPF that were examined. Faecal coliforms were detected in 75/79 (94.9%) of fresh grasses, 19/26 (73.1%) of silages and 36/121 (29.8%) of processed feeds. Coliphages were detected in 63/79 (79.7%) and 18/26 (69.2%) of fresh grasses and silages, respectively. Coliphages were isolated at a significantly lower prevalence of 5% (6/121) from processed feeds. Although stx(2)-phage was isolated from the enrichment of a single grass sample, stx-phages were not detected in any of the silage or processed feeds. We did not detect stx(1)-phage in any of the samples collected. CONCLUSIONS: Pastures have the potential to act as transmission vectors for stx-harbouring E. coli for grazed livestock. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study to report on the prevalence of E. coli harbouring stx genes, faecal coliforms, coliphages and stx-harbouring bacteriophages in a range of feedstuffs destined for consumption by UK livestock. This study provides information on the risk of feeds to the spread of stx-phages between livestock and/or the environment. [TOP OF PAGE]

  165. Evaluation of a microcolony detection method and phage assay for rapid detection of Mycobacterium tuberculosis in sputum samples. Irfan,S., Hasan,R., Kanji,A., Hassan,Q., Azam,I. (2006). S. E. Asian J. Trop. Med. Pub. Health 37:1187-1195. Early and rapid diagnosis of tuberculosis is necessary for both treatment and control of the disease. This study evaluated two microcolony observation techniques based on liquid and solid media and a mycobacteriophage assay, to evaluate their effectiveness in the diagnosis of pulmonary TB compared with a standard culture (BACTEC 460 and LJ medium). Middlebrook7H9 (M7H9) broth based on microcolony determination detected 57/61 positives cultures (n = 200) with a sensitivity of 93.4% and a specificity of 87.1%. M7H11 agar detected 57/62 positive cultures (n = 198) with a sensitivity of 91.9% and a specificity of 89.7%. The mycobacteriophage assay detected 98/143 (68.5%) of positive samples. The time to positivity was 48 hours in the mycobacteriophage assay versus 7 days in both the M7H9 broth and M7H11 agar. The costs in comparison with the culture (BACTEC 460 and LJ) were 33% and 48% for the microcolony and mycobacteriophage methods, respectively. Microcolony methods were rapid and cost effective compared to standard cultures. The mycobacteriophage assay, despite its lower sensitivity, has a short turn around time, and may be recommended as a screening test in countries with a low prevalence of tuberculosis. [TOP OF PAGE]

  166. Evolutionary genomics of nucleo-cytoplasmic large DNA viruses. Iyer,L.M., Balaji,S., Koonin,E.V., Aravind,L. (2006). Virus Res. 117:156-184. A previous comparative-genomic study of large nuclear and cytoplasmic DNA viruses (NCLDVs) of eukaryotes revealed the monophyletic origin of four viral families: poxviruses, asfarviruses, iridoviruses, and phycodnaviruses [Iyer, L.M., Aravind, L., Koonin, E.V., 2001. Common origin of four diverse families of large eukaryotic DNA viruses. J. Virol. 75 (23), 11720-11734]. Here we update this analysis by including the recently sequenced giant genome of the mimiviruses and several additional genomes of iridoviruses, phycodnaviruses, and poxviruses. The parsimonious reconstruction of the gene complement of the ancestral NCLDV shows that it was a complex virus with at least 41 genes that encoded the replication machinery, up to four RNA polymerase subunits, at least three transcription factors, capping and polyadenylation enzymes, the DNA packaging apparatus, and structural components of an icosahedral capsid and the viral membrane. The phylogeny of the NCLDVs is reconstructed by cladistic analysis of the viral gene complements, and it is shown that the two principal lineages of NCLDVs are comprised of poxviruses grouped with asfarviruses and iridoviruses grouped with phycodnaviruses-mimiviruses. The phycodna-mimivirus grouping was strongly supported by several derived shared characters, which seemed to rule out the previously suggested basal position of the mimivirus [Raoult, D., Audic, S., Robert, C., Abergel, C., Renesto, P., Ogata, H., La Scola, B., Suzan, M., Claverie, J.M. 2004. The 1.2-megabase genome sequence of Mimivirus. Science 306 (5700), 1344-1350]. These results indicate that the divergence of the major NCLDV families occurred at an early stage of evolution, prior to the divergence of the major eukaryotic lineages. It is shown that subsequent evolution of the NCLDV genomes involved lineage-specific expansion of paralogous gene families and acquisition of numerous genes via horizontal gene transfer from the eukaryotic hosts, other viruses, and bacteria (primarily, endosymbionts and parasites). Amongst the expansions, there are multiple families of predicted virus-specific signaling and regulatory domains. Most NCLDVs have also acquired large arrays of genes related to ubiquitin signaling, and the animal viruses in particular have independently evolved several defenses against apoptosis and immune response, including growth factors and potential inhibitors of cytokine signaling. The mimivirus displays an enormous array of genes of bacterial provenance, including a representative of a new class of predicted papain-like peptidases. It is further demonstrated that a significant number of genes found in NCLDVs also have homologs in bacteriophages, although a vertical relationship between the NCLDVs and a particular bacteriophage group could not be established. On the basis of these observations, two alternative scenarios for the origin of the NCLDVs and other groups of large DNA viruses of eukaryotes are considered. One of these scenarios posits an early assembly of an already large DNA virus precursor from which various large DNA viruses diverged through an ongoing process of displacement of the original genes by xenologous or non-orthologous genes from various sources. The second scenario posits convergent emergence, on multiple occasions, of large DNA viruses from small plasmid-like precursors through independent accretion of similar sets of genes due to strong selective pressures imposed by their life cycles and hosts. [TOP OF PAGE]

  167. Investigation of bacteriophage MS2 viral dynamics using model discrimination analysis and the implications for phage therapy. Jain,R., Knorr,A.L., Bernacki,J., Srivastava,R. (2006). Biotechnol. Prog. 22:1650-1658. Lytic phages infect their bacterial hosts, use the host machinery to replicate, and finally lyse and kill their hosts, releasing progeny phages. Various mathematical models have been developed that describe these phage-host viral dynamics. The aim of this study was to determine which of these models best describes the viral dynamics of lytic RNA phage MS2 and its host Escherichia coli C-3000. Experimental data consisted of uninfected and infected bacterial cell densities, free phage density, and substrate concentration. Parameters of various models were either determined directly through other experimental techniques or estimated using regression analysis of the experimental data. The models were evaluated using a Bayesian-based model discrimination technique. Through model discrimination it was shown that phage-resistant cells inhibited the growth of phage population. It was also shown that the uninfected bacterial population was a quasispecies consisting of phage-sensitive and phage-resistant bacterial cells. When there was a phage attack the phage-sensitive cells died out and the phage-resistant cells were selected for and became the dominant strain of the bacterial population. [TOP OF PAGE]

  168. Diversity of cyanophages infecting the heterocystous filamentous cyanobacterium Nodularia isolated from the brackish Baltic Sea. Jenkins,C.A., Hayes,P.K. (2006). J. Mar. Bio. Assoc. UK 86:529-536. A collection of 17 cyanophage isolates able to infect the heterocystous, filamentous cyanobacterium Nodularia spumigena has been established from the Baltic Sea. These cyanophages have been characterized based on their morphology, cross infectivity and genetic structure. Short fragments (450bp) of the gene encoding the major capsid protein (g23) were amplified and sequenced from several isolates, and the encoded protein was found to be 99% identical across all the N. spumigena-specific cyanophages tested. These results suggest that the Nodularia-specific cyanophages are very closely related. However, these cyanophages were found to be diverse in terms of their morphology and host range. Cyanophages belonging to two families within the order Caudovirales, Myoviridae and Siphoviridae, were included in the collection of isolates. The cyanophage particles are large in comparison with cyanophages previously isolated from the marine environment, with the largest capsid measuring 127×122×888nm. Host ranges of the cyanophage isolates varied, some being able to infect up to five genotypically distinct strains of Nodularia spumigena, while others were very specific, infecting only one strain. We conclude that Nodularia-specific cyanophages form a diverse community in surface waters during summer and autumn months and that they may play a role both in the transfer of genetic information between Nodularia lineages and in promoting changes in the genetic structure of the host population. [TOP OF PAGE]

  169. Modeling the role of bacteriophage in the control of cholera outbreaks. Jensen,M.A., Faruque,S.M., Mekalanos,J.J., Levin,B.R. (2006). Proc. Natl. Acad. Sci. USA 103:4652-4657. Cholera is a waterborne diarrheal disease that continues to plague the developing world. Individuals become infected by consuming water from reservoirs contaminated by virulent strains of the bacterium Vibrio cholerae. Epidemiological and environmental observations of a cholera outbreak in Dhaka, Bangladesh, suggest that lytic bacteriophage specific for V. cholerae may limit the severity of cholera outbreaks by killing bacteria present in the reservoir and in infected individuals. To quantify this idea and generate testable hypotheses, we analyzed a mathematical model that combines the epidemiology of cholera with the population dynamics of the bacteria and phage. Under biologically reasonable conditions, we found that vibriophage can ameliorate cholera outbreaks. If phage predation limits bacterial density before an outbreak, a transient reduction in phage density can disrupt that limitation, and subsequent bacterial growth can initiate a cholera outbreak. The severity of the outbreak depends on the density of phage remaining in the reservoir. If the outbreak is initiated instead by a rise in bacterial density, the introduction of phage can reduce the severity of the outbreak and promote its decline. In both situations, the magnitude of the phage effect depends mainly on vibrio growth and phage mortality rates; the lower the rates, the greater the effect. Our analysis also suggests that either bacteria in the environmental reservoir are hyperinfectious or most victims ingest bacteria amplified in food or drinking water contaminated by environmental water carrying few viable V. cholerae. Our theoretical results make a number of empirically testable predictions. [TOP OF PAGE]

  170. Natural and anthropogenic forcing on the dynamics of virioplankton in the Yangtze river estuary. Jiao,N., Zhao,Y., Luo,T., Wang,X. (2006). J. Mar. Bio. Assoc. UK 86:543-550. Seasonal investigation of virus dynamics by flow cytometry was conducted in the Yangtze river estuarine area in April, August, November 2002 and February 2003, and a supplemental investigation in the inner estuary and downstream of the river was conducted in October 2005. The majority of the total viral abundance was bacteriophage and only 5.4% of the total was algal virus. Total viral abundance varied with season and location, ranging from 6.75×105-1.68×107 particles/ml, and the virus:bacterium ratio (VBR) ranged from 1.52 to 72.02 with a mean of 8.7. In the present study, viral abundance peaked in both the summer and the winter, unlike the typical seasonal pattern reported in the literature, in which viral abundance peaks in the summer when bacterial hosts are also at their most abundant. However, the driving forces for the two peaks reported here were totally different, the summer viral abundance peak coupled with the development of bacterial hosts which were controlled largely by temperature year-round and by trophic state occasionally, while the winter one seemed to be multi-factor controlled. The host-phage interaction was no longer predominant in control of the winter viral abundance as bacterial abundance was lowest in this season. The winter low temperature would help maintain a high viral abundance as high temperatures might increase viral inactivation and viral decay; the VBR peak values actually occurred in the winter. More importantly, the high virus-containing freshwater discharge in winter due to a higher proportion of anthropogenic sewage relative to low natural flooding in winter run-off, turned out to be the first factor contributing to the high winter viral abundance and VBR values. In addition, the variation of intrusion of warm and relatively oligotrophic water from oceanic currents played a role alternating the distribution patterns of temperature, salinity and trophic conditions and consequently the distribution patterns of virus and bacteria seasonally and spatially. Dynamics of virus in the Yangtze river estuarine area is thus characterized by distinct seasonal and spatial variations due to natural forcing and by pronounced alternation of the regular patterns due to anthropogenic impacts. [TOP OF PAGE]

  171. Major technological advances and trends in cheese. Johnson,M.E., Lucey,J.A. (2006). J. Dairy Sci. 89:1174-1178. Over the last 25 yr, cheese production in the United States has more than doubled with most of the increase due to production in the western states. Processing large volumes of milk into cheese has necessitated changes in vat size and design, reliance on computer software, and milk standardization, including use of membrane concentration of milk either at the cheese plant or on the farm. There has been increased interest in specialty cheeses including cheese made from sheep, goat, and organic milks. In addition, membrane processing of whey into various value-added components has become routine. Changes in cheese manufacturing protocols have resulted in a reduction of the manufacturing time and the necessity for consistent and reliable starter activity. Major advances in the genetics of microorganisms have not only resulted in widespread use of fermentation-produced chymosin but also in starter bacteria with improved resistance to bacteriophage infection. Genomics and proteomics have increased the likelihood of the development of nonstarter adjuncts with specific enzymatic activity. Indeed, the use of adjunct microorganisms to produce cheese with a unique flavor profile or to produce cheese with more consistent or better quality flavor has gained almost universal acceptance. [TOP OF PAGE]

  172. Bacteriophage-mediated competition in Bordetella bacteria. Joo,J., Gunny,M., Cases,M., Hudson,P., Albert,R., Harvill,E. (2006). Proc. R. Soc. Lond. B Biol. Sci. 273:1843-1848. Apparent competition between species is believed to be one of the principal driving forces that structure ecological communities, although the precise mechanisms have yet to be characterized. Here we develop a model system that isolates phage-mediated interactions by neutralizing resource competition with a large excess of nutrients, and consists of two genetically identical Bordetella strains that differ only in that one is the carrier of phage and the other is susceptible to the phage. We observe and quantify the competitive advantage of the bacterial strain bearing the prophage in both invading and in resisting invasion by the bacterial strain sensitive to the phage, and use our experimental measurements to develop a mathematical model of phage-mediated competition. The model predicts, and experimental evidence confirms, that the competitive advantage conferred by the lysogenic phage depends only on the phage pathology on the sensitive bacterial strain and is independent of other phage and host parameters, such as the infection-causing contact rate, the spontaneous and infection-induced lysis rates and the phage burst size. This work combines experimental and mathematical approaches to the study of phage-driven competition, and provides an experimentally tested framework for evaluation of the effects of pathogens/parasites on interspecific competition. [TOP OF PAGE]

  173. Unstable lysogeny and pseudolysogeny in Vibrio harveyi siphovirus-like phage 1. Khemayan,K., Pasharawipas,T., Puiprom,O., Sriurairatana,S., Suthienkul,O., Flegel,T.W. (2006). Appl. Environ. Microbiol. 72:1355-1363. Exposure of Vibrio harveyi (strain VH1114) to V. harveyi siphovirus-like phage 1 (VHS1) resulted in the production of a low percentage of lysogenized clones of variable stability. These were retrieved most easily as small colonies within dot plaques. Analysis revealed that VHS1 prophage was most likely carried by VH1114 as an episome rather than integrated into the host chromosome. In the late exponential growth phase, lysogenized VH1114 continuously produced VHS1 but also gave rise to a large number of cured progeny. The absence of phage DNA in the cured progeny was confirmed by the absence of VHS1 DNA in Southern blot and PCR assays. Curiously, these very stable, cured subclones did not show the parental phenotype of clear plaques with VHS1 but instead showed turbid plaques, both in overlaid lawns and in dot plaque assays. This phenotypic difference from the original parental isolate suggested that transient lysogeny by VHS1 had resulted in a stable genetic change in the cured clones. Such clones may be called pseudolysogens (i.e., false lysogens), since they have undergone transient lysogeny and have retained some resistance to full lytic phage development, despite the loss of viable or detectable prophage. [TOP OF PAGE]

  174. The genetic basis of thermal reaction norm evolution in lab and natural phage population. Khies,J.L., Izem,R., Supler,K.L., Kingsolver,J.G., Burch,C.L. (2006). PLoS Biol. 4:e207 Two major goals of laboratory evolution experiments are to integrate from genotype to phenotype to fitness, and to understand the genetic basis of adaptation in natural populations. Here we demonstrate that both goals are possible by re-examining the outcome of a previous laboratory evolution experiment in which the bacteriophage G4 was adapted to high temperatures. We quantified the evolutionary changes in the thermal reaction norms—the curves that describe the effect of temperature on the growth rate of the phages—and decomposed the changes into modes of biological interest. Our analysis indicated that changes in optimal temperature accounted for almost half of the evolutionary changes in thermal reaction norm shape, and made the largest contribution toward adaptation at high temperatures. Genome sequencing allowed us to associate reaction norm shape changes with particular nucleotide mutations, and several of the identified mutations were found to be polymorphic in natural populations. Growth rate measures of natural phage that differed at a site that contributed substantially to adaptation in the lab indicated that this mutation also underlies thermal reaction norm shape variation in nature. In combination, our results suggest that laboratory evolution experiments may successfully predict the genetic bases of evolutionary responses to temperature in nature. The implications of this work for viral evolution arise from the fact that shifts in the thermal optimum are characterized by tradeoffs in performance between high and low temperatures. Optimum shifts, if characteristic of viral adaptation to novel temperatures, would ensure the success of vaccine development strategies that adapt viruses to low temperatures in an attempt to reduce virulence at higher (body) temperatures. [TOP OF PAGE]

  175. Alternative methods to limit extracellular bacterial activity for enumeration of intracellular bacteria. Kim,H.J., Kim,E.Y., Hong,Y., Rhee,J.H., Choy,H.E. (2006). J. Microbiol. Meth. 64:17-26. The gentamicin survival assay, a method routinely used to estimate bacterial infection of eukaryotic host cells, depends on the presumed limited penetration of gentamicin across the eukaryotic cell membrane. However, some studies have suggested that gentamicin may in fact enter eukaryotic cells and kill intracellular bacteria. In this study we devised alternative methods to enumerate intracellular Salmonellae using a lytic bacteriophage, SP6, and an amino acid auxotroph, Pro- mutant, which replicates selectively within host cells in the presence of its uptake inhibitor, 3,4-dehydro-L-proline. The conventional gentamicin survival assay was systematically compared with the alternative methods for the enumeration of intracellular Salmonellae. We found that gentamicin decreases the survival of intracellular Salmonellae when added to extracellular media at concentrations above 20 microg/ml. The alternative methods do not suffer from this disadvantage, suggesting that they should be used to replace the gentamicin survival assay. In addition, the proline auxotroph method could be applied to detect bacterial release from host cells. [TOP OF PAGE]

  176. Evolution of complexity in the viral world: The dawn of a new vision. Koonin,E.V., Dolja,V.V. (2006). Virus Res. 117:1-4. Recent sequencing of the genomes of numerous large viruses provide for unprecedented opportunities to study the emergence and evolution of complexity in the virus world. This special issue of Virus Research explores trends in the evolution of complex genomes in most major classes of viruses. [TOP OF PAGE]

  177. A putative RNA-interference-based immune system in prokaryotes: the epitome of prokaryotic genome diversity. Koonin,E.V., Makarova,K.S., Grishin,N.V., Wolf,Y.I. (2006). pp. 39-64. In In Logan,N.A., Lappin-Scott,H.M., and Oyston,P.C.F. (eds.), Prokaryotic Diversity: Mechanisms and Significance. Cambridge University Press, Cambridge, UK. [TOP OF PAGE]

  178. [Substantiation for the model value of somatic coliphage T2 in virological control of water preparation technology risk assessment]. Korchak,G.I., Skorokhod,I.N., Surmasheva,E.V. (2006). Gigiena i Sanitariia 37-39. [TOP OF PAGE]

  179. [Ambivalent bacteriophages of different species active on Escherichia coli K12 and Salmonella sps. strains]. Krylov,V.N., Miller,S., Rachel,R., Biebl,M., Pletneva,E.A., Shuetz,M., Krylov,S.V., Shaburova,O.V. (2006). Genetika 42:159-168. A study was made of several bacteriophages (including phages U2 and LB related to T-even phages of Escherichia coli) that grow both on E. coli K12 and on some Salmonella strains. Such phages were termed ambivalent. T-even ambivalent phages (U2 and LB) are rare and have a limited number of hosts among Salmonella strains. U2 and LB are similar to canonical E. coli-specific T-even phages in morphological type and size of the phage particle and in reaction with specific anti-T4 serum. Phages U2 and LB have identical sets of structural proteins, some of which are similar in size to structural proteins of phages T2 and T4. DNA restriction patterns of phages U2 and LB differ from each other and from those of T2 and T4. Still, DNAs of all four phages have considerable homology. Unexpectedly, phages U2 and LB grown on Salmonella bungori were unstable during centrifugation in a CsCl gradient. Ambivalent bacteriophages were found in species other than T-even phages and were similar in morphotype to lambdoid and other E. coli phages. One of the ambivalent phages was highly similar to well-known Felix01, which is specific for Salmonella. Ambivalent phages can be used to develop a new set for phage typing in Salmonella. An obvious advantage is that ambivalent phages can be reproduced in the E. coli K12 laboratory strain, which does not produce active temperate phages. Consequently, the resulting typing phage preparation is devoid of an admixture of temperate phages, which are common in Salmonella. The presence of temperate phages in phage-typing preparations may cause false-positive results in identifying specific Salmonella strains isolated from the environment or salmonellosis patients. Ambivalent phages are potentially useful for phage therapy and prevention of salmonellosis in humans and animals. [TOP OF PAGE]

  180. Emergence of biofilm-forming subpopulations upon exposure of Escherichia coli to environmental bacteriophages. Lacqua,A., Wanner,O., Colangelo,T., Martinotti,M.G., Landini,P. (2006). Appl. Environ. Microbiol. 72:956-959. Exposure of Escherichia coli MG1655 to environmental bacteriophages results in rapid selection for phage-tolerant subpopulations displaying increased biofilm formation. Analysis of one phage-tolerant strain revealed large amounts of the DNA-binding Dps protein in the outer membrane protein and production of fimbria-like structures. In dps and fimA mutant derivatives of MG1655, no selection of phage-tolerant bacteria upon exposure to bacteriophages occurred, suggesting a role for Dps and type I pili in bacteriophage tolerance. [TOP OF PAGE]

  181. FDA approves use of bacteriophages to be added to meat and poultry products. Lang,L.H. (2006). Gastroenterology 131:1370 [first paragraph] In the Federal Register of August 18, 2006, the US Food and Drug Administration (FDA) announced that it had approved the use of a bacteriophage preparation made from 6 individually purified phages (LMP-102) to be used on ready-to-eat (RTE) meat and poultry products as an antimicrobial agent against Listeria monocytogenes. The ruling came in response to a food additive petition submitted in 2002 from Intralytix, Inc. (Baltimore, MD), the biotech company that produces the bacteriophage. [TOP OF PAGE]

  182. Induction of temperate cyanophage AS-1 by heavy metal--copper. Lee,L.H., Lui,D., Platner,P.J., Hsu,S.F., Chu,T.C., Gaynor,J.J., Vega,Q.C., Lustigman,B.K. (2006). BMC Microbiol. 6:17 BACKGROUND: It has been reported that some marine cyanophage are temperate and can be induced from a lysogenic phase to a lytic phase by different agents such as heavy metals. However, to date no significant reports have focused on the temperate nature of freshwater cyanophage/cyanobacteria. Previous experiments with cyanophage AS-1 and cyanobacteria Anacystis nidulans have provided some evidence that AS-1 may have a lysogenic life cycle in addition to the characterized lytic cycle. RESULTS: In this study, the possible temperate A. nidulans was treated with different concentrations of heavy metal-copper. CuSO4 with concentrations of 3.1 x 10(-3) M, 3.1 x 10(-4) M, 3.1 x 10(-5) M and 3.1 x 10(-6) M were used to detect the induction of AS-1 from A. nidulans. The population of the host, unicellular cyanobacteria Anacystis nidulans, was monitored by direct count and turbidity while the amount of virus produced was derived from plaque forming units (PFU) by a direct plating method. The ratio of AS-1 release from A. nidulans was also determined. From these results it appears that AS-1 lysogenic phage can be induced by copper at concentrations from 3.1 x 10(-6) M to 3.1 x 10(-4) M. Maximal phage induction occurred at 6 hours after addition of copper, with an optimal concentration of 3.1 x 10(-6) M. CONCLUSION: Cu2+ is a significant inducer for lysogenic cyanobacterial cells and consequently would be a potential control agent in the cyanobacteria population in fresh water ecosystems. [TOP OF PAGE]

  183. Plasmids and prophages in Baltic Sea bacterioplankton isolates. Leitet,C., Riemann,L., Hagström,Ä. (2006). J. Mar. Bio. Assoc. UK 86:567-575. Plasmids and phages influence bacterial phenotype and may serve as vectors for transferring genes between bacteria. In the present study, we examined 130 marine bacterioplankton isolates for the presence of plasmids and prophages. Samples were obtained in spring, summer and autumn in the Baltic Sea proper. Plasmids and inducible prophages were found in 19% and 28% of the isolates, respectively. During spring, plasmids and prophages were 41-55% and 30% more common compared to the summer and autumn measurements and prevalence varied up to five-fold between bacterial phylogenetic groups, with the highest plasmid prevalence found in Bacteriodetes (41%), and lysogeny being common in a-, b-, and g-Proteobacteria (32-50%). Plasmid genome sizes ranged from 1.5-15kb with most in the 2.1-4.0kb size-range. No plasmids showed identity to the broad-host-range incompatibility groups N and P. Phage genomes ranged in size from 8-87kb, with 57% being 35-45kb in size. Strain typing of phages with similar genome sizes by means of DP-RAPD (degenerated primer randomly amplified polymorphic DNA) showed that all were different (except two that were not resolved). In PFGE (pulsed-field gel electrophoresis) 34% of the lysates produced multiple bands. Transmission electron microscopy suggested that these originated from several phage morphotypes indicating that polylysogeny is common. The widespread distribution of small cryptic plasmids as well as of lysogeny and polylysogeny in Baltic Sea bacterioplankton may have important implications for bacterial phenotype and for lateral gene transfer; hence, the ecological significance of these vectors in marine environments requires further study. [TOP OF PAGE]

  184. [Removal of coliphages by wastewater treatment processes]. Li,M., Hu,H.Y., Zhang,X., Shen,H. (2006). Huan Jing Ke Xue 27:80-84. The concentrations of somatic coliphages (SC) and F-specific RNA bacteriophages in effluent of three wastewater treatment plants in Beijing city were detected. Somatic coliphages and F-RNA bacteriophages in source wastewater were 6.25 x 10(3) - 1.34 x 10(4) PFU x mL(-1) and 2.4 x 10 - 2.4 x 10(3) PFU x mL(-1) respectively, and the corresponding average removal rates were 72.45% - 99.89 % and 57.84% - 93.06% by the wastewater processes, and which were lower than that of faecal coliforms. Biological aerated stage appeared to be the most efficient step in reducing the numbers of phages in wastewater, but not obviously in sand filter. The result of predicted concentrations of enteroviruses according to concentrations of F-RNA bacteriophages in water show that there are 0.65 - 15.8 PFU x L(-1) of the enteroviruses in final effluent. [TOP OF PAGE]

  185. Structural constraints and mutational bias in the evolutionary restoration of a severe deletion in RNA phage MS2. Licis,N., van Duin,J. (2006). J. Mol. Evol. 63:314-329. A 4-nucleotide (nt) deletion was made in the 36-nt-long intercistronic region separating the coat and replicase genes of the single-stranded RNA phage MS2. This region is the focus of several RNA structures conferring high fitness. One such element is the operator hairpin, which, in the course of infection, will bind a coat-protein dimer, thereby precluding further replicase synthesis and initiating encapsidation. Another structure is a long-distance base pairing (MJ) controlling replicase expression. The 4-nt deletion does not directly affect the operator hairpin but it disrupts the MJ pairing. Its main effect, however, is a frame shift in the overlapping lysis gene. This gene starts in the upstream coat gene, runs through the 36-nt-long intercistronic region, and ends in the downstream replicase cistron. Here we report and interpret the spectrum of solutions that emerges when the crippled phage is evolved. Four different solutions were obtained by sequencing 40 plaques. Three had cured the frame shift in the lysis gene by inserting one nt in the loop of the operator hairpin causing its inactivation. Yet these low-fitness revertants could further improve themselves when evolved. The inactivated operator was replaced by a substitute and thereafter these revertants found several ways to restore control over the replicase gene. To allow for the evolutionary enrichment of low-probability but high-fitness revertants, we passaged lysate samples before plating. Revertants obtained in this way also restored the frame shift, but not at the expense of the operator. By taking larger and larger lysates samples for such bulk evolution, ever higher-fitness and lower-frequency revertants surfaced. Only one made it back to wild type. As a rule, however, revertants moved further and further away from the wild-type sequence because restorative mutations are, in the majority of cases, selected for their capacity to improve the phenotype by optimizing one of several potential alternative RNA foldings that emerge as a result of the initial deletion. This illustrates the role of structural constraints which limit the path of subsequent restorative mutations. [TOP OF PAGE]

  186. Two novel bacteriophages of thermophilic bacteria isolated from deep-sea hydrothermal fields. Liu,B., Wu,S., Song,Q., Zhang,X., Xie,L. (2006). Curr. Microbiol. 53:163-166. Bacteriophages of thermophiles are of great interest due to their important roles in many biogeochemical and ecological processes. However, no virion has been isolated from deep-sea thermophilic bacteria to date. In this investigation, two lytic bacteriophages (termed Bacillus virus W1 and Geobacillus virus E1) of thermophilic bacteria were purified from deep-sea hydrothermal fields in the Pacific for the first time. Bacillus virus W1 (BVW1) obtained from Bacillus sp. w13, had a long tail (300nm in length and 15 nm in width) and a hexagonal head (70 nm in diameter). Another virus, Geobacillus virus E1 (GVE1) from Geobacillus sp. E26323, was a typical Siphoviridae phage with a hexagonal head (130 nm in diameter) and a tail (180 nm in length and 30 nm in width). The two phages contained double-stranded genomic DNAs. The genomic DNA sizes of BVW1 and GVE1 were estimated to be about 18 and 41 kb, respectively. Based on SDS-PAGE of purified virions, six major proteins were revealed for each of the two phages. The findings in our study will be very helpful to realize the effect of virus on thermophiles as well as the communities in deep-sea hydrothermal fields. [TOP OF PAGE]

  187. Protein repertoire of double-stranded DNA bacteriophages. Liu,J., Glazko,G., Mushegian,A. (2006). Virus Res. 117:68-80. The complexity and diversity of phage gene sets, which are produced by rapid evolution of phage genomes and rampant gene exchanges among phages, hamper the efforts to decipher the evolutionary relationships between individual phage proteins and reconstruct the complete set of evolutionary events leading to the known phages. To start unraveling the natural history of phages, we built the phage orthologous groups (POGs), a natural system of phage protein families that includes 6378 genes from 164 complete genome sequences of double-stranded DNA bacteriophages. Phage proteomes have high POG coverage: on average, 39 genes per phage genome belong to POGs, which is close to half of all genes in most phages. In an agreement with the notion of phage role in horizontal gene transfer, we see many cases of likely gene exchange between phages and their microbial hosts. At the same time, about 80% of all POGs are highly specific to phage genomes and are not commonly found in microbial genomes, indicating coherence and large degree of evolutionary independence of phage gene sets. The information on orthologous genes is essential for evolutionary classification of known bacteriophages and for reconstruction of ancestral phage genomes. [TOP OF PAGE]

  188. Occurrence of bacterial indicators and bacteriophages infecting enteric bacteria in groundwater in different geographical areas. Lucena,F., Ribas,F., Duran,A.E., Skraber,S., Gantzer,C., Campos,C., Moron,A., Calderon,E., Jofre,J. (2006). J. Appl. Microbiol. 101:96-102. AIMS: The aim of this research was to determine the suitability of coliphages (bacteriophages) for assessing the microbial quality of groundwater. METHODS AND RESULTS: The number of several bacterial indicators (faecal coliforms, Escherichia coli, enterococci and spores of sulfite-reducing clostridia) and bacteriophages (somatic coliphages, F-specific RNA bacteriophages and bacteriophages infecting Bacteroides fragilis) were determined in groundwater of aquifers in various geographical areas. Results show that the relative abundance, determined as percentages of positive detections, of the bacterial indicators and bacteriophages varies depending on the aquifer. CONCLUSIONS: A single bacterial indicator may not be enough to assess microbiological quality in certain aquifers. One bacterial indicator and a bacteriophage parameter provide more information than two bacterial indicators. SIGNIFICANCE AND IMPACT OF THE STUDY: Coliphages (CPH) provide different information from that provided by bacterial indicators on the microbial quality of groundwater in different geographical areas. Easy, fast and inexpensive methods for the detection of CPH are feasible in both industrialized and developing countries. [TOP OF PAGE]

  189. [The presence of bacteriophages in human feces and their potential importance]. Lusiak-Szelachowska,M., Weber-Dabrowska,B., Gorski,A. (2006). Polski merkuriusz lekarski 21:381-383. Bacteriophages are widely distributed throughout the environment as well as in the bodies of humans and animals (feces, urine, saliva, sputum). Higher presence of Escherichia coli phages compared with Bacteroides fragilis and Salmonella phages was noticed in the feces of healthy human individuals and patients, mainly those with gastro-intestinal tract diseases. A strict correlation exists between the number of bacteria and of phages in the feces of healthy individuals as well as of patients with different diseases. The presence of phages in human feces correlates with the character of the coexisting disease. The frequency of phages in the feces depends on the different indicator bacterial host strains and the numbers of indicator strains. The role of bacteriophages in protecting against pathogenic microorganisms and controlling bacterial flora in the human organism is of major significance. [TOP OF PAGE]

  190. Sequence and comparative genomic analysis of lactococcal bacteriophages jj50, 712 and P008: evolutionary insights into the 936 phage species. Mahony,J., Deveau,H., Mc Grath,S., Ventura,M., Canchaya,C., Moineau,S., Fitzgerald,G.F., van Sinderen,D. (2006). FEMS Microbiol. Lett. 261:253-261. The complete genome sequences of three lactococcal 936-type bacteriophages, 712, jj50 and P008, were determined. Comparative genomic analysis of these phages with the previously sequenced 936-type phages, sk1 and bIL170, reveals a strict conservation of the overall genetic organization of this geographically diverse phage group. Genetic divergence was mainly observed in the early expressed region of the phage genomes, where a number of deletions, exchanges and insertions appear to have occurred. These genetic differences may be responsible for the observed differential sensitivity to the lactococcal DNA injection blocking protein, Sie(2009), and the abortive infection system, AbiA. [TOP OF PAGE]

  191. The use of bacteriophages for monitoring the microbiological quality of sewage sludge. Mandilara,G., Mavridou,A., Lambiri,M., Vatopoulos,A., Rigas,F. (2006). Environ. Technol. 27:367-375. The use of bacteriophages as potential indicators of faecal pollution has recently been studied. The correlation of the number of bacterial indicators and the presence of three groups of bacteriophages, namely somatic coliphages (SOMCPH), F-RNA specific phages (FRNAPH) and phages of Bacteroides fragilis (BFRPH), in raw and treated sludge is presented in this study. Raw and anaerobically digested sewage sludge samples from two wastewater treatment plants in Athens were collected on a monthly basis, over a 2-year period, and analyzed for total coliforms, E. coli, intestinal enterococci and the three groups of bacteriophages. A clear correlation between the number of bacterial indicators and the presence of bacteriophages was observed. E. coli concentrations of > or =10(3) cfus g(-1) and <10(3) cfus g(-1) comprise a threshold for the presence of FRNAPH and BFRPH, respectively. Likewise, intestinal enterococci concentrations of > or =10(4) cfus g(-1) and <10(3) cfus g(-1) comprise a threshold for the presence of FRNAPH and BFRPH, respectively. In the case of SOMCPH, it was not possible to define a threshold, since they were detected with the lowest observed indicator concentrations in all samples. [TOP OF PAGE]

  192. Correlation between bacterial indicators and bacteriophages in sewage and sludge. Mandilara,G.D., Smeti,E.M., Mavridou,A.T., Lambiri,M.P., Vatopoulos,A.C., Rigas,F.P. (2006). FEMS Microbiol. Lett. 263:119-126. The use of bacteriophages as potential indicators of faecal pollution has recently been studied. The correlation of the number of bacterial indicators and the presence of three groups of bacteriophages, namely somatic coliphages (SOMCPH), F-RNA-specific phages (FRNAPH) and phages of Bacteroides fragilis (BFRPH), in raw and treated wastewater and sludge is presented in this study. Raw and treated wastewater and sewage sludge samples from two wastewater treatment plants in Athens were collected on a monthly basis, over a 2-year period, and analysed for total coliforms, Escherichia coli, intestinal enterococci and the three groups of bacteriophages. A clear correlation between the number of bacterial indicators and the presence of bacteriophages was observed. SOMCPH may be used as additional indicators, because of their high densities and resistance to various treatment steps. [TOP OF PAGE]

  193. Phages of cyanobacteria. Mann,N.H. (2006). pp. 517-533. In In Calendar,R. and Abedon,S.T. (eds.), The Bacteriophages. Oxford University Press, Oxford. [first paragraph] The scientific importance of the phages of cyanobacteria (cyanophages) is intimately associated with the ecological significance of their hosts. Cyanobacteria are arguably the most diverse and widely distributed group of eubacteria on the planet and play central roles in major biogeochemical processes, such as the carbon and nitrogen cycles. Cyanobacteria exist in a wide range of freshwater and marine environments, ranging from thermophilic to psycrophilic, and terrestrial environments, including those subject to periodic desiccation. By virtue of their higher plant-like oxygenic photosynthetic apparatus they contribute significantly to the maintenance of the Earth's atmosphere, in terms of both oxygen production and carbon dioxide fixation. Consequently, the ability of cyanophages to determine the population structures and genetic diversity of cyanobacteria, as well as potentially influence the dynamics of biogeochemical processes, gives them a unique ecological significance. [TOP OF PAGE]

  194. Multiplication of therapeutically administered bacteriophages in Pseudomonas aeruginosa infected patients. Marza,J.A.S., Soothill,J.S., Boydell,P., Collyns,T.A. (2006). Burns 32:644-646. [first paragraph] Antibiotic resistant strains of bacteria are an increasing problem and Pseudomonas aeruginosa is one of the most resistant species [1]. No new classes of anti-pseudomonal agent have been developed for over 30 years. It is time to consider alternative strategies such as bacteriophage (phage) therapy. [TOP OF PAGE]

  195. Temperate and lytic cyanophages from the Gulf of Mexico. McDaniel,L.D., delaRosa,M., Paul,J.H. (2006). J. Mar. Bio. Assoc. UK 86:517-527. The unicellular cyanobacterial species Synechococcus and Prochlorococcus are known to be vital components of marine ecosystems, especially in the vast oligotrophic areas. Lytic cyanophages infecting unicellular phytoplankton are prevalent and have been demonstrated to act as important constraints on community composition contributing to the seasonal succession in genotypes. Lysogeny in Synechococcus has been documented experimentally in natural environments by prophage induction. At this time it is completely unknown how prevalent lysogeny is among Synechococcus populations. This study was performed to document important features such as size, morphology and the incidence of the T4-like capsid portal protein gene (g20) in a group of lytic Synechococcus cyanophages (35 isolates) isolated from the Gulf of Mexico. A group of Synechococcus isolates (24 strains) were isolated concurrently to investigate the virulence and cross-infectivity of the lytic cyanophages and to determine the frequency of lysogeny by detection of inducible prophage. The host range of the cyanophages toward these Synechococcus strains ranged from 1 of 25 (host of isolation only) to 17 of 25 (68%). Of the 35 cyanophage isolates the large majority were myoviruses (94%) and only two (6%) were of the podovirus type. The expected polymerase chain reaction product for g20 was detected in 20 of the phages (63%). The presence of a detectable g20 was associated with low-infectivity cyanophages at the 90% confidence interval. The Synechococcus strains varied in their resistance to lytic infection from 11% to resistance to all of the phage isolates utilized in testing. The prevalence of inducible prophage-like particles was determined in the Synechococcus strains using mitomycin C and enumerating viruses by epifluorescence microscopy. A statistically significant increase in viruses was detected in 11 of the strains (46%) in response to mitomycin C. There was no observed relationship between the occurrence of prophage induction in the Synechococcus isolates and their resistance to lytic infection. One putative lysogen was induced by continuous high light and contained a prophage-like particle with a single-stranded DNA (ssDNA) genome. Such a prophage-like particle is unlike any prophage described to date, implying that the process of lysogeny is unique in certain marine Synechococcus strains. [TOP OF PAGE]

  196. Enhanced contrast of bacteriophage plaques in Salmonella with ferric ammonium citrate and sodium thiosulfate (FACST) and tetrazolium red (TZR). McLaughlin,M.R., Balaa,M.F. (2006). J. Microbiol. Meth. 65:318-323. Visualization of bacteriophage plaques may be enhanced by addition of ferric ammonium citrate and sodium thiosulfate (FACST) or 2,3,5-triphenyltetrazolium chloride (tetrazolium red, TZR) to the soft agar layer of a traditional bacteriophage plaque assay. Background color from these reagents improved contrast between clear plaques and turbid host lawns in trypticase soy agar (TSA) plates. Enhancement by FACST is based on reaction with hydrogen sulfide gas (H2S) produced by some strains of bacteria and was tested here using H2S+ and H2S- strains of Salmonella enterica subsp. enterica with a bacteriophage (Podoviridae) isolated from swine lagoon effluent. Only the H2S+ strain produced dark brown-black color in FACST-amended agar. Both strains showed bright pinkish-red color in TZR-amended agar. Color intensity for both reagents decreased with decreasing concentrations of the reagents. Contrast in FACST-amended plates appeared greater than that with TZR, but diminished after 12 h, while contrast in TZR-amended plates remained constant. At the concentrations tested, neither reagent affected plaque counts in the H2S+ strain. The FACST should be useful in bacteriophage plaque assays with H2S+ strains of Salmonella and other H2S+ bacteria. [TOP OF PAGE]

  197. Isolation of Salmonella bacteriophages from swine effluent lagoons. McLaughlin,M.R., Balaa,M.F., Sims,J., King,R. (2006). J. Environ. Qual. 35:522-528. Bacteriophages (phages) associated with Salmonella were collected from nine swine manure lagoons in Mississippi. Phages were isolated by an enrichment protocol or directly from effluent. For enrichment, chloroform-treated samples were filtered (0.22 mum) and selectively enriched by adding a cocktail of Salmonella strains in trypticase soy broth. After overnight incubation at 35 degrees C, chloroform was added and samples stored at 5 degrees C. Enriched samples were tested by double agar layer (DAL) plaque assay against individual Salmonella isolates. Phage titers of 2.9 x 10(8) to 2.1 x 10(9) plaque forming units (pfu) per mL were produced, but estimation of phage titers in lagoons was not possible. For direct isolation, effluent was clarified by centrifugation, filtered (0.22 microm), and used in DAL plaque assays to select single-plaque isolates for 15 Salmonella strains. Plaque counts varied among Salmonella strains and lagoons. The most sensitive strain for direct phage recovery was ATCC 13311. Phage titers estimated by direct isolation with ATCC 13311 ranged among lagoons from 12 to 148 pfu per mL. In limited host range tests, 66 isolates recovered by the enrichment protocol produced plaques only on Enteritidis and Typhimurium strains of Salmonella and none produced plaques on lagoon isolates of Citrobacter, Escherichia, Proteus, Providencia, or Serratia. Electron microscopy (EM) showed purified enrichment isolates had Podoviridae morphology (tailless 50-nm icosahedral heads with tail spikes). Electron microscopy of clarified concentrated effluent showed 5.5:1 tailless to tailed phages. The isolated phages have potential as typing reagents, specific indicators, and biocontrol agents of Salmonella. [TOP OF PAGE]

  198. Factors affecting iron sulfide-enhanced bacteriophage plaque assays in Salmonella. McLaughlin,M.R. (2006). J. Microbiol. Meth. 67:611-615. Reaction of ferric ions with hydrogen sulfide (H(2)S) enhances contrast of phage plaques in H(2)S+ Salmonella, but contrast diminishes in weak H(2)S+ strains. H(2)S was affected by concentrations of peptones, glucose, ferric ammonium citrate (FAC) and sodium thiosulfate (ST), and by FAC:ST ratio, temperature, pH, air, and host strain. Increasing peptone levels was most important for improving contrast in weak H(2)S+ strains. [TOP OF PAGE]

  199. Phage therapy. Merril,C.R., Scholl,D., Adhya,S. (2006). pp. 725-741. In In Calendar,R. and Abedon,S.T. (eds.), The Bacteriophages. Oxford University Press, Oxford. [first paragraph] The ability of bacteriophage (phage) to replicate exponentially and lyse pathogenic strains of bacteria suggests that they should play a vital role in our armamentarium for the treatment of infectious diseases. However, in spite of an initial enthusiasm, early clinical applications resulted in a negative shift of opinion concerning the therapeutic potential of phage. There are a number of factors that may have been responsible for this rejection of the use of phage as antibacterial therapeutic agents, particularly in countries that require certification based on the results of efficacy and pharmacokinetic studies in animals and humans. These factors include an initial lack of understanding of the relatively narrow host range of phage and an inability to purify phage preparations from bacteria products and debris. These contaminating materials often include bacterial exo- and endotoxins along with bacterial cellular components that tend to inactivate phage preparations when they are stored without further purification (60). Another major factor that affected the development of phage therapy was the successful introduction of antibiotics effective against a broad range of bacterial strains. With such antibiotics physicians could often successfully treat infections even before they determined the causative bacterial strain. The narrow host range of phage made duplication of such a practice questionable at best. [TOP OF PAGE]

  200. Viral lysis of bacteria: an important source of dissolved amino acids and cell wall compounds. Middelboe,M., Jørgensen,N.O.G. (2006). J. Mar. Bio. Assoc. UK 86:605-612. Viral infection of bacteria causes release of dissolved organic matter (DOM), which is available for bacterial uptake. In aquatic environments, this virus-mediated transformation of living cells into dissolved and colloidal organic matter may be a quantitatively important process in the pelagic recycling of carbon and nutrients, but little is known about the amount, composition, or bioavailability of viral lysates. By using a model system of a marine bacterium (Cellulophaga sp.) and a virus specific to this bacterium, the present study provides a first quantification of the input of dissolved free and combined amino acids (DFAA and DCAA) and bacterial cell wall compounds following viral lysis. The DCAA constituted 51-86% of the total virus-mediated organic carbon release of 1087-1825?gCl-1 (estimated biomass of the lysed bacteria), whereas DFAA and glucosamine each accounted for 2-3% of total lysate-C. The viral particles themselves constituted 4-6% of the released organic carbon, and altogether, the applied analyses thus identified 53-92% of the released lysates. Approximately 12% of the identified compounds were derived from bacterial cell wall peptidoglycan, including various D-isomers of DFAA and DCAA, glucosamine and diaminopimelic acid (DAPA). Although a portion of this cell wall material may have entered the pool of refractory material, a significant fraction of some peptidoglycan-derived components, e.g. 83% of the released D-DFAA, were removed from the dissolved phase during the last part of the incubations, suggesting that part of the cell wall material were utilized by the developing virus-resistant Cellulophaga population. Therefore, we suggest that virus-mediated DOM is a source of a variety of organic compounds, which contribute significantly to the pool of rapidly recycling material in the ocean. [TOP OF PAGE]

  201. A temporal and spatial investigation of cyanophage abundance in the Gulf of Aqaba, Red Sea. Millard,A.D., Mann,N.H. (2006). J. Mar. Bio. Assoc. UK 86:507-515. The aim of this study was to determine the abundance of cyanophages over an annual cycle in the Red Sea from the period April 1999 to December 1999 at a range of depths. Cyanophage numbers from 71 water samples were determined by the use of plaque assays using four different Synechococcus strains. The results indicate that cyanophage are found throughout the water column from surface waters to depths of 150m, with a discrete maximum in the number of cyanophages in the summer months of July, August and September at a depth of 30m. Eighty-seven cyanophages were isolated and characterized in terms of host range, genome size and possession of a myoviral portal vertex gene. Cyanophages were found to infect multiple strains of Synechococcus from different phylogenetic clades. The genome sizes of cyanophages were also found to be bigger than previously estimated. [TOP OF PAGE]

  202. Marine phages. Miller,R.V. (2006). pp. 534-544. In In Calendar,R. and Abedon,S.T. (eds.), The Bacteriophages. Oxford University Press, Oxford. [first three paragarphs] Two decades ago marine bacteriophages were unimportant to microbial ecologists. After all, bacteriophages could only be important in certain environments where their concentrations were high enough to have an effect on bacterial populations. In most microbiologists' minds, this limited them to just a few ecosystems such as waste treatment facilities, cheese and yogurt production facilities, and the microbiology laboratory. It certainly did not include marine environments! Of course, specific phages such as PM2 were of interest (18, 87; see also chapter 14) because of their cytology and molecular microbiology. The few studies that had addressed the frequency of viruses in the oceans, however, showed that marine bacteriophages were not prevalent enough to affect marine ecosystems. ¶ Two important developments in the 1980's changed this picture and stimulated interest in aquatic bacteriophages. The first arose from concerns originating from the newly emerging environmental biotechnology industry's use of genetically engineered microorganisms. Many feared that the recombinant sequences would escape to naturally occurring bacteria by horizontal gene exchange (36). Soon it was demonstrated that transduction was a viable mechanism for genetic exchange in aquatic environments (27, 37, 47, 68, 70, 72, 73). Still, phages were considered to be too infrequent in the aquatic environment to make this a real possibility. The second development, however, eliminated this objection and clearly demonstrated the importance of bacteriophages in marine environments. In 1989, Bratbak, Heldal and others (5, 8, 11) demonstrated that in many aquatic environments bacteriophages were present in very high concentrations and that they often exceeded by one to two orders of magnitude the concentrations of bacterioplankton that were their hosts. This report was quickly followed by several confirmatory publication (42, 64) that made it clear that bacteriophages are indeed real players in the ecology of marine habitats. ¶ This chapter is designed to provide the reader with an up-to-date and informed overview of the current understanding of the abundance of bacteriophages in aquatic, especially marine, environments. It is meant to demonstrate that bacterial viruses are important members of these ecosystems. It will explore the current controversy over the role of bacteriophages in the microbial loops of marine food webs (12). More detailed accounts of the subject can be found in several recent reviews (10, 20, 63, 79, 84, 100). See also chapter 32, which reviews cyanophages, the viruses of cyanobacteria. [TOP OF PAGE]

  203. Resistance development of bacterial biofilms against bacteriophage attack. Moons,P., Werckx,W., Van Houdt,R., Aertsen,A., Michiels,C.W. (2006). Communications in agricultural and applied biological sciences 71:297-300. [TOP OF PAGE]

  204. Relative number of generations of hosts and parasites does not influence parasite local adaptation in coevolving populations of bacteria and phages. Morgan,A.D., Buckling,A. (2006). J. Evol. Biol. 19:1956-1963. A potential consequence of host-parasite coevolution in spatially structured populations is parasite local adaptation: local parasites perform better than foreign parasites on their local host populations. It has been suggested that the generally shorter generation times of parasites compared with their hosts contributes to parasites, rather than hosts, being locally adapted. We tested the hypothesis that relative generation times of hosts and parasites affect local adaptation of hosts and parasites, using the bacterium Pseudomonas fluorescens and a lytic phage as host and parasite, respectively. Generation times were not directly manipulated, but instead one of the coevolving partners was regularly removed and replaced with a population from an earlier time point. Thus, one partner underwent more generations than the other. Manipulations were carried out at both early and later periods of coevolutionary interactions. At early stages of coevolution, host and parasites that underwent relatively more generations displayed higher levels of resistance and infectivity, respectively. However, the relative number of generations that bacteria and phages underwent did not change the level of local adaptation relative to control populations. This is likely because generalist hosts and parasites are favoured during early stages of coevolution, preventing local adaptation. By contrast, at later stages manipulations had no effect on either average levels of resistance or infectivity, or alter the level of local adaptation relative to the controls, possibly because traits other than resistance and infectivity were under strong selection. Taken together, these data suggest that the relative generation times of hosts and parasites may not be an important determinant of local adaptation in this system. [TOP OF PAGE]

  205. Quantifying the significance of phage attack on starter cultures: a mechanistic model for population dynamics of phage and their hosts isolated from fermenting sauerkraut. Mudgal,P., Breidt,F.J., Lubkin,S.R., Sandeep,K.P. (2006). Appl. Environ. Microbiol. 72:3908-3915. We investigated the possibility of using starter cultures in sauerkraut fermentation and thereby reducing the quantity of salt used in the process. This, in turn, would reduce the amount of waste salt that would enter in our water resources. Phage, naturally present in sauerkraut fermentation, could potentially affect the starter cultures introduced. Thus, a mechanistic mathematical model was developed to quantify the growth kinetics of the phage and starter cultures. The model was validated by independent experiments with two Leuconostoc mesenteroides strains isolated from sauerkraut and their corresponding phage. Model simulations and experimental evidence showed the presence of phage-resistant cell populations in starter cultures which replaced phage-sensitive cells, even when the initial phage density (P(0)) and multiplicity of infection (MOI) were low (P(0) < 1 x 10(3) PFU/ml; MOI < 10(-4)) in the MRS media. Based on the results of model simulation and parameter optimization, it was suggested that the kinetic parameters of phage-host interaction, especially the adsorption rate, vary with the initial phage and host densities and with time. The model was validated in MRS broth. Therefore, the effects of heterogeneity and other environmental factors, such as temperature and pH, should be considered to make the model applicable to commercial fermentations. [TOP OF PAGE]

  206. Viruses as pathogens of marine organisms—from bacteria to whales. Munn,C.B. (2006). J. Mar. Bio. Assoc. UK 86:453-467. Viruses are the most abundant members of marine ecosystems and play an enormous role in ocean processes through their interactions with all types of marine organisms. This short review provides examples of the dramatic increase in our knowledge of the diversity of marine viruses as pathogens of bacteria, protists, molluscs, crustaceans, cnidaria, reptiles, fish and mammals. Several examples are provided showing evidence of evolution of new strains, changes in virulence, and transfer of viruses between ecosystems. The natural and anthropogenic causes of these shifts are discussed. Despite considerable advances in recent years, knowledge of the importance of viruses in many important groups of marine organisms is lacking or incomplete. Suggestions for future investigations necessary to understand the dynamics of biogeochemical processes and the impacts of disease in our oceans are proposed. [TOP OF PAGE]

  207. Enteric viruses in inlet and outlet samples from sewage treatment plants. Myrmel,M., Berg,E.M.M., Grinde,B., Rimstad,E. (2006). J. Water Health 4:197-209. Samples collected every two weeks from the inlet and outlet of three sewage treatment plants were screened for the presence of noro-, rota-, astro-, adeno-, hepatitis A- and circoviruses by (RT)-nested PCR, and for F-specific bacteriophages by isolation in Escherichia coli Famp. Plants A and B were secondary treatment plants and plant C used primary treatment. Noroviruses were detected in 43%, 53% and 24% of the inlet samples and 26%, 40% and 21% of the outlet samples from plants A, B and C, respectively. Astroviruses, rotaviruses and adenoviruses were more prevalent. Adenoviruses were detected in 96% of inlet and 94% of outlet samples, supporting the potential of these viruses as indicators of viral contamination from sewage. Hepatitis A virus and circoviruses were found only rarely. Reduction of infective viral particles during sewage treatment was evaluated using F-specific bacteriophages. The phages were reduced by, respectively, 99%, 87% and 0% in plants A, B and C, which corresponded to the observed differences in reduction of norovirus positive samples between the same plants. The study shows that the high viral load in sewage results in a discharge to the environment of a large amount of virus despite sewage treatment. On the other hand, the advantage of a more advanced treatment is demonstrated. [TOP OF PAGE]

  208. Dinoflagellate-infecting viruses. Nagasaki,K., Tomaru,Y., Shirai,Y., Takao,Y., Mizumoto,H. (2006). J. Mar. Bio. Assoc. UK 86:469-474. Dinoflagellates (Dinophyceae) are considered to be one of the most abundant and diverse groups of phytoplankton; however, the viral impact on dinoflagellates was not studied until recently. This review shows the present information concerning the viruses infecting dinoflagellates and the ecology relationships between the host and the virus. So far, two viruses have been isolated and characterized: a large DNA virus (HcV: Heterocapsa circularisquama virus) and a small RNA virus (HcRNAV: H. circularisquama RNA virus); both of which are infectious to the harmful bloom-forming dinoflagellate H. circularisquama. [TOP OF PAGE]

  209. Male-specific coliphages as indicators of thermal inactivation of pathogens in biosolids. Nappier,S.P., Aitken,M.D., Sobsey,M.D. (2006). Appl. Environ. Microbiol. 72:2471-2475. Male-specific (F+) coliphages have been proposed as a candidate indicator of fecal contamination and of virus reduction in waste treatment. However, in this and earlier work with a laboratory thermophilic anaerobic digester, a heat-resistant fraction of F+ coliphage populations indigenous to municipal wastewater and sludge was evident. We therefore isolated coliphages from municipal wastewater sludge and from biosolid samples after thermophilic anaerobic digestion to evaluate the susceptibility of specific groups to thermal inactivation. Similar numbers of F+ DNA and F+ RNA coliphages were found in untreated sludge, but the majority of isolates in digested biosolids were group I F+ RNA phages. Separate experiments on individual isolates at 53 degrees C confirmed the apparent heat resistance of group I F+ RNA coliphages as well as the susceptibility of group III F+ RNA coliphages. Although few F+ DNA coliphages were recovered from the treated biosolid samples, thermal inactivation experiments indicated heat resistance similar to that of group I F+ RNA phages. Hence, F+ DNA coliphage reductions during thermophilic anaerobic digestion are probably related to mechanisms other than thermal inactivation. Further studies should focus on the group III F+ RNA coliphages as potential indicators of reductions of heat-resistant pathogens in thermal processes for sludge treatment. [TOP OF PAGE]

  210. UV disinfection of wastewater effluents for unrestricted irrigation. Nasser,A.M., Paulman,H., Sela,O., Ktaitzer,T., Cikurel,H., Zuckerman,I., Meir,A., Aharoni,A., Adin,A. (2006). Water Sci. Technol. 54:83-88. Wastewater reuse in arid regions is important for the production of a water resource to be utilised for non-potable purposes and to prevent the environmental transmission of disease-causing agents. This study was conducted to evaluate the effect of water quality on the comparative disinfection efficiency of viruses, bacteria and spores by UV irradiation. Furthermore, the microbial quality of effluent produced by coagulation, high rate filtration (HRF) and either UV irradiation or chlorination was determined. Using low pressure collimated beam, a UV dose of 80 mWs/cm2 was needed to achieve a 3-log10 inactivation of either rotavirus SA-11 or coliphage MS2, whereas over 5-log10 inactivation of E. coli was reached with a dose of only 20 mWs/cm2. B. subtilis inactivation was found to be linear up to a dose of 40 mWs/cm2 and then a tailing up to a UV dose of 120 mWs/cm2 was observed. It is worth noting that effluent turbidity of < 5 NTU did not influence the inactivation efficiency of UV irradiation. Operation of a pilot plant to treat secondary effluent by coagulation, HRF and UV disinfection at a UV dose of 80 mWs/cm2 resulted in the production of high quality effluent in compliance with the Israel standards for unrestricted irrigation (< 10 CFU/100 mL faecal coliform and turbidity of < 5 NTU). Sulphite reducing clostridia (SRC) were found to be more resistant than coliphages and F coliform for UV irradiation. The results of this study indicated that UV disinfection is suitable for the production of effluents for unrestricted irrigation of food crops. [TOP OF PAGE]

  211. Genetic diversity among five T4-like bacteriophages. Nolan,J.M., Petrov,V., Bertrand,C., Krisch,H.M., Karam,J.D. (2006). Virol. J. 3:30 BACKGROUND: Bacteriophages are an important repository of genetic diversity. As one of the major constituents of terrestrial biomass, they exert profound effects on the earth's ecology and microbial evolution by mediating horizontal gene transfer between bacteria and controlling their growth. Only limited genomic sequence data are currently available for phages but even this reveals an overwhelming diversity in their gene sequences and genomes. The contribution of the T4-like phages to this overall phage diversity is difficult to assess, since only a few examples of complete genome sequence exist for these phages. Our analysis of five T4-like genomes represents half of the known T4-like genomes in GenBank. RESULTS: Here, we have examined in detail the genetic diversity of the genomes of five relatives of bacteriophage T4: the Escherichia coli phages RB43, RB49 and RB69, the Aeromonas salmonicida phage 44RR2.8t (or 44RR) and the Aeromonas hydrophila phage Aeh1. Our data define a core set of conserved genes common to these genomes as well as hundreds of additional open reading frames (ORFs) that are nonconserved. Although some of these ORFs resemble known genes from bacterial hosts or other phages, most show no significant similarity to any known sequence in the databases. The five genomes analyzed here all have similarities in gene regulation to T4. Sequence motifs resembling T4 early and late consensus promoters were observed in all five genomes. In contrast, only two of these genomes, RB69 and 44RR, showed similarities to T4 middle-mode promoter sequences and to the T4 motA gene product required for their recognition. In addition, we observed that each phage differed in the number and assortment of putative genes encoding host-like metabolic enzymes, tRNA species, and homing endonucleases. CONCLUSION: Our observations suggest that evolution of the T4-like phages has drawn on a highly diverged pool of genes in the microbial world. The T4-like phages harbour a wealth of genetic material that has not been identified previously. The mechanisms by which these genes may have arisen may differ from those previously proposed for the evolution of other bacteriophage genomes. [TOP OF PAGE]

  212. The newly isolated lytic bacteriophages st104a and st104b are highly virulent against Salmonella enterica. O'Flynn,G., Coffey,A., Fitzgerald,G.F., Ross,R.P. (2006). J. Appl. Microbiol. 101:251-259. AIMS: To screen Irish faecal samples from a variety of sources with a view to isolating novel anti-Salmonella phages and to subsequently evaluate their lytic capability. METHODS AND RESULTS: Two novel anti-Salmonella phages st104a and st104b were isolated from a screening programme based on their lytic capability. The phages produced significantly larger plaques (2 mm) on the chosen indicator Salmonella enterica strain, DPC6046, when compared with the well-known control phage, Felix 01 (0.5 mm). Both phages st104a and st104b were found to have a broad host range within the Salm. enterica species. During in vitro trials, both phages (st104a and st104b) reduced Salm. enterica numbers more than 99% within 1 h. In vivo studies, involving the addition of the phage to porcine gastric juice (pH 2.5) demonstrated that phage st104a and phage Felix 01 were capable of surviving (10 and 30% survival respectively) the acidic conditions, unlike st104b, which was undetectable after 2 h exposure. CONCLUSIONS: Two novel lytic anti-Salmonella phages were isolated and characterized. SIGNIFICANCE AND IMPACT OF THE STUDY: With the exception of phage Felix 01, there has been relatively little phage therapy work performed using lytic Salmonella phage. In this study, the lytic phages st104a and st104b were isolated as a result of a faecal screening programme. Subsequently, phage st104a was found to have potential for biocontrol of Salm. enterica numbers if administered orally to pigs given their survival in porcine gastric juice, whereas, phage st104b may have potential in reducing cell numbers if applied by alternative approaches. [TOP OF PAGE]

  213. Horizontal gene transfer and its role in the emergence of new phenotypes. Osborn,A.M. (2006). pp. 275-293. In In Logan,N.A., Lappin-Scott,H.M., and Oyston,P.C.F. (eds.), Prokaryotic Diversity: Mechanisms and Significance. Cambridge University Press, Cambridge, UK. [TOP OF PAGE]

  214. Viral burst size of heterotrophic prokaryotes in aquatic systems. Parada,V., Herndl,G.J., Weinbauer,M.G. (2006). J. Mar. Bio. Assoc. UK 86:613-621. Viral burst size (BS), i.e. the number of viruses released during cell lysis, is a critical parameter for assessing the ecological and biogeochemical role of viruses in aquatic systems. Burst size is typically estimated by enumerating the viral particles in bacteria using transmission electron microscopy. Here, we review the average BS reported for different aquatic systems, present several hypotheses on the control of the BS and evaluate whether there are relationships between BS and bacterial activity parameters across systems. Based on reports from a variety of different aquatic environments, we calculated a mean BS of 24 and 34 for marine and freshwater environments, respectively. Generally, the BS increased with the trophic status of the environment and with the percentage of infected cells in marine populations. When diel dynamics were investigated or averages from large-scale environments were used, BS was positively related to bacterial production but no trend was detectable across systems. The across systems' finding that BS was significantly related to the frequency of infected cells (FIC) could be due to co-infection or superinfection. At any given site, BS seems to be influenced by a number of factors such as the size of the host cell and the viruses, the metabolic activity of the host and phage and host diversity. Thus, based on the available data collected over the past two decades on a variety of aquatic systems, some relations between BS and bacterial variables were detectable. [TOP OF PAGE]

  215. Microbial inactivation by microwave radiation in the home environment. Park,D.K., Bitton,G., Melker,R. (2006). J. Environ. Health 69:17-24. The study reported here looked at the survival of microorganisms (heterotrophic plate counts, total coliforms, E. coli, and bacterial spores) in a consumer-type microwave oven. Kitchen sponges, scrubbing pads, and syringes were experimentally contaminated with wastewater and subsequently exposed to microwave radiation. At 100 percent power level, it was found that the heterotrophic plate count (i.e., total bacterial count) of the wastewater was reduced by more that 99 percent within 1 to 2 minutes, and the total coliform and E. coli were totally inactivated after 30 seconds of microwave radiation. Bacterial phage MS2 was totally inactivated within 1 to 2 minutes. Spores of Bacillus cereus were more resistant than the other microorganisms tested, and were completely eradicated only after 4-minute irradiation. Similar inactivation rates were obtained in wastewater-contaminated scrubbing pads. Microorganisms attached to plastic syringes were more resistant to microwave irradiation than those associated with kitchen sponges or scrubbing pads. It took 10 minutes for total inactivation of the heterotrophic plate count and 4 minutes for total inactivation of total coliform and E. coli. A 4-log reduction of phage MS2 was obtained after 2 minutes; 97.4 percent reduction was observed after 12 minutes. The authors also observed a higher inactivation of B. cereus spores in syringes placed in a ceramic container than of spores in syringes placed in a glass container. This finding could have some implications for the design of containers to be used in exposure of medical devices to microwave radiation. The article discusses the implications of these findings for consumer safety in the home environment. [TOP OF PAGE]

  216. Flow cytometric analysis of virus-like particles and heterotrophic bacteria within coral-associated reef water. Patten,N.L., Seymour,J.R., Mitchell,J.G. (2006). J. Mar. Bio. Assoc. UK 86:563-566. Using flow cytometry, two distinct populations of virus-like particles (VLP) and heterotrophic bacteria were defined within the 12cm water layer immediately overlying healthy, diseased and dead acroporid corals. Bacterial abundances were similar in overlying water for all coral types, however, VLP were 30% higher above diseased corals than healthy or dead corals. Mean virus to bacteria ratios (VBR) were up to 30% higher above diseased corals than above healthy or dead coral or in distant water. Concomitant with increasing VLP concentrations within 5cm of coral surfaces, VBR distributions were generally highest above healthy and diseased coral and depressed above dead coral. These results suggest fundamental shifts in the VLP and bacterial community in water associated with diseased corals. [TOP OF PAGE]

  217. Origin and evolution of overlapping genes in the family Microviridae. Pavesi,A. (2006). J. Gen. Virol. 87:1013-1017. The possibility of creating novel genes from pre-existing sequences, known as overprinting, is a widespread phenomenon in small viruses. Here, the origin and evolution of gene overlap in the bacteriophages belonging to the family Microviridae have been investigated. The distinction between ancestral and derived frames was carried out by comparing the patterns of codon usage in overlapping and non-overlapping genes. By this approach, a gradual increase in complexity of the phage genome--from an ancestral state lacking gene overlap to a derived state with a high density of genetic information--was inferred. Genes encoding less-essential proteins, yet playing a role in phage growth and diffusion, were predicted to be novel genes that originated by overprinting. Evaluation of the rates of synonymous and non-synonymous substitution yielded evidence for overlapping genes under positive selection in one frame and purifying selection in the alternative frame. [TOP OF PAGE]

  218. Variable pleiotropic effects from mutations at the same locus hamper prediction of fitness from a fitness component. Pepin,K.M., Samuel,M.A., Wichman,H.A. (2006). Genetics 172:2047-2056. The relationship of genotype, fitness components, and fitness can be complicated by genetic effects such as pleiotropy and epistasis and by heterogeneous environments. However, because it is often difficult to measure genotype and fitness directly, fitness components are commonly used to estimate fitness without regard to genetic architecture. The small bacteriophage fX174 enables direct evaluation of genetic and environmental effects on fitness components and fitness. We used 15 mutants to study mutation effects on attachment rate and fitness in six hosts. The mutants differed from our lab strain of fX174 by only one or two amino acids in the major capsid protein (gpF, sites 101 and 102). The sites are variable in natural and experimentally evolved fX174 populations and affect phage attachment rate. Within the limits of detection of our assays, all mutations were neutral or deleterious relative to the wild type; 11 mutants had decreased host range. While fitness was predictable from attachment rate in most cases, 3 mutants had rapid attachment but low fitness on most hosts. Thus, some mutations had a pleiotropic effect on a fitness component other than attachment rate. In addition, on one host most mutants had high attachment rate but decreased fitness, suggesting that pleiotropic effects also depended on host. The data highlight that even in this simple, well-characterized system, prediction of fitness from a fitness component depends on genetic architecture and environment. [TOP OF PAGE]

  219. A peptidoglycan hydrolase motif within the mycobacteriophage TM4 tape measure protein promotes efficient infection of stationary phase cells. Piuri,M., Hatfull,G.F. (2006). Mol. Microbiol. 62:1569-1585. The predominant morphotype of mycobacteriophage virions has a DNA-containing capsid attached to a long flexible non-contractile tail, features characteristic of the Siphoviridae. Within these phage genomes the tape measure protein (tmp) gene can be readily identified due to the well-established relationship between the length of the gene and the length of the phage tail--because these phages typically have long tails, the tmp gene is usually the largest gene in the genome. Many of these mycobacteriophage Tmp's contain small motifs with sequence similarity to host proteins. One of these motifs (motif 1) corresponds to the Rpf proteins that have lysozyme activity and function to stimulate growth of dormant bacteria, while the others (motifs 2 and 3) are related to proteins of unknown function, although some of the related proteins of the host are predicted to be involved in cell wall catabolism. We show here that motif 3-containing proteins have peptidoglycan-hydrolysing activity and that while this activity is not required for phage viability, it facilitates efficient infection and DNA injection into stationary phase cells. Tmp's of mycobacteriophages may thus have acquired these motifs in order to avoid a selective disadvantage that results from changes in peptidoglycan in non-growing cells. [TOP OF PAGE]

  220. Functional origins of fitness effect-sizes of compensatory mutations in the DNA bacteriophage fX174. Poon,A.F.Y., Chao,L. (2006). Evolution 60:2032-2043. Epistasis is an important and poorly understood aspect of mutations and strongly influences the evolutionary impact of genetic variation on adaptation and fitness. Although recent studies have begun to characterize the distribution of epistatic effects between mutations affecting fitness, there is currently a lack of empirical information on the underlying biological causes of these epistatic interactions. What are the functional constraints that determine the effectiveness of a compensatory mutation at restoring fitness? We have measured the effect-sizes of 52 compensatory mutations affecting nine different deleterious mutations in the major capsid and spike proteins of the DNA bacteriophage fX174. On average, an experimentally detectable compensatory mutation recovers about two-thirds of the fitness cost of the preceding deleterious mutation. Variation in fitness effect-sizes is only weakly associated with measures of the distance separating the deleterious and compensatory mutations in the amino acid sequence or the folded protein structure. However, there is a strong association of fitness effect-size with the correlation in the effects of the mutations on the biochemical properties of amino acids. A compensatory mutation has the largest effect-size, on average, when both the compensatory and deleterious mutations have radical effects on the overall biochemical make-up of the amino acids. By examining the relative contributions of specific biochemical properties to variation in fitness effect-size, we find that the area and charge of amino acids have a major influence, which suggests that the complexity of the amino acid phenotype is simplified by selection into a reduced number of phenotypic components. [TOP OF PAGE]

  221. Microbiological quality of groundwater sources used by rural communities in Limpopo Province, South Africa. Potgieter,N., Mudau,L.S., Maluleke,F.R.S. (2006). Water Sci. Technol. 54:371-377. A survey of the microbiological quality of water from 194 boreholes (97 privately owned and 97 communal boreholes) in the rural Thitale-Hlanganani area of the Limpopo Province, South Africa was carried out between August 2002 and August 2003. Very little information on the microbiological quality of privately-owned boreholes in rural communities is available raising concerns about the safety of these groundwater supplies. In this study, levels of total coliforms, thermotolerant (faecal) coliforms, faecal enterococci, Clostridium perfringens (vegetative cells and spores) and somatic coliphages were determined for community and privately-owned borehole water. The average counts for total coliforms, faecal coliforms, faecal enterococci and Clostridium perfringens exceeded the South African recommended guideline limits of 0-10 counts.100 ml(-1) for total coliforms and 0 counts. 100 ml(-1) for faecal coliforms, faecal enterococci and Clostridium perfringens respectively. Comparisons between the levels of indicator bacteria present in private and communal boreholes during dry seasons indicated a statistical difference for faecal enterococci bacteria (p = 0.005) and Clostridium perfringens (p = 0.08). Comparisons between the levels of indicator bacteria present in private and communal boreholes during rainy seasons indicated statistical differences between total coliforms (p = 0.05), faecal coliforms (p = 0.03) and Clostridium perfringens (p = 0.009) bacteria. No significant differences in the presence of somatic coliphages in both private and communal borehole water were see [sic]. The results indicated the need for environmental impact assessment studies to monitor the microbiological quality of groundwater sources in rural communities. [TOP OF PAGE]

  222. A naturally occurring novel allele of Escherichia coli outer membrane protein A reduces sensitivity to bacteriophage. Power,M.L., Ferrari,B.C., Littlefield-Wyer,J., Gordon,D.M., Slade,M.B., Veal,D.A. (2006). Appl. Environ. Microbiol. 72:7930-7932. A novel Escherichia coli outer membrane protein A (OmpA) was discovered through a proteomic investigation of cell surface proteins. DNA polymorphisms were localized to regions encoding the protein's surface-exposed loops which are known phage receptor sites. Bacteriophage sensitivity testing indicated an association between bacteriophage resistance and isolates having the novel ompA allele. [TOP OF PAGE]

  223. Evolutionary genomics of archaeal viruses: Unique viral genomes in the third domain of life. Prangishvili,D., Garrett,R.A., Koonin,E.V. (2006). Virus Res. 117:52-67. In terms of virion morphology, the known viruses of archaea fall into two distinct classes: viruses of mesophilic and moderately thermophilic Eueryarchaeota closely resemble head-and-tail bacteriophages whereas viruses of hyperthermophilic Crenarchaeota show a variety of unique morphotypes. In accord with this distinction, the sequenced genomes of euryarchaeal viruses encode many proteins homologous to bacteriophage capsid proteins. In contrast, initial analysis of the crenarchaeal viral genomes revealed no relationships with bacteriophages and, generally, very few proteins with detectable homologs. Here we describe a re-analysis of the proteins encoded by archaeal viruses, with an emphasis on comparative genomics of the unique viruses of Crenarchaeota. Detailed examination of conserved domains and motifs uncovered a significant number of previously unnoticed homologous relationships among the proteins of crenarchaeal viruses and between viral proteins and those from cellular life forms and allowed functional predictions for some of these conserved genes. A small pool of genes is shared by overlapping subsets of crenarchaeal viruses, in a general analogy with the metagenome structure of bacteriophages. The proteins encoded by the genes belonging to this pool include predicted transcription regulators, ATPases implicated in viral DNA replication and packaging, enzymes of DNA precursor metabolism, RNA modification enzymes, and glycosylases. In addition, each of the crenarchaeal viruses encodes several proteins with prokaryotic but not viral homologs, some of which, predictably, seem to have been scavenged from the crenarchaeal hosts, but others might have been acquired from bacteria. We conclude that crenarchaeal viruses are, in general, evolutionarily unrelated to other known viruses and, probably, evolved via independent accretion of genes derived from the hosts and, through more complex routes of horizontal gene transfer, from other prokaryotes. [TOP OF PAGE]

  224. Evidence of host-virus co-evolution in tetranucleotide usage patterns of bacteriophages and eukaryotic viruses. Pride,D.T., Wassenaar,T.M., Ghose,C., Blaser,M.J. (2006). BMC Genom. 7:8 BACKGROUND: Virus taxonomy is based on morphologic characteristics, as there are no widely used non-phenotypic measures for comparison among virus families. We examined whether there is phylogenetic signal in virus nucleotide usage patterns that can be used to determine ancestral relationships. The well-studied model of tail morphology in bacteriophage classification was used for comparison with nucleotide usage patterns. Tetranucleotide usage deviation (TUD) patterns were chosen since they have previously been shown to contain phylogenetic signal similar to that of 16S rRNA. RESULTS: We found that bacteriophages have unique TUD patterns, representing genomic signatures that are relatively conserved among those with similar host range. Analysis of TUD-based phylogeny indicates that host influences are important in bacteriophage evolution, and phylogenies containing both phages and their hosts support their co-evolution. TUD-based phylogeny of eukaryotic viruses indicates that they cluster largely based on nucleic acid type and genome size. Similarities between eukaryotic virus phylogenies based on TUD and gene content substantiate the TUD methodology. CONCLUSION: Differences between phenotypic and TUD analysis may provide clues to virus ancestry not previously inferred. As such, TUD analysis provides a complementary approach to morphology-based systems in analysis of virus evolution. [TOP OF PAGE]

  225. Genomewide screens for Escherichia coli genes affecting growth of T7 bacteriophage. Qimron,U., Marintcheva,B., Tabor,S., Richardson,C.C. (2006). Proc. Natl. Acad. Sci. USA 103:19039-19044. Use of bacteriophages as a therapy for bacterial infection has been attempted over the last century. Such an endeavor requires the elucidation of basic aspects of the host-virus interactions and the resistance mechanisms of the host. Two recently developed bacterial collections now enable a genomewide search of the genetic interactions between Escherichia coli and bacteriophages. We have screened >85% of the E. coli genes for their ability to inhibit growth of T7 phage and >90% of the host genes for their ability to be used by the virus. In addition to identifying all of the known interactions, several other interactions have been identified. E. coli CMP kinase is essential for T7 growth, whereas overexpression of the E. coli uridine/cytidine kinase inhibits T7 growth. Mutations in any one of nine genes that encode enzymes for the synthesis of the E. coli lipopolysaccharide receptor for T7 adsorption leads to T7 resistance. Selection of T7 phage that can recognize these altered receptors has enabled the construction of phage to which the host is 100-fold less resistant. [TOP OF PAGE]

  226. Coliphage hsa as a model for antiviral studies/spectrum by some indigenous bacteriocin like inhibitory substances (BLIS). Qureshi,H., Saeed,S., Ahmed,S., Rasool,S.A. (2006). Pakist. J. Pharm. Sci. 19:182-185. Coliphage HSA was isolated from a raw sewage sample (collected from a local sewage treatment plant). The phage was analyzed by spot and tube lysis followed by plaque assay. Phage titre (plaque forming units i.e. PFU) was found to be 4.2 x 103 PFU/mL. Further purification of the phage was achieved by acid-precipitation method. Genomic identification of the coliphage HSA (done by fluorescent staining using acridine orange) revealed it to be a dsDNA bacterial virus. Staphylococcin188, Enterocins AAR-71, AAR-74, and Erwiniocin NA4 were screened for their antiphage activity by plaque assay. Accordingly, all the bacteriocin preparations possess demonstrable antiphage activity witnessed as a reduction in PFU after treatment. In the case of Staphylococcin 188, the number dropped up to 40 PFU/mL, Enterocin AAR-71 and Erwiniocin NA4 treatment reduced it to a zero PFU level, while Enterocin AAR-74 could reduce PFU to 50 (after addition of a constant volume, 500uL, of each of the crude bacteriocin preparations). Transmission Electron Microscopy studies revealed the phage to have an icosahedral head with a long tail and tail fibers. [TOP OF PAGE]

  227. Aquatic viruses: the emerging story. Raven,J.A. (2006). J. Mar. Bio. Assoc. UK 86:449-451. It is likely that all living organisms can be infected by one or more viruses. One of the latest higher taxa to be converted from 'no characterized viruses' to 'well characterized viruses' are the diatoms (Bacillariophyceae, Heterokontophyta) with the recent publication of three papers characterizing an ssRNA and a ssDNA virus from two genera (Chaetoceros and Rhizosolenia) of marine planktonic diatom (Nagasaki et al., 2004, 2005; Bettarel et al., 2005). It would have been strange if viruses had not been able to exploit the dominant, in terms of global primary production, photosynthetic organisms in the ocean (assimilating perhaps as much as 20 Pg inorganic C into organic C per year), despite the less than completely convincing arguments assembled by Raven & Waite (2004) as to possible anti-viral defences unique to diatoms. [TOP OF PAGE]

  228. Isolation and characterization of a new T-even bacteriophage, CEV1, and determination of its potential to reduce Escherichia coli O157:H7 levels in sheep. Raya,R.R., Varey,P., Oot,R.A., Dyen,M.R., Callaway,T.R., Edrington,T.S., Kutter,E.M., Brabban,A.D. (2006). Appl. Environ. Microbiol. 72:6405-6410. Bacteriophage CEV1 was isolated from sheep resistant to Escherichia coli O157:H7 colonization. In vitro, CEV1 efficiently infected E. coli O157:H7 grown both aerobically and anaerobically. In vivo, sheep receiving a single oral dose of CEV1 showed a 2-log-unit reduction in intestinal E. coli O157:H7 levels within 2 days compared to levels in the controls. [TOP OF PAGE]

  229. Phage for rapid detection and control of bacterial pathogens in food. Rees,C.E.D., Dodd,C.E.R. (2006). Adv. Appl. Microbiol. 59:159-186. [first paragraph] In recent years there has been a revival of interest in the use of phage to treat bacterial infections, and research using phage therapy to overcome the problem of increasing levels of antibiotic resistance has become widely publicized (Sulakvelidze and Kutter, 2005). Accordingly, the idea that phage could be applied to food products as biocontrol agents has also received more interest among researchers. However, those working in the dairy industry are only too aware of the potential for these viruses to destroy populations of bacteria in milk-starter cultures. On the other hand, the use of phage is nothing new in the field of bacterial characterization, and phage typing schemes are routinely used in the subtyping of isolates of organisms such as Salmonella enterica (Anderson et al., 1977; Laszlo and Csorian, 1988), Staphylococcus aureus (Blair and Williams, 1961), Listeria monocytogenes (Rocourt, 1996), Vibrio cholerae (Basu and Mukerjee, 1968), and Escherichia coli O157:H7 (Khakhria et al., 1990). Over the last 10 years much work has been carried out to develop phage-based methods for rapid detection of pathogens in foods and, although the only commercially available test is that for detection of Mycobacterium tuberculosis in human sputum samples (Mole and Maskell, 2001), many new tests and applications for detection of pathogens in foods are currently being developed. This chapter will provide an overview of the recent developments in these fields and look at some new areas that may be developed into practical applications in the future. [TOP OF PAGE]

  230. Linking bacteriophage infection to quorum sensing signalling and bioluminescent bioreporter monitoring for direct detection of bacterial agents. Ripp,S., Jegier,P., Birmele,M., Johnson,C.M., Daumer,K.A., Garland,J.L., Sayler,G.S. (2006). J. Appl. Microbiol. 100:488-499. AIM: To incorporate into the lambda phage genome, a luxI-based acyl-homoserine lactone (AHL) synthase genetic construct and exploit the autoamplified power of quorum sensing to translate a phage infection event into a chemical signature detectable by a lux-based bioluminescent bioreporter, with focus towards facile detection of microbial pathogens. METHODS AND RESULTS: The luxI gene from Vibrio fischeri was inserted into the lambda phage genome to construct a model phage-based biosensor system for the general detection of Escherichia coli. The AHL signalling molecules synthesized upon phage infection are detected by an AHL-specific bioluminescent bioreporter based on the luxCDABE gene cassette of V. fischeri. The assay generates target-specific visible light signals with no requisite addition of extraneous substrate. This binary reporter system was able to autonomously respond to lambda phage infection events at target E. coli concentrations ranging from 1 x 10(8) to 1 CFU ml(-1) within 1.5-10.3 h, respectively, in pure culture. When assayed against artificially contaminated lettuce leaf washings, detection within an E. coli inoculum range from 1 x 10(8) to 130 CFU ml(-1) was achieved within 2.6-22.4 h, respectively. CONCLUSIONS: The initial feasibility of binary phage-based reporter assays indicates that quorum sensing can be used to translate a phage infection event into an autoamplified chemical signature. SIGNIFICANCE AND IMPACT OF STUDY: With further modification, binary phage-based reporter assays may be capable of rapidly and cost effectively detecting pathogenic agents at very low population densities. [TOP OF PAGE]

  231. Horizontal gene transfer and the evolution of microvirid coliphage genomes. Rokyta,D.R., Burch,C.L., Caudle,S.B., Wichman,H.A. (2006). J. Bacteriol. 188:1134-1142. Bacteriophage genomic evolution has been largely characterized by rampant, promiscuous horizontal gene transfer involving both homologous and nonhomologous source DNA. This pattern has emerged through study of the tailed double-stranded DNA (dsDNA) phages and is based upon a sparse sampling of the enormous diversity of these phages. The single-stranded DNA phages of the family Microviridae, including fX174, appear to evolve through qualitatively different mechanisms, possibly as result of their strictly lytic lifestyle and small genome size. However, this apparent difference could reflect merely a dearth of relevant data. We sought to characterize the forces that contributed to the molecular evolution of the Microviridae and to examine the genetic structure of this single family of bacteriophage by sequencing the genomes of microvirid phage isolated on a single bacterial host. Microvirids comprised 3.5% of the detectable phage in our environmental samples, and sequencing yielded 42 new microvirid genomes. Phylogenetic analysis of the genes contained in these and five previously described microvirid phages identified three distinct clades and revealed at least two horizontal transfer events between clades. All members of one clade have a block of five putative genes that are not present in any member of the other two clades. Our data indicate that horizontal transfer does contribute to the evolution of the microvirids but is both quantitatively and qualitatively different from what has been observed for the dsDNA phages. [TOP OF PAGE]

  232. Host range of 14 mycobacteriophages in Mycobacterium ulcerans and seven other mycobacteria including Mycobacterium tuberculosis--application for identification and susceptibility testing. Rybniker,J., Kramme,S., Small,P.L. (2006). J. Med. Microbiol. 55:37-42. The host range of well-characterized mycobacteriophages, such as D29 and TM4, has been determined, together with that of more recently isolated mycobacteriophages, in Mycobacterium ulcerans, Mycobacterium tuberculosis, Mycobacterium bovis BCG, Mycobacterium avium, Mycobacterium marinum, Mycobacterium scrofulaceum, Mycobacterium fortuitum and Mycobacterium chelonae. Here, a set of virulent phages for M. ulcerans, a pathogen with a dramatic increase of incidence over the last decade, is demonstrated. In this work, a mycobacteriophage replication assay was adapted for the identification and rifampicin-susceptibility testing of M. ulcerans. Mycobacteriophages have generated a number of useful tools and enabled insights into mycobacterial genetics. With regard to the neglected pathogen M. ulcerans, the findings presented in this work allow the application of a large range of phage-based vectors and markers. The potential of phage therapy can now be evaluated for this extracellular pathogen. [TOP OF PAGE]

  233. Seasonal variations in virus-host populations in Norwegian coastal waters: focusing on the cyanophage community infecting marine Synechococcus spp. Sandaa,R.A., Larsen,A. (2006). Appl. Environ. Microbiol. 72:4610-4618. Viruses are ubiquitous components of the marine ecosystem. In the current study we investigated seasonal variations in the viral community in Norwegian coastal waters by pulsed-field gel electrophoresis (PFGE). The results demonstrated that the viral community was diverse, displaying dynamic seasonal variation, and that viral populations of 29 different sizes in the range from 26 to 500 kb were present. Virus populations from 260 to 500 kb and dominating autotrophic pico- and nanoeukaryotes showed similar dynamic variations. Using flow cytometry and real-time PCR, we focused in particular on one host-virus system: Synechococcus spp. and cyanophages. The two groups covaried throughout the year and were found in the highest amounts in fall with concentrations of 7.3 x 10(4) Synechococcus cells ml(-1) and 7.2 x 10(3) cyanophage ml(-1). By using primers targeting the g20 gene in PCRs on DNA extracted from PFGE bands, we demonstrated that cyanophages were found in a genomic size range of 26 to 380 kb. The genetic richness of the cyanophage community, determined by denaturing gradient gel electrophoresis (DGGE) of PCR-amplified g20 gene fragments, revealed seasonal shifts in the populations, with one community dominating in spring and summer and a different one dominating in fall. Phylogenetic analysis of the sequences originating from PFGE and DGGE bands grouped the sequences into three groups, all with homology to cyanomyoviruses present in cultures. Our results show that the cyanophage community in Norwegian coastal waters is dynamic and genetically diverse and has a surprisingly wide genomic size range. [TOP OF PAGE]

  234. WO bacteriophage transcription in Wolbachia-infected Culex pipiens. Sanogo,Y.O., Dobson,S.L. (2006). Insect Biochem. Mol. Biol. 36:80-85. Bacteriophages are commonly found in association with free-living bacteria, both as exogenic phages (virions) and as prophages integrated into the bacterial genome. In contrast, the observation of bacteriophages associated with obligate intracellular bacteria has been described infrequently. An exception is provided by Wolbachia endosymbionts, which harbor multiple phage elements that have been designated as WO phage. Wolbachia are maternally inherited bacteria that occur in the cytoplasm of many invertebrates, where they often manipulate host reproduction. Previously, the WO phage orf7 locus and ankyrin repeat-encoding genes have been observed to represent sources of genetic diversity between Wolbachia (wPip) strains infecting mosquitoes of the Culex pipiens complex and have been suggested as potential participants in the reproductive manipulations. We have characterized WO phage associated with multiple Wolbachia-infected Culex strains and an uninfected strain using electron microscopy and RT-PCR. For each strain, different developmental stages were examined for transcription of three WO phage orf7 genes. The results provide evidence for the presence of both actively transcribed virions and inactive prophages. Variable orf7 transcription patterns are observed in comparisons of differing Cx. pipiens strains. Variability includes both mosquito stage-specific and sexually dimorphic orf7 expression patterns. This report provides additional support for the hypothesis that bacteriophages play an important role in Wolbachia and host evolution. [TOP OF PAGE]

  235. Microscale patchiness of virioplankton. Seymour,J.R., Seuront,L., Doubell,M., Waters,R.L., Mitchell,J.G. (2006). J. Mar. Bio. Assoc. UK 86:551-561. The microscale spatial distributions of viruses were investigated in three contrasting environments including oligotrophic open ocean, eutrophic coastal and estuarine habitats. The abundances of two discrete populations of both viruses and heterotrophic bacteria were measured at spatial resolutions of between 1 and 5cm using purpose-designed microscale sampling equipment and flow cytometric sample analysis. Within open water samples, virus distributions were characterized by non-normal distributions and by 'hotspots' in abundance where concentrations varied by up to 17-fold. In contrast to patterns generally observed at larger spatiotemporal scales, there was no correlation between bacterial and viral abundance or correspondence between bacteria and virus hotspots within these samples. Consequently, strong hotspots and gradients in the virus:bacteria ratio (VBR) were also apparent within samples. Within vertical profiles taken from above the sediment-water interface within a temperate mangrove estuary, distributions of planktonic viruses were characterized by gradients in abundance, with highest concentrations observed within the 1-2cm immediately above the sediment surface, and virus distributions were correlated to bacterial abundance (P<0.01). The patterns observed in these contrasting habitats indicate that microscale patchiness of virus abundance may be a common feature of the marine environment. This form of heterogeneity may have important implications for virus-host dynamics and subsequently influence microbial trophodynamics and nutrient cycling in the ocean. [TOP OF PAGE]

  236. Isolation and characterization of Listeria monocytogenes isolates from ready-to-eat foods in Florida. Shen,Y., Liu,Y., Zhang,Y., Cripe,J., Conway,W., Meng,J., Hall,G., Bhagwat,A.A. (2006). Appl. Environ. Microbiol. 72:5073-5076. Of 3,063 ready-to-eat food samples tested, 91 (2.97%) were positive for Listeria monocytogenes, and lineage 1 strains outnumbered lineage 2 strains 57 to 34. Seventy-one isolates (78%) exhibited multiple antibiotic resistance, and an L. monocytogenes-specific bacteriophage cocktail lysed 65 of 91 (71%) isolates. Determining phage, acid, and antibiotic susceptibility phenotypes enabled us to identify differences among strains which were otherwise indistinguishable by conventional methods. [TOP OF PAGE]

  237. Application of bacteriophages to control intestinal Escherichia coli O157:H7 levels in ruminants. Sheng,H., Knecht,H.J., Kudva,I.T., Hovde,C.J. (2006). Appl. Environ. Microbiol. 72:5359-5366. A previously characterized O157-specific lytic bacteriophage KH1 and a newly isolated phage designated SH1 were tested, alone or in combination, for reducing intestinal Escherichia coli O157:H7 in animals. Oral treatment with phage KH1 did not reduce the intestinal E. coli O157:H7 in sheep. Phage SH1 formed clear and relatively larger plaques on lawns of all 12 E. coli O157:H7 isolates tested and had a broader host range than phage KH1, lysing O55:H6 and 18 of 120 non-O157 E. coli isolates tested. In vitro, mucin or bovine mucus did not inhibit bacterial lysis by phage SH1 or KH1. A phage treatment protocol was optimized using a mouse model of E. coli O157:H7 intestinal carriage. Oral treatment with SH1 or a mixture of SH1 and KH1 at phage/bacterium ratios > or = 10(2) terminated the presence of fecal E. coli O157:H7 within 2 to 6 days after phage treatment. Untreated control mice remained culture positive for >10 days. To optimize bacterial carriage and phage delivery in cattle, E. coli O157:H7 was applied rectally to Holstein steers 7 days before the administration of 10(10) PFU SH1 and KH1. Phages were applied directly to the rectoanal junction mucosa at phage/bacterium ratios calculated to be > or = 10(2). In addition, phages were maintained at 10(6) PFU/ml in the drinking water of the phage treatment group. This phage therapy reduced the average number of E. coli O157:H7 CFU among phage-treated steers compared to control steers (P < 0.05); however, it did not eliminate the bacteria from the majority of steers. [TOP OF PAGE]

  238. Genomic and phylogenetic analysis of a single-stranded RNA virus infecting Rhizosolenia setigera (Stramenopiles: Bacillariophyceae). Shirai,Y., Takao,Y., Mizumoto,H., Tomaru,Y., Honda,D., Nagasaki,K. (2006). J. Mar. Bio. Assoc. UK 86:475-483. We report the first complete genome sequence of the marine diatom-infecting, positive-sense single-stranded RNA (ssRNA) virus, Rhizosolenia setigera RNA virus (RsRNAV). The genome is 8877 nucleotides (nt), polyadenylated, lacking a cap structure, and has two large open reading frames (ORFs): ORF-1 (4818 nt), a polyprotein gene coding for replicases, e.g. RNA helicase, RNA-dependent RNA polymerase (RdRp); and ORF-2 (2883 nt), a polyprotein gene coding for structural proteins. The ORFs are separated by a 323nt intergenic region (IGR), flanked by a 624nt 5?-untranslated region (UTR) and a 229 nt 3?-UTR. The deduced amino acid sequences for ORF-1 and ORF-2 respectively show considerable similarities to the non-structural and structural proteins of a marine raphidophyte-infecting virus HaRNAV (Heterosigma akashiwo RNA virus). Phylogenetic analyses of concatenated amino acid sequences of RNA helicase and RdRp domains supported the monophyly of RsRNAV, HaRNAV and a marine protist-infecting virus SssRNAV (Schizochytrium single-stranded RNA virus) with moderate bootstrap values of 79–83%, but not at the family level, whilst their monophyly was only weakly supported (50–55%) in the phylogenetic tree based on RdRp whole domain. As a result, comparison of the genome organization and sequence suggests RsRNAV is not a member of any currently defined virus family. In the RdRp tree, the positive-sense ssRNA viruses infecting Stramenopiles (RsRNAV, HaRNAV and SssRNAV) and Alveolata (HcRNAV (Heterocapsa circularisquama RNA virus)) were categorized into phylogenetically distant clades, which suggests a host/virus coevolution. Our study supports the hypothesis that a diverse array of ssRNA viruses exists in marine environments. [TOP OF PAGE]

  239. Bacteriophage origins of mitochondrial replication and transcription proteins. Shutt,T.E., Gray,M.W. (2006). Trends Genet. 22:90-95. Mounting evidence suggests that key components of the mitochondrial transcription and replication apparatus are derived from the T-odd lineage of bacteriophage rather than from an alpha-Proteobacterium, as the endosymbiont hypothesis would predict. We propose that several mitochondrial replication genes were acquired together from an ancestor of T-odd phage early in the evolution of the eukaryotic cell, at the time of the mitochondrial endosymbiosis. We further propose that at a later stage the single-subunit RNA polymerase, originally acquired for mitochondrial DNA replication, was co-opted to serve in mitochondrial transcription. [TOP OF PAGE]

  240. Inhibition of bacteriophage M13 replication with esterified milk proteins. Sitohy,M., Chobert,J.M., Karwowska,U., Gozdzicka-Jozefiak,A., Haertle,T. (2006). J. Ag. Food Chem. 54:3800-3806. Esterified milk proteins [methylated (Met) or ethylated (Et) a-lactalbumin (ALA), b-lactoglobulin (BLG), and b-casein (BCN)], unmodified native milk proteins, and native basic proteins (calf thymus histone and hen egg white lysozyme) were tested for their antiviral activity against the bacteriophage M13 and for their influence on its replication (except BCN). All esterified milk proteins showed an antiviral activity against the bacteriophage M13, proportional to the extent of esterification and, hence, to the increased basicity of the modified proteins. Antiviral activity of 100% Met-BLG disappeared after its pepsinolysis but not after its trypsinolysis. The antiviral activity of Met-BLG was much higher than that of native basic proteins (histone and lysozyme). One hundred percent Met-BLG and 73% Et-BLG inhibited the replication of bacteriophage M13 completely, whereas 60% Met-ALA inhibited phage replication partially. Calf thymus histone inhibited the replication of bacteriophage M13 at a lower extent (20%) than Met- and Et-BLG (100% inhibition). Protein concentration, pH, and concentration of the Escherichia coli culture in the preincubation medium of the virus were other factors influencing antiviral activity. Interactions of esterified proteins with the phage DNA (phenol extracted) followed the same pattern as observed during studies of the inhibition of the phage replication: Met-BLG > Et-BLG > or = Met-ALA. [TOP OF PAGE]

  241. Phage therapy: facts and fiction. Skurnik,M., Strauch,E. (2006). Int. J. Med. Microbiol. 296:5-14. Recent examples of the use of bacteriophages in controlling bacterial infections are presented, some of which show therapeutic promise. The therapeutic use of bacteriophages, possibly in combination with antibiotics, may be a valuable approach. However, it is also quite clear that the safe and controlled use of phage therapy will require detailed information on the properties and behavior of specific phage-bacterium systems, both in vitro and especially in vivo. In vivo susceptibility of bacterial pathogens to bacteriophages is still largely poorly understood and future research on more phage-bacterium systems has to be undertaken to define the requirements for successful phage treatments. [TOP OF PAGE]

  242. High pressure-induced inactivation of Qb coliphage and c2 phage in oysters and in culture media. Smiddy,M., Kelly,A.L., Patterson,M.F., Hill,C. (2006). Int. J. Food Microbiol. 106:105-110. High pressure (HP) treatment inactivates bacteria in shellfish, but its effects on viruses in shellfish have not yet been determined, although viral illness is frequently associated with shellfish consumption. The aim of this study was to investigate the baroresistance of two bacteriophage viruses, Qb coliphage and c2 phage, in oysters and in culture media. High numbers (>or=10(7) ml(-1) or g(-1)) of both phages were obtained in culture media and in oysters. Samples were HP treated at 200-800 MPa at 20 degrees C for up to 30 min. Little or no inactivation of either phage was observed in oysters or in culture media after treatment at <or=400 MPa. High levels of inactivation of both phages in oysters and in culture medium were observed following treatment at 500-700 MPa. Titres of both phages were reduced to non-detectable levels (up to 8 log inactivation) in oysters and in GM17 broth (for c2 phage) after treatment at 800 MPa. The level of Qbeta coliphage in tryptone soya broth with yeast extract (10(10) PFU ml(-1)) was reduced by approximately 7 log units following treatment of 800 MPa. Levels of inactivation of both phages in oysters were similar to those in culture media. Increasing the duration of treatment at 550 or 600 MPa increased the level of inactivation of both phages in oysters. HP treatment may effectively inactivate phage in shellfish but HP-induced inactivation of human enteric viruses in oysters needs to be studied directly, to more accurately assess the ability of this technology to inactivate these viruses. [TOP OF PAGE]

  243. Bacteriophages: how bacterial spores capture and protect phage DNA. Sonenshein,A.L. (2006). Curr. Biol. 16:R14-R16 A recent study explains how bacterial spores capture and protect phage DNA, which remains free in the host cytoplasm but is unable to initiate the virulence pathway that leads to lysis of actively growing bacterial cells. [TOP OF PAGE]

  244. Viruses of Archaea. Stedman,K.M., Prangishvilli,D., Zillig,W. (2006). pp. 499-516. In In Calendar,R. and Abedon,S.T. (eds.), The Bacteriophages. Oxford University Press, Oxford. [first two paragraphs] The Archaea (previously known as archaebacteria) were originally identified as such by Carl Woese in the late 1970s (121) and are now accepted as one of the three major divisions or domains of life, along with the Bacteria and the Eukarya. They have recently been shown by molecular techniques to be ubiquitous and may even be the dominant organisms in the open ocean (22, 45). Phylogenetically the Archaea have been split into four major phyla: the Euryarchaeota, the Crenarchaeota, the Korarchaeota and the Nanoarchaeota. The Euryarchaeota comprise the methanogens (6), the extreme halophiles and the extremely thermophilic Thermococcales (29). All isolated members of the Crenarchaeota are extreme thermophiles, however, some as yet uncultured members appear to be mesophilic or even psychrophilic, e.g in marine, environments (28). Korarchaea are not yet represented by any cultured organisms (8, 56, 87). The recently discovered phylum Nanoarchaeota is represented to date by only one apparently parasitic organism (38). ¶ In comparison to viruses of Bacteria and Eukarya, relatively little work has been done on viruses of Archaea. Most characterized viruses of Euryarchaea have typical "head and tail" morphology, similar to that of many bacteriophages, and belong to the known families Myoviridae and Siphoviridae. By contrast, all characterized viruses of Crenarchaeota have unusual morphotypes, and novel viral families had to be introduced for their classification. [TOP OF PAGE]

  245. Biogeographical diversity of archaeal viruses. Stedman,K.M., Clore,A., Combet-Blanc,Y. (2006). pp. 131-143. In In Logan,N.A., Lappin-Scott,H.M., and Oyston,P.C.F. (eds.), Prokaryotic Diversity: Mechanisms and Significance. Cambridge University Press, Cambridge, UK. [first paragraph] Biogeography, or the spatial distribution of biological diversity, has been studied since Darwin and Wallace in the 1800s. Their studies, and most later studies, concentrated on macroscopic organisms, mostly animals and plants, and many differences between species were observed, often correlated with geographical isolation. The theoretical basis for these differences was established later and is still being refined. The theories of island biogeography have been extremely influential in many fields of biology (Bell et al., 2005). Critical to biogeographical studies are comparable organisms from different locations with quantifiable diversity, often sequence diversity. [TOP OF PAGE]

  246. F+ RNA coliphage typing for microbial source tracking in surface waters. Stewart-Pullaro,J., Daugomah,J.W., Chestnut,D.E., Graves,D.A., Sobsey,M.D., Scott,G.I. (2006). J. Appl. Microbiol. 101:1015-1026. AIMS: The utility of coliphages to detect and track faecal pollution was evaluated using South Carolina surface waters that exceeded State faecal coliform standards. METHODS AND RESULTS: Coliphages were isolated from 117 surface water samples by single agar layer (SAL) and enrichment presence/absence (EP/A) methods. Confirmed F+ RNA coliphages were typed for microbial source tracking using a library-independent approach. Concentrations of somatic coliphages using 37 and 44.5 degrees C incubation temperatures were found to be significantly different and the higher temperature may be more specific for faecal contamination. The EP/A technique detected coliphages infecting Escherichia coli Famp in 38 (66%) of the 58 surface water samples negative for F+ coliphages by the SAL method. However, coliphages isolated by EP/A were found to be less representative of coliphage diversity within a sample. Among the 2939 coliphage isolates tested from surface water and known source samples, 813 (28%) were found to be F+ RNA. The majority (94%) of surface water F+ RNA coliphage isolates typed as group I. Group II and/or III viruses were identified from 14 surface water stations, the majority of which were downstream of wastewater discharges. These sites were likely contaminated by human-source faecal pollution. CONCLUSIONS: The results suggest that faecal contamination in surface waters can be detected and source identifications aided by coliphage analyses. SIGNIFICANCE AND IMPACT OF THE STUDY: This study supports the premise that coliphage typing can provide useful, but not absolute, information to distinguish human from animal sources of faecal pollution. Furthermore, the comparison of coliphage isolation methods detailed in this study should provide valuable information to those wishing to incorporate coliphage detection into water quality assessments. [TOP OF PAGE]

  247. Sequence variation among group III F-specific RNA coliphages from water samples and swine lagoons. Stewart,J.R., Vinje,J., Oudejans,S.J.G., Scott,G.I., Sobsey,M.D. (2006). Appl. Environ. Microbiol. 72:1226-1230. Typing of F-specific RNA (FRNA) coliphages has been proposed as a useful method for distinguishing human from animal fecal contamination in environmental samples. Group II and III FRNA coliphages are generally associated with human wastes, but several exceptions have been noted. In the present study, we have genotyped and partially sequenced group III FRNA coliphage field isolates from swine lagoons in North Carolina (NC) and South Carolina (SC), along with isolates from surface waters and municipal wastewaters. Phylogenetic analysis of a region of the 5' end of the maturation protein gene revealed two genetically different group III FRNA subclusters with 36.6% sequence variation. The SC swine lagoon isolates were more closely related to group III prototype virus M11, whereas the isolates from a swine lagoon in NC, surface waters, and wastewaters grouped with prototype virus Qb. These results suggest that refining phage genotyping systems to discriminate M11-like phages from Qb-like phages would not necessarily provide greater discriminatory power in distinguishing human from animal sources of pollution. Within the group III subclusters, nucleotide sequence diversity ranged from 0% to 6.9% for M11-like strains and from 0% to 8.7% for Qb-like strains. It is demonstrated here that nucleotide sequencing of closely related FRNA strains can be used to help track sources of contamination in surface waters. A similar use of phage genomic sequence information to track fecal pollution promises more reliable results than phage typing by nucleic acid hybridization and may hold more potential for field applications. [TOP OF PAGE]

  248. Evolution of bacteriophages infecting encapsulated bacteria: lessons from Escherichia coli K1-specific phages. Stummeyer,K., Schwarzer,D., Claus,H., Vogel,U., Gerardy-Schahn,R., Muhlenhoff,M. (2006). Mol. Microbiol. 60:1123-1135. Bacterial capsules are not only important virulence factors, but also provide attachment sites for bacteriophages that possess capsule degrading enzymes as tailspike proteins. To gain insight into the evolution of these specialized viruses, we studied a panel of tailed phages specific for Escherichia coli K1, a neuroinvasive pathogen with a polysialic acid capsule. Genome sequencing of two lytic K1-phages and comparative analyses including a K1-prophage revealed that K1-phages did not evolve from a common ancestor. By contrast, each phage is related to a different progenitor type, namely T7-, SP6-, and P22-like phages, and gained new host specificity by horizontal uptake of an endosialidase gene. The new tailspikes emerged by combining endosialidase domains with the capsid binding module of the respective ancestor. For SP6-like phages, we identified a degenerated tailspike protein which now acts as versatile adaptor protein interconnecting tail and newly acquired tailspikes and demonstrate that this adapter utilizes an N-terminal undecapeptide interface to bind otherwise unrelated tailspikes. Combining biochemical and sequence analyses with available structural data, we provide new molecular insight into basic mechanisms that allow changes in host specificity while a conserved head and tail architecture is maintained. Thereby, the present study contributes not only to an improved understanding of phage evolution and host-range extension but may also facilitate the on purpose design of therapeutic phages based on well-characterized template phages. [TOP OF PAGE]

  249. Engineered bacteriophage-defence systems in bioprocessing. Sturino,J.M., Klaenhammer,T.R. (2006). Nat. Rev. Microbiol. 4:395-404. Bacteriophages (phages) have the potential to interfere with any industry that produces bacteria as an end product or uses them as biocatalysts in the production of fermented products or bioactive molecules. Using microorganisms that drive food bioprocesses as an example, this review will describe a set of genetic tools that are useful in the engineering of customized phage-defence systems. Special focus will be given to the power of comparative genomics as a means of streamlining target selection, providing more widespread phage protection, and increasing the longevity of these industrially important bacteria in the bioprocessing environment. [TOP OF PAGE]

  250. Prevalence and evolution of core photosystem II genes in marine cyanobacterial viruses and their hosts. Sullivan,M.B., Lindell,D., Lee,J.A., Thompson,L.R., Bielawski,J.P., Chisholm,S.W. (2006). PLoS Biol. 4:e234 Cyanophages (cyanobacterial viruses) are important agents of horizontal gene transfer among marine cyanobacteria, the numerically dominant photosynthetic organisms in the oceans. Some cyanophage genomes carry and express host-like photosynthesis genes, presumably to augment the host photosynthetic machinery during infection. To study the prevalence and evolutionary dynamics of this phenomenon, 33 cultured cyanophages of known family and host range and viral DNA from field samples were screened for the presence of two core photosystem reaction center genes, psbA and psbD. Combining this expanded dataset with published data for nine other cyanophages, we found that 88% of the phage genomes contain psbA, and 50% contain both psbA and psbD. The psbA gene was found in all myoviruses and Prochlorococcus podoviruses, but could not be amplified from Prochlorococcus siphoviruses or Synechococcus podoviruses. Nearly all of the phages that encoded both psbA and psbD had broad host ranges. We speculate that the presence or absence of psbA in a phage genome may be determined by the length of the latent period of infection. Whether it also carries psbD may reflect constraints on coupling of viral- and host-encoded PsbA-PsbD in the photosynthetic reaction center across divergent hosts. Phylogenetic clustering patterns of these genes from cultured phages suggest that whole genes have been transferred from host to phage in a discrete number of events over the course of evolution (four for psbA, and two for psbD), followed by horizontal and vertical transfer between cyanophages. Clustering patterns of psbA and psbD from Synechococcus cells were inconsistent with other molecular phylogenetic markers, suggesting genetic exchanges involving Synechococcus lineages. Signatures of intragenic recombination, detected within the cyanophage gene pool as well as between hosts and phages in both directions, support this hypothesis. The analysis of cyanophage psbA and psbD genes from field populations revealed significant sequence diversity, much of which is represented in our cultured isolates. Collectively, these findings show that photosynthesis genes are common in cyanophages and that significant genetic exchanges occur from host to phage, phage to host, and within the phage gene pool. This generates genetic diversity among the phage, which serves as a reservoir for their hosts, and in turn influences photosystem evolution. [TOP OF PAGE]

  251. Development of a rapid strain differentiation method for methicillin-resistant Staphylococcus aureus isolated in Japan by detecting phage-derived open-reading frames. Suzuki,M., Tawada,Y., Kato,M., Hori,H., Mamiya,N., Hayashi,Y., Nakano,M., Fukushima,R., Katai,A., Tanaka,T., Hata,M., Matsumoto,M., Takahashi,M., Sakae,K. (2006). J. Appl. Microbiol. 101:938-947. AIMS: To develop a rapid genotyping method for investigating outbreaks of methicillin-resistant strains of Staphylococcus aureus (MRSA) isolated in Japan. METHODS AND RESULTS: Isolates were genotyped by detecting the keeping pattern of 16 open-reading frames (ORFs), a process we call phage ORF typing (POT). Thirteen of the ORFs were selected from phage genomes and one from a genomic island SaGIm in the genome of strain Mu50. The other two ORFs, one from Tn554 and one from staphylococcal cassette chromosome mec (SCCmec) type II, were used as strain markers. Three hundred and sixty-eight isolates from five hospitals were classified into 133 types by POT, whereas they were classified into 139 types by pulsed-field gel electrophoresis (PFGE) subtyping. The discriminatory power of POT (D=0.989) was equal to that of PFGE subtyping (D=0.986). CONCLUSIONS: MRSA isolates collected in Japan can be genotyped by detecting the keeping pattern of phage-derived ORFs with a discriminatory power equal to that of PFGE subtyping. SIGNIFICANCE AND IMPACT OF THE STUDY: MRSA isolates can be genotyped rapidly by detecting phage-derived ORFs. As particular pandemic clones can be found in a specific region, a typing method localized to a pandemic clone may be effective for the rapid genotyping of MRSA during outbreaks. [TOP OF PAGE]

  252. Complete nucleotide sequence and genome organization of a single-stranded RNA virus infecting the marine fungoid protist Schizochytrium sp. Takao,Y., Mise,K., Nagasaki,K., Okuno,T., Honda,D. (2006). J. Gen. Virol. 87:723-733. The complete nucleotide sequence of the genomic RNA of a marine fungoid protist-infecting virus (Schizochytrium single-stranded RNA virus; SssRNAV) has been determined. The viral RNA is single-stranded with a positive sense and is 9,018 nt in length [excluding the 3' poly(A) tail]. It contains two long open reading frames (ORFs), which are separated by an intergenic region of 92 nt. The 5' ORF (ORF1) is preceded by an untranslated leader sequence of 554 nt. The 3' large ORF (ORF2) and an additional ORF (ORF3) overlap ORF2 by 431 nt and are followed by an untranslated region of 70 nt [excluding the 3' poly(A) tail]. The deduced amino acid sequences of ORF1 and ORF2 products show similarity to non-structural and structural proteins of dicistroviruses, respectively. However, Northern blot analysis suggests that SssRNAV synthesizes subgenomic RNAs to translate ORF2 and ORF3, showing that the translation mechanism of downstream ORFs is distinct from that of dicistroviruses. Furthermore, although considerable similarities were detected by using a blast genome database search, phylogenetic analysis based on both the nucleotide and amino acid sequences of the putative RNA-dependent RNA polymerase (RdRp) and the RNA helicase suggests that SssRNAV is phylogenetically distinct from other virus families. Therefore, it is concluded that SssRNAV is not a member of any currently defined virus family and belongs to a novel, unrecognized virus group. [TOP OF PAGE]

  253. Impact of iron particles in groundwater on the UV inactivation of bacteriophages MS2 and T4. Templeton,M.R., Andrews,R.C., Hofmann,R. (2006). J. Appl. Microbiol. 101:732-741. AIMS: To investigate the impact of iron particles in groundwater on the inactivation of two model viruses, bacteriophages MS2 and T4, by 254-nm ultraviolet (UV) light. METHODS AND RESULTS: One-litre samples of groundwater with high iron content (from the Indianapolis Water Company, mean dissolved iron concentration 1.3 mg l(-1)) were stirred vigorously while exposed to air, which oxidized and precipitated the dissolved iron. In parallel samples, ethylenediaminetetra-acetic acid (EDTA) was added to chelate the iron and prevent formation of iron precipitate. The average turbidity in the samples without EDTA (called the 'raw' samples) after 210 min of stirring was 2.7 +/- 0.1 NTU while the average turbidity of the samples containing EDTA (called the 'preserved' samples) was 1.0 +/- 0.1 NTU. 'Raw' and 'preserved' samples containing bacteriophage MS2 were exposed to 254-nm UV light at doses of 20, 40, or 60 mJ (cm(2))(-1), while samples containing bacteriophage T4 were exposed to 2 or 5 mJ (cm(2))(-1), using a low pressure UV collimated beam. The UV inactivation of both phages in the 'raw' groundwater was lower than in the EDTA-'preserved' groundwater to a statistically significant degree (alpha = 0.05), due to the association of phage with the UV-absorbing iron precipitate particles. A phage elution technique confirmed that a large fraction of the phage that survived the UV exposures were particle-associated. CONCLUSIONS: Phages that are associated with iron oxide particles in groundwater are shielded from UV light to a measurable and statistically significant degree at a turbidity level of 2.7 NTU when the phage particle association is induced under experimental conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: While the particle association of the phage in this study was induced experimentally, the findings provide further evidence that certain particles in natural waters and wastewaters (e.g. iron oxide particles) may have the potential to shield viruses from UV light. [TOP OF PAGE]

  254. [Microenvironment of positive pressure powered air purifying medical protective equipment]. Tian,F., Cheng,G.x., Wang,Z., Yang,J.q., Yang,J., Liu,S.j. (2006). Chin. J. Indust. Hyg. Occup. Dis. 24:151-153. OBJECTIVE: To study the filtration efficiency of a positive pressure powered air purifying medical protective equipment and the effect of the flow rate on the microenvironment of the equipment. METHODS: The filtration efficiency of high efficiency particulate air (HEPA) filter was measured with the biologic aerosol of simulating virus (Escherichia coli bacteriophage f(2)). The simulation work was done at the walk rate of 4 km/h in summer. The effect of the flow rate on the oxygen content, the carbon dioxide content, the temperature and the humidity of the microenvironment of the equipment was investigated. The clinical experiments were conducted in three appointed hospital for fighting against SARS. RESULTS: The HEPA filter could filtrate 99.99% simulating viruses in the air. When the flow rate ranged from 75 to 125 L/min, the microenvironment parameters of the equipment were: the oxygen content was between 19.6% and 20.1% (the physiological safety limit is more than 14.6%); the carbon dioxide content ranged from 0.43% to 0.57% (the physiological safety limit is less than 1.0%); the temperature was between 32.0 degrees C to 32.2 degrees C; the humidity ranged from 49.7% to 59.4% (the physiological safety limit is the temperature 31 degrees C and the humidity 85% or temperature 38 degrees C and humidity 50%). Each microenvironment parameter met the demand of a healthy person under the normal workload. In the clinical experiments, the doctors wearing the equipment who performed the tracheotomy for a SARS patient in a deep coma were not infected. CONCLUSION: The medical protective equipment can protect the doctor and nurse in SARS contaminated areas effectively and improve their work conditions. [TOP OF PAGE]

  255. Bacteriophages and pathogenicity: more than just providing a toxin? Tinsley,C.R., Bille,E., Nassif,X. (2006). Microbes Infect. 8:1365-1371. An increasing number of pathogenicity factors carried by bacteriophages have been discovered. This review considers bacteriophage-bacterium interaction and its relation to disease processes. We discuss the search for new bacteriophage-associated pathogenicity factors, with emphasis on recent advances brought by the use of genomic sequence data and the techniques of genomic epidemiology. [TOP OF PAGE]

  256. Identification of adsorption inhibition, restriction/modification and abortive infection type phage resistance systems in Lactococcus lactis strains. Tukel,C., Sanlibaba,P., Ozden,B., Akcelik,M. (2006). Acta Biol. Hung. 57:377-385. 98 Lactococcus lactis strains were isolated from traditional fermented milk products in Turkey tested against 60 lactococcal lytic phages to determine their resistance levels. While 82 L. lactis strains were sensitive against lactic phages at different levels, 16 L. lactis strains showed resistance to all phages tested. Types of phage resistance among 16 L. lactis strains were identified as phage adsorption inhibition in eight strains, restriction/modification in six strains and abortive infection (heat sensitive phage resistance) in two strains, using three broad-spectrum phages phi pll 98-32, phi pld 67-42 and phi pld 67-44. [TOP OF PAGE]

  257. Hybridisation of F+ RNA coliphages detected in shellfish samples with oligonucleotide probes to assess the origin of microbiological pollution of shellfish. Vantarakis,A., Venieri,D., Komninou,G., Papapetropoulou,M. (2006). Water Sci. Technol. 54:219-223. Current measures for controlling the public health risks associated with bivalve molluscan shellfish consumption rely on the use of Escherichia coli to indicate the sanitary quality of shellfish harvesting areas. However, it has been demonstrated that E. coli is an inadequate indicator of the viral risk associated with shellfish. An alternative indicator, male-specific B+ coliphages, have been investigated as viral indicators of faecal contamination that may provide source-specific information for impacted environmental waters. This study compared the distribution of E. coli and F+ RNA bacteriophages in shellfish grown in harvesting areas of Greece and also examined the presence and proportions of the different subgroups of F+ RNA coliphages in shellfish. F+ RNA bacteriophages were present in shellfish at higher concentrations than E. coli. Elevated numbers of F+ RNA bacteriophages observed in the winter concur with the known increased viral risk associated with shellfish harvested at that time of year in Greece. The majority of F+ RNA coliphages detected in shellfish samples belonged to group IV which indicated the possible presence of animal faecal material in sample harvesting areas. Phages of groups II and III (human waste and human faecal material, respectively) were present at low levels. Finally, 8% of the phages hybridised were found to belong to group I. The presence of group IV showed seasonal distribution (more in winter, less in summer) whereas the other groups did not show any difference. Monitoring of F+ coliphage subgroups may indicate the presence and major sources of microbial inputs to surface waters; however, environmental effects on the relative occurrence of different groups need to be considered. [TOP OF PAGE]

  258. Transport of coliphage PRD1 in a surface flow constructed wetland. Vidales-Contreras,J.A., Gerba,C.P., Karpiscak,M.M., Acuna-Askar,K., Chaidez-Quiroz,C. (2006). Water Environ. Res. 78:2253-2260. A tracer study was conducted in a 3-ha surface flow constructed wetland to analyze transport performance of PRD1, an enteric virus model. The convection-dispersion equation (CDE), including a first-order reaction model, adequately simulated transport performance of PRD1 in the wetland under an average hydraulic loading rate of 82 mm/d. Convective velocity (v) and longitudinal dispersion coefficient (D) were estimated by modeling a conservative tracer (bromide) pulse through the wetland. Both PRD1 and bromide were simultaneously added to the entering secondary treated wastewater effluent. The mass of bromide and PRD1 recovered was 76 and 16%, respectively. The PRD1 decay rate was calculated to be 0.3/day. The findings of this study suggest that the CDE model and analytical moment equations represent a suitable option to characterize virus transport performance in surface flow constructed wetlands. [TOP OF PAGE]

  259. Bacteriophage gene products and pathogenesis. Wagner,PL., Waldor,M.K. (2006). pp. 710-724. In In Calendar,R. and Abedon,S.T. (eds.), The Bacteriophages. Oxford University Press, Oxford. Bacteriophages contribute to the virulence of many bacterial pathogens, largely by encoding the structural genes for virulence factors. The most widely recognized phage-encoded virulence factors are bacterial exotoxins, which account for the characteristic clinical manifestations of a number of human diseases caused by bacterial infections. The previous edition of this chapter focused primarily on phage-encoded toxins, but in the intervening years, two themes have emerged (169). First, phages are increasingly recognized for encoding genes that contribute to other aspects of bacterial pathogenesis in addition to toxin production. In fact, phage-encoded gene products contribute to virtually every facet of bacterial pathogenicity, from attachment and invasion to immune evasion and transmission among humans. Second, while phages are like other mobile genetic elements in that they disseminate virulence genes among bacterial populations, phages have unique properties that enable them to contribute to bacterial pathogenesis by mechanisms other than transduction as well. For example, virion particles may themselves contain pathogenic components (12, 13); and prophage induction, through gene amplification, transcriptional up-regulation and phage-mediated lysis, can contribute to production and release of virulence factors from bacterial cells (167, 168). Owing to these developments, the contribution of bacteriophages to bacterial pathogenesis can no longer be conceived of simply as transduction of toxin genes that are regulated by the host bacterium. This chapter presents a summary of bacteriophage involvement in bacterial pathogenesis, in which we consider (i) the nature of the bacterial virulence properties altered by phages, (ii) the basic mechanisms by which phages alter these properties, and (iii) the regulation of the phage-related virulence property by the phage and/or by its host bacterium. [TOP OF PAGE]

  260. Recovery of temperate Desulfovibrio vulgaris bacteriophage using a novel host strain. Walker,C.B., Stolyar,S.S., Pinel,N., Yen,H.C., He,Z., Zhou,J., Wall,J.D., Stahl,D.A. (2006). Environ. Microbiol. 8:1950-1959. A novel sulfate-reducing bacterium (strain DePue) closely related to Desulfovibrio vulgaris ssp. vulgaris strain Hildenborough was isolated from the sediment of a heavy-metal impacted lake using established techniques. Although few physiological differences between strains DePue and Hildenborough were observed, pulse-field gel electrophoresis (PFGE) revealed a significant genome reduction in strain DePue. Comparative whole-genome microarray and polymerase chain reaction analyses demonstrated that the absence of genes annotated in the Hildenborough genome as phage or phage-related contributed to the significant genome reduction in strain DePue. Two morphotypically distinct temperate bacteriophage from strain Hildenborough were recovered using strain DePue as a host for plaque isolation. [TOP OF PAGE]

  261. Lysis timing and bacteriophage fitness. Wang,I.-N. (2006). Genetics 172:17-26. The effect of lysis timing on bacteriophage (phage) fitness has received little theoretical or experimental attention. Previously, the impact of lysis timing on phage fitness was studied using a theoretical model based on the marginal value theorem from the optimal foraging theory. An implicit conclusion of the model is that, for any combination of host quantity and quality, an optimal time to lyse the host would exist so that the phage fitness would be the highest. To test the prediction, an array of isogenic l-phages that differ only in their lysis times was constructed. For each phage strain, the lysis time, burst size, and fitness (growth rate) were determined. The result showed that there is a positive linear relationship between lysis time and burst size. Moreover, the strain with an intermediate lysis time has the highest fitness, indicating the existence of an optimal lysis time. A mathematical model is also constructed to describe the population dynamics of phage infection. Computer simulations using parameter values derived from phage l-infection also showed an optimal lysis time. However, both the optimum and the fitness are different from the experimental result. The evolution of phage lysis timing is discussed from the perspectives of multiple infection and life-history trait evolution. [TOP OF PAGE]

  262. Use of bacteriophage in the treatment of experimental animal bacteremia from imipenem-resistant Pseudomonas aeruginosa. Wang,J., Hu,B., Xu,M., Yan,Q., Liu,S., Zhu,X., Sun,Z., Reed,E., Ding,L., Gong,J., Li,Q.Q., Hu,J. (2006). International journal of molecular medicine 17:309-317. The emergence of antibiotic-resistant bacterial strains still remains a significant problem for antimicrobial chemotherapy in the clinic. Bacterial viruses (bacteriophages or phages) have been suggested to be used as alternative therapeutic agents for bacterial infections. However, the efficacy of phage therapy in treating drug-resistant infections in humans is uncertain. Therefore in the present study, we examined the effectiveness of phages in the treatment of imipenem-resistant Pseudomonas aeruginosa (IMPR-Pa) infection in an experimental mouse model. Twenty-nine strains of phage were isolated from our hospital sewage, and phage OA392 was representatively used for all testing because it has lytic activity against a wide range of clinical isolates of IMPR-Pa. We found that intraperitoneal (i.p.) injections of one IMPR-Pa strain (3 x 10(7) CFU) caused bacteremia and all mice died within 24 h. A single i.p. inoculation of the phage strain (MOI > or =0.01) at up to 60 min after the bacterial challenge was sufficient to rescue 100% of the animals. This lifesaving effect coincided with the rapid appearance of OA392 in the circulation (within 2 h after i.p. injection), which remained at substantially higher levels for up to 48 h until the bacteria were eradicated. However, the survival rates of the mice dropped to approximately 50% and 20% when the same dose of this purified phage preparation was administered at 180 min and 360 min, respectively, after IMPR-Pa infections. In addition, we demonstrated that the ability of this phage to rescue bacteremic animals was due to the functional capabilities of the phage and not to a non-specific immune effect. The protection from death occurred only in animals inoculated with bacteria-specific virulent phage strains. When the heat-inactivated phages were used, the survival rate of the infected mice was dramatically reduced to 20% or lower. Moreover, the levels of the antibody against the phage were not significantly changed at the time when the bacteremic animals were protected by the active phages. Finally, our observations revealed that the inoculation of the mice with high-doses of OA392 alone produced no adverse effects attributable to the phage. These data indicate that phages can save animals from pernicious P. aeruginosa infections and suggest that phage therapy may be potentially used as a stand-alone therapy for patients with IMPR-Pa infections. [TOP OF PAGE]

  263. Therapeutic effectiveness of bacteriophages in the rescue of mice with extended spectrum beta-lactamase-producing Escherichia coli bacteremia. Wang,J., Hu,B., Xu,M., Yan,Q., Liu,S., Zhu,X., Sun,Z., Tao,D., Ding,L., Reed,E., Gong,J., Li,Q.Q., Hu,J. (2006). International journal of molecular medicine 17:347-355. The emergence of multidrug-resistant bacteria has become a global crisis. Accumulating evidence shows that bacteriophages (phages) can rescue animals from a variety of lethal infections and be effective in treating drug-resistant infections in humans. Enterobacteriaceae, producing extended spectrum beta-lactamase enzymes (ESBLs), are resistant to a broad range of beta-lactamase antibiotics. One of the most common ESBL-producing gram-negative bacilli in Enterobacteriaceae is Escherichia coli. Since ESBL-producing E. coli poses a formidable challenge in the management of critically ill patients with bacterial infections, we undertook this study to explore the possible therapeutic utility of phages to control ESBL-producing E. coli infections. The phage O9882 used in this study was isolated from our hospital sewage and has lytic activity against a broad range of clinical isolates of ESBL-producing E. coli. ESBL-producing E. coli strains (n=30) were isolated in the clinic, and one of them was used to induce bacteremia in a murine model. Bacteremia was established by intraperitoneal (i.p.) injection of 3 x 10(7) CFU/ml, the minimum lethal dose (MLD) of bacterium in this animal model. Mice infected with the MLD of this strain alone died within 14 h, whereas a single i.p. inoculation of O9882 (MOI > or =10(-4)) given 40 min after the bacterial challenge led to 100% survival at 24-168 h, compared to 0% survival of saline-treated controls. Protection was obtained even when administration of the phage was delayed up to 60 min after the bacterial infection and the survival rate of infected animals was 60% at 168 h. Furthermore, it was shown that the therapeutic efficacy of O9882 in lethal systemic infection in our model is due to the functional capability of the phage and not the nonspecific immune effects. Our data both in vitro and in vivo revealed that: i) the protection of mice from death occurred only in animals infected with selected bacterial strains and the virulent phage specific to them; ii) when the phages were heat-inactivated, survival of the infected mice was strikingly decreased to 0; and iii) the level of antibody against the phage was not substantially elevated when the bacteremic animals were protected by the phage. The present findings indicate that phages can effectively rescue our mouse model from bacteremia and death, and thus provide the rationale and framework to evaluate the therapeutical efficacy of lytic phages against fatal ESBL-producing E. coli infections in humans. [TOP OF PAGE]

  264. Phage in the time of cholera. Weitz,J.S., Hartman,H. (2006). The Lancet infectious diseases 6:257-258. [first paragraph] Bacteriophage (bacterial viruses) were heralded as revolutionary therapeutic agents soon after the discovery by Felix d'Herelle in 1917 of an "invisible microbe" capable of lysing bacteria.1 Bacteriophage appeared to be effi cient killers of their bacterial hosts- we now know that their life history is far more complex than fi rst assumed2-and so the effort to use phage as curatives or prophylaxis spread quickly to research institutes in Europe, North America, and Asia.3 d'Herelle himself spearheaded many of these eff orts, the most famous of which was the initiation of an extensive campaign to use phage in the treatment and prevention of cholera in colonial India. The authors of one such study4 conclude by noting that "the results establish sufficient probability in favour of a significant effect of the administration of bacteriophage to form a basis of practical policy in the treatment and prevention of cholera in villages". [TOP OF PAGE]

  265. Marine and freshwater cyanophages in a Laurentian Great Lake: evidence from infectivity assays and molecular analyses of g20 genes. Wilhelm,S.W., Carberry,M.J., Eldridge,M.L., Poorvin,L., Saxton,M.A., Doblin,M.A. (2006). Appl. Environ. Microbiol. 72:4957-4963. While it is well established that viruses play an important role in the structure of marine microbial food webs, few studies have directly addressed their role in large lake systems. As part of an ongoing study of the microbial ecology of Lake Erie, we have examined the distribution and diversity of viruses in this system. One surprising result has been the pervasive distribution of cyanophages that infect the marine cyanobacterial isolate Synechococcus sp. strain WH7803. Viruses that lytically infect this cyanobacterium were identified throughout the western basin of Lake Erie, as well as in locations within the central and eastern basins. Analyses of the gene encoding the g20 viral capsid assembly protein (a conservative phylogenetic marker for the cyanophage) indicate that these viruses, as well as amplicons from natural populations and the ballast of commercial ships, are related to marine cyanophages but in some cases form a unique clade, leaving questions concerning the native hosts of these viruses. The results suggest that cyanophages may be as important in freshwater systems as they are known to be in marine systems. [TOP OF PAGE]

  266. Environmental factors that influence the transition from lysogenic to lytic existence in the fHSIC/Listonella pelagia marine phage-host system. Williamson,S.J., Paul,J.H. (2006). Microb. Ecol. 52:217-225. The marine phage varphiHSIC has been previously reported to enter into a pseudolysogenic-like interaction with its host Listonella pelagia. This phage-host system displays behaviors that are characteristic of both pseudolysogeny and lysogeny including a high rate of spontaneous induction and chromosomal integration of the prophage. To determine what parameters may influence the transition from lysogenic to lytic existence in the varphiHSIC/L. pelagia phage-host system, cultures of this organism were incubated under different environmental conditions, while host cell growth and bacteriophage production were monitored. The environmental parameters tested included salinity, temperature, a rapid temperature shift, and degree of culture aeration. The highest titers of phage were produced by HSIC-1a cells grown in high-salinity nutrient artificial seawater media (67 ppt with a natural salinity equivalent of 57 ppt) or those cultured in highly aerated nutrient artificial seawater media (cultures shaken at 300 rpm). Conversely, the lowest titers of phage were produced under low salinity or rate of aeration. In general, conditions that stimulated growth resulted in greater lytic phage production, whereas slow growth favored lysogeny. These results indicate that elevated salinity and aeration influenced the switch from lysogenic to lytic existence for the phage varphiHSIC. These results may have implications for environmental controls of the lysogenic switch in natural populations of marine bacteria. [TOP OF PAGE]

  267. Phylogenetic analysis of PgV-102P, a new virus from the English Channel that infects Phaeocystis globosa. Wilson,W.H., Schroeder,D.C., Ho,J., Canty,M. (2006). J. Mar. Bio. Assoc. UK 86:485-490. A new virus that infects the harmful algal bloom-forming microalga Phaeocystis globosa was isolated from surface water in the English Channel off the coast of Plymouth, UK, in May 2001. Phylogenetic analysis of the DNA polymerase gene revealed the virus isolate, designated PgV-102P, belongs to the family Phycodnaviridae, a group of large double-stranded DNA viruses known to infect algae. Basic characterization of PgV-102P revealed it was a lytic virus with a relatively slow culture lysis period of 10-days. The genome size (176kbp) and capsid diameter (98nm) of PgV-102P fall at the bottom end of the range expected for phycodnaviruses. Interestingly, PgV-102P did not cluster with other P. globosa viruses; instead, it was more closely related to other prymnesioviruses that infect the marine prymnesiophyte Chrysochromulina brevifilum. We discuss the effectiveness of DNA polymerase as a diagnostic marker. Although it is ideal for determining what family or even genus an algal virus belongs to, it is clear that the DNA polymerase gene does not have sufficient resolution when looking for relationships within algal virus genera. [TOP OF PAGE]

  268. Targeting antibacterial agents by using drug-carrying filamentous bacteriophages. Yacoby,I., Shamis,M., Bar,H., Shabat,D., Benhar,I. (2006). Antimicrob. Agents Chemother. 50:2087-2097. Bacteriophages have been used for more than a century for (unconventional) therapy of bacterial infections, for half a century as tools in genetic research, for 2 decades as tools for discovery of specific target-binding proteins, and for nearly a decade as tools for vaccination or as gene delivery vehicles. Here we present a novel application of filamentous bacteriophages (phages) as targeted drug carriers for the eradication of (pathogenic) bacteria. The phages are genetically modified to display a targeting moiety on their surface and are used to deliver a large payload of a cytotoxic drug to the target bacteria. The drug is linked to the phages by means of chemical conjugation through a labile linker subject to controlled release. In the conjugated state, the drug is in fact a prodrug devoid of cytotoxic activity and is activated following its dissociation from the phage at the target site in a temporally and spatially controlled manner. Our model target was Staphylococcus aureus, and the model drug was the antibiotic chloramphenicol. We demonstrated the potential of using filamentous phages as universal drug carriers for targetable cells involved in disease. Our approach replaces the selectivity of the drug itself with target selectivity borne by the targeting moiety, which may allow the reintroduction of nonspecific drugs that have thus far been excluded from antibacterial use (because of toxicity or low selectivity). Reintroduction of such drugs into the arsenal of useful tools may help to combat emerging bacterial antibiotic resistance. [TOP OF PAGE]

  269. Male-specific RNA coliphages detected by plaque assay and RT-PCR in tropical river waters and animal fecal matter. Yee,S.Y.F., Fong,N.Y., Fong,G.T., Tak,O.J., Hui,G.T., Su Ming,Y. (2006). International Journal of Environmental Health Research 16:59-68. Male-specific RNA coliphages (FRNA) have been recommended as indicators of fecal contamination and of the virological quality of water. In this study, 16 river water and 183 animal fecal samples were examined for the presence of FRNA coliphages by a plaque assay using Salmonella typhimurium WG49 and WG25 to differentiate between male-specific and somatic phages, a RNase spot test to differentiate between DNA and RNA phages and a reverse transcriptase-polymerase chain reaction (RT-PCR) for the specific identification of FRNA phages. The overall recovery rate for F-specific coliphages was 8.0%. (4.4% from animal fecal matter and 50% from river water samples). Plaque counts were generally low (< 6 x 10(2) pfu per g feces or ml water), with FRNA (6.5%) and Male-specific DNA coliphages (FDNA) (7.0%) phages occurring at almost equal frequencies. The RT-PCR was positive in all FRNA plaques and was able to identify FRNA phages in mixed populations of FRNA, FDNA and somatic phages. [TOP OF PAGE]

  270. Isolation and characterization of a cyanophage infecting the toxic cyanobacterium Microcystis aeruginosa. Yoshida,T., Takashima,Y., Tomaru,Y., Shirai,Y., Takao,Y., Hiroishi,S., Nagasaki,K. (2006). Appl. Environ. Microbiol. 72:1239-1247. We isolated a cyanophage (Ma-LMM01) that specifically infects a toxic strain of the bloom-forming cyanobacterium Microcystis aeruginosa. Transmission electron microscopy showed that the virion is composed of anisometric head and a tail complex consisting of a central tube and a contractile sheath with helical symmetry. The morphological features and the host specificity suggest that Ma-LMM01 is a member of the cyanomyovirus group. Using semi-one-step growth experiments, the latent period and burst size were estimated to be 6 to 12 h and 50 to 120 infectious units per cell, respectively. The size of the phage genome was estimated to be ca. 160 kbp using pulse-field gel electrophoresis; the nucleic acid was sensitive to DNase I, Bal31, and all 14 restriction enzymes tested, suggesting that it is a linear double-stranded DNA having a low level of methylation. Phylogenetic analyses based on the deduced amino acid sequences of two open reading frames coding for ribonucleotide reductase alpha- and beta-subunits showed that Ma-LMM01 forms a sister group with marine and freshwater cyanobacteria and is apparently distinct from T4-like phages. Phylogenetic analysis of the deduced amino acid sequence of the putative sheath protein showed that Ma-LMM01 does not form a monophyletic group with either the T4-like phages or prophages, suggesting that Ma-LMM01 is distinct from other T4-like phages that have been described despite morphological similarity. The host-phage system which we studied is expected to contribute to our understanding of the ecology of Microcystis blooms and the genetics of cyanophages, and our results suggest the phages could be used to control toxic cyanobacterial blooms. [TOP OF PAGE]

  271. Evolutionary design on a budget: robustness and optimality of bacteriophage T7. You,L., Yin,J. (2006). Systems Biol. 153:46-52. Exploring how biological systems have been 'designed' by evolution to achieve robust behaviours is now a subject of increasing research effort. Yet, it still remains unclear how environmental factors may contribute to this process. This issue is addressed by employing a detailed computer model for the intracellular growth of phage T7. More than 150 000 in silico T7 mutants were generated and the rates and efficiencies of their growth in two host environments, namely, a realistic environment that offered finite host resources for the synthesis of phage functions and a hypothetical environment where the phage was supplied infinite host resources, were evaluated. Results revealed two key properties of phage T7. First, T7 growth was overall robust with respect to perturbations in its parameters, but fragile with respect to changes in the ordering of its genetic elements. Secondly, the wild-type T7 had close to optimal fitness in the finite environment. Furthermore, a strong correlation was found between fitness and growth efficiency in the finite environment. The results underscore the potential importance of the environment in shaping robust design of a biological system. In particular, the strong correlation between fitness and growth efficiency suggests that T7 may have evolved to maximise its growth rate by minimising waste of finite resources. [TOP OF PAGE]

  272. Isolation and characterization of Thermus bacteriophages. Yu,M.X., Slater,M.R., Ackermann,H.W. (2006). Arch. Virol. 151:663-679. One-hundred-fifteen bacteriophage strains were isolated from alkaline hot springs in Iceland, New Zealand, Russia (Kamchatka), and the U.S.A. The phages belonged to the Myoviridae, Siphoviridae, Tectiviridae, and Inoviridae families. Over 50% of isolates were isometric or filamentous. One type of siphovirus had giant tails of over 800 nm in length. Phages were further characterized by host range, genome size, DNA restriction endonuclease digestion patterns, and temperature and pH sensitivity. Myoviruses and tectiviruses had a worldwide distribution. Most phages were narrowly host-specific and all were highly resistant against heating and alkaline and acidic pH. This is the first time that tectiviruses and filamentous phages are reported for bacteria of the Thermus-Deinococcus phylum. The presence of tectiviruses, inoviruses, and myoviruses is attributed to acquisition from ancestral gamma-proteobacteria by horizontal gene transfer. [TOP OF PAGE]

  273. Isolation and characterization of a Lactobacillus fermentum temperate bacteriophage from Chinese yogurt. Zhang,X., Kong,J., Qu,Y. (2006). J. Appl. Microbiol. 101:857-863. AIMS: The aim of this study was to investigate the properties of temperate bacteriophage of Lactobacillus fermentum, based on its morphology, restriction patterns, protein profile and the impact on the growth of host strain. METHODS AND RESULTS: With Mitomycin C, seven temperate phages were induced from Lactobacilli derived from Chinese yogurt. The temperate phages induced belong to the most common Bradley's group B, having hexagonal head and long, noncontractile tail. They were furthermore confirmed to be the same bacteriophage by identical restriction patterns. SDS-PAGE profile showed that the phage studied had one major structure protein about 31.9 kDa. The presence of the prophage influenced the cell shape and colony size of its lysogenic strain. CONCLUSIONS: The phage obtained had similar, but not complete identical properties with other L. fermentum phages reported. It influenced the growth behaviour of its lysogenic strain. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides some information about bacteriophages occurring in the Chinese yoghurt manufacture and contributes to our knowledge on the bacteriophage diversity in the dairy industry. [TOP OF PAGE]

  274. Why bacteriophage encode exotoxins and other virulence factors. Abedon,S.T., LeJeune,J.T. (2005). Evol. Bioinf. Online 1:97-110. This study considers gene location within bacteria as a function of genetic element mobility. Our emphasis is on prophage encoding of bacterial virulence factors (VFs). At least four mechanisms potentially contribute to phage encoding of bacterial VFs: (i) Enhanced gene mobility could result in greater VF gene representation within bacterial populations. We question, though, why certain genes but not others might benefit from this mobility. (ii) Epistatic interactions—between VF genes and phage genes that enhance VF utility to bacteria—could maintain phage genes via selection acting on individual, VF-expressing bacteria. However, is this mechanism sufficient to maintain the rest of phage genomes or, without gene co-regulation, even genetic linkage between phage and VF genes? (iii) Phage could amplify VFs during disease progression by carrying them to otherwise commensal bacteria colocated within the same environment. However, lytic phage kill bacteria, thus requiring assumptions of inclusive fitness within bacterial populations to explain retention of phage-mediated VF amplification for the sake of bacterial utility. Finally, (iv) phage-encoded VFs could enhance phage Darwinian fitness, particularly by acting as ecosystem-modifying agents. That is, VF-supplied nutrients could enhance phage growth by increasing the density or by improving the physiology of phage-susceptible bacteria. Alternatively, VF-mediated break down of diffusion-inhibiting spatial structure found within the multicellular bodies of host organisms could augment phage dissemination to new bacteria or to environments. Such phage-fitness enhancing mechanisms could apply particularly given VF expression within microbiologically heterogeneous environments, ie, ones where phage have some reasonable potential to acquire phage-susceptible bacteria. [TOP OF PAGE]

  275. 2004 ASM Conference on the New Phage Biology: the 'Phage Summit'. Adhya,S., Black,L., Friedman,D., Hatfull,G., Kreuzer,K., Merril,C., Oppenheim,A., Rohwer,F., Young,R. (2005). Mol. Microbiol. 55:1300-1314. In August, more than 350 conferees from 24 countries attended the ASM Conference on the New Phage Biology, in Key Biscayne, Florida. This meeting, also called the Phage Summit, was the first major international gathering in decades devoted exclusively to phage biology. What emerged from the 5 days of the Summit was a clear perspective on the explosive resurgence of interest in all aspects of bacteriophage biology. The classic phage systems like [lambda] and T4, reinvigorated by structural biology, bioinformatics and new molecular and cell biology tools, remain model systems of unequalled power and facility for studying fundamental biological issues. In addition, the New Phage Biology is also populated by basic and applied scientists focused on ecology, evolution, nanotechnology, bacterial pathogenesis and phagebased immunologics, therapeutics and diagnostics, resulting in a heightened interest in bacteriophages per se , rather than as a model system. Besides constituting another landmark in the long history of a field begun by d'Herelle and Twort during the early 20th century, the Summit provided a unique venue for establishment of new interactive networks for collaborative efforts between scientists of many different backgrounds, interests and expertise. [TOP OF PAGE]

  276. Induction of Shiga toxin-converting prophage in Escherichia coli by high hydrostatic pressure. Aertsen,A., Faster,D., Michiels,C.W. (2005). Appl. Environ. Microbiol. 71:1155-1162. Since high hydrostatic pressure is becoming increasingly important in modern food preservation, its potential effects on microorganisms need to be thoroughly investigated. In this context, mild pressures (<200 MPa) have recently been shown to induce an SOS response in Escherichia coli MG1655. Due to this response, we observed a RecA- and LexA-dependent induction of lambda prophage upon treating E. coli lysogens with sublethal pressures. In this report, we extend this observation to lambdoid Shiga toxin (Stx)-converting bacteriophages in MG1655, which constitute an important virulence trait in Stx-producing E. coli strains (STEC). The window of pressures capable of inducing Stx phages correlated well with the window of bacterial survival. When pressure treatments were conducted in whole milk, which is known to promote bacterial survival, Stx phage induction could be observed at up to 250 MPa in E. coli MG1655 and at up to 300 MPa in a pressure-resistant mutant of this strain. In addition, we found that the intrinsic pressure resistance of two types of Stx phages was very different, with one type surviving relatively well treatments of up to 400 MPa for 15 min at 20°C. Interestingly, and in contrast to UV irradiation or mitomycin C treatment, pressure was not able to induce Stx prophage or an SOS response in several natural Stx-producing STEC isolates. [TOP OF PAGE]

  277. Genotoxicity of the Yamuna River water at Okhla (Delhi), India. Aleem,A., Malik,A. (2005). Ecotoxicology and Environmental Safety 61:404-412. Water samples from the Yamuna River at Okhla (Delhi), India, were concentrated using XAD resins (XAD-4 and XAD-8) and liquid-liquid extraction procedures. Gas chromatographic analysis of liquid-liquid extracted water samples revealed the presence of the pesticides DDT, BHC, dieldrin, endosulfan, aldrin, 2,4-D, dimethoate, methyl parathion, and malathion at concentrations of 14, 25, 2.1, 114, 0.9, 0.6, 0.9, 1.7, and 1.9 ng/L, respectively. The genotoxicity of the extracted water samples was evaluated with the Ames Salmonella/mammalian microsome test, DNA repair-defective mutants, and bacteriophage lambda systems. The results of the Salmonella test demonstrated that the XAD-concentrated water samples had maximum mutagenicity with the TA98 strain both with and without metabolic activation. However, the liquid-liquid-extracted water samples were also found to be mutagenic with one or more of the Ames tester strains, but to a lesser extent as compared with XAD extracts. The damage brought about in the DNA repair-defective mutants in the presence of XAD-concentrated water samples was found to be markedly high as compared with that liquid-liquid-extracted water samples at the dose level of 20 microl/mL culture. All mutants invariably exhibited significant declines in their colony-forming units as compared with their isogenic wild-type counterparts. Survival decreased by 86.3 and 75.5% in the polA- strain after 6 h of treatment with XAD-concentrated and liquid-liquid-extracted water samples, respectively. A significant decrease was also observed in the survival of bacteriophage lambda when treated with the test samples. Mutagenic responses of the liquid-liquid-extracted water samples may not necessarily reflect the mutagenicity of existing pesticides in the test water, because some other organic pollutants might accompany the pesticides in the extract. [TOP OF PAGE]

  278. Comparison of bacteriophage and enteric virus removal in pilot scale activated sludge plants. Arraj,A., Bohatier,J., Laveran,H., Traore,O. (2005). J. Appl. Microbiol. 98:516-524. AIMS: The aim of this experimental study was to determine comparatively the removal of two types of bacteriophages, a somatic coliphage and an F-specific RNA phage and of three types of enteric viruses, hepatitis A virus (HAV), poliovirus and rotavirus during sewage treatment by activated sludge using laboratory pilot plants. METHODS AND RESULTS: The cultivable simian rotavirus SA11, the HAV HM 175/18f cytopathic strain and poliovirus were quantified by cell culture. The bacteriophages were quantified by plaque formation on the host bacterium in agar medium. In each experiment, two pilots simulating full-scale activated sludge plants were inoculated with viruses at known concentrations, and mixed liquor and effluent samples were analysed regularly. In the mixed liquor, liquid and solid fractions were analysed separately. The viral behaviour in both the liquid and solid phases was similar between pilots of each experiment. Viral concentrations decreased rapidly following viral injection in the pilots. Ten minutes after the injections, viral concentrations in the liquid phase had decreased from 1.0 +/- 0.4 log to 2.2 +/- 0.3 log. Poliovirus and HAV were predominantly adsorbed on the solid matters of the mixed liquor while rotavirus was not detectable in the solid phase. In our model, the estimated mean log viral reductions after 3-day experiment were 9.2 +/- 0.4 for rotavirus, 6.6 +/- 2.4 for poliovirus, 5.9 +/- 3.5 for HAV, 3.2 +/- 1.2 for MS2 and 2.3 +/- 0.5 for fX174. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates that the pilots are useful models to assess the removal of infectious enteric viruses and bacteriophages by activated sludge treatment. Our results show the efficacy of the activated sludge treatment on the five viruses and suggest that coliphages could be an acceptable indicator of viral removal in this treatment system. [TOP OF PAGE]

  279. Correlation of Campylobacter bacteriophage with reduced presence of hosts in broiler chicken ceca. Atterbury,R.J., Dillon,E., Swift,C., Connerton,P.L., Frost,J.A., Dodd,C.E.R., Rees,C.E.D., Connerton,I.F. (2005). Appl. Environ. Microbiol. 71:4885-4887. Campylobacter jejuni and Campylobacter-specific bacteriophage were enumerated from broiler chicken ceca selected from 90 United Kingdom flocks (n = 205). C. jejuni counts in the presence of bacteriophage (mean log(10) 5.1 CFU/g) were associated with a significant (P < 0.001) reduction compared to samples with Campylobacter alone (mean log(10) 6.9 CFU/g). [TOP OF PAGE]

  280. Fate of Escherichia coli O157 and detection of stx phage during fermentation of maize, an animal feedstuff. Avery,S.M., Walters,L.D., Hutchison,M.L. (2005). Lett. Appl. Microbiol. 40:99-105. AIMS: The fate of inoculated Escherichia coli O157, stx phages and the physico-chemical properties of maize were studied during laboratory-scale fermentation by naturally occurring lactic acid bacteria. METHODS AND RESULTS: Before fermentation, chopped maize was inoculated with 6.2 log(10) CFU g(-1) of a five-isolate mixture of E. coli O157. After fermentation, the silage contained 70.6 g kg(-1) dry matter (DM) lactic acid and 12.8 g kg(-1) DM acetic acid and was pH 4.0. Levels of E. coli O157 fell rapidly, and none of the five isolates could be recovered from the fermenting maize after 8 days. Using a resuscitation step did not consistently enhance recovery of E. coli O157. Stx phages were not isolated from the fermenting maize at any time. Pulsed-field gel electrophoresis (PFGE) genotyping showed that E. coli O157 2975 and 64a/01 survived better than the other three isolates studied. Escherichia coli O157 isolate 1474/00 was particularly sensitive to the laboratory procedures used to harvest the inocula and contaminate the maize. Some colonies recovered during the fermentation had one to three band alterations compared with the initial PFGE genotypes. SIGNIFICANCE AND IMPACT OF THE STUDY: None of the five different E. coli O157 genotypes survived maize fermentation. Fermentation of maize produces an animal feedstuff that is unlikely to contain E. coli O157 or stx phages. [TOP OF PAGE]

  281. Mosaic prophages with horizontally acquired genes account for the emergence and diversification of the globally disseminated M1T1 clone of Streptococcus pyogenes. Aziz,R.K., Edwards,R.A., Taylor,W.W., Low,D.E., McGeer,A., Kotb,M. (2005). J. Bacteriol. 187:3311-3318. The recrudescence of severe invasive group A streptococcal (GAS) diseases has been associated with relatively few strains, including the M1T1 subclone that has shown an unprecedented global spread and prevalence and high virulence in susceptible hosts. To understand its unusual epidemiology, we aimed to identify unique genomic features that differentiate it from the fully sequenced M1 SF370 strain. We constructed DNA microarrays from an M1T1 shotgun library and, using differential hybridization, we found that both M1 strains are 95% identical and that the 5% unique M1T1 clone sequences more closely resemble sequences found in the M3 strain, which is also associated with severe disease. Careful analysis of these unique sequences revealed three unique prophages that we named M1T1.X, M1T1.Y, and M1T1.Z. While M1T1.Y is similar to phage 370.3 of the M1-SF370 strain, M1T1.X and M1T1.Z are novel and encode the toxins SpeA2 and Sda1, respectively. The genomes of these prophages are highly mosaic, with different segments being related to distinct streptococcal phages, suggesting that GAS phages continue to exchange genetic material. Bioinformatic and phylogenetic analyses revealed a highly conserved open reading frame (ORF) adjacent to the toxins in 18 of the 21 toxin-carrying GAS prophages. We named this ORF paratox, determined its allelic distribution among different phages, and found linkage disequilibrium between particular paratox alleles and specific toxin genes, suggesting that they may move as a single cassette. Based on the conservation of paratox and other genes flanking the toxins, we propose a recombination-based model for toxin dissemination among prophages. We also provide evidence that a minor population of the M1T1 clonal isolates have exchanged their virulence module on phage M1T1.Y, replacing it with a different module identical to that found on a related M3 phage. Taken together, the data demonstrate that mosaicism of the GAS prophages has contributed to the emergence and diversification of the M1T1 subclone. [TOP OF PAGE]

  282. Common ancestry of herpesviruses and tailed DNA bacteriophages. Baker,M.L., Jiang,W., Rixon,F.J., Chiu,W. (2005). J. Virol. 79:14967-14970. Comparative analysis of capsid protein structures in the eukaryote-infecting herpesviruses (Herpesviridae) and the prokaryote-infecting tailed DNA bacteriophages (Caudovirales) revealed a characteristic fold that is restricted to these two virus lineages and is indicative of common ancestry. This fold not only serves as a major architectural element in capsid stability but also enables the conformational flexibility observed during viral assembly and maturation. On the basis of this and other emerging relationships, it seems increasingly likely that the very diverse collection of extant viruses may have arisen from a relatively small number of primordial progenitors. [TOP OF PAGE]

  283. Diversity of phage integrases in Enterobacteriaceae: development of markers for environmental analysis of temperate phages. Balding,C., Bromley,S.A., Pickup,R.W., Saunders,J.R. (2005). Environ. Microbiol. 7:1558-1567. Viruses are the most abundant biological entities in aquatic systems. Temperate bacteriophages have enormous influences on microbial diversity, genetic exchange and bacterial population dynamics. However, development of molecular tools for their detection in the environment has been problematic. The integrase gene is used here as a molecular marker to analyse the diversity of temperate bacteriophages in a population of freshwater bacteria. Interrogation of the GenBank database revealed 32 non-cryptic enteric phage integrase sequences, leading to the development of a suite of 11 degenerate primer sets specific to the extant sequences elucidated. Application of these primer sets to enterobacterial isolates recovered from a freshwater pond and the temperate phages induced from them revealed a number of diverse integrase genes, including novel integrase-like sequences not represented in the databases. This highlights the potential of utilizing the integrase gene family as a marker for phage diversity. [TOP OF PAGE]

  284. Occurrence and correlations between coliphages and anthropogenic viruses in the Massachusetts Bay using enrichment and ICC-nPCR. Ballester,N.A., Fontaine,J.H., Margolin,A.B. (2005). J. Water Health 3:59-68. We evaluated a two-step enrichment procedure to detect coliphages and an integrated cell culture-nested polymerase chain reaction (ICC-nPCR) to detect human astrovirus, enteroviruses, rotavirus and adenovirus type 40 and 41 in marine water samples collected by the Massachusetts Water Resource Authority (MWRA). MWRA has been monitoring its receiving waters for coliphages, anthropogenic viruses and indicator bacteria in order to evaluate the impact of Boston's Deer Island Sewage Treatment Plant discharge. Coliphages and enteric viruses were originally assayed using single agar overlay and most probable number cell culture (MPN) methods, respectively. Reanalysis of these samples for enteric viruses by ICC-nPCR demonstrated that 46% were positive for at least one virus compared with 23% with the MPN method. Use of the enrichment method showed a 47% increase in the detection of male specific and somatic coliphages compared with the single agar overlay method. Correlations between the presence of coliphages, enteric viruses and indicator bacteria were based on proximity to the treatment plant discharge, seasonal variations and site levels. The presence of enteric viruses was significantly correlated to coliphages but not to indicator bacteria. Preliminary comparative results demonstrate that effective and efficient monitoring of anthropogenic contamination can be achieved using these more sensitive and specific techniques. [TOP OF PAGE]

  285. Rituximab inhibits the in vivo primary and secondary antibody response to a neoantigen, bacteriophage phiX174. Bearden,C.M., Agarwal,A., Book,B.K., Vieira,C.A., Sidner,R.A., Ochs,H.D., Young,M., Pescovitz,M.D. (2005). Am. J. Transpl. 5:50-57. The response to primary immunization in patients treated with Rituximab (RIT) is not clear. We studied the in vivo antibody response of chronic renal failure (CRF) patients to the neoantigen bacteriophage phiX174 given alone or after ablation with RIT. Eighteen CRF subjects received two immunizations with phiX174 separated by 6 weeks. Nine subjects received a single dose of RIT. The intensity and immunoglobulin isotype of the antibody response (K(v)) were measured post-infusion. In addition, three subjects previously immunized and treated with RIT underwent a third and fourth immunization with phiX174 and a tetanus control 2 years later. RIT significantly decreased peak K(v) responses when compared to both historic non-CRF controls and to CRF subjects. CRF itself decreased peak K(v) responses compared to non-CRF controls. Percent-ratio of anti-phage IgM to IgG was significantly decreased in RIT treated subjects. One of three subjects treated with RIT was found to have developed a partial B cell tolerance to phiX174 administration 2 years later. RIT decreases antibody production and isotype switching to neoantigens and might be useful to prevent antibody response to therapeutic drugs and to newly transplanted organs. [TOP OF PAGE]

  286. Microbiological assessment of ambient waters and proposed water sources for restoration of a Florida wetland. Betancourt,W.Q., Rose,J.B. (2005). J. Water Health 3:89-100. This study evaluated the microbial quality of reclaimed and storm water as proposed sources for restoration of a Florida wetland. Bacterial indicators, bacteriophages and waterborne pathogenic microorganisms (Cryptosporidium, Giardia and infectious enteric viruses) were analysed during a 1-year period in order to determine potential public health risks associated with exposure to the proposed water sources for restoration. Ambient waters within the wetland (four active water wells and four major lakes) were included in the study in order to determine the microbial water quality before restoration. Storm water and lakes had the highest level of microbial contamination. Much lower levels of microbial indicators and waterborne pathogens were found in reclaimed water and groundwater. Pathogen occurrence in groundwater was intermittent. Owing to the small percentage of source waters (3.3%) migrating to the water wells, ambient concentration of microbial constituents in surface and groundwater could dominate microbial risk. The results of this study indicate that, in the light of the uncertainties involved in computing average Cryptosporidium concentrations, additional characterization of the current ambient water quality should be ongoing prior to restoration. [TOP OF PAGE]

  287. Isolation and characterization of a small nuclear inclusion virus infecting the diatom Chaetoceros c.f. gracilis. Bettarel,Y., Kan,J., Wang,K., Cooney,S., Williamson,K., Chen,F., Wommack,E., Coats,W. (2005). Aquat. Microb. Ecol. 40:103-114. A novel virus (Chaetoceros nuclear inclusion virus: CspNIV) causing lysis of a culture of the diatom Chaetoceros cf. gracilis was isolated from the Chesapeake Bay, USA, in April 2003. Transmission electron microscopy of ultrathin sections of infected C. cf. gracilis revealed that CspNIV proliferates within the nucleus and forms paracrystalline arrays corresponding to the alignment of icosahedral viral particles of about 25 nm diameter. CspNIV shows some strong similarities with Heterosigma akashiwo nuclear inclusion virus (HaNIV) (cf. Lawrence et al. 2001; J Phycol 37: 216-222). The latent period of CspNIV is <24 h. The most widespread occurrence of Chaetoceros viruses in Chesapeake Bay was recorded in April 2003, ca. 1 mo after the winter-spring Chaetoceros bloom. However, results indicate that CspNIV remains infectious in surface water of the bay no longer than 1 mo after the disappearance of its host. Thus, our results reinforce the idea that microalgae are also sensitive to viruses other than those belonging to the family Phycodnaviridae. Furthermore, discovery and initial description of the infection process and ecology of CspNIV expands the breadth of phytoplankton shown to be susceptible to viral attack to include a ubiquitous diatom genera. [TOP OF PAGE]

  288. Low consumption of virus-sized particles by heterotrophic nanoflagellates in two lakes of the French Massif central. Bettarel,Y., Sime-Ngando,T., Amblard,C., Bouvy,M. (2005). Aquat. Microb. Ecol. 39:205-209. Seasonal and depth-related variability in the grazing activity of heterotrophic nanoflagellates (HNF) on viruses was examined in the oligo-mesotrophic Lake Pavin and in the eutrophic Lake Aydat, French Massif Central, between May and November 2000. Ingestion rates (IR) were determined using 50 nm diameter fluorescent microspheres, as virus analogues. In both lakes, highest grazing activities on virus-sized particles were recorded in the metalimnion, at the beginning and the end of the thermal stratification period. Estimated IRs in Lake Pavin (mean = 0.4 viruses HNF[-1] h[-1], CV = 38.0%) and in Lake Aydat (mean = 0.3 viruses HNF[-1] h[-1], CV = 35.6%) were not significantly different, in contrast to clearance rates (CR), which were significantly higher in the oligomesotrophic (2.3 x 10[-2] nl HNF[-1] h[-1]) than in the eutrophic lake (0.7 × 10[-2] nl HNF[-1] h[-1]). CRs for viruses were correlated with CRs for bacteria in Lake Aydat but not in Lake Pavin, suggesting a greater abundance within the HNF assemblages of virus-sized particle feeders in the less productive lake. We estimated that 4.1 and 0.8% of viral production were grazed by HNF in Lake Pavin and Lake Aydat, respectively. Finally, although viruses seem to represent a minor food source for HNF (i.e. compared to bacteria), they may not be inconsequential in their diet, especially in oligotrophic lakes. [TOP OF PAGE]

  289. A broad-host-range, generalized transducing phage (SN-T) acquires 16S rRNA genes from different genera of bacteria. Beumer,A., Robinson,J.B. (2005). Appl. Environ. Microbiol. 71:8301-8304. Genomic analysis has revealed heterogeneity among bacterial 16S rRNA gene sequences within a single species; yet the cause(s) remains uncertain. Generalized transducing bacteriophages have recently gained recognition for their abundance as well as their ability to affect lateral gene transfer and to harbor bacterial 16S rRNA gene sequences. Here, we demonstrate the ability of broad-host-range, generalized transducing phages to acquire 16S rRNA genes and gene sequences. Using PCR and primers specific to conserved regions of the 16S rRNA gene, we have found that generalized transducing phages (D3112, UT1, and SN-T), but not specialized transducing phages (D3), acquired entire bacterial 16S rRNA genes. Furthermore, we show that the broad-host-range, generalized transducing phage SN-T is capable of acquiring the 16S rRNA gene from two different genera: Sphaerotilus natans, the host from which SN-T was originally isolated, and Pseudomonas aeruginosa. In sequential infections, SN-T harbored only 16S rRNA gene sequences of the final host as determined by restriction fragment length polymorphism analysis. The frequency of 16S rRNA gene sequences in SN-T populations was determined to be 1 x 10(-9) transductants/PFU. Our findings further implicate transduction in the horizontal transfer of 16S rRNA genes between different species or genera of bacteria. [TOP OF PAGE]

  290. A chromosomally integrated bacteriophage in invasive meningococci. Bille,E., Zahar,J.R., Perrin,A., Morelle,S., Kriz,P., Jolley,K.A., Maiden,M.C.J., Dervin,C., Nassif,X., Tinsley,C.R. (2005). J. Exp. Med. 201:1905-1913. Cerebrospinal meningitis is a feared disease that can cause the death of a previously healthy individual within hours. Paradoxically, the causative agent, Neisseria meningitidis, is a common inhabitant of the human nasopharynx, and as such, may be considered a normal, commensal organism. Only in a small proportion of colonized people do the bacteria invade the bloodstream, from where they can cross the blood-brain barrier to cause meningitis. Furthermore, most meningococcal disease is caused by bacteria belonging to only a few of the phylogenetic groups among the large number that constitute the population structure of this genetically variable organism. However, the genetic basis for the differences in pathogenic potential remains elusive. By performing whole genome comparisons of a large collection of meningococcal isolates of defined pathogenic potential we brought to light a meningococcal prophage present in disease-causing bacteria. The phage, of the filamentous family, excises from the chromosome and is secreted from the bacteria via the type IV pilin secretin. Therefore, this element, by spreading among the population, may promote the development of new epidemic clones of N. meningitidis that are capable of breaking the normal commensal relationship with humans and causing invasive disease. [TOP OF PAGE]

  291. Influence of water chemistry and travel distance on bacteriophage PRD-1 transport in a sandy aquifer. Blanford,W.J., Brusseau,M.L., Jim Yeh,T.C., Gerba,C.P., Harvey,R. (2005). Water Res. 39:2345-2357. Experiments were conducted to evaluate the impact of groundwater chemistry and travel distance on the transport and fate behavior of PRD-1, a bacteriophage employed as a surrogate tracer for pathogenic enteric viruses. The experiments were conducted in the unconfined aquifer at the United States Geological Survey Cape Cod Toxic-Substances Hydrology Research Site in Falmouth, Massachusetts. The transport behavior of bromide (Br(-)) and PRD-1 were evaluated in a sewage-effluent contaminated zone and a shallower uncontaminated zone at this site. Several multilevel sampling devices located along a 13-m transect were used to collect vertically discrete samples to examine longitudinal and vertical variability of PRD-1 retardation and attenuation. The concentration of viable bacteriophage in the aqueous phase decreased greatly during the first few meters of transport. This decrease is attributed to a combination of colloid filtration (attachment) and inactivation. The removal was greater (10(-12) relative recovery) and occurred within the first meter for the uncontaminated zone, whereas it was lesser (10(-9) relative recovery) and occurred over 4m in the contaminated zone. The lesser removal observed for the contaminated zone is attributed to the influence of sorbed and dissolved organic matter, phosphate, and other anions, which are present in higher concentrations in the contaminated zone, on PRD-1 attachment. After the initial decrease, the aqueous PRD-1 concentrations remained essentially constant in both zones for the remainder of the tests (total travel distances of 13 m), irrespective of variations in geochemical properties within and between the two zones. The viable, mobile PRD-1 particles traveled at nearly the rate of bromide, which was used as a non-reactive tracer. The results of this study indicate that a small fraction of viable virus particles may persist in the aqueous phase and travel significant distances in the subsurface environment. [TOP OF PAGE]

  292. Viral production, decay rates, and life strategies along a trophic gradient in the North Adriatic Sea. Bongiorni,L., Magagnini,M., Armeni,M., Noble,R., Danovaro,R. (2005). Appl. Environ. Microbiol. 71:6644-6650. Although the relationships between trophic conditions and viral dynamics have been widely explored in different pelagic environments, there have been few attempts at independent estimates of both viral production and decay. In this study, we investigated factors controlling the balance between viral production and decay along a trophic gradient in the north Adriatic basin, providing independent estimates of these variables and determining the relative importance of nanoflagellate grazing and viral life strategies. Increasing trophic conditions induced an increase of bacterioplankton growth rates and of the burst sizes. As a result, eutrophic waters displayed highest rates of viral production, which considerably exceeded observed rates of viral decay (up to 2.9 x 10(9) VLP liter(-1) h(-1)). Viral decay was also higher in eutrophic waters, where it accounted for ca. 40% of viral production, and dropped significantly to 1.3 to 10.7% in oligotrophic waters. These results suggest that viral production and decay rates may not necessarily be balanced in the short term, resulting in a net increase of viruses in the system. In eutrophic waters nanoflagellate grazing, dissolved-colloidal substances, and lysogenic infection were responsible together for the removal of ca. 66% of viral production versus 17% in oligotrophic waters. Our results suggest that different causative agents are primarily responsible for the removal of viruses from the water column in different trophic conditions. Factors other than those considered in the past might shed light on processes responsible for the removal and/or decay of viral particles from the water column. [TOP OF PAGE]

  293. Mobile DNA in obligate intracellular bacteria. Bordenstein,S.R., Reznikoff,W.S. (2005). Nat. Rev. Microbiol. 3:688-699. The small genomes of obligate intracellular bacteria are often presumed to be impervious to mobile DNA and the fluid genetic processes that drive diversification in free-living bacteria. Categorized by reductive evolution and streamlining, the genomes of some obligate intracellular bacteria manifest striking degrees of stability and gene synteny. However, recent findings from complete genome sequences of obligate intracellular species and their mobile genetic associates favour the abandonment of these wholesale terms for a more complex and tantalizing picture. [TOP OF PAGE]

  294. [The potential use of bacteriophages in view of the current antibiotic therapy crisis]. Borysowski,J., Weber-Dabrowska,B., Gorski,A. (2005). Polskie Archiwum Medycyny Wewnetrznej 113:73-78. [TOP OF PAGE]

  295. Evolution of foodborne pathogens via temperate bacteriophage-mediated gene transfer. Brabban,A.D., Hite,E., Callaway,T.R. (2005). Foodborne Pathog. Dis. 2:287-303. Temperate bacteriophages have always been central to the evolution of bacteria, although their importance has been consistently underestimated compared to transformation and conjugation. In the last 20 years, as more gene and genome sequences have become available and researchers have more accurately determined bacteriophage populations in the environment, we are gaining a clearer picture of their role in the past and potential role in the future. The transductive and lysogenic capacities of this class of bacteriophages have contributed to the evolution and shaping of emerging foodborne pathogenic bacteria through the dissemination of virulence and antibiotic resistance genes. For example, the genome sequences of Shigella dysenteriae, Escherichia coli O157:H7, and the Stx encoding bacteriophages demonstrate the critical role bacteriophage-mediated gene transfer events played in the evolution of these high-profile human pathogens. In this review, we describe the basic genetic exchange mechanisms mediated by temperate bacteriophages and how these mechanisms have been central to the dissemination of virulence genes, such as toxins and antibiotics from one species to another (the shiga-like toxins, and multiple antibiotic resistance dissemination in Salmonella are used as specific examples). Data demonstrating the role of bacteriophages in the spread of antimicrobial resistance in bacteria, including interspecies transduction, are also presented. That temperate bacteriophages play a role in the on-going evolution of emerging pathogenic bacteria is obvious, but it is also clearly an on-going process with a breadth that must be appreciated as well as studied further if we are to be able to foresee what new challenges will arise to imperil food safety. [TOP OF PAGE]

  296. Bacteriophage WO in Wolbachia infecting terrestrial isopods. Braquart-Varnier,C., Greve,P., Felix,C., Martin,G. (2005). Biochem. Biophys. Res. Com. 337:580-585. Wolbachia are maternally inherited intracellular alpha-proteobacteria that infect a wide range of arthropods. They are associated with a number of different reproductive phenotypes in arthropods and nematodes. In isopod crustacean, Wolbachia are responsible for feminization of genetic males in many species, and for cytoplasmic incompatibility in two species. In this paper, we report the first detection of phage WO from Wolbachia infecting terrestrial isopods. All Wolbachia strains tested in this study were infected with phage WO. Based on the orf7 phage sequence, we identified three different phage sequences in four Wolbachia strains. The phage of Wolbachia infecting Armadillidium vulgare seems to be not active, unlike other phages WO previously described in arthropods. [TOP OF PAGE]

  297. Here a virus, there a virus, everywhere the same virus? Breitbart,M., Rohwer,F. (2005). Trends Microbiol. 13:278-284. There are an estimated 10(31) viruses on Earth, most of which are phages that infect bacteria. Metagenomic analyses have shown that environmental viral communities are incredibly diverse. There are an estimated 5000 viral genotypes in 200 liters of seawater and possibly a million different viral genotypes in one kilogram of marine sediment. By contrast, some culturing and molecular studies have found that viruses move between different biomes. Together, these findings suggest that viral diversity could be high on a local scale but relatively limited globally. Also, by moving between environments, viruses can facilitate horizontal gene transfer. [TOP OF PAGE]

  298. Phage ecology and bacterial pathogenesis. Breitbart,M., Rohwer,F., Abedon,S.T. (2005). pp. 66-91. In In Waldor,M.K., Friedman,D.I., and Adhya,S.L. (eds.), Phages: Their Role in Bacterial Pathogenesis and Biotechnology. ASM Press, Washington DC. [first paragraph] Bacteriophages (phages) are the virues of bacteria. The impact of phages on bacterial pathogenesis may be divided into two major themes, transduction and predation. (i) Phages can move genes, including genes encoding bacterial virulence factors (VFs), between bacteria. This movement can occur via generalized or specialized transduction. (ii) Phages can also virulently attack bacteria. This predation can modify the structure of bacterial communities, selecting for bacteria that are resistant to phage infection. Depending on the nature of a phage infection-e.g., lytic versus lysogenic infection or infection of a bacterial pathogen versus infection of a competitor in the normal flora-phages may either negatively or positively affect bacterial pathogenicity. An understanding of the phage impact on bacterial pathogenesis consequently requires not just knowledge of VF expression but also an understanding of phage transduction and propagation in the environment. In this chapter, we take a phage-centered view of the ecology of the phage-bacterium relationship, looking in particular for unappreciated subtleties that might impact pathogen formation, disease progression, or the phage-induced destruction of bacterial populations. [TOP OF PAGE]

  299. Results with phages. Bricelj,M. (2005). <None Specified> [TOP OF PAGE]

  300. Thermostability of landscape phage probes. Brigati,J.R., Petrenko,V.A. (2005). Analyt. Bioanalyt. Chem. 382:1346-1350. Immunoassays have traditionally relied on antibodies as diagnostic probes. Their use outside of a laboratory, however, may be problematic because antibodies are often unstable in severe environmental conditions. Environmental monitoring requires thermostable probes, such as landscape phage, that carry thousands of foreign peptides on their surfaces, are superior to antibodies, and can operate in non-controlled conditions. While parent wild-type phage are known to be extremely stable in various media at high temperatures, no work has been done to demonstrate the stability of landscape phage probes. We examined the thermostability of a landscape phage probe and a monoclonal antibody specific for b-galactosidase in parallel in an enzyme-linked immunosorbent assay (ELISA) format. They were both stable for greater than six months at room temperature, but at higher temperatures the antibody degraded more rapidly than the phage probe. Phage retained detectable binding ability for more than six weeks at 63 degrees C, and three days at 76 degrees C. The activation energy of phage degradation was determined to be 1.34 x 10(5) J/mol. These results confirm that phage probes are highly thermostable and can function even after exposure to high temperatures during shipping, storage and operation. [TOP OF PAGE]

  301. Artificial neural network prediction of viruses in shellfish. Brion,G., Viswanathan,C., Neelakantan,T.R., Lingireddy,S., Girones,R., Lees,D., Allard,A., Vantarakis,A. (2005). Appl. Environ. Microbiol. 71:5244-5253. A database was probed with artificial neural network (ANN) and multivariate logistic regression (MLR) models to investigate the efficacy of predicting PCR-identified human adenovirus (ADV), Norwalk-like virus (NLV), and enterovirus (EV) presence or absence in shellfish harvested from diverse countries in Europe (Spain, Sweden, Greece, and the United Kingdom). The relative importance of numerical and heuristic input variables to the ANN model for each country and for the combined data was analyzed with a newly defined relative strength effect, which illuminated the importance of bacteriophages as potential viral indicators. The results of this analysis showed that ANN models predicted all types of viral presence and absence in shellfish with better precision than MLR models for a multicountry database. For overall presence/absence classification accuracy, ANN modeling had a performance rate of 95.9%, 98.9%, and 95.7% versus 60.5%, 75.0%, and 64.6% for the MLR for ADV, NLV, and EV, respectively. The selectivity (prediction of viral negatives) was greater than the sensitivity (prediction of viral positives) for both models and with all virus types, with the ANN model performing with greater sensitivity than the MLR. ANN models were able to illuminate site-specific relationships between microbial indicators chosen as model inputs and human virus presence. A validation study on ADV demonstrated that the MLR and ANN models differed in sensitivity and selectivity, with the ANN model correctly identifying ADV presence with greater precision. [TOP OF PAGE]

  302. The use of bacteriophages in eliminating polyresistant strains of Staphylococcus aureus and Streptococcus agalactiae. Brnakova,Z., Farkasovska,J., Godany,A. (2005). Folia Microbiol (Praha) 50:187-194. Temperate bacteriophages were induced in and released from isolates of Staphylococcus aureus and Streptococcus agalactiae using mitomycin C. Various specific indicator cultures were tested for providing clear plaques after phage infection. Specific lytic mixture of bacteriophages was prepared using the induced, modified and laboratory variants of phages. Under laboratory conditions, the mixture eliminated all isolates from the tested collection of microorganisms. The restriction barrier of some bacterial isolates to bacteriophage infection was overcome either by UV irradiation or in vitro modification of bacteriophage DNA with specific methyltransferases. Conjugative R plasmids, capable of replication in G+ and G- bacteria, were detected and isolated from S. aureus and S. agalactiae antibiotic-resistant strains. [TOP OF PAGE]

  303. The effect of a bacteriophage on diversification of the opportunistic bacterial pathogen, Pseudomonas aeruginosa. Brockhurst,M.A., Buckling,A., Rainey,P.B. (2005). Proc. Roy. Soc. Lond. B 272:1385-1391. Pseudomonas aeruginosa is an opportunistic human pathogen that colonizes the lungs of cystic fibrosis (CF) patients. CF lungs often contain a diverse range of P. aeruginosa phenotypes, some of which are likely to contribute to the persistence of infection, yet the causes of diversity are unclear. While the ecological heterogeneity of the lung environment and therapeutic regimes are probable factors, a role for parasitic bacteriophage cannot be ruled out. Parasites have been implicated as a key ecological variable driving the evolution of diversity in host populations. PP7 drove cycles of morphological diversification in host populations of P. aeruginosa due to the de novo evolution of small-rough colony variants that coexisted with large diffuse colony morph bacteria. In the absence of phage, bacteria only displayed the large diffuse colony morphology of the wild-type. Further assays revealed there to be two distinct types of resistant bacteria; these had very different ecological phenotypes, yet each carried a cost of resistance. [TOP OF PAGE]

  304. Relative value of surrogate indicators for detecting pathogens in lakes and reservoirs. Brookes,J.D., Hipsey,M.R., Burch,M.D., Regel,R.H., Linden,L.G., Ferguson,C.M., Antenucci,J.P. (2005). Environ. Sci. Technol. 39:8614-8621. This study investigated the relative behavior of pathogens, fecal indicator organisms, and particles of varying size during transport through a reservoir following a storm event inflow in Myponga Reservoir, South Australia. During the inflow, samples were collected from the river and at various locations within the reservoir to determine the fate and transport of microroganisms as they progressed through the water body. Microbiological analysis included the indicator organisms Escherichia coli, enterococci, Clostridium perfringens, aerobic spores, and somatic coliphages, the protozoan pathogens Cryptosporidium spp. and Giardia spp., and the potential physical surrogates of pathogen contamination including particle size and turbidity. Of the microbial indicator groups, C. perfringens spores were the most highly correlated with Cryptosporidium spp. concentrations (Spearman Rho = 0.58), closely followed by enterococci (Spearman Rho = 0.57). Cryptosporidium spp. oocysts were predominantly associated with small sized particles (range of 14.3-27.7 microm). All of the microbial indicator groups tested were associated with larger sized particle ranges (> 63.3 microm) except C. perfringens spores which were associated with particles in the size range of 45.5-63.3 microm. Although indicators may rank correlate with Cryptosporidium spp., the variation in settling rates of different microorganisms has significant implications for the use of surrogates to estimate pathogen attenuation within reservoirs. For example, concentrations of Cryptosporidium spp. oocysts were reduced by a factor of 3 on reaching the dam wall, whereas enterococci were reduced by a factor of 10. [TOP OF PAGE]

  305. Human volunteers receiving Escherichia coli phage T4 orally: a safety test of phage therapy. Bruttin,A., Brüssow,H. (2005). Antimicrob. Agents Chemother. 49:2874-2878. Fifteen healthy adult volunteers received in their drinking water a lower Escherichia coli phage T4 dose (10(3) PFU/ml), a higher phage dose (10(5) PFU/ml), and placebo. Fecal coliphage was detected in a dose-dependent way in volunteers orally exposed to phage. All volunteers receiving the higher phage dose showed fecal phage 1 day after exposure; this prevalence was only 50% in subjects receiving the lower phage dose. No fecal phage was detectable a week after a 2-day course of oral phage application. Oral phage application did not cause a decrease in total fecal E. coli counts. In addition, no substantial phage T4 replication on the commensal E. coli population was observed. No adverse events related to phage application were reported. Serum transaminase levels remained in the normal range, and neither T4 phage nor T4-specific antibodies were observed in the serum of the subjects at the end of the study. This is, to our knowledge, the first safety test in the recent English literature which has measured the bioavailability of oral phage in humans and is thus a first step to the rational evaluation of phage therapy for diarrheal diseases. [TOP OF PAGE]

  306. Genomics and the evolution of tailed phages. Brüssow,H., Kutter,E. (2005). pp. 91-128. In In Kutter,E. and Sulakvelidze,A. (eds.), Bacteriophages: Biology and Application. CRC Press, Boca Raton, Florida. [first paragarph (but may not be final version)] Large numbers of bacteriophages have been found wherever potential host bacteria are found; e.g., in soils, lakes, hot springs and deep sea vents, or associated with commensal bacteria inhabiting plants and animals; phage ecology is discussed in some depth in chapter 6. About 10 times as many phages as bacteria have been detected in seawater samples, leading to estimates of about 10(32) phages on earth (Bergh, et al. 1989; Wommack and Colwell 2000). Phages may be incredibly varied in their properties; e.g., their hosts, genetic content, regulatory mechanisms, physiological effects, and most of the genes in the larger phages correspond to nothing yet described in the currently available databases. There is wide interest in how viruses arose; i.e, whether they are representatives of early pre-cellular forms of life or are sophisticated forms of selfish genes derived from modern genomes, how they acquired their special properties and genes, and how they relate to each other and to cellular genomes. The evidence so far indicates that the tailed phages, at least, are of very ancient origin. As discussed below, some phage-encoded enzymes like T4 thymidylate synthase appear to have diverged from the precursor of their bacterial and eukaryotic relatives before those two diverged from each other. So far, we have only begun to examine an infinitesimal sample of the phages infecting common, culturable bacteria. We can only look with fascination at the variety of tailed phages in aqueous and soil habitats, where most of the bacteria cannot yet be cultured to use as hosts for isolating phages. The various groups of tailless phages have been less widely studied; they seem to be less numerous, but they also are harder to distinguish from nonviral elements and algal viruses in the environment. [TOP OF PAGE]

  307. Phage therapy: the Escherichia coli experience. Brüssow,H. (2005). Microbiology (Reading) 151:2133-2140. Phages have been proposed as natural antimicrobial agents to fight bacterial infections in humans, in animals or in crops of agricultural importance. Phages have also been discussed as hygiene measures in food production facilities and hospitals. These proposals have a long history, but are currently going through a kind of renaissance as documented by a spate of recent reviews. This review discusses the potential of phage therapy with a specific example, namely Escherichia coli. [TOP OF PAGE]

  308. Phage ecology. Brüssow,H., Kutter,E. (2005). pp. 129-164. In In Kutter,E. and Sulakvelidze,A. (eds.), Bacteriophages: Biology and Application. CRC Press, Boca Raton, Florida. [first three paragarphs (but may not be final version)] Felix d'Herelle, co-discoverer of phage, had a strikingly modern approach to biology. Nearly one hundred years ago, he used living organisms to control pests (diarrhea-causing bacteria to halt locust epidemics) and disease (phage therapy of diarrheal diseases). His approaches reflected ecological insights before this branch of biology became an established scientific discipline. In fact, one might have predicted that phage research would become a springboard for studies of microbial ecology. Instead, studies of phage ecology were largely ignored and phage research became the cradle of molecular biology. This turn in the history of biological research is not explained by any critical technical breakthrough, but rather by a number of biographical reasons in the lives of a handful of scientists. The second generation of Western phage researchers concentrated on a few phages from E. coli, the workhorse of bacterial genetics, in order to better understand the basic nature of phages and of the phage infection process and use this knowledge to explore fundamental aspects of biology at the molecular level. Phage ecology was not within their conceptual framework. The diversity of phages was better appreciated by medical microbiologists, who used phages for the typing of clinical isolates of bacterial pathogens. However, in that field phages were exclusively used as tools without intrinsic interest in their ecology or molecular characteristics. Consequently, the first monograph on the distribution and behaviour of bacterial viruses in the environment appeared only in 1987 (Goyal). ¶ Since the appearance of Goyal's book, the study of phage ecology has changed fundamentally. One drastic reminder of the importance of phage in the ecosystem was the surprising discovery of very large numbers of phage-like particles in the ocean (Bergh, et al. 1989). Phage ecology quickly became an intensively investigated branch of marine microbiology, as documented by a recent review listing hundreds of publications, mostly from the last decade (Wommack and Colwell 2000). Recently, general reviews have appeared on various major aspects of phage ecology (Abedon 2005; Ashelford, et al. 2003; Azam and Worden 2004; Breitbart, et al. 2002; 2003; Chibani-Chennoufi, et al. 2004; Paul and Kellogg 2000; Suttle 2000b). ¶ Scientists in two fields have developed a particularly keen interest in phage ecology. One is the food industry, where fermentation techniques are used to transform milk, vegetables or meat into processed foods like cheese, sauerkraut or salami. This food production relies either on spontaneous fermentation or, in the case of milk, on fermentation initiated by the addition of industrial bacterial starters. Phages that infect these starters are the major cause of fermentation failures in the dairy industry (chapter 10). The high economic losses associated with phage infection there motivated intense research into phages from lactic acid bacteria, which are the major dairy starter organisms. As the dairy factory is a man-made environment, it did not so much attract the interest of ecologists, but rather that of more technologically oriented microbiologists, focusing on the design of efficient starter rotation systems and the construction of phage-resistant starter cells. In doing that, dairy microbiologists had to investigate the factory ecology of phage infections, leading to large systematic collections of dairy phages. However, these phages were characterized more by sequence analysis and molecular biology than by classical ecological approaches. The other emerging field is linked to the rekindling of interest in phage therapy, as discussed in chapters 13 and 14. The successful application of this approach to the growing problem of antibiotic resistance depends on the availability of large collections of phages and a detailed knowledge of phage-host interactions in different physiological compartments that necessitates sound ecological knowledge of the interactions between the phage, bacteria and plant or animal host. It is also increasingly extending to interest in potential applications against plant pathogens, bacteria in biofilms and other complex real-world situations. [TOP OF PAGE]

  309. Phage biology. Calendar,R., Inman,R. (2005). pp. 18-36. In In Waldor,M.K., Friedman,D.I., and Adhya,S.L. (eds.), Phages: Their Role in Bacterial Pathogenesis and Biotechnology. ASM Press, Washington DC. [TOP OF PAGE]

  310. Bacteriophage P100 for control of Listeria monocytogenes in foods: genome sequence, bioinformatic analyses, oral toxicity study, and application. Carlton,R.M., Noordman,W.H., Biswas,B., de Meester,E.D., Loessner,M.J. (2005). Regulatory toxicology and pharmacology : RTP 43:301-312. Listeria monocytogenes is an opportunistic foodborne pathogen responsible for Listeriosis, a frequently fatal infection. This investigation represents a comprehensive approach to characterize and evaluate the broad host range, strictly virulent phage P100, which can infect and kill a majority of Listeria monocytogenes strains. First, the complete nucleotide sequence (131,384 basepairs) of the genome of P100 was determined, predicted to encode 174 gene products and 18 tRNAs. Bioinformatic analyses revealed that none of the putative phage proteins has any homologies to genes or proteins of Listeria or any other bacteria which are known or suspected to be toxins, pathogenicity factors, antibiotic resistance determinants, or any known allergens. Next, a repeated dose oral toxicity study in rats was conducted, which did not produce any abnormal histological changes, morbidity or mortality. Therefore, no indications for any potential risk associated with using P100 as a food additive were found. As proof of concept, and to determine the parameters for application of P100 to foods sensitive to Listeria contamination, surface-ripened red-smear soft cheese was produced. Cheeses were contaminated with low concentrations of L. monocytogenes at the beginning of the ripening period, and P100 was applied to the surface during the rind washings. Depending on the time points, frequency and dose of phage applications, we were able to obtain a significant reduction (at least 3.5 logs) or a complete eradication of Listeria viable counts, respectively. We found no evidence for phage resistance in the Listeria isolates recovered from samples. Taken together, our results indicate that P100 can provide an effective and safe measure for the control of Listeria contamination in foods and production equipment. [TOP OF PAGE]

  311. Comparative genomics and evolution of the tailed-bacteriophages. Casjens,S.R. (2005). Curr. Opin. Mirobiol. 8:451-458. The number of completely sequenced tailed-bacteriophage genomes that have been published increased to more than 125 last year. The comparison of these genomes has brought their highly mosaic nature into much sharper focus. Furthermore, reports of the complete sequences of about 150 bacterial genomes have shown that the many prophage and parts thereof that reside in these bacterial genomes must comprise a significant fraction of Earth's phage gene pool. These phage and prophage genomes are fertile ground for attempts to deduce the nature of viral evolutionary processes, and such analyses have made it clear that these phage have enjoyed a significant level of horizontal exchange of genetic information throughout their long histories. The strength of these evolutionary deductions rests largely on the extensive knowledge that has accumulated during intensive study into the molecular nature of the life cycles of a few 'model system' phages over the past half century. Recent molecular studies of phages other than these model system phages have made it clear that much remains to be learnt about the variety of lifestyle strategies utilized by the tailed-phage. [TOP OF PAGE]

  312. Microbial partitioning to settleable particles in stormwater. Characklis,G.W., Dilts,M.J., Simmons,O.D., Likirdopulos,C.A., Krometis,L.A., Sobsey,M.D. (2005). Water Res. 39:1773-1782. The degree to which microbes in the water column associate with settleable particles has important implications for microbial transport in receiving waters, as well as for microbial removal via sedimentation (i.e. detention basins). The partitioning behavior of several bacterial, protozoan and viral indicator organisms is explored in three urban streams under both storm and dry weather conditions. The fraction of organisms associated with settleable particles in stormwater is estimated through use of a centrifugation technique which is calibrated using suspensions of standard particles (e.g., glass, latex). The fraction of organisms associated with settleable particles varies by type of microbe, and the partitioning behavior of each organism generally changes between dry weather and storm conditions. Bacterial indicator organisms (fecal coliforms, Escherichia coli, enterococci) exhibited relatively consistent behavior, with an average of 20-35% of organisms associated with these particles in background samples and 30-55% in storm samples. Clostridium perfringens spores exhibited the highest average level of particle association, with storm values varying from 50% to 70%. Results related to total coliphage partitioning were more variable, with 20-60% associated with particles during storms. These estimates should be valuable in surface water quality modeling efforts, many of which currently assume that all microbes exist as free (unattached) organisms. [TOP OF PAGE]

  313. Phage associated bacteriocins reveal a novel mechanism for bacteriocin diversification in Klebsiella. Chavan,M., Rafi,H., Wertz,J., Goldstone,C., Riley,M.A. (2005). J. Mol. Evol. 60:546-556. Ninety-six isolates of Klebsiella pneumoniae and K. oxytoca were recovered from wild mammals in Australia. 14.6% of these bacteria produce killing phenotypes that suggest the production of bacteriocin toxins. Cloning and sequencing of the gene clusters encoding two of these killing phenotypes revealed two instances of a bacteriocin associated with a bacteriophage gene, the first such genetic organization described. The newly identified klebicin C gene cluster was discovered in both K. pneumoniae and K. oxytoca. The newly identified klebicin D gene cluster was detected in K. oxytoca. Protein sequence comparisons and phylogenetic inference suggest that klebicin C is most closely related to the rRNase group of colicins (such as colicin E4), while klebicin D is most closely related to the tRNase group of colicins (such as colicin D). The klebicin C and D gene clusters have similar genetic and regulatory organizations. In both cases, an operon structure is inferred consisting of a phage-associated open reading frame and klebicin activity and associated immunity genes. This novel bacteriophage/bacteriocin organization may provide a novel mechanism for the generation of bacteriocin diversity in Klebsiella. [TOP OF PAGE]

  314. Population fitness and the regulation of Escherichia coli genes by bacterial viruses. Chen,Y., Golding,I., Sawai,S., Guo,L., Cox,E.C. (2005). PLoS Biol. 3:e229 Temperate bacteriophage parasitize their host by integrating into the host genome where they provide additional genetic information that confers higher fitness on the host bacterium by protecting it against invasion by other bacteriophage, by increasing serum resistance, and by coding for toxins and adhesion factors that help the parasitized bacterium invade or evade its host. Here we ask if a temperate phage can also regulate host genes. We find several different host functions that are down-regulated in lysogens. The pckA gene, required for gluconeogenesis in all living systems, is regulated directly by the principal repressor of many different temperate prophage, the cI protein. cI binds to the regulatory region of pckA, thereby shutting down pckA transcription. The pckA regulatory region has target sequences for many other temperate phage repressors, and thus we suggest that down-regulation of the host pckA pathway increases lysogen fitness by lowering the growth rate of lysogens in energy-poor environments, perhaps as an adaptive response to the host predation system or as an aspect of lysogeny that must be offset by down-regulating pckA. [TOP OF PAGE]

  315. Information theory based T7-like promoter models: classification of bacteriophages and differential evolution of promoters and their polymerases. Chen,Z., Schneider,T.D. (2005). Nucleic Acids Research 33:6172-6187. Molecular information theory was used to create sequence logos and promoter models for eight phages of the T7 group: T7, jA1122, T3, jYeO3-12, SP6, K1-5, gh-1 and K11. When these models were used to scan the corresponding genomes, a significant gap in the individual information distribution was observed between functional promoter sites and other sequences, suggesting that the models can be used to identify new T7-like promoters. When a combined 76-site model was used to scan the eight phages, 108 of the total 109 promoters were found, while none were found for other T7-like phages, jKMV, P60, VpV262, SIO1, PaP3, Xp10, P-SSP7 and Ppu40, indicating that these phages do not belong to the T7 group. We propose that the T7-like transcription system, which consists of a phage-specific RNA polymerase and a set of conserved T7-like promoters, is a hallmark feature of the T7 group and can be used to classify T7-like phages. Phylogenetic trees of the T7-like promoter models and their corresponding RNA polymerases are similar, suggesting that the eight phages of the T7 group can be classified into five subgroups. However the SP6-like polymerases have apparently diverged from other polymerases more than their promoters have diverged from other promoters. [TOP OF PAGE]

  316. Different inactivation behaviors of MS-2 phage and Escherichia coli in TiO2 photocatalytic disinfection. Cho,M., Chung,H., Choi,W., Yoon,J. (2005). Appl. Environ. Microbiol. 71:270-275. Despite a wealth of experimental evidence concerning the efficacy of the biocidal action associated with the TiO(2) photocatalytic reaction, our understanding of the photochemical mechanism of this particular biocidal action remains largely unclear. It is generally accepted that the hydroxyl radical (.OH), which is generated on the surface of UV-illuminated TiO(2), plays the main role. However, our understanding of the exact mode of action of the hydroxyl radical in killing microorganisms is far from complete, and some studies report that other reactive oxygen species (ROS) (H(2)O(2) and O(2).(-), etc.) also play significant roles. In particular, whether hydroxyl radicals remain bound to the surface or diffuse into the solution bulk is under active debate. In order to examine the exact mode of action of ROS in inactivating the microorganism, we tested and compared the levels of photocatalytic inactivation of MS-2 phage and Escherichia coli as representative species of viruses and bacteria, respectively. To compare photocatalytic microbial inactivation with the photocatalytic chemical degradation reaction, para-chlorobenzoic acid, which rapidly reacts with a hydroxyl radical with a diffusion-limited rate, was used as a probe compound. Two different hydroxyl radical scavengers, tert-butanol and methanol, and an activator of the bulk phase hydroxyl radical generation, Fe(2+), were used to investigate their effects on the photocatalytic mode of action of the hydroxyl radical in inactivating the microorganism. The results show that the biocidal modes of action of ROS are very different depending on the specific microorganism involved, although the reason for this is not clear. It seems that MS-2 phage is inactivated mainly by the free hydroxyl radical in the solution bulk but that E. coli is inactivated by both the free and the surface-bound hydroxyl radicals. E. coli might also be inactivated by other ROS, such as O(2).(-) and H(2)O(2), according to the present results. [TOP OF PAGE]

  317. Phage abortive infection in lactococci: variations on a theme. Chopin,M.C., Chopin,A., Bidnenko,E. (2005). Curr. Opin. Mirobiol. 8:473-479. Abortive infection (Abi) systems, also called phage exclusion, block phage multiplication and cause premature bacterial cell death upon phage infection. This decreases the number of progeny particles and limits their spread to other cells allowing the bacterial population to survive. Twenty Abi systems have been isolated in Lactococcus lactis, a bacterium used in cheese-making fermentation processes, where phage attacks are of economical importance. Recent insights in their expression and mode of action indicate that, behind diverse phenotypic and molecular effects, lactococcal Abis share common traits with the well-studied Escherichia coli systems Lit and Prr. Abis are widespread in bacteria, and recent analysis indicates that Abis might have additional roles other than conferring phage resistance. [TOP OF PAGE]

  318. War is peace--dispatches from the bacterial and phage killing fields. Comeau,A.M., Krisch,H.M. (2005). Curr. Opin. Mirobiol. 8:488-494. Large-scale sequence analyses of phage and bacteria have provided new insights into the diverse and multifaceted interactions of these genomes. Such interactions are important because they determine the partitioning of a large fraction of global biomass. Furthermore, the struggle between phage and bacteria has had a significant impact on the evolution of the biosphere. This competition for resources has created an enormous pool of genetic diversity. Eons of horizontal genetic transfer have permitted the entire biosphere to directly benefit from a bargain-basement source of evolutionary innovation. [TOP OF PAGE]

  319. A persistent, productive, and seasonally dynamic vibriophage population within Pacific oysters (Crassostrea gigas). Comeau,A.M., Buenaventura,E., Suttle,C.A. (2005). Appl. Environ. Microbiol. 71:5324-5331. In an effort to understand the relationship between Vibrio and vibriophage populations, abundances of Vibrio spp. and viruses infecting Vibrio parahaemolyticus (VpVs) were monitored for a year in Pacific oysters and water collected from Ladysmith Harbor, British Columbia, Canada. Bacterial abundances were highly seasonal, whereas high titers of VpVs (0.5 x 10(4) to 11 x 10(4) viruses cm(-3)) occurred year round in oysters, even when V. parahaemolyticus was undetectable (< 3 cells cm(-3)). Viruses were not detected (<10 ml(-1)) in the water column. Host-range studies demonstrated that 13 VpV strains could infect 62% of the V. parahaemolyticus strains from oysters (91 pairings) and 74% of the strains from sediments (65 pairings) but only 30% of the water-column strains (91 pairings). Ten viruses also infected more than one species among V. alginolyticus, V. natriegens, and V. vulnificus. As winter approached and potential hosts disappeared, the proportion of host strains that the viruses could infect decreased by approximately 50% and, in the middle of winter, only 14% of the VpV community could be plated on summer host strains. Estimates of virus-induced mortality on V. parahaemolyticus indicated that other host species were required to sustain viral production during winter when the putative host species was undetectable. The present study shows that oysters are likely one of the major sources of viruses infecting V. parahaemolyticus in oysters and in the water column. Furthermore, seasonal shifts in patterns of host range provide strong evidence that the composition of the virus community changes during winter. [TOP OF PAGE]

  320. Bacteriophage penetration in vertebrates. Dabrowska,K., Switala-Jelen,K., Opolski,A., Weber-Dabrowska,B., Gorski,A. (2005). J. Appl. Microbiol. 98:7-13. Bacteriophages are viruses that infect bacteria. They are the most numerous life forms on earth. As antibiotic resistance is becoming an increasingly worldwide challenge, bacteriophages as potential antimicrobial agents are being more intensively explored. Some very important questions involve their ability to penetrate higher organisms, as this determines potential phage activity in antibacterial treatment. Higher organisms are widely exposed to bacteriophages, which penetrate them quite freely. Bacteriophage activity can be influenced by specific antibodies which, together with the nonspecific immune system, can contribute to their rapid clearance from the organism. Bacteriophages can also interact directly with mammalian cells and even play a role in the development of some nonbacterial diseases, although they are not able to multiply in these cells. All aspects of the interaction between phages and higher organism are of interest and importance for further medical and biochemical applications. [TOP OF PAGE]

  321. Viruses, prokaryotes and DNA in the sediments of a deep-hypersaline anoxic basin (DHAB) of the Mediterranean Sea. Danovaro,R., Corinaldesi,C., Dell'Anno,A., Fabiano,M., Corselli,C. (2005). Environ. Microbiol. 7:586-592. Viral and prokaryote abundance were investigated in a deep-hypersaline anoxic basin of the Eastern Mediterranean Sea (DHAB Atalante basin at c. 3000 m depth). This system was compared with two nearby deep-sea sites characterized by oxic conditions. Viral abundance and virus to prokaryote abundance ratio in hypersaline anoxic sediments displayed values close to those reported in oxic sites. The analysis of vertical profiles of viral abundance in the Atalante basin revealed the lack of significant changes with depth in the sediment, suggesting that benthic viruses in these anoxic and hypersaline conditions are preserved or resistant to decay. The anoxic basin displayed also very high concentrations of labile organic components (proteins and lipids) and extracellular DNA. These findings suggest that the DHAB sediments represent a reservoir for long-term preservation of benthic viruses and nucleic acids. [TOP OF PAGE]

  322. Virus removal in a pilot-scale 'advanced' pond system as indicated by somatic and F-RNA bacteriophages. Davies-Colley,R.J., Craggs,R.J., Park,J., Sukias,J.P.S., Nagels,J.W., Stott,R. (2005). Water Sci. Technol. 51:107-110. Advanced pond systems (APS), incorporating high-rate ponds, algal settling ponds, and maturation ponds, typically achieve better and more consistent disinfection as indicated by Escherichia coli than conventional waste stabilisation ponds. To see whether this superior disinfection extends also to enteric viruses, we studied the removal of somatic phages ('model' viruses) in a pilot-scale APS treating sewage. Measurements through the three aerobic stages of the APS showed fairly good removal of somatic phage in the summer months (2.2 log reduction), but much less effective removal in winter (0.45 log reduction), whereas E. coli was removed efficiently (> 4 logs) in both seasons. A very steep depth-gradient of sunlight inactivation of somatic phage in APS pond waters (confined in silica test tubes) is consistent with inactivation mainly by solar UVB wavelengths. Data for F-RNA phage suggests involvement of longer UV wavelengths. These findings imply that efficiency of virus removal in APS will vary seasonally with variation in solar UV radiation. [TOP OF PAGE]

  323. Survival of viruses on fresh produce, using MS2 as a surrogate for norovirus. Dawson,D.J., Paish,A., Staffell,L.M., Seymour,I.J., Appleton,H. (2005). J. Appl. Microbiol. 98:203-209. AIMS: To study the survival and removal of viruses from fresh fruit and vegetables using the bacteriophage MS2 as a potential surrogate for noroviruses. METHOD AND RESULTS: Survival of MS2 in buffer and on fresh produce was studied at 4, 8 and 22 degrees C. At 4 and 8 degrees C a reduction of <1 log10 was observed after 50 days in buffer; however a reduction in excess of 1 log10 occurred within 9 days at 22 degrees C. Similar results were obtained with fresh produce with virus survival times exceeding the shelf life of the produce. In washing experiments, using a chlorine wash (100 ppm), in all but one case <1.5 log10 MS2 bacteriophage was removed from fruit and vegetables. The mean across all produce types was 0.89 log10. With potable water, reduction was lower (0.3 log mean across all produce types). CONCLUSIONS: MS2 survived for prolonged periods, both in buffer and on fresh produce, at temperatures relevant to chilled foods. It was not removed effectively by chlorine washing. SIGNIFICANCE AND IMPACT OF THE STUDY: Bacteriophage MS2 has been evaluated as a potential surrogate for noroviruses on fresh produce. Experimental results together with current knowledge of norovirus resistance and survival indicate that MS2 could be used as an effective surrogate in future evaluations. [TOP OF PAGE]

  324. Report on an ICTV-sponsored symposium on Virus Evolution. Desselberger,U. (2005). Arch. Virol. 150:629-635. A symposium on Virus Evolution, sponsored by the International Committee on Taxonomy of Viruses (ICTV), was held at the 23rd Annual Meeting of the American Society for Virology (ASV) in Montreal, Canada on July 10, 2004. It was organized by Ann Palmenberg (University of Madison-Wisconsin) and Andrew Ball (President, ICTV; University of Alabama at Birmingham) and was supported by Academic Press/Elsevier, Bristol Myers Squibb, The University of Alabama at Birmingham School of Medicine, US National Biodefense Analysis and Countermeasures Center,Wyeth Lederle Vaccines, and the ASV. [TOP OF PAGE]

  325. Bacteriophage-mediated protein delivery into the central nervous system and its application in immunopharmacotherapy. Dickerson,T.J., Kaufmann,G.F., Janda,K.D. (2005). Expert Opin. Biol. Ther. 5:773-781. Cocaine addiction continues to be a major health and social problem in spite of governmental efforts devoted towards educating the public in the dangers of illicit drug use. A variety of pharmacotherapies and psychosocial programmes have been proposed in an effort to provide a method for alleviating the physical and psychological symptoms of cocaine abuse. Unfortunately, these methods have been met with limited success, illustrating a critical need for new effective approaches for the treatment of cocaine addiction. The authors have recently disclosed an alternative cocaine abuse treatment strategy using intranasal administration of an engineered filamentous bacteriophage displaying cocaine-sequestering antibodies on its surface. These phage particles are an effective vector for central nervous system penetration and are capable of binding cocaine, thereby blocking its behavioural effects in a rodent model. [TOP OF PAGE]

  326. Enumeration and diversity of campylobacters and bacteriophages isolated during the rearing cycles of free-range and organic chickens. El Shibiny,A., Connerton,P.L., Connerton,I.F. (2005). Appl. Environ. Microbiol. 71:1259-1266. Campylobacters and Campylobacter-specific bacteriophages were isolated and enumerated during the rearing cycle of free-range (56 days) and organic chickens (73 days) at 3-day intervals from hatching until slaughter. In both flocks Campylobacter jejuni was the initial colonizer but Campylobacter coli was detected more frequently from 5 weeks of age. The diversity of the Campylobacter isolates was examined by pulsed-field gel electrophoresis of SmaI-digested genomic DNA and antimicrobial resistance typing. Bacteriophages were isolated from 51% (19 of 37 birds) of Campylobacter-positive organic birds (log10 2.5 to log10 5.7 PFU/g of cecal contents). The bacteriophages were all typical group III Campylobacter bacteriophages in terms of genomic size but could be characterized in terms of their host range and placed into five different groups. In contrast to the organic birds, anti-Campylobacter activity (bacteriocin-like) was observed in 26% (10 of 38 birds) of Campylobacter-positive free-range birds, and only one bacteriophage was isolated. Appearance of either bacteriophages or anti-Campylobacter activity was associated with changes in the levels of colonization and the predominant genotypes and species isolated. The frequency and potential influence of naturally occurring bacteriophages and/or inhibitory substances on the diversity and fluctuations of populations of campylobacters have not previously been reported in either free-range or organic chickens. [TOP OF PAGE]

  327. Self-limiting nature of seasonal cholera epidemics: Role of host-mediated amplification of phage. Faruque,S.M., Islam,M.J., Ahmad,Q.S., Faruque,A.S.G., Sack,D.A., Nair,G.B., Mekalanos,J.J. (2005). Proc. Natl. Acad. Sci. USA 102:6119-6124. Phage predation of Vibrio cholerae has recently been reported to be a factor that influences seasonal epidemics of cholera in Bangladesh. To understand more about this phenomenon, we studied the dynamics of the V. cholerae-phage interaction during a recent epidemic in Dhaka. Because the outbreak strain causing this epidemic was resistant to multiple antibiotics, including streptomycin, we used a selective medium containing streptomycin to monitor accurately the abundance of this strain in the environment. The changing prevalence in the environment of the epidemic V. cholerae O1 strain and a particular lytic cholera phage (JSF4) to which it was sensitive was measured every 48-72 h for 17 weeks. We also monitored the incidence of phage excretion in stools of 387 cholera patients during the epidemic. The peak of the epidemic was preceded by high V. cholerae prevalence in the environment and was followed by high JSF4 phage levels as the epidemic ended. The buildup to the phage peak in the environment coincided with increasing excretion of the same phage in the stools of cholera patients. These results suggest that patients toward the end of the epidemic ingested both JSF4 phage and the outbreak V. cholerae strain. Host-mediated phage amplification during the cholera epidemic likely contributed to increased environmental phage abundance, decreased load of environmental V. cholerae and, hence, the collapse of the epidemic. Thus, in vivo phage amplification in patients and subsequent phage predation in the environment may explain the self-limiting nature of seasonal cholera epidemics in Bangladesh. [TOP OF PAGE]

  328. Seasonal epidemics of cholera inversely correlate with the prevalence of environmental cholera phages. Faruque,S.M., Naser,I.B., Islam,M.J., Faruque,A.S.G., Ghosh,A.N., Nair,G.B., Sack,D.A., Mekalanos,J.J. (2005). Proc. Natl. Acad. Sci. USA 102:1702-1707. The relationship among (i) the local incidence of cholera, (ii) the prevalence in the aquatic environment of Vibrio cholerae, and (iii) bacterial viruses that attack potentially virulent O1 and O139 serogroup strains of this organism (cholera phages) was studied in Dhaka, Bangladesh. Over nearly a 3-year period, we found that significantly more environmental water samples contained either a phage or a phage-susceptible V. cholerae strain than both (P < 0.00001). The number of cholera patients varied seasonally during this period and frequently coincided with the presence of pathogenic V. cholerae strains in water samples that otherwise lacked detectable cholera phages. Interepidemic periods were characterized by water samples containing cholera phages but no viable bacteria. Our data support the conclusion that cholera phages can influence cholera seasonality and may also play a role in emergence of new V. cholerae pandemic serogroups or clones. [TOP OF PAGE]

  329. High pressure mediated lysogenic conversion of Escherichia coli with Shiga-toxin encoding bacteriophage. Faster,D., Aertsen,A., Michiels,C.W. (2005). Communications in agricultural and applied biological sciences 70:131-134. [TOP OF PAGE]

  330. Marine T4-type bacteriophages, a ubiquitous component of the dark matter of the biosphere. Filee,J., Tetart,F., Suttle,C.A., Krisch,H.M. (2005). Proc. Natl. Acad. Sci. USA 102:12471-12476. Tailed bacteriophages are the most abundant biological entities in marine environments. However, most of these marine phages are uncharacterized because few of their hosts have been cultivated. To learn more about such phages, we designed a set of degenerate PCR primers for phage T4 g23, which encodes the major capsid protein in all of the T4-type phages, an important family of the tailed phage. These primers were used to amplify g23-related sequences from diverse marine environments (fjords and bays of British Columbia, the eastern Gulf of Mexico, and the western Arctic Ocean) revealing a remarkable level of molecular diversity, which in some cases was correlated with morphological variation of the virions. Phylogenetic analysis showed that although some of these sequences were closely related to well studied subgroups of the T4-type phage, such as the T-evens, the majority of them belong to five previously uncharacterized subgroups. These data indicate that the host range of T4-type phages is much broader than previously imagined and that the laboratory isolate T4 belongs to a phage family that is extraordinarily widespread and diverse in the biosphere. [TOP OF PAGE]

  331. Viral proteins functioning in organelles: a cryptic origin? Filee,J., Forterre,P. (2005). Trends Microbiol. 13:510-513. Although mitochondria derive from alpha-proteobacteria, many proteins acting in this organelle did not originate from bacteria. In particular, phylogenetic evidence indicates that RNA polymerase, DNA polymerase and DNA primase--with homologues encoded by T3/T7-like bacteriophages--have replaced the ancestral proteins of bacterial origin. To date, there was no clear explanation for this puzzling observation. Bacterial genomics has now revealed the presence of cryptic prophages that are related to T3/T7 in several genomes of proteobacteria. We propose that such a prophage was present in the ancestral alpha-proteobacterium at the origin of mitochondria and that RNA polymerase, DNA polymerase and DNA primase encoded by this prophage replaced the original bacterial enzymes to function in mitochondria. Another T3/T7 viral-like RNA polymerase is functional in the chloroplast, indicating that a strong selection pressure has favored replacement of some cellular proteins by viral proteins in organelle evolution. [TOP OF PAGE]

  332. Oral treatment with bacteriophages reduces the concentration of Salmonella Enteritidis PT4 in caecal contents of broilers. Fiorentin,L., Vieira,N.D., Barioni,W.J. (2005). Avian pathology : journal of the W. V. P. A 34:258-263. Bacteriophages isolated from free-range chickens were tested as a therapeutic agent for reducing the concentration of Salmonella enterica serovar Enteritidis phage type 4 (S. Enteritidis PT4) in caeca of broilers. One-day-old broilers infected with S. Enteritidis PT4 by a seeder bird method were orally treated on the seventh day of age with a mixture of 10(11) plaque-forming units of each of three bacteriophages. Five days after treatment the bacteriophage-treated group showed a reduction of 3.5 orders of magnitude on colony-forming units of S. Enteritidis PT4 per gram of caecal content. Samples collected at 10, 15, 20 and 25 days after treatment revealed that treated birds still had lower colony-forming units of S. Enteritidis PT4 per gram of caecal content. These data gave us compelling evidence that a mixture of bacteriophages may be efficacious in reducing S. Enteritidis PT4 concentration in broilers' caeca and therefore reducing contamination of poultry products by this food-borne pathogen. [TOP OF PAGE]

  333. The coexistence of Escherichia coli serotype O157:H7 and its specific bacteriophage in continuous culture. Fischer,C.R., Yoichi,M., Unno,H., Tanji,Y. (2005). FEMS Microbiol. Lett. 241:171-177. For the development of phage therapy, systematic understanding mechanisms of bacteriophage resistance will be required. We describe a new strain of Escherichia coli O157:H7, named MuL, which stably co-exists with the O157:H7-specific lytic bacteriophage PP01. Chemostat cultures of E. coli O157:H7 infected with PP01 showed unchanging cell concentration, but phage concentrations which increased by 10(8) PFU mL(-1). However, the latent period, burst size, and growth rate of MuL were the same as in a PP01-susceptible strain. The binding rate of PP01 to the cell surface was diminished 8.5-fold in MuL. By observation of the binding of fluorescently labeled O157:H7-specific phage to individual MuL cells, we found that clonal MuL cultures were heterogeneous in their ability to bind bacteriophage. 15% of the MuL population was completely resistant to PP01 infection. MuL also co-existed with bacteriophages unrelated to PP01. Broad-range phage resistance by clonal heterogeneity represents a new class of bacteria-phage interactions. [TOP OF PAGE]

  334. Structural and functional similarities between the capsid proteins of bacteriophages T4 and HK97 point to a common ancestry. Fokine,A., Leiman,P.G., Shneider,M.M., Ahvazi,B., Boeshans,K.M., Steven,A.C., Black,L.W., Mesyanzhinov,V.V., Rossmann,M.G. (2005). Proc. Natl. Acad. Sci. USA 102:7163-7168. Gene product (gp) 24 of bacteriophage T4 forms the pentameric vertices of the capsid. Using x-ray crystallography, we found the principal domain of gp24 to have a polypeptide fold similar to that of the HK97 phage capsid protein plus an additional insertion domain. Fitting gp24 monomers into a cryo-EM density map of the mature T4 capsid suggests that the insertion domain interacts with a neighboring subunit, effecting a stabilization analogous to the covalent crosslinking in the HK97 capsid. Sequence alignment and genetic data show that the folds of gp24 and the hexamer-forming capsid protein, gp23*, are similar. Accordingly, models of gp24* pentamers, gp23* hexamers, and the whole capsid were built, based on a cryo-EM image reconstruction of the capsid. Mutations in gene 23 that affect capsid shape map to the capsomer's periphery, whereas mutations that allow gp23 to substitute for gp24 at the vertices modify the interactions between monomers within capsomers. Structural data show that capsid proteins of most tailed phages, and some eukaryotic viruses, may have evolved from a common ancestor. [TOP OF PAGE]

  335. Mobile genetic elements: the agents of open source evolution. Frost,L.S., Leplae,R., Summers,A.O., Toussaint,A. (2005). Nat. Rev. Microbiol. 3:722-732. Horizontal genomics is a new field in prokaryotic biology that is focused on the analysis of DNA sequences in prokaryotic chromosomes that seem to have originated from other prokaryotes or eukaryotes. However, it is equally important to understand the agents that effect DNA movement: plasmids, bacteriophages and transposons. Although these agents occur in all prokaryotes, comprehensive genomics of the prokaryotic mobile gene pool or 'mobilome' lags behind other genomics initiatives owing to challenges that are distinct from cellular chromosomal analysis. Recent work shows promise of improved mobile genetic element (MGE) genomics and consequent opportunities to take advantage - and avoid the dangers - of these 'natural genetic engineers'. This review describes MGEs, their properties that are important in horizontal gene transfer, and current opportunities to advance MGE genomics. [TOP OF PAGE]

  336. Evolution of Listeria populations in food samples undergoing enrichment culturing. Gnanou Besse,N., Audinet,N., Kerouanton,A., Colin,P., Kalmokoff,M. (2005). Int. J. Food Microbiol. 104:123-134. The isolation of Listeria monocytogenes from food is carried out using a double enrichment. It is believed that the double enrichment can allow the overgrowth of Listeria innocua in samples where both species are present. In this study, we have evaluated the impact of overgrowth between Listeria species and strains during each step of the enrichment process. The effect of factors minimizing interactions between strains or phage inhibitory effects has also been estimated. In an artificially contaminated food undergoing enrichment, overgrowth could result from competitive interactions between Listeria spp. resulting from the production of bacteriocins and bacteriophage at high initial contamination levels (>10(4) cfu/g), but not at lower levels (50-100 cfu/g) as generally found in contaminated foods. At high levels of inoculation, the competitive effect could be reduced by solidification of the selective broths, to limit the diffusion of the inhibitors. Overgrowth resulting from differences in growth rate occurred independent of the initial contamination level. However, in naturally contaminated foods undergoing enrichment, there were no absolute correlations between growth rates or inhibitory profiles in terms of strain evolution during enrichment. In fact, Listeria strains which were predominant in the original sample in most cases remained the dominant strains at the end of the enrichment, although the relative proportion of any given strain could change significantly over the enrichment process. Additional factors which have yet to be identified impact on the evolution of Listeria in the two-step enrichment process. Analysis of strain evolution in eight naturally contaminated foods has indicated that the second enrichment step in Fraser broth can be reduced from 48 to 24 h without impacting on the recovery of L. monocytogenes. Our limited survey of naturally contaminated foods also demonstrated that maximum recovery of L. monocytogenes and other Listeria strains was found following 24 h incubation in 1/2 Fraser Broth. This finding suggests that it may be possible to shorten the current two-step isolation method further without reducing method sensitivity. [TOP OF PAGE]

  337. Effect of phage infection on toxin production by Clostridium difficile. Goh,S., Chang,B.J., Riley,T.V. (2005). J. Med. Microbiol. 54:129-135. Infection with Clostridium difficile and subsequent production of toxins A and B may result in C. difficile-associated diarrhoea and pseudomembranous colitis in hospital patients. The effect of four temperate phages, obtained by induction of clinical C. difficile isolates, on toxin production by C. difficile was determined. None of these phages converted a lysogenized non-toxigenic C. difficile strain to toxin production. One of the accessory toxin genes, tcdE, was detected in three phages, phiC2, phiC6 and phiC8; however, the non-repeating regions of tcdA and tcdB encoding the enzymic domains were not carried on phage DNA. Phage infection of toxigenic strains increased toxin B production in four of six lysogens, although the level of tcdB transcription as determined by real-time RT-PCR was not significantly altered. However, levels of toxin A transcription in two lysogens were significantly altered without any corresponding differences in toxin A production. [TOP OF PAGE]

  338. Isolation and characterization of temperate bacteriophages of Clostridium difficile. Goh,S., Riley,T.V., Chang,B.J. (2005). Appl. Environ. Microbiol. 71:1079-1083. The lack of information on bacteriophages of Clostridium difficile prompted this study. Three of 56 clinical C. difficile isolates yielded double-stranded DNA phages fC2, fC5, fC6, and fC8 upon induction. Superinfection and DNA analyses revealed relatedness between the phages, while partial sequencing of phiC2 showed nucleotide homology to the sequenced C. difficile strain CD630. [TOP OF PAGE]

  339. The potential role of endogenous bacteriophages in controlling invading pathogens. Gorski,A., Weber-Dabrowska,B. (2005). Cell. Mol. Life Sci. 62:511-519. Bacteriophages (phages) are omnipresent in our environment, and recent studies highlight their potential impact on the microbial world. Phages can also be present in mammalian organisms, including man (intestines, oral cavity, urine, sputum and serum). Data are available which suggest that those endogenous phages could play an important role in eliminating bacteria and regulating the body ecosystem. Furthermore, our most recent findings suggest that phages can exert immunosuppressive action in the gut, helping control local inflammatory and autoimmune reactions, and demonstrate anticancer activity. We hypothesize that phages could act in concert with the immune system in immunosurveillance against bacteria, viruses and cancer. [TOP OF PAGE]

  340. Bacteriophage control of foodborne bacteria. Greer,G.G. (2005). J. Food Prot. 68:1102-1111. Bacteriophages are measurable components of the natural microflora in the food production continuum from the farm to the retail outlet. Phages are remarkably stable in these environments and are readily recovered from soil, sewage, water, farm and processing plant effluents, feces, and retail foods. Purified high-titer phage lysates have been used for the species-specific control of bacteria during the pre- and postharvest phases of food production and storage. For example, the inhibition of the phytopathogens Erwinia amylovara and Xanthomonas campestris has reduced the incidence of diseases such as fire blight in apples and bacterial spot of tomato and peaches. Research on preslaughter treatment of food animals has demonstrated phage control of salmonellosis in chickens, enteropathogenic Escherichia coli infections in calves, piglets, and lambs, and E. coli O157:H7 shedding by beef cattle. Phages have also been applied to control the growth of pathogens such as Listeria monocytogenes, Salmonella, and Campylobacter jejuni in a variety of refrigerated foods such as fruit, dairy products, poultry, and red meats. Phage control of spoilage bacteria (e.g., Pseudomonas spp. and Brochothrix thermosphacta) in raw chilled meats can result in a significant extension of storage life. Phage biocontrol strategies for food preservation have the advantages of being self-perpetuating, highly discriminatory, natural, and cost-effective. Some of the drawbacks of biopreservation with phages are a limited host range, the requirement for threshold numbers of the bacterial targets, phage-resistant mutants, and the potential for the transduction of undesirable characteristics from one bacterial strain to another. Most research to date has involved experimentally infected plants and animals or artificially inoculated foods. This technology must be transferred to the field and to commercial environments to assess the possibility of controlling natural contaminants under more realistic production and processing conditions. [TOP OF PAGE]

  341. Determining the performance of a commercial air purification system for reducing airborne contamination using model micro-organisms: a new test methodology. Griffiths,W.D., Bennett,A., Speight,S., Parks,S. (2005). J. Hosp. Infect. 61:242-247. The performance of a duct-mounted air disinfection system, designed to reduce airborne pathogens in the hospital environment, was determined using a new testing methodology. The methodology places the equipment in a test duct, a microbial aerosol is generated and then sampled simultaneously before and after the test system. This allows a percentage efficiency value to be calculated. The air disinfection system is a novel chemical-coated filter and ultraviolet (UV) radiation air purification system, operating at a flow rate of 500 m(3)/h, against aerosols of MS2 phage and Mycobacterium vaccae (surrogates of viral and mycobactericidal pathogens). A three UV lamp system was effective against airborne phages, removing an average of 97.34% of the aerosolized challenge. With the UV component switched off, the average efficiency dropped to 61.46%. This demonstrates that the chemical-coated filter component plays a more significant role than the UV radiation in destroying phages. When six UV lamps were used, the system was able to remove mycobacteria with an efficiency exceeding 99.99%. This test methodology can be used to assess manufacturers' claims of efficacy of equipment against airborne micro-organisms in the hospital environment. [TOP OF PAGE]

  342. The origin and evolution of human pathogens. Groisman,E.A., Casadesus,J. (2005). Mol. Microbiol. 56:1-7. What are the genetic origins of human pathogens? An international group of scientists discussed this topic at a workshop that took place in late October 2004 in Baeza (Spain). Focusing primarily on bacterial pathogens, they examined the role that pathogenicity islands and bacteriophages play on determining the virulence properties that distinguish closely related members of a given species, such as host range and tissue specificity. They also discussed an instance in which closely related bacterial species differ in the production of a cell surface modification mediating resistance to an antibiotic as a result of the disparate regulation of homologous genes. In certain pathogens, genes normally carrying out housekeeping functions may adopt new functions, whereas in other organisms, genes that respond to stresses associated with non-host environments are silenced during infection to prevent the expression of products that interfere with the normal colonization process. The adaptive behaviour of certain pathogens relies on gene variation at certain loci that by virtue of containing polymeric repeats in regulatory or coding regions, can generate variants that may or may not express products that modify the cell surface of the organism. The meeting also addressed the properties of ORFan genes, which have no homologues in the sequence databases, as well as the creation of genes de novo by duplication and divergence. [TOP OF PAGE]

  343. Resistance of corynebacterial strains to infection and lysis by corynephage BFK 20. Halgasova,N., Majtan,T., Ugorcakova,J., Timko,J., Bukovska,G. (2005). J. Appl. Microbiol. 98:184-192. AIMS: Defence mechanisms of the corynebacterial strains against corynephage BFK 20, which causes lysis of Brevibacterium flavum CCM 251. METHODS AND RESULTS: We tested adsorption of the phage BFK 20 to the corynebacterial cell surface. We observed strong adsorption ranging from ca 79 to 93% on the cells of B. flavum ATCC strains, but only ca 76% for B. flavum CCM 251. Minor adsorption for Brevibacterium lactofermentum BLOB (ca 13%) and no adsorption for Corynebacterium glutamicum RM3 were determined. BFK 20 infection had no significant effect on growth and viability of C. glutamicum and B. lactofermentum, but significantly influenced growth and viability of B. flavum ATCC 21127, 21128 and 21474. Cell growth stopped in short time after infection but with no lysis. Brevibacterium flavum CCM 251 cell growth was arrested too and lysis occurred. The Southern hybridization confirmed the presence of significant amount of BFK 20 DNA in samples from B. flavum CCM 251 and B. flavum ATCC strains after BFK 20 infection. Only weak hybridization signal was detected for DNA from infected cells of B. lactofermentum BLOB and no signal for C. glutamicum RM3. CONCLUSIONS: Based on the above results we suggest presence of a mechanism leading to abortive infection in B. flavum ATCC 21127, 21128 and 21474. In B. lactofermentum BLOB and C. glutamicum RM3 the adsorption barrier is more likely. SIGNIFICANCE AND IMPACT OF THE STUDY: This study increases the knowledge on defence mechanisms of corynebacteria against bacteriophages. [TOP OF PAGE]

  344. The viriosphere, diversity, and genetic exchange within phage communities. Hambly,E., Suttle,C.A. (2005). Curr. Opin. Mirobiol. 8:444-450. Natural phage communities are reservoirs of the greatest uncharacterized genetic diversity on Earth. Yet, identical phage sequences can be found in extremely different environments, which implies that there is wide circulation of viral genes among distantly related host populations. Further evidence of genetic exchange among phage and host communities is the presence in phage of genes coding for proteins that are essential for photosynthesis. These observations support the idea that a primary role of host populations in phage ecology and evolution is to serve as vectors for genetic exchange. [TOP OF PAGE]

  345. Impact of phages on two-species bacterial communities. Harcombe,W.R., Bull,J.J. (2005). Appl. Environ. Microbiol. 71:5254-5259. A long history of experimental work has shown that addition of bacteriophages to a monoculture of bacteria leads to only a temporary depression of bacterial levels. Resistant bacteria usually become abundant, despite reduced growth rates relative to those of phage-sensitive bacteria. This restoration of high bacterial density occurs even if the phages evolve to overcome bacterial resistance. We believe that the generality of this result may be limited to monocultures, in which the resistant bacteria do not face competition from bacterial species unaffected by the phage. As a simple case, we investigated the impact of phages attacking one species in a two-species culture of bacteria. In the absence of phages, Escherichia coli B and Salmonella enterica serovar Typhimurium were stably maintained during daily serial passage in glucose minimal medium (M9). When either of two E. coli-specific phages (T7 or T5) was added to the mixed culture, E. coli became extinct or was maintained at densities that were orders of magnitude lower than those before phage introduction, even though the E. coli densities with phage reached high levels when Salmonella was absent. In contrast, the addition of a phage that attacked only Salmonella (SP6) led to transient decreases in the bacterial number whether E. coli was absent or present. These results suggest that phages can sometimes, although not always, provide long-term suppression of target bacteria. [TOP OF PAGE]

  346. Evolutionary robustness of an optimal phenotype: re-evolution of lysis in a bacteriophage deleted for its lysin gene. Heineman,R.H., Molineux,I.J., Bull,J.J. (2005). J. Mol. Evol. 61:181-191. Optimality models are frequently used to create expectations about phenotypic evolution based on the fittest possible phenotype. However, they often ignore genetic details, which could confound these expectations. We experimentally analyzed the ability of organisms to evolve towards an optimum in an experimentally tractable system, lysis time in bacteriophage T7. T7 lysozyme helps lyse the host cell by degrading its cell wall at the end of infection, allowing viral escape to infect new hosts. Artificial deletion of lysozyme greatly reduced fitness and delayed lysis, but after evolution both phenotypes approached wild-type values. Phage with a lysis-deficient lysozyme evolved similarly. Several mutations were involved in adaptation, but most of the change in lysis timing and fitness increase was mediated by changes in gene 16, an internal virion protein not formerly considered to play a role in lysis. Its muralytic domain, which normally aids genome entry through the cell wall, evolved to cause phage release. Theoretical models suggest there is an optimal lysis time, and lysis more rapid or delayed than this optimum decreases fitness. Artificially constructed lines with very rapid lysis had lower fitness than wild-type T7, in accordance with the model. However, while a slow-lysing line also had lower fitness than wild-type, this low fitness resulted at least partly from genetic details that violated model assumptions. [TOP OF PAGE]

  347. Bacteriophage evolution and the role of phages in host evolution. Hendrix,R.W. (2005). pp. 55-65. In In Waldor,M.K., Friedman,D.I., and Adhya,S.L. (eds.), Phages: Their Role in Bacterial Pathogenesis and Biotechnology. ASM Press, Washington DC. [first paragraph] The tailed double-stranded DNA bacteriophages have been evolving for perhaps 3 billion years or more, but it is only in very recent years that we have come to the beginning of a real understanding of the genetic mechanisms behind that evolution as well as an appreciation for the major role that phages have in the evolution of their bacterial hosts. Because the host range of a typical phage is narrow, estimates of the abundance of phages in the environment based on the number of plaques formed on a few bacterial strains that can be grown in the laboratory have been low by many orders of magnitude. It was only when environmental samples were examined directly by electron microscopy that it became clear not only that tailed phages are remarkably abundant in theenvironment but that they probably constitute a numerical majority of organisms on the planet. In the first such measurements (2), the concentration of particles with characteristic morphology of tailed phages in Norwegian fjord water was about 10(7) per mel. Subsequent measurements from several other environmental sources have found similarly large, and in some cases, substantially larger numbers (31). Estimates of the total global population of phages can be made from these measurements and from the sizes of the different environmental compartments, and estimates are on the order of 10(31) total viral particles. This is a truly astronomical number, in that 10(31) tailed phages, laid end to end, would extend into space to a distance of 200 million light years. In all of the environmental samples examined, there were roughly 5 to 10 phage particles for every bacterial cell, which is the basis for the claim above that phage are a majority of the organisms on Earth. [TOP OF PAGE]

  348. Genome comparison of Pseudomonas aeruginosa large phages. Hertveldt,K., Lavigne,R., Pleteneva,E., Sernova,N., Kurochkina,L., Korchevskii,R., Robben,J., Mesyanzhinov,V., Krylov,V.N., Volckaert,G. (2005). J. Mol. Biol. 354:536-545. Pseudomonas aeruginosa phage EL is a dsDNA phage related to the giant fKZ-like Myoviridae. The EL genome sequence comprises 211,215 bp and has 201 predicted open reading frames (ORFs). The EL genome does not share DNA sequence homology with other viruses and micro-organisms sequenced to date. However, one-third of the predicted EL gene products (gps) shares similarity (Blast alignments of 17-55% amino acid identity) with fKZ proteins. Comparative EL and fKZ genomics reveals that these giant phages are an example of substantially diverged genetic mosaics. Based on the position of similar EL and phiKZ predicted gene products, five genome regions can be delineated in EL, four of which are relatively conserved between EL and fKZ. Region IV, a 17.7 kb genome region with 28 predicted ORFs, is unique to EL. Fourteen EL ORFs have been assigned a putative function based on protein similarity. Assigned proteins are involved in DNA replication and nucleotide metabolism (NAD+-dependent DNA ligase, ribonuclease HI, helicase, thymidylate kinase), host lysis and particle structure. EL-gp146 is the first chaperonin GroEL sequence identified in a viral genome. Besides a putative transposase, EL harbours predicted mobile endonucleases related to H-N-H and LAGLIDADG homing endonucleases associated with group I intron and intein intervening sequences. [TOP OF PAGE]

  349. Use of a specific bacteriophage treatment to reduce Salmonella in poultry products. Higgins,J.P., Higgins,S.E., Guenther,K.L., Huff,W., Donoghue,A.M., Donoghue,D.J., Hargis,B.M. (2005). Poult. Sci. 84:1141-1145. Bacteriophages represent a group of viruses that specifically infect and replicate in bacteria and could potentially be used to reduce recovery of Salmonella from poultry carcasses. Bacteriophages were isolated from municipal wastewater in the presence of Salmonella enteritidis phage type 13A (SE). In the first 2 experiments, commercially processed broiler carcass rinse water was pooled and divided. The addition of 10(10) pfu/mL of a single bacteriophage (PHL 4) with selected concentrations of SE reduced (P < 0.05) frequency of SE recovered as compared with the control rinse water sample. In experiments 3 and 4, broiler carcasses were intentionally inoculated with SE, sprayed with selected concentrations of PHL 4, and rinsed for SE enrichment and isolation. Application of 5.5 mL of 10(8) or 10(10) pfu/mL of PHL 4 reduced (P < 0.05) the frequency of SE recovery as compared with controls. In experiments 5 and 6, commercially processed turkeys were rinsed with water containing 72 wild-type bacteriophages isolated against SE, which were amplified in SE, or the Salmonella isolated antemortem from drag swabs from the flock selected for in-plant treatment, or a combination of bacteriophages amplified by each bacterial host. All bacteriophage treatments reduced (P < 0.05) frequency of Salmonella recovery as compared with controls. Sufficient concentrations of an appropriate bacteriophage, or a bacteriophage mixture, can significantly reduce recoverable Salmonella from carcass rinses. [TOP OF PAGE]

  350. Transport of MS2 phage, Escherichia coli, Clostridium perfringens, Cryptosporidium parvum, and Giardia intestinalis in a gravel and a sandy soil. Hijnen,W.A.M., Brouwer-Hanzens,A.J., Charles,K.J., Medema,G.J. (2005). Environ. Sci. Technol. 39:7860-7868. To define protection zones around groundwater abstraction wells and safe setback distances for artificial recharge systems in water treatment, quantitative information is needed about the removal of microorganisms during soil passage. Column experiments were conducted using natural soil and water from an infiltration site with fine sandy soil and a river bank infiltration site with gravel soil. The removal of phages, bacteria, bacterial spores, and protozoan (oo)-cysts was determined at two velocities and compared with field data from the same sites. The microbial elimination rate (MER) in both soils was generally >2 log, but MER in the gravel soil was higher than that in the fine sandy soil. This was attributed to enhanced attachment, related to higher metal-hydroxides content. From the high sticking efficiencies (>1) and the low influence of flow rate on MER it was deduced that straining played a significant role in the removal of Escherichia coli and Cryptosporidium parvum oocysts in the gravel soil. Lower removal of oocysts than the 4-5 times smaller E. coli and spores in the fine sand indicates that the contribution of straining is variable and needs further attention in transport models. Thus, simple extrapolation of grain size and particle size to the extent of microbial transport underground is inappropriate. Finally, the low MER of indigenous E. coli and Clostridium perfringens observed in the soil columns as well as under field conditions and the second breakthrough peak found for Cryptosporidium and spores in the fine sandy soil upon a change in the feedwater pH indicate a significant role of detachment and retardation to microbial transport and the difficulty of extrapolation of quantitative column test results to field conditions. [TOP OF PAGE]

  351. Effects of decreased resource availability, protozoan grazing and viral impact on a structure of bacterioplankton assemblage in a canyon-shaped reservoir. Hornak,K., Masin,M., Jezbera,J., Bettarel,Y., Nedoma,J., Sime-Ngando,T., Simek,K. (2005). FEMS Microbiol. Ecol. 52:315-327. We conducted a transplant experiment to elucidate the effects of different levels of grazing pressure, nutrient availability, especially phosphorus, and the impact of viruses on the changes in the structure of bacterioplankton assemblage in a meso-eutrophic reservoir. A sample taken from the nutrient-rich inflow part of the reservoir was size-fractionated and incubated in dialysis bags in both inflow and dam area. The structure of bacterial assemblage was examined by fluorescence in situ hybridization using oligonucleotide probes with different levels of specificity. In terms of the relative proportions of different bacterial groups, we found very few significant changes in the bacterioplankton composition after transplanting the treatments to the nutrient-poor dam area. However, we observed marked shifts in morphology and biomass towards the development of filaments, flocs and "vibrio-like" morphotypes of selected probe-defined groups of bacteria induced by increased grazing pressure. Despite the very high abundances of viruses in all the treatments, their effects on bacterioplankton were rather negligible. [TOP OF PAGE]

  352. Bacteriophages as biocontrol agents in food. Hudson,J.A., Billington,C., Carey-Smith,G., Greening,G. (2005). J. Food Prot. 68:426-437. Bacteriophages possess attributes that appear to be attractive to those searching for novel ways to control foodborne pathogens and spoilage organisms. These phages have a history of safe use, can be highly host specific, and replicate in the presence of a host. Campylobacter, Salmonella, and Listeria monocytogenes and various spoilage organisms have responded to phage control on some foods. However, the use of phages as biocontrol agents is complicated by factors such as an apparent requirement for a threshold level of host before replication can proceed and by suboptimal performance, at best, at temperatures beneath the optimum for the host. This review is a summary of the information on these issues and includes brief descriptions of alternative phage-based strategies for control of foodborne pathogens. [TOP OF PAGE]

  353. Alternatives to antibiotics: utilization of bacteriophage to treat colibacillosis and prevent foodborne pathogens. Huff,W.E., Huff,G.R., Rath,N.C., Balog,J.M., Donoghue,A.M. (2005). Poult. Sci. 84:655-659. Bacteriophages are viruses that infect and kill bacteria. Bacteriophage do not infect animal and plant cells, which makes them a potentially safe alternative to antibiotics. We have been conducting research on the efficacy of bacteriophage to prevent and treat colibacillosis in poultry. Bacteriophages that were lytic to a non-motile, serotype 02 isolate of Escherichia coli were isolated from municipal wastewater treatment plants and poultry processing plants. This E. coli isolate is pathogenic to poultry, causing severe respiratory and systemic infections. Two bacteriophage isolates were selected for use in studies designed to determine the efficacy of these bacteriophage to prevent and treat severe colibacillosis in poultry. Colibacillosis was induced by injecting 6 x 10(4) cfu of E. coli into the thoracic air sac when birds were 1 wk of age. Initial studies demonstrated that mortality was significantly reduced from 85 to 35% when the challenge culture was mixed with equal titers of bacteriophage, and the birds were completely protected when the challenge culture was mixed with 10 pfu of bacteriophage. In subsequent studies, we have shown that an aerosol spray of bacteriophage given to birds prior to this E. coli challenge could significantly reduce mortality even when given 3 d prior to the E. coli challenge. Our research on treating colibacillosis in poultry has demonstrated that an intramuscular injection of bacteriophage given 24 or 48 h after the birds were challenged rescued the birds from this severe E. coli infection. We have demonstrated that bacteriophage can be used to prevent and treat colibacillosis in poultry and may provide an effective alternative to antibiotic use in animal production. [TOP OF PAGE]

  354. Organization of the CTX prophage in environmental isolates of Vibrio mimicus. Islam,M.S., Rahman,M.Z., Khan,S.I., Mahmud,Z.H., Ramamurthy,T., Nair,G.B., Sack,R.B., Sack,D.A. (2005). Microbiology and Immunology 49:779-784. The organization of the CTX prophage in environmental strains of Vibrio mimicus was investigated. Sixteen hundred non-sucrose fermenting vibrios were examined for ctx gene by hybridization. Out of 1,600 isolates, 6 V. mimicus isolates contained ctxA gene. The organization of CTX prophage was determined by RFLP using ctxA probe. The CTX prophage integrated at a single site in V. mimicus genome which was present as a single copy flanked by at least a single RS element. Ribotype pattern revealed that a particular clone of V. mimicus acquired the CTXPhi in the aquatic environment. This study demonstrated that V. mimicus could act as a reservoir of CTXPhi in the aquatic environment. [TOP OF PAGE]

  355. Estimates of protozoan- and viral-mediated mortality of bacterioplankton in Lake Bourget (France). Jacquet,S., Domaizon,I., Personnic,S., Pradeep Ram,A.S., Hedal,M., Duhamel,S., Sime-Ngando,T. (2005). Freshw. Biol. 50:627-645. 1. We performed three, 1-week in situ experiments in March-April (expt 1), May (expt 2) and August (expt 3) 2003 in order to assess protozoan and virus-induced mortality of heterotrophic bacteria in a French lake. Viral and bacterial abundances were obtained using flow cytometry (FCM) while protozoa were counted using epifluorescence microscopy (EFM). ¶ 2. A dilution approach, applied to pretreated grazer-free samples, allowed us to estimate that viral lysis could be responsible for 60% (expt 1), 35% (expt 2) and 52% (expt 3) of daily heterotrophic bacterial mortality. Flagellate (both mixotrophic and heterotrophic) grazing in untreated samples, was responsible for 56% (expt 1), 63% (expt 2) and 18% (expt 3) of daily heterotrophic bacteria removal. ¶ 3. These results therefore suggest that both viral lysis and flagellate grazing had a strong impact on bacterial mortality, and this impact varied seasonally. ¶ 4. From parallel transmission electron microscopy (TEM) analysis, we found that the burst size (i.e. the number of viruses potentially released per lysed cell) ranged from nine to 25 (expt 1), 10 to 35 (expt 2) and eight to 25 (expt 3). The percentage of infected heterotrophic bacteria was 5.7% (expt 1), 3.4% (expt 2) and 5.7% (expt 3) so that the calculated percentage of bacterial mortality induced by viruses was 6.3% (expt 1), 3.7% (expt 2) and 6.3% (expt 3). ¶ 5. It is clear that the dilution-FCM and TEM methods yielded different estimates of viral impact, although both methods revealed an increased impact of viruses during summer. [TOP OF PAGE]

  356. The use of a novel biodegradable preparation capable of the sustained release of bacteriophages and ciprofloxacin, in the complex treatment of multidrug-resistant Staphylococcus aureus-infected local radiation injuries caused by exposure to Sr90. Jikia,D., Chkhaidze,N., Imedashvili,E., Mgaloblishvili,I., Tsitlanadze,G., Katsarava,R., Glenn Morris,J.J., Sulakvelidze,A. (2005). Clinical and experimental dermatology 30:23-26. In December 2001, three Georgian lumberjacks from the village of Lia were exposed to a strontium-90 source from two Soviet-era radiothermal generators they found near their village. In addition to systemic effects, two of them developed severe local radiation injuries which subsequently became infected with Staphylococcus aureus. After hospitalization in Tbilisi, Georgia, the patients were treated with various medications, including antibiotics and topical ointments; however, wound healing was only moderately successful, and their S. aureus infection could not be eliminated. Approximately 1 month after hospitalization, treatment with PhagoBioDerm (a wound-healing preparation consisting of a biodegradable polymer impregnated with ciprofloxacin and bacteriophages) was initiated. Purulent drainage stopped within 2-7 days. Clinical improvement was associated with rapid (7 days) elimination of the aetiologic agent, a strain of S. aureus resistant to many antibiotics (including ciprofloxacin), but susceptible to the bacteriophages contained in the PhagoBioDerm preparation. [TOP OF PAGE]

  357. Survival of a Shiga toxin-encoding bacteriophage in a compost model. Johannessen,G.S., James,C.E., Allison,H.E., Smith,D.L., Saunders,J.R., McCarthy,A.J. (2005). FEMS Microbiol. Lett. 245:369-375. Bacteriophages that carry the Shiga toxin gene (stx) represent an additional hazard in cattle manure-based fertilizers in that their survival could lead to toxigenic conversion of Escherichia coli and other bacteria post-composting. A Stx-phage in which the Shiga toxin (stx2) gene was inactivated by insertion of a chloramphenicol resistance gene was used in combination with a rifampicin-resistant E. coli host where RecA is constitutively activated so that all infectious phage particles could be enumerated by plaque assay. PCR-based confirmation methods and the additional application of a host enrichment protocol ensured that very low numbers of surviving bacteriophage could be detected and unequivocally identified. Stx-bacteriophage numbers declined rapidly over the first 48h and none could be detected after 3 days. The host enrichment method was applied after 6 days and no bacteriophages were recovered. While addition of fresh E. coli cells at intervals after the compost temperature had reduced below 40°C demonstrated that E. coli growth could be supported in the compost, Stx-phages or their lysogens were never detected. Here, we demonstrate that composting animal manure for 40 days during which a temperature of >60°C is maintained for at least 5 days is effective at removing both E. coli and a model infectious Stx-encoding bacteriophage. [TOP OF PAGE]

  358. Review of factors affecting microbial survival in groundwater. John,D.E., Rose,J.B. (2005). Environ. Sci. Technol. 39:7345-7356. This review quantitatively examines a number of published studies that evaluated survival and inactivation of public-health-related microorganisms in groundwater. Information from reviewed literature is used to express microbial inactivation in terms of log10 decline per day for comparison to other studies and organisms. The geometric mean value for inactivation rates for coliphage, poliovirus, echovirus, coliform bacteria, enterococci, and Salmonella spp. were similar at approximately 0.07-0.1 log10 day(-1), while geometric mean inactivation rates for hepatitis A virus, coxsackievirus, and phage PRD-1 were somewhat less at 0.02-0.04 log10 day(-1). Viruses show a temperature dependency with greater inactivation at greater temperatures; however this occurs largely at temperatures greater than 20 degrees C. Coliform bacteria die off in groundwater does not show the temperature dependency that viruses show, likely indicating a complex interplay of inactivation and reproduction subject to influences from native groundwater organisms, temperature, and water chemistry. The presence of native microorganisms seems to negatively impact E. coli survival more so than viruses, but in most cases, nonsterile conditions led to a greater inactivation for viruses also. The effect of attachment to solid surfaces appears to be virus-type-dependent, with PRD-1 more rapidly inactivated as a result of attachment and hepatitis A and poliovirus survival prolonged when attached. [TOP OF PAGE]

  359. Bacteriophage-based tests for the detection of Mycobacterium tuberculosis in clinical specimens: a systematic review and meta- analysis. Kalantri,S., Pai,M., Pascopella,L., Riley,L., Reingold,A. (2005). BMC Infect. Dis. 5:59 BACKGROUND: Sputum microscopy, the most important conventional test for tuberculosis, is specific in settings with high burden of tuberculosis and low prevalence of non tuberculous mycobacteria. However, the test lacks sensitivity. Although bacteriophage-based tests for tuberculosis have shown promising results, their overall accuracy has not been systematically evaluated. METHODS: We did a systematic review and meta-analysis of published studies to evaluate the accuracy of phage-based tests for the direct detection of M. tuberculosis in clinical specimens. To identify studies, we searched Medline, EMBASE, Web of science and BIOSIS, and contacted authors, experts and test manufacturers. Thirteen studies, all based on phage amplification method, met our inclusion criteria. Overall accuracy was evaluated using forest plots, summary receiver operating (SROC) curves, and subgroup analyses. RESULTS: The data suggest that phage-based assays have high specificity (range 0.83 to 1.00), but modest and variable sensitivity (range 0.21 to 0.88). The sensitivity ranged between 0.29 and 0.87 among smear-positive, and 0.13 to 0.78 among smear-negative specimens. The specificity ranged between 0.60 and 0.88 among smear-positive and 0.89 to 0.99 among smear-negative specimens. SROC analyses suggest that overall accuracy of phage-based assays is slightly higher than smear microscopy in direct head-to-head comparisons. CONCLUSION: Phage-based assays have high specificity but lower and variable sensitivity. Their performance characteristics are similar to sputum microscopy. Phage assays cannot replace conventional diagnostic tests such as microscopy and culture at this time. Further research is required to identify methods that can enhance the sensitivity of phage-based assays without compromising the high specificity. [TOP OF PAGE]

  360. Diel infection of a cyanobacterium by a contractile bacteriophage. Kao,C.C., Green,S., Stein,B., Golden,S.S. (2005). Appl. Environ. Microbiol. 71:4276-4279. Light was found to strongly influence the infection of a freshwater cyanobacterium (Synechococcus elongatus PCC 7942) by a contractile DNA phage named AS-1. Phage progeny production was correlated with the amount of light in the laboratory and occurred in a diel pattern under natural light. At least one effect of light on AS-1 infection is at the level of adsorption. [TOP OF PAGE]

  361. Bacteriophages: the viruses for all seasons of molecular biology. Karam,J.D. (2005). Virol. J. 2:19 Bacteriophage research continues to break new ground in our understanding of the basic molecular mechanisms of gene action and biological structure. The abundance of bacteriophages in nature and the diversity of their genomes are two reasons why phage research brims with excitement. The pages of Virology Journal will reflect the excitement of the "New Phage Biology.". [TOP OF PAGE]

  362. Genome-wide comparison of phage M13-infected vs. uninfected Escherichia coli. Karlsson,F., Malmborg-Hager,A.C., Albrekt,A.S., Borrebaeck,C.A.K. (2005). Can. J. Microbiol. 51:29-35. To identify Escherichia coli genes potentially regulated by filamentous phage infection, we used oligonucleotide microarrays. Genome-wide comparison of phage M13-infected and uninfected E. coli, 2 and 20 min after infection, was performed. The analysis revealed altered transcription levels of 12 E. coli genes in response to phage infection, and the observed regulation of phage genes correlated with the known in vivo pattern of M13 mRNA species. Ten of the 12 host genes affected could be grouped into 3 different categories based on cellular function, suggesting a coordinated response. The significantly upregulated genes encode proteins involved in reactions of the energy-generating phosphotransferase system and transcription processing, which could be related to phage transcription. No genes belonging to any known E. coli stress response pathways were scored as upregulated. Furthermore, phage infection led to significant downregulation of transcripts of the bacterial genes gadA, gadB, hdeA, gadE, slp, and crl. These downregulated genes are normally part of the host stress response mechanisms that protect the bacterium during conditions of acid stress and stationary phase transition. The phage-infected cells demonstrated impaired function of the oxidative and the glutamate-dependent acid resistance systems. Thus, global transcriptional analysis and functional analysis revealed previously unknown host responses to filamentous phage infection. [TOP OF PAGE]

  363. Barriers to coliphage infection of commensal intestinal flora of laboratory mice. Kasman,L.M. (2005). Virol. J. 2:34 BACKGROUND: Growth characteristics of coliphage viruses indicate that they are adapted to live with their Eschericia coli hosts in the intestinal tract. However, coliphage experimentally introduced by ingestion persist only transiently if at all in the gut of humans and other animals. This study attempted to identify the barriers to long term establishment of exogenous coliphage in the gastrointestinal (GI) tracts of laboratory mice. Intestinal contents were screened for the presence of coliphage and host bacteria, and strains of E. coli bacteria from different segments of the GI tract were tested for susceptibility to six common laboratory coliphages. RESULTS: Contrary to expectations, coliphage were not evident in the GI tracts of laboratory mice, although they were occasionally detected in feces. Commensal flora showed extreme variability within groups of mice despite identical handling and diet. Less than 20% of 48 mice tested carried E. coli in their gut, and of 22 commensal E. coli strains isolated and tested, 59% were completely resistant to infection by lambda, M13, P1, T4, T7, and PhiX174 coliphage. Lysogeny could not be demonstrated in the commensal strains as mitomycin C failed to induce detectable phage. Pre-existing immunity to phages was not evident as sera and fecal washes did not contain significant antibody titers to six laboratory phage types. CONCLUSION: Lack of sufficient susceptible host bacteria seems to be the most likely barrier to establishment of new coliphage infections in the mouse gut. [TOP OF PAGE]

  364. Structure of an archaeal virus capsid protein reveals a common ancestry to eukaryotic and bacterial viruses. Khayat,R., Tang,L., Larson,E.T., Lawrence,C.M., Young,M., Johnson,J.E. (2005). Proc. Natl. Acad. Sci. USA 102:18944-18949. Archaea and their viruses are poorly understood when compared with the Eukarya and Bacteria domains of life. We report here the crystal structure of the major capsid protein (MCP) of the Sulfolobus turreted icosahedral virus, an archaeal virus isolated from an acidic hot spring (pH 2-4, 72-92 degrees C) in Yellowstone National Park. The structure is nearly identical to the MCP structures of the eukaryotic Paramecium bursaria Chlorella virus, and the bacteriophage PRD1, and shows a common fold with the mammalian adenovirus. Structural analysis of the capsid architecture, determined by fitting the subunit into the electron cryomicroscopy reconstruction of the virus, identified a number of key interactions that are akin to those observed in adenovirus and PRD1. The similar capsid proteins and capsid architectures strongly suggest that these viral capsids originated and evolved from a common ancestor. Hence, this work provides a previously undescribed example of a viral relationship spanning the three domains of life (Eukarya, Bacteria, and Archaea). The MCP structure also provides insights into the stabilizing forces required for extracellular hyperthermophilic proteins to tolerate high-temperature hot springs. [TOP OF PAGE]

  365. The receptor of an oyster juice-borne coliphage OJ367 in the outer membrane of Salmonella derby. Ko,Y.T. (2005). J. Microbiol. Immunol. Infect. = Wei Mian Yu Gan Ran Za Zhi 38:399-408. The objective of this study was to identify the receptor of OJ367, an oyster juice-borne bacteriophage, in Salmonella derby ATCC 6960. The crude receptor outer membrane (OM) fraction was prepared and examined from the total cell envelope (TCE) by differential extraction with N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid (HEPES)-MgCl2 and then with Triton-HEPES-ethylenediamine tetra-acetic acid buffers. The OM proteins (Omps) were isolated by diethylaminoethyl column chromatography to screen for receptor activity. A 45-kDa protein belonging to a minor Omp species, with phage neutralization ability, was eluted in a homogeneous form. It was a non-peptidoglycan-associated protein which was digestible by trypsin. Lipopolysaccharide had no influence on its receptor activity when coexistent in the diethylaminoethyl column fractions. An S. derby mutant resistant to lysis by phage OJ367 was isolated. The mutant not only showed decreased receptor activity in vitro when its TCE was tested but had an altered Omp profile. This implied that the 45-kDa Omp is involved as a receptor in coliphage binding; however, this role is affected by the expression of other Omps. [TOP OF PAGE]

  366. Actinophages as indicators of actinomycete taxa in marine environments. Kurtboke,D.I. (2005). Antonie van Leeuwenhoek J. Microbiol. 87:19-28. It is necessary to continue to screen for new metabolites and evaluate the potential of less known and new bacterial taxa so that new and improved compounds for future use against drug-resistant bacteria or for chemical modification may be developed. There has been considerable interest in the detection and identification of marine microorganisms since they have been reported to produce bioactive compounds ranging from antitumour to antibacterial and antiviral agents. In this study, an improved technique that involves the exploitation of marine actinophages as indicators of the marine actinomycete taxa and uses marine bacteriophages as tools to reduce the numbers of common marine bacteria, which impedes the growth of rare actinomycetes on isolation plates, has been applied. This technique reduced the numbers of colony forming units of unwanted bacteria on isolation plates and hence increased the chances of detecting novel marine actinomycete genera for isolation and subsequent screening for antiviral activity. [TOP OF PAGE]

  367. Bacteriophages: Biology and Application. Kutter,E., Sulakvelidze,A. (2005). CRC Press, Boca Raton, FL.[TOP OF PAGE]

  368. In situ measurements of viral particles diffusion inside mucoid biofilms. Lacroix-Gueu,P., Briandet,R., leveque-Fort,S., Bellon-Fontaine,M.N., Fountaine-Aupart,M.P. (2005). C. R. Biol. 328:1065-1072. Fluorescence correlation spectroscopy (FCS) under two-photon excitation was used successfully to characterize the diffusion properties of model virus particles (bacteriophages) in bacterial biofilm of Stenotrophonas maltophilia. The results are compared to those obtained with fluorescent latex beads used as a reference. The FCS data clearly demonstrated the possibility for viral particles to penetrate inside the exopolymeric matrix of mucoid biofilms, and hence to benefit from its protective effect toward antimicrobials (antibiotics and biocides). Microbial biofilms should hence be considered as potential reservoirs of pathogenic viruses, and are probably responsible for numerous persistent viral infections. [TOP OF PAGE]

  369. Biofilms in an urban water distribution system: measurement of biofilm biomass, pathogens and pathogen persistence within the Greater Stockholm Area, Sweden. Langmark,J., Storey,M.V., Ashbolt,N.J., Stenstrom,T.A. (2005). Water Sci. Technol. 52:181-189. Distribution pipe biofilms can provide sites for the concentration of a wide range of microbial pathogens, thereby acting as a potential source of continual microbial exposure and furthermore can affect the aesthetic quality of water. In a joint project between Stockholm Water, the MISTRA "Sustainable Urban Water" program, the Swedish Institute for Infectious Disease Control and the Royal Technical University, Stockholm, the aim of the current study was to investigate biofilms formed in an urban water distribution system, and quantify the impact of such biofilms on potential pathogen accumulation and persistence within the Greater Stockholm Area, Sweden. When used for primary disinfection, ultra-violet (UV) treatment had no measurable influence on biofilm formation within the distribution system when compared to conventional chlorination. Biofilms produced within a model pilot-plant were found to be representative to those that had formed within the larger municipal water distribution system, demonstrating the applicability of the novel pilot-plant for future studies. Polystyrene microspheres (1.0 microm) and Salmonella bacteriophages demonstrated their ability to accumulate and persist within the model pilot-plant system, where the means of primary disinfection (UV-treatment, chlorination) had no influence on such phenomena. With the exception of aeromonads, potential pathogens and faecal indicators could not be detected within biofilms from the Stockholm water distribution system. Results from this investigation may provide information for water treatment and distribution management strategies, and fill key data gaps that presently hinder the refinement of microbial risk models. [TOP OF PAGE]

  370. Accumulation and fate of microorganisms and microspheres in biofilms formed in a pilot-scale water distribution system. Langmark,J., Storey,M.V., Ashbolt,N.J., Stenstrom,T.A. (2005). Appl. Environ. Microbiol. 71:706-712. The accumulation and fate of model microbial "pathogens" within a drinking-water distribution system was investigated in naturally grown biofilms formed in a novel pilot-scale water distribution system provided with chlorinated and UV-treated water. Biofilms were exposed to 1-mum hydrophilic and hydrophobic microspheres, Salmonella bacteriophages 28B, and Legionella pneumophila bacteria, and their fate was monitored over a 38-day period. The accumulation of model pathogens was generally independent of the biofilm cell density and was shown to be dependent on particle surface properties, where hydrophilic spheres accumulated to a larger extent than hydrophobic ones. A higher accumulation of culturable legionellae was measured in the chlorinated system compared to the UV-treated system with increasing residence time. The fate of spheres and fluorescence in situ hybridization-positive legionellae was similar and independent of the primary disinfectant applied and water residence time. The more rapid loss of culturable legionellae compared to the fluorescence in situ hybridization-positive legionellae was attributed to a loss in culturability rather than physical desorption. Loss of bacteriophage 28B plaque-forming ability together with erosion may have affected their fate within biofilms in the pilot-scale distribution system. The current study has demonstrated that desorption was one of the primary mechanisms affecting the loss of microspheres, legionellae, and bacteriophage from biofilms within a pilot-scale distribution system as well as disinfection and biological grazing. In general, two primary disinfection regimens (chlorination and UV treatment) were not shown to have a measurable impact on the accumulation and fate of model microbial pathogens within a water distribution system. [TOP OF PAGE]

  371. Photosynthesis genes in marine viruses yield proteins during host infection. Lindell,D., Jaffe,J.D., Johnson,Z.I., Church,G.M., Chisholm,S.W. (2005). Nature 438:86-89. Cyanobacteria, and the viruses (phages) that infect them, are significant contributors to the oceanic 'gene pool'. This pool is dynamic, and the transfer of genetic material between hosts and their phages probably influences the genetic and functional diversity of both. For example, photosynthesis genes of cyanobacterial origin have been found in phages that infect Prochlorococcus and Synechococcus, the numerically dominant phototrophs in ocean ecosystems. These genes include psbA, which encodes the photosystem II core reaction centre protein D1, and high-light-inducible (hli) genes. Here we show that phage psbA and hli genes are expressed during infection of Prochlorococcus and are co-transcribed with essential phage capsid genes, and that the amount of phage D1 protein increases steadily over the infective period. We also show that the expression of host photosynthesis genes declines over the course of infection and that replication of the phage genome is a function of photosynthesis. We thus propose that the phage genes are functional in photosynthesis and that they may be increasing phage fitness by supplementing the host production of these proteins. [TOP OF PAGE]

  372. Lysogeny, prophage induction, and lysogenic conversion. Little,J.W. (2005). pp. 37-54. In In Waldor,M.K., Friedman,D.I., and Adhya,S.L. (eds.), Phages: Their Role in Bacterial Pathogenesis and Biotechnology. ASM Press, Washington DC. Temperate phages can carry genes that affect the phenotype and behavior of their bacterial host. These genes can be considered extra genetic material in that they are not necessarily for viral lytic growth or for the lysogenic lifestyle. In this chapter, these extra genes will be termed "foreign genes" for ease of reference. Foreign genes include genes for various toxins that have pathogenic effects. The expression of toxin genes has been documented to occur in two different phases of the viral life cycle. The primary goal of this chapter is to describe circumstances under which foreign genes can be expressed. We will first consider the life cycle of temperate phages. With this background, we will then describe how foreign genes can be expressed in the lysogenic state. We will then turn to a more detailed description of a particular temperate phage, l, with an emphasis on the regulatory mechanisms that are best understood for this phage. This description will facilitate an understanding of how foreign genes can be expressed during the process of prophage induction, and a particular example will be descrbed. [TOP OF PAGE]

  373. Bacteriophage therapy to reduce Campylobacter jejuni colonization of broiler chickens. Loc Carrillo,C., Atterbury,R.J., El Shibiny,A., Connerton,P.L., Dillon,E., Scott,A., Connerton,I.F. (2005). Appl. Environ. Microbiol. 71:6554-6563. Colonization of broiler chickens by the enteric pathogen Campylobacter jejuni is widespread and difficult to prevent. Bacteriophage therapy is one possible means by which this colonization could be controlled, thus limiting the entry of campylobacters into the human food chain. Prior to evaluating the efficacy of phage therapy, experimental models of Campylobacter colonization of broiler chickens were established by using low-passage C. jejuni isolates HPC5 and GIIC8 from United Kingdom broiler flocks. The screening of 53 lytic bacteriophage isolates against a panel of 50 Campylobacter isolates from broiler chickens and 80 strains isolated after human infection identified two phage candidates with broad host lysis. These phages, CP8 and CP34, were orally administered in antacid suspension, at different dosages, to 25-day-old broiler chickens experimentally colonized with the C. jejuni broiler isolates. Phage treatment of C. jejuni-colonized birds resulted in Campylobacter counts falling between 0.5 and 5 log10 CFU/g of cecal contents compared to untreated controls over a 5-day period postadministration. These reductions were dependent on the phage-Campylobacter combination, the dose of phage applied, and the time elapsed after administration. Campylobacters resistant to bacteriophage infection were recovered from phage-treated chickens at a frequency of <4%. These resistant types were compromised in their ability to colonize experimental chickens and rapidly reverted to a phage-sensitive phenotype in vivo. The selection of appropriate phage and their dose optimization are key elements for the success of phage therapy to reduce campylobacters in broiler chickens. [TOP OF PAGE]

  374. Use of rotavirus virus-like particles as surrogates to evaluate virus persistence in shellfish. Loisy,F., Atmar,R.L., Le Saux,J.C., Cohen,J., Caprais,M.P., Pommepuy,M., Le Guyader,F.S. (2005). Appl. Environ. Microbiol. 71:6049-6053. Rotavirus virus-like particles (VLPs) and MS2 bacteriophages were bioaccumulated in bivalve mollusks to evaluate viral persistence in shellfish during depuration and relaying under natural conditions. Using this nonpathogenic surrogate virus, we were able to demonstrate that about 1 log10 of VLPs was depurated after 1 week in warm seawater (22 degrees C). Phage MS2 was depurated more rapidly (about 2 log10 in 1 week) than were VLPs, as determined using a single-compartment model and linear regression analysis. After being relayed in the estuary under the influence of the tides, VLPs were detected in oysters for up to 82 days following seeding with high levels of VLPs (concentration range between 10(10) and 10(9) particles per g of pancreatic tissue) and for 37 days for lower contamination levels (10(5) particles per g of pancreatic tissue). These data suggest that viral particles may persist in shellfish tissues for several weeks. [TOP OF PAGE]

  375. Rapid detection of viruses using electrical biochips and anti-virion sera. Los,M., Los,J.M., Blohm,L., Spillner,E., Grunwald,T., Albers,J., Hintsche,R., Wegrzyn,G. (2005). Lett. Appl. Microbiol. 40:479-485. AIMS: Rapid detection and quantification of viruses is crucial in clinical practice, veterinary medicine, agriculture, basic research as well as in biotechnological factories. However, although various techniques were described and are currently used, development of more rapid, more sensitive and quantitative methods seems to be still important. METHODS AND RESULTS: Here we describe a method for rapid detection of viruses (using bacteriophages as model viruses), based on electrical biochip array technology with the use of antibodies against capsid proteins. CONCLUSIONS: Using the procedure developed in this work, we were able to detect 2 x 10(4) virions on the chip. The whole assay procedure takes c. 50 min and the assay is quantitative. SIGNIFICANCE AND IMPACT OF THE STUDY: This procedure may be useful in various approaches, including detection of bacteriophage contamination in bioreactors and possibly detection of toxin gene-bearing phages or other viruses in food samples. [TOP OF PAGE]

  376. Sequence analysis of the Lactobacillus plantarum bacteriophage FJL-1. Lu,Z., Altermann,E., Breidt,F., Predki,P., Fleming,H.P., Klaenhammer,T.R. (2005). Gene 348:45-54. The complete genomic sequence of a Lactobacillus plantarum virulent phage FJL-1 was determined. The phage possesses a linear, double-stranded, DNA genome consisting of 36,677 bp with a G+C content of 39.36%. A total of 52 possible open reading frames (ORFs) were identified. According to N-terminal amino acid sequencing and bioinformatic analyses, proven or putative functions were assigned to 21 ORFs (41%), including 5 structural protein genes. The FJL-1 genome shows functionally related genes clustered together in a genome structure composed of modules for DNA replication, DNA packaging, head and tail morphogenesis, and lysis. This type of modular genomic organization was similar to several other phages infecting lactic acid bacteria. The structural gene maps revealed that the order of the head and tail genes is highly conserved among the genomes of several Siphoviridae phages, allowing the assignment of probable functions to certain uncharacterized ORFs from phage FJL-1 and other Siphoviridae phages. [TOP OF PAGE]

  377. Effects of diverse environmental conditions on fLC3 prophage stability in Lactococcus lactis. Lunde,M., Aastveit,A.H., Blatny,J.M., Nes,I.F. (2005). Appl. Environ. Microbiol. 71:721-727. The effects of various growth conditions on spontaneous fLC3 prophage induction in Lactococcus lactis subsp. cremoris IMN-C1814 was analyzed with a half fraction of a 4(4) factorial experimental design. The four factors included in the study were nutrient availability, acidity, osmolarity, and temperature, each applied at four levels. These environmental factors are related to the fermentation processes in the dairy industry, in which bacteriophage attacks on sensitive starter strains are a constant threat to successful fermentation processes. The frequency of spontaneous fLC3 induction was determined by quantitative analyses of restored DNA attachment sites (attB) on the bacterial chromosomes in a population of lysogenic cells. Statistical analysis revealed that all four environmental factors tested affected fLC3 prophage stability and that the environmental factors were involved in interactions (interactions exist when the effect of one factor depends on the level of another factor). The spontaneous fLC3 induction frequency varied from 0.08 to 1.76%. In general, the induction frequency remained at the same rate or decreased when level 1 to 3 of the four environmental factors was applied. At level 4, which generally gave the least favorable growth conditions, the induction frequency was either unchanged, decreased, or increased, depending on the type of stress. It appeared that the spontaneous induction frequency was independent of the growth behavior of the host. It was the environmental growth conditions that were the decisive factor in induction frequency. [TOP OF PAGE]

  378. [Comparison between bacteriophage-based assay and BACTEC-960 system in detection of ethambutol resistance in Mycobacterium tuberculosis]. Ma,X.W., Hu,Z.Y., Wang,J., Zhang,Y.R., Cui,Z.L., Cao,X.Y., Weng,X.H. (2005). Zhonghua nei ke za zhi [Chinese journal of internal medicine] 44:202-205. OBJECTIVE: To set up phage amplified biologically assay (PhaB) for rapid detection ethambutol (EMB) resistance and to evaluate the use of PhaB in the detection of EMB resistance. METHODS: To detect the EMB resistance of 138 clinical isolates of Mycobacterium tuberculosis (MTB) by PhaB and compare it with the results of BACTEC-960 system. The minimal inhibitory concentration (MIC) was detected for all the discrepant isolates. RESULTS: Of all the 138 strains of MTB clinical isolates, 114 strains were EMB-susceptible and 24 strains were EMB-resistant with BACTEC-960 system while 118 strains were EMB-susceptible and 20 strains were EMB-resistant with PhaB. 112 of the 138 strains were EMB-susceptible and 18 strains were EMB-resistant with the two methods. The concordant isolates in determination of EMB resistance were 130 strains in the two methods and the concordance rate was 94.2%. The disconcordant isolates were 8 strains and the discrepancy rate was 5.8%. The sensitivity, specificity, positive and negative predictive value as well as overall accuracy for the PhaB assay was 75.0% (18/24), 98.2% (112/114), 90.0% (18/20), 94.9% (112/118) and 94.2% (130/138) respectively if the judgment standard was adopted by BACTEC-960 method. CONCLUSIONS: The PhaB assay can be used for detection of EMB resistance in isolates of MTB easily and quickly in three days. This method do not need special instrument and may be used in rapid screening method for EMB resistance of MTB. [TOP OF PAGE]

  379. Characterization of phages virulent for Sarothamnus scoparius bradyrhizobia. Malek,W., Sajnaga,E., Wdowiak-Wrobel,S., Studzinska,B., Icka,I.S., Nosalewicz,I., Slomka,M., Tatara,A., Gawron,A. (2005). Curr. Microbiol. 51:244-249. Four virulent phages--fDl, fTl, fCYT21, and fOS6, infective on Sarothamnus scoparius rhizobia--were isolated from the soil and characterized for morphology, host range, rate of adsorption to bacterial cells, and genome size. New phages were separated into two morphological families: Siphoviridae with long, noncontractile tails (fDl, fTl) and Myoviridae with long, contractile tails (fCYT21, fOS6). They were also classified into two groups by a host specificity. One of them included viruses (fDl and fTl) that lysed S. scoparius bradyrhizobia and Bradyrhizobium sp. (Lupinus) strain Dl, and the second one comprised phages (fCYT21 and fOS6) that parasitized only Scotch broom native microsymbionts. Phages specific for S. scoparius rhizobia were differentiated not only by morphology and host range but also by a genome size that was in the range from 47,583 to 60,098 b.p. [TOP OF PAGE]

  380. The third age of phage. Mann,N.H. (2005). PLoS Biol. 3:e182 The third age of phage has begun with the recognition that phages may be key to the great planetary biogeochemical cycles and represent the greatest potential genetic resource in the biosphere. [TOP OF PAGE]

  381. Lysis-deficient bacteriophage therapy decreases endotoxin and inflammatory mediator release and improves survival in a murine peritonitis model. Matsuda,T., Freeman,T.A., Hilbert,D.W., Duff,M., Fuortes,M., Stapleton,P.P., Daly,J.M. (2005). Surgery 137:639-646. BACKGROUND: Lysis-deficient (LyD) bacteriophages (phages) kill bacteria without endotoxin (Et) release. This may minimize systemic cytokine responses and limit inflammation in bacterial sepsis. We determined the effects of t amber A3 T4 LyD and virulent wild-type (WT) phages on mouse bacterial peritonitis. METHODS: Balb/c mice were injected with B40sul Escherichia coli, treated intraperitoneally with LyD, WT, or a beta-lactam antibiotic [latamoxef sodium (LMOX)], and followed for survival. We measured Et release, tumor necrosis factor (TNF)-alpha and interleukin (IL)-6, as well as bacterial counts and peritoneal exudative cells (PECs) in peritoneal lavage fluid at 6 and 12 hours after infection. RESULTS: LyD mice showed significantly greater survival compared with other groups. Et levels were significantly lower in the LyD mice at 6 and 12 hours after infection. TNF-alpha and IL-6 levels were lower in LyD mice compared with control (untreated) mice at 12 hours. Compared with controls, bacteria counts in peritoneal lavage fluid were lower in all treatment groups (LyD, WT, or LMOX) at 6 and 12 hours. PEC counts were highest in LyD mice at 6 hours but significantly lower than that in WT phage- and LMOX-treated mice at 12 hours. CONCLUSIONS: LyD phage therapy significantly improves survival and attenuates the systemic effects of bacterial sepsis by minimizing Et release and pro-inflammatory mediators in murine bacterial peritonitis. Further studies may find phage therapy useful in treating peritonitis and multidrug-resistant bacterial infections. [TOP OF PAGE]

  382. Bacteriophage therapy: a revitalized therapy against bacterial infectious diseases. Matsuzaki,S., Rashel,M., Uchiyama,J., Sakurai,S., Ujihara,T., Kuroda,M., Ikeuchi,M., Tani,T., Fujieda,M., Wakiguchi,H., Imai,S. (2005). Journal of infection and chemotherapy : official journal of the Japan Society of Chemotherapy 11:211-219. Bacteriophage (phage) therapy involves using phages or their products as bioagents for the treatment or prophylaxis of bacterial infectious diseases. Much evidence in support of the effectiveness of phage therapy against bacterial infectious diseases has accumulated since 1980 from animal model studies conducted in Western countries. Reports indicate that appropriate administration of living phages can be used to treat lethal infectious diseases caused by gram-negative bacteria, such as Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii, Klebsiella pneumoniae, Vibrio vulnificus, and Salmonella spp., and gram-positive bacteria, such as Enterococcus faecium and Staphylococcus aureus. The phage display system and genetically modified nonreplicating phages are also effective for treatment of Helicobacter pylori and P. aeruginosa, respectively. In addition to phage particles per se, purified phage-encoded peptidoglycan hydrolase (lysin) is also reported to be effective for the treatment of bacterial infectious diseases caused by gram-positive bacteria such as Streptococcus pyogenes, S. pneumoniae, Bacillus anthracis, and group B streptococci. All phage lysins that have been studied to date exhibit immediate and strong bacteriolytic activity when applied exogenously. Furthermore, phage-coded inhibitors of peptidoglycan synthesis (protein antibiotics), search methods for novel antibacterial agents using phage genome informatics, and vaccines utilizing phages or their products are being developed. Phage therapy will compensate for unavoidable complications of chemotherapy such as the appearance of multidrug resistance or substituted microbism. [TOP OF PAGE]

  383. Effect of nutrient addition and environmental factors on prophage induction in natural populations of marine Synechococcus species. McDaniel,L., Paul,J.H. (2005). Appl. Environ. Microbiol. 71:842-850. A series of experiments were conducted with samples collected in both Tampa Bay and the Gulf of Mexico to assess the impact of nutrient addition on cyanophage induction in natural populations of Synechococcus sp. The samples were virus reduced to decrease the background level of cyanophage and then either left untreated or amended with nitrate, ammonium, urea, or phosphate. Replicate samples were treated with mitomycin C to stimulate cyanophage induction. In five of the nine total experiments performed, cyanophage induction was present in the non-nutrient-amended control samples. Stimulation of cyanophage induction in response to nutrient addition (phosphate) occurred in only one Tampa Bay sample. Nutrient additions caused a decrease in lytic (or control) phage production in three of three offshore stations, in one of three estuarine experiments, and in a lysogenic marine Synechococcus in culture. These results suggest that the process of cyanophage induction as an assay of Synechococcus lysogeny was not inorganically nutrient limited, at least in the samples examined. More importantly, it was observed that the level of cyanophage induction (cyanophage milliliter(-1)) was inversely correlated to Synechococcus and cyanophage abundance. Thus, the intensity of the prophage induction response is defined by ambient population size and cyanophage abundance. This corroborates prior observations that lysogeny in Synechococcus is favored during times of low host abundance. [TOP OF PAGE]

  384. Use of phages in therapy and bacterial detection. McKinstry,M., Edgar,R. (2005). pp. 430-440. In In Waldor,M.K., Friedman,D.I., and Adhya,S.L. (eds.), Phages: Their Role in Bacterial Pathogenesis and Biotechnology. ASM Press, Washington DC. [TOP OF PAGE]

  385. CTXf and Vibrio cholerae: exploring a newly recognized type of phage-host cell relationship. McLeod,S.M., Kimsey,H.H., Davis,B.M., Waldor,M.K. (2005). Mol. Microbiol. 57:347-356. The genes encoding cholera toxin, one of the principal virulence factors of the diarrhoeal pathogen Vibrio cholerae, are part of the genome of CTXf, a filamentous bacteriophage. Thus, CTXf has played a critical role in the evolution of the pathogenicity of V. cholerae. Unlike the well-studied F pilus-specific filamentous coliphages, CTXf integrates site-specifically into its host chromosome and forms stable lysogens. Here we focus on the CTXf life cycle and, in particular, on recent studies of the mechanism of CTXf integration and the factors that govern lysogeny. These and other processes illustrate the remarkable dependence of CTXf on host-encoded factors. [TOP OF PAGE]

  386. Mycobacteriophage and their application to disease control. McNerney,R., Traore,H. (2005). J. Appl. Microbiol. 99:223-233. The resurgence of tuberculosis and emergence of drug resistant disease has stimulated fresh research into mycobacteriophage. Studies are currently underway to develop phage-based tools for therapeutic and diagnostic use. Previous attempts at mycobacteriophage therapy in experimentally infected animals were not successful and alternative strategies of phage delivery that enable killing of intracellular bacteria are required. Replication of mycobacteriophage provides a simple means of detecting viable bacteria and good progress has been made towards the development of new phage-based diagnostic tools. When screening isolates for resistance to the major antituberculosis drug rifampicin phage-based tests have been shown to have high sensitivity. For the diagnosis of pulmonary tuberculosis evaluation studies indicate that current phage tests are not as sensitive as traditional culture methods. Further trials are needed to determine whether they might have a role in the detection of smear negative tuberculosis. A second generation of phage tests are under development following the construction of luciferase reporter phage. Preliminary data suggests they may offer rapid detection of mycobacteria and simple screening for drug resistance. The potential of mycobacteriophage to detect and treat other mycobacterial diseases remains largely unexplored. The resurgence of tuberculosis and emergence of drug resistant disease has stimulated fresh research into mycobacteriophage. Studies are currently underway to develop phage-based tools for therapeutic and diagnostic use. Previous attempts at mycobacteriophage therapy in experimentally infected animals were not successful and alternative strategies of phage delivery that enable killing of intracellular bacteria are required. Replication of mycobacteriophage provides a simple means of detecting viable bacteria and good progress has been made towards the development of new phage-based diagnostic tools. When screening isolates for resistance to the major antituberculosis drug rifampicin phage-based tests have been shown to have high sensitivity. For the diagnosis of pulmonary tuberculosis evaluation studies indicate that current phage tests are not as sensitive as traditional culture methods. Further trials are needed to determine whether they might have a role in the detection of smear negative tuberculosis. A second generation of phage tests are under development following the construction of luciferase reporter phage. Preliminary data suggests they may offer rapid detection of mycobacteria and simple screening for drug resistance. The potential of mycobacteriophage to detect and treat other mycobacterial diseases remains largely unexplored. [TOP OF PAGE]

  387. Bacterial viruses against viruses pathogenic for man? Miedzybrodzki,R., Fortuna,W., Weber-Dabrowska,B., Gorski,A. (2005). Virus Res. 110:1-8. In this review, we discuss possible models of bacteriophage-virus interactions. The first is based on the mechanism by which phages may interact indirectly with viruses. Its essence is that bacteriophage-derived nucleic acid may inhibit pathogenic virus infection. It seems that this phenomenon can be partly explained on the basis of interferon induction. We also discuss a study by Borecky's group (conducted over two decades ago) which provided some clinical data on the effectiveness of the application of native bacteriophage RNA in the treatment of viral infections. The second interaction model is based on the direct competition of bacteriophages and viruses for cellular receptors for viral cell-entry. The use of bacteriophages as inducers or displayers of antibodies with antiviral action is considered as the third model. In this part of the article, we also discuss other data and hypotheses on conceivable interactions between bacterial and animal viruses. ¶ As our current supply of antiviral drugs is quite limited, using natural agents such as bacteriophages as a weapon against pathogenic viruses could be an attractive and cost-efficient alternative, and further studies are urgently needed to test this possibility. [TOP OF PAGE]

  388. Enteroviruses and bacteriophages in bathing waters. Moce-Llivina,L., Lucena,F., Jofre,J. (2005). Appl. Environ. Microbiol. 71:6838-6844. A new procedure for detecting and counting enteroviruses based on the VIRADEN method applied to 10 liters of seawater was examined. It improved the efficiency of detection by taking into account both the number of positive isolations and numbers found with traditional methods. It was then used to quantify viruses in bathing waters. A number of bacterial indicators and bacteriophages were also tested. Cultivable enteroviruses were detected in 55% of the samples, most of which complied with bacteriological criteria. In contrast, viral genomes were only detected in 20% of the samples by reverse transcription-PCR. Somatic coliphages outnumbered all other indicators. F-specific RNA phages were detected in only 15% of the samples, whereas phages infecting Bacteroides thetaiotaomicron were detected in 70% of samples. A numerical relationship between the numbers of enteroviruses and the numbers of enterococci and somatic coliphages was observed. In situ inactivation experiments showed that viruses persisted significantly longer than the bacterial indicators. Only somatic coliphages and bacteriophages infecting Bacteroides persisted longer than the viruses. These results explain the numbers of enteroviruses and indicators in bathing waters attending the numbers usually found in sewage in the area. Somatic coliphages show a very good potential to predict the risk of viruses being present in bathing waters. [TOP OF PAGE]

  389. Control of bacteriophages in industrial ferments. Moineau,S., Lévesque,C. (2005). pp. 285-296. In In Kutter,E. and Sulakvelidze,A. (eds.), Bacteriophages: Biology and Application. CRC Press, Boca Raton, Florida. [first paragarph (but may not be final version)] Bacteria are used in a variety of industrial fermentation processes because of their ability to convert a wide variety of substrates into complex products or specific molecules. In principle, any industries and technological processes relying on bacterial fermentation are vulnerable to bacteriophage infection, and phages represent a constant threat of serious economic losses for those industries. The literature describing such problems goes back several decades (reviewed by Ackermann and DuBow 1987; Ogata 1980; Wünsche 1989), and faulty fermentations due to phages also are well-recognized in many modern biotechnological industries commercializing/using various bacterial fermentation products (Table 1). However, many other cases of phage contamination are not reported in scientific publications, and microbiologists learn of them primarily via confidential reports and personal communications (Ackermann and Moineau, unpublished data). ¶ One industry that has openly acknowledged phage infection of their bacterial strains is the dairy industry. In fact, dairy microbiologists have attempted for more than 70 years to eliminate, or at least to bring under better control, the bacteriophages that interfere with the manufacture of many fermented milk products (Moineau, et al. 2002). As a result, faulty milk fermentations are, undoubtedly, the best-documented examples of virulent bacteriophage infections in the industrial settings. The purpose of this chapter is to present an overview of the current strategies used by the dairy industry to curtail bacteriophage attacks. The general approaches described here are likely to be effective in many other, similar fermentation processes (Table 1). Additional, more specific information about the ecology of "industrial" phages, including phages relevant to the dairy industry and to various non-dairy food fermentation processes (e.g., the preparation of sauerkraut), is presented in Chapter 6 of this book. Also, comprehensive reviews about dairy phages and their control strategies have recently been published (Boucher and Moineau 2001; Coffey and Ross 2002). [TOP OF PAGE]

  390. Evolution of mutational robustness in an RNA virus. Montville,R., Froissart,R., Remold,S.K., Tenaillon,O., Turner,P.E. (2005). PLoS Biol. 3:e381 Mutational (genetic) robustness is phenotypic constancy in the face of mutational changes to the genome. Robustness is critical to the understanding of evolution because phenotypically expressed genetic variation is the fuel of natural selection. Nonetheless, the evidence for adaptive evolution of mutational robustness in biological populations is controversial. Robustness should be selectively favored when mutation rates are high, a common feature of RNA viruses. However, selection for robustness may be relaxed under virus co-infection because complementation between virus genotypes can buffer mutational effects. We therefore hypothesized that selection for genetic robustness in viruses will be weakened with increasing frequency of co-infection. To test this idea, we used populations of RNA phage f6 that were experimentally evolved at low and high levels of co-infection and subjected lineages of these viruses to mutation accumulation through population bottlenecking. The data demonstrate that viruses evolved under high co-infection show relatively greater mean magnitude and variance in the fitness changes generated by addition of random mutations, confirming our hypothesis that they experience weakened selection for robustness. Our study further suggests that co-infection of host cells may be advantageous to RNA viruses only in the short term. In addition, we observed higher mutation frequencies in the more robust viruses, indicating that evolution of robustness might foster less-accurate genome replication in RNA viruses. [TOP OF PAGE]

  391. Enumeration of bacteriophages in water by different laboratories of the European Union in two interlaboratory comparison studies. Mooijman,K.A., Ghameshlou,Z., Bahar,M., Jofre,J., Havelaar,A.H. (2005). J. Virol. Meth. As part of a European project on bacteriophages in bathing waters two interlaboratory comparison studies were carried out (May 1997 and March 1998). During these studies phage reference materials as well as naturally polluted standard samples were analysed in 16 European laboratories. Three groups of bacteriophages were tested using standardised methods: somatic coliphages, F-specific RNA-phages and phages of Bacteroides fragilis. Many of the participating laboratories applied one or more of the phage methods for the first time, after a one-week training session in a central laboratory. Nevertheless, the values of repeatability (r=1.35-1.38 calculated on log10-scale) and reproducibility (R=1.52-2.04 calculated on log10-scale) when analysing phage reference materials were close to the theoretical optimum for a Poisson distribution. When analysing the naturally polluted samples more variation in results within and between laboratories was found (r=1.63-2.34; R=3.10-5.72), in comparison with the results obtained with the pure phage reference materials. [TOP OF PAGE]

  392. The effect of migration on local adaptation in a coevolving host-parasite system. Morgan,A.D., Gandon,S., Buckling,A. (2005). Nature 437:253-256. Antagonistic coevolution between hosts and parasites in spatially structured populations can result in local adaptation of parasites; that is, the greater infectivity of local parasites than foreign parasites on local hosts. Such parasite specialization on local hosts has implications for human health and agriculture. By contrast with classic single-species population-genetic models, theory indicates that parasite migration between subpopulations might increase parasite local adaptation, as long as migration does not completely homogenize populations. To test this hypothesis we developed a system-specific mathematical model and then coevolved replicate populations of the bacterium Pseudomonas fluorescens and a parasitic bacteriophage with parasite only, with host only or with no migration. Here we show that patterns of local adaptation have considerable temporal and spatial variation and that, in the absence of migration, parasites tend to be locally maladapted. However, in accord with our model, parasite migration results in parasite local adaptation, but host migration alone has no significant effect. [TOP OF PAGE]

  393. Phage variation: understanding the behaviour of an accidental pathogen. Moxon,E.R., Jansen,V.A. (2005). Trends Microbiol. 13:563-565. Understanding why carriers of meningococci occasionally develop invasive disease is a major challenge. Individual strains of meningococci are extremely variable and undergo dynamic changes in DNA content and organization. This heterogeneity of meningococcal populations might enhance the fitness of this human-restricted bacterium. The recent discovery of a meningococcal bacteriophage and its associations to disease is an intriguing example of this variability and could contribute to a better understanding of microbial commensal and virulence behaviour. [TOP OF PAGE]

  394. Genetic diversity of marine Synechococcus and co-occurring cyanophage communities: evidence for viral control of phytoplankton. Muhling,M., Fuller,N.J., Millard,A., Somerfield,P.J., Marie,D., Wilson,W.H., Scanian,D.J., Post,A.F., Joint,I., Mann,N.H. (2005). Environ. Microbiol. 7:499-508. Unicellular cyanobacteria of the genus Synechococcus are a major component of the picophytoplankton and make a substantial contribution to primary productivity in the oceans. Here we provide evidence that supports the hypothesis that virus infection can play an important role in determining the success of different Synechococcus genotypes and hence of seasonal succession. In a study of the oligotrophic Gulf of Aqaba, Red Sea, we show a succession of Synechococcus genotypes over an annual cycle. There were large changes in the genetic diversity of Synechococcus , as determined by restriction fragment length polymorphism analysis of a 403- bp rpoC1 gene fragment, which was reduced to one dominant genotype in July. The abundance of co-occurring cyanophage capable of infecting marine Synechococcus was determined by plaque assays and their genetic diversity was determined by denaturing gradient gel electrophoresis analysis of a 118-bp g20 gene fragment. The results indicate that both abundance and genetic diversity of cyanophage covaried with that of Synechococcus . Multivariate statistical analyses show a significant relationship between cyanophage assemblage structure and that of Synechococcus . These observations are consistent with cyanophage infection being a major controlling factor in picophytoplankton succession. [TOP OF PAGE]

  395. Kinetics of the thermal inactivation of the Lactococcus lactis bacteriophage P008. Muller-Merbach,M., Neve,H., Hinrichs,J. (2005). J. Dairy Res. 72:281-286. The thermal resistance of the lactococcal bacteriophage P008 was investigated between 55 and 80 degrees C. Inactivation kinetics revealed an order of reaction above 1 and could be determined by a non-1st-order regression model. Phage inactivation was influenced by the medium (milk and Ca-M17-broth). Within the investigated temperature range, milk had a protective effect on phage P008. This was reflected in the rate constant and in the activation energy. Thermal phage inactivation studies reported in literature were re-analysed using non-1st-order regression. The obtained kinetic parameters showed that phage P008 belongs to the most heat resistant lactococcal phages investigated so far. [TOP OF PAGE]

  396. Bacteriophages may bias outcome of bacterial enrichment cultures. Muniesa,M., Blanch,A.R., Lucena,F., Jofre,J. (2005). Appl. Environ. Microbiol. 71:4269-4275. Enrichment cultures are widely used for the isolation of bacteria in clinical, biotechnological, and environmental studies. However, competition, relative growth rates, or inhibitory effects may alter the outcome of enrichment cultures, causing the phenomenon known as enrichment bias. Bacteriophages are a major component in many microbial systems, and it abounds in natural settings. This abundance means that bacteriophages are likely to be present in many laboratory enrichment cultures. Our hypothesis was that bacteriophages present in the sample might bias the enriched subpopulation, since it can infect and lyse the target bacteria during the enrichment step once the bacteria reach a given density. Here we show that the presence of bacteriophages in Salmonella and Shigella enrichment cultures produced a significant reduction (more than 1 log unit) in the number of these bacteria compared with samples in which bacteriophages had been reduced by filtration through 0.45-microm non-protein-binding membranes. Furthermore, our data indicate that the Salmonella biotypes isolated after the enrichment culture change if bacteriophages are present, thus distorting the results of the analysis. [TOP OF PAGE]

  397. Inactivation of bacteriophages by thermal and high-pressure treatment. Müller-Merbach,M., Rauscher,T., Hinrichs,J. (2005). Int. Dairy J. Dairy companies commonly experience fermentation failures due to bacteriophages that are spread mainly by milk, whey or air. Heat or high-pressure treatment may potentially reduce the phage titre, but further knowledge about the inactivation kinetics is desirable. Inactivation experiments were carried out with the commonly occurring lactococcal phages P001 and P008. Phage suspensions in calcium-enriched M17-broth were heated at 55-80 °C, or high-pressure treated at up to 600 MPa. Kinetic analysis showed that the order of inactivation reaction was above 1; thus, inactivation kinetics were approximated by a non-linear regression model. The Arrhenius parameters, rate constant, k(p,T), and activation energy, EA (for heat treatments), and the volume of activation, DV# (for pressure treatments) were calculated. Both measured and calculated results indicate that phage P008 was the more heat- and pressure-resistant of the two. By combining the results from heat and pressure inactivations, a pressure-temperature diagram for phage P008 was established. [TOP OF PAGE]

  398. [Molecular ecology of microalgal viruses] [text in Japanese]. Nagasaki,K., Takao,Y., Shirai,Y., Mizumoto,H., Tomaru,Y. (2005). Uirusu 55:127-132. A great amount of virus particles exist in natural waters. Each virion is considered to have its own ecological role, affecting the maintenance and fluctuation of aquatic ecosystems. We have been studying viruses infectious to micro-plankton, especially those infecting phytoplankton. Red tides are caused by drastic increase in abundance of plankton. We succeeded in elucidating that viral infection is one of the most important factors determining the dynamics and termination of algal blooms by means of field survey and molecular experiments. In addition, we demonstrated that the interrelationship between viruses and their hosts are highly complicated, and might be determined by the molecular-structural difference of viral capsids among distinct virus ecotypes. Furthermore, in the process of our investigation on various aquatic algal viruses, their importance as genetic sources has also been suggested. In order to deeply understand the mechanism of aquatic ecosystem, more intensive studies as for aquatic viruses are urgently required. [TOP OF PAGE]

  399. Predation, death, and survival in a biofilm: Bdellovibrio investigated by atomic force microscopy. Nunez,M.E., Martin,M.O., Chan,P.H., Spain,E.M. (2005). Colloids and surfaces. B, Biointerfaces 42:263-271. Biofilms are complex microbial communities that are resistant to attack by bacteriophages and to removal by drugs and chemicals. Here we use atomic force microscopy (AFM) to image the attack on Escherichia coli biofilms by Bdellovibrio bacteriovorus 109J. Bdellovibrio is a small, predatory bacterium that invades and devours other Gram-negative bacteria. We demonstrate that under dilute nutrient conditions, bdellovibrios can prevent the formation of simple bacterial biofilms and destroy established biofilms; under richer conditions the prey bacteria persist and are not eradicated, but may be shifted toward solution populations. Using AFM we explore these bacterial interactions with more detail and accuracy than available by more traditional staining assays or optical microscopy. AFM also allows us to investigate the nanoscale morphological changes of the predator, especially those related to motility. This demonstration of Bdellovibrio's successful predation in a biofilm inspires us to consider ways that it might be used productively for industrial, medical, agricultural, and biodefensive purposes. [TOP OF PAGE]

  400. Potential of the polyvalent anti-Staphylococcus bacteriophage K for control of antibiotic-resistant staphylococci from hospitals. O'Flaherty,S., Ross,R.P., Meaney,W., Fitzgerald,G.F., Elbreki,M.F., Coffey,A. (2005). Appl. Environ. Microbiol. 71:1836-1842. The increasing prevalence of antibiotic-resistant staphylococci has prompted the need for antibacterial controls other than antibiotics. In this study, a lytic bacteriophage (phage K) was assessed in vitro for its ability to inhibit emerging drug-resistant Staphylococcus aureus strains from hospitals and other species of Staphylococcus isolated from bovine infections. In in vitro inhibitory assays, phage K lysed a range of clinically isolated methicillin-resistant S. aureus (MRSA) strains, S. aureus with heterogeneous vancomycin resistance and vancomycin resistance, and teicoplanin-resistant strains. In these assays, 14 of the MRSA strains were initially only weakly sensitive to this phage. However, propagation of phage K on these less-sensitive strains resulted in all 14 being sensitive to the modified phages. The results enforce the principle that, while certain target bacteria may be relatively insensitive to lytic phage, this can be overcome by obtaining modified phage variants from passage of the phage through the insensitive strains. Model in situ hand wash studies using a phage-enriched wash solution resulted in a 100-fold reduction in staphylococcal numbers on human skin by comparison with numbers remaining after washing in phage-free solution. Infusion of the phage into a nonimmunogenic bismuth-based cream resulted in strong anti-Staphylococcus activity from the cream on plates and in broth. [TOP OF PAGE]

  401. Inhibition of bacteriophage K proliferation on Staphylococcus aureus in raw bovine milk. O'Flaherty,S., Coffey,A., Meaney,W.J., Fitzgerald,G.F., Ross,R.P. (2005). Lett. Appl. Microbiol. 41:274-279. AIMS: To assess the ability of staphylococcal bacteriophage K to inhibit Staphylococcus aureus in raw milk. METHODS AND RESULTS: The ability of bacteriophage (phage) to replicate in milk is important in situations where phage might be used as a therapeutic for bovine mastitis. Phage K was able to replicate normally, leading to elimination of the host culture in milk, which had been previously heat-treated. When raw milk was used under identical conditions, the phages were unable to replicate. Phage adsorption assays were performed and these demonstrated that adsorption of phage was significantly reduced in the raw milk while it was restored in the heat-treated sample (86.50% compared with 99.96% adsorption respectively). When confocal microscopy with a Live/Dead Bac light staining system was employed, it was observed that in raw milk S. aureus formed clusters associated with fat globules, while in heat-treated milk, bacterial agglutination had not occurred. CONCLUSIONS: Raw milk inhibits staphylococcal phage K proliferation. Significance and Impact of the Study: This observation has implications for the exploitation of staphylococcal therapeutic phage in milk. [TOP OF PAGE]

  402. Isolation and characterisation of two anti-staphylococcal bacteriophages specific for pathogenic Staphylococcus aureus associated with bovine infections. O'Flaherty,S., Ross,R.P., Flynn,J., Meaney,W.J., Fitzgeral,G.F., Coffey,A. (2005). Lett. Appl. Microbiol. 41:482-486. Aims: The aim of this study was to isolate and characterize bacteriophages against bovine Staphylococcus aureus associated with mastitis.
    Methods and Results: We describe the isolation of two anti-staphylococcal phages namely DW2 and CS1 from farmyard slurry. Both phages were characterized by electron microscopy and restriction analysis and shown to belong to the Siphoviridae family. CS1 and DW2 were lytic for representatives of all three clonal groups of Irish mastitis-associated staphylococci. These phages were compared with the previously characterized Myoviridae phage K. Infusion of a cocktail of all three phages at 10(8) PFU ml(-1) into live cow teats resulted in no detectable increase in somatic cell counts in milks indicating that the phages did not irritate the animal.
    Conclusion: Two new anti-staphylococcal phages CS1 and DW2 were isolated and characterized and tested for immunogenicity in animal teats.
    Significance and Impact of the Study: The phages isolated in this study are active against pathogenic S. aureus and may be incorporated into teat-dips or teat-washes as a non-antibiotic prophylaxis against staphylococcal bovine mastitis. [TOP OF PAGE]

  403. Seasonal virus removal by alternative onsite wastewater treatment systems. Olson,M.R., Axler,R.P., Hicks,R.E., Henneck,J.R., McCarthy,B.J. (2005). J. Water Health 3:139-155. Viral contamination of public waters is a leading health concern around the world, including in Minnesota where cold climate, abundant onsite systems on poor or thin soils, and abundant surface water resources present a significant risk of wastewater pathogens reaching sensitive water sources. Three alternative onsite treatment systems, a sand filter, peat filter and subsurface-flow constructed wetland (CW) at a field research site were evaluated for seasonal virus removal by seeding each with MS2 bacteriophage. The sand and peat filters and CW removed 2.7, 7.0, and 1.4 log10 of MS2, respectively, during summer and 1.8 and 6.9 log for the sand and peat filter during winter (CW not seeded). Somatic coliphage reductions for the sand filter, peat filter and CW were 2.9, 3.5, 1.0 log10 in summer, and 1.5, 2.8, 0.7 log10 during winter, respectively over a 3 year period. During this period, fecal coliform log10 reductions were 2.9, 4.6, 2.0 in summer for the sand and peat filters and CW, and 2.0, 4.6, 1.6 in winter. The peat filter was the most effective system for removing MS2, somatic coliphage and fecal coliforms during both winter and summer but all systems removed > 90% of viruses throughout the year. [TOP OF PAGE]

  404. Removal of micro-organisms in a small-scale hydroponics wastewater treatment system. Ottoson,J., Norstrom,A., Dalhammar,G. (2005). Lett. Appl. Microbiol. 40:443-447. Aims: To measure the microbial removal capacity of a small-scale hydroponics wastewater treatment plant. Methods and Results: Paired samples were taken from untreated, partly-treated and treated wastewater and analysed for faecal microbial indicators, i.e. coliforms, Escherichia coli, enterococci, Clostridium perfringens spores and somatic coliphages, by culture based methods. Escherichia coli was never detected in effluent water after >5.8-log removal. Enterococci, coliforms, spores and coliphages were removed by 4.5, 4.1, 2.3 and 2.5 log respectively. Most of the removal (60-87%) took place in the latter part of the system because of settling, normal inactivation (retention time 12.7 d) and sand filtration. Time-dependent log-linear removal was shown for spores (k = -0.17 log d(-1), r(2) = 0.99). Conclusions: Hydroponics wastewater treatment removed micro-organisms satisfactorily. Significance and Impact of the Study: Investigations on the microbial removal capacity of hydroponics have only been performed for bacterial indicators. In this study it has been shown that virus and (oo)cyst process indicators were removed and that hydroponics can be an alternative to conventional wastewater treatment. [TOP OF PAGE]

  405. Bacteriophage-based tests for tuberculosis. Pai,M., Kalantri,S.P. (2005). Indian Journal of Medical Microbiology 23:149-150. [TOP OF PAGE]

  406. Filtration and transport of Bacillus subtilis spores and the F-RNA phage MS2 in a coarse alluvial gravel aquifer: implications in the estimation of setback distances. Pang,L., Close,M., Goltz,M., Noonan,M., Sinton,L. (2005). J. Contam. Hydrol. 77:165-194. Filtration of Bacillus subtilis spores and the F-RNA phage MS2 (MS2) on a field scale in a coarse alluvial gravel aquifer was evaluated from the authors' previously published data. An advection-dispersion model that is coupled with first-order attachment kinetics was used in this study to interpret microbial concentration vs. time breakthrough curves (BTC) at sampling wells. Based on attachment rates (katt) that were determined by applying the model to the breakthrough data, filter factors (f) were calculated and compared with f values estimated from the slopes of log (cmax/co) vs. distance plots. These two independent approaches resulted in nearly identical filter factors, suggesting that both approaches are useful in determining reductions in microbial concentrations over transport distance. Applying the graphic approach to analyse spatial data, we have also estimated the f values for different aquifers using information provided by some other published field studies. The results show that values of f, in units of log (cmax/co) m(-1), are consistently in the order of 10(-2) for clean coarse gravel aquifers, 10(-3) for contaminated coarse gravel aquifers, and generally 10(-1) for sandy fine gravel aquifers and river and coastal sand aquifers. For each aquifer category, the f values for bacteriophages and bacteria are in the same order-of-magnitude. The f values estimated in this study indicate that for every one-log reduction in microbial concentration in groundwater, it requires a few tens of meters of travel in clean coarse gravel aquifers, but a few hundreds of meters in contaminated coarse gravel aquifers. In contrast, a one-log reduction generally only requires a few meters of travel in sandy fine gravel aquifers and sand aquifers. Considering the highest concentration in human effluent is in the order of 10(4) pfu/l for enteroviruses and 10(6) cfu/100 ml for faecal coliform bacteria, a 7-log reduction in microbial concentration would comply with the drinking water standards for the downgradient wells under natural gradient conditions. Based on the results of this study, a 7-log reduction would require 125-280 m travel in clean coarse gravel aquifers, 1.7-3.9 km travel in contaminated coarse gravel aquifers, 33-61 m travel in clean sandy fine gravel aquifers, 33-129 m travel in contaminated sandy fine gravel aquifers, and 37-44 m travel in contaminated river and coastal sand aquifers. These recommended setback distances are for a worst-case scenario, assuming direct discharge of raw effluent into the saturated zone of an aquifer. Filtration theory was applied to calculate collision efficiency (alpha) from model-derived attachment rates (katt), and the results are compared with those reported in the literature. The calculated alpha values vary by two orders-of-magnitude, depending on whether collision efficiency is estimated from the effective particle size (d10) or the mean particle size (d50). Collision efficiency values for MS-2 are similar to those previously reported in the literature (e.g. ) [DeBorde, D.C., Woessner, W.W., Kiley, QT., Ball, P., 1999. Rapid transport of viruses in a floodplain aquifer. Water Res. 33 (10), 2229-2238]. However, the collision efficiency values calculated for Bacillus subtilis spores were unrealistic, suggesting that filtration theory is not appropriate for theoretically estimating filtration capacity for poorly sorted coarse gravel aquifer media. This is not surprising, as filtration theory was developed for uniform sand filters and does not consider particle size distribution. Thus, we do not recommend the use of filtration theory to estimate the filter factor or setback distances. Either of the methods applied in this work (BTC or concentration vs. distance analyses), which takes into account aquifer heterogeneities and site-specific conditions, appear to be most useful in determining filter factors and setback distances. [TOP OF PAGE]

  407. Georgia: an unlikely stronghold for bacteriophage therapy. Parfitt,T. (2005). Lancet 365:2166-2167. The increasing failure of antibiotics to combat infections like multi-drug resistant Staphylococcus aureus has renewed interest in a long-forgotten treatment developed over 60 years ago in ex-Soviet Georgia. Tom Parfitt travelled to Tbilisi to witness the revival of bacteriophage therapy. [TOP OF PAGE]

  408. Marine phage genomics: what have we learned? Paul,J.H., Sullivan,M.B. (2005). Curr. Opin. Biotechnol. 16:299-307. Marine phages are the most abundant and diverse form of life on the planet, and their genomes have been described as the largest untapped reservoir of genomic information. To date, however, the complete genome sequences of only 17 marine phage are known. Nevertheless, these genomes have revealed some interesting features, including the presence of photosynthetic genes in cyanophage and common patterns of genomic organization. Intriguing findings are also being made from studies of the uncultivated marine viral community genome ('metavirome'). The greatest challenge in interpreting the biology of these phages, and for making comparisons with their terrestrial counterparts, is the high proportion of unidentifiable open reading frames (~60%). Future studies are likely to focus on sequencing more marine phage genomes from disparate hosts and diverse environments and on further basic studies of the biology of existing marine phages. [TOP OF PAGE]

  409. Method for isolation of Bacteroides bacteriophage host strains suitable for tracking sources of fecal pollution in water. Payan,A., Ebdon,J., Taylor,H., Gantzer,C., Ottoson,J., Papageorgiou,G.T., Blanch,A.R., Lucena,F., Jofre,J., Muniesa,M. (2005). Appl. Environ. Microbiol. 71:5659-5662. Bacteriophages infecting Bacteroides are potentially a good tool for fecal source tracking, but different Bacteroides host strains are needed for different geographic areas. A feasible method for isolating Bacteroides host strains for phages present in human fecal material is described. Useful strains were identified for application in Spain and the United Kingdom. One strain, GA-17, identified as Bacteroides thetaiotaomicron, was tested in several locations in Europe with excellent performance in Southern Europe. [TOP OF PAGE]

  410. Vibrio vulnificus load reduction in oysters after combined exposure to Vibrio vulnificus--specific bacteriophage and to an oyster extract component. Pelon,W., Luftig,R.B., Johnston,K.H. (2005). J. Food Prot. 68:1188-1191. Oysters infected with Vibrio vulnificus can present a serious health risk to diabetic, immunocompromised, and iron-deficient individuals. Numerous studies have been conducted with the goal of eliminating this organism from raw oysters. We utilized two natural oyster-associated components: pooled Vibrio vulnificus-specific bacteriophage and an extract of the eastern oyster (Crassostrea virginica) that contains an antimicrobial component we named anti-Vibrio vulnificus factor, which is bactericidal for V. vulnificus. Although each component alone can reduce V. vulnificus numbers independently, the simultaneous use of both components in an in vitro system successfully more effectively reduced V. vulnificus bacterial loads. [TOP OF PAGE]

  411. Diagnostic yield of fast plaque TB test for rapid detection of Mycobacterium in tuberculosis suspects. Phulpoto,M.A., Qayyum,S., Rizvi,N., Khuhawar,S.M. (2005). Journal of the Pakistan Medical Association 55:57-60. OBJECTIVE: To compare the diagnostic yield of FAST Plaque TB test with the conventional methods for detection of Mycobacterium tuberculosis in sputum of Tuberculosis suspects at Jinnah Postgraduate Medical Center Karachi Pakistan. METHODS: A comparative study of diagnostic yield of FAST Plaque TB test with the culture and ZN staining, conducted from January to June 2004. RESULTS: The study was completed on 48 samples, 31 (64.58%) male and 17 females (35.42%). Half of the cases were sputum positive. Culture positive was in 17 (35.41%) and negative in 28 (58.3%) wereas 3 (6.25%) were contaminated. FAST Plaque TB test was positive in 16 (33.33%) and negative in 32 (66.6%) specimens. Out of 17 culture positive, 2 (11.7%) were negative and in 28 culture negative, 1 (3.57%) specimen was positive for FAST Plaque TB test. Out of 24 smear positive, 11 (45.83%) were negative and in 24 smear negative, 3 (12.5%) were positive, for FAST Plaque TB test. Compared to culture it has sensitivity of 86.23% and specificity of 96.42%, positive predictive value of 93.75% and negative predictive value of 93.1%. CONCLUSION: FAST Plaque TB test is a simple test that can detect viable mycobacterium in 2 days. It has a good sensitivity and specificity. The cost is three times less than the other available tests like PCR. Thus it can be useful in the diagnosis of tuberculosis as an adjunct to sputum microscopy in endemic countries. [TOP OF PAGE]

  412. The rate of compensatory mutation in the DNA bacteriophage fX174. Poon,A., Chao,L. (2005). Genetics 170:989-999. A compensatory mutation occurs when the fitness loss caused by one mutation is remedied by its epistatic interaction with a second mutation at a different site in the genome. This poorly understood biological phenomenon has important implications, not only for the evolutionary consequences of mutation, but also for the genetic complexity of adaptation. We have carried out the first direct experimental measurement of the average rate of compensatory mutation. An arbitrary selection of 21 missense substitutions with deleterious effects on fitness was introduced by site-directed mutagenesis into the bacteriophage fX174. For each deleterious mutation, we evolved 8-16 replicate populations to determine the frequency at which a compensatory mutation, instead of the back mutation, was acquired to recover fitness. The overall frequency of compensatory mutation was approximately 70%. Deleterious mutations that were more severe were significantly more likely to be compensated for. Furthermore, experimental reversion of deleterious mutations revealed that compensatory mutations have deleterious effects in a wild-type background. A large diversity of intragenic compensatory mutations was identified from sequencing fitness-recovering genotypes. Subsequent analyses of intragenic mutation diversity revealed a significant degree of clustering around the deleterious mutation in the linear sequence and also within folded protein structures. Moreover, a likelihood analysis of mutation diversity predicts that, on average, a deleterious mutation can be compensated by about nine different intragenic compensatory mutations. We estimate that about half of all compensatory mutations are located extragenically in this organism. [TOP OF PAGE]

  413. Phage bacteriolysis, protistan bacterivory potential, and bacterial production in a freshwater reservoir: coupling with temperature. Pradeep R.,A.S., Boucher,D., Sime-Ngando,T., Debroas,D., Romagoux,J.C. (2005). Microb. Ecol. 50:64-72. Phage abundance and infection of bacterioplankton were studied from March to November 2003 in the Sep Reservoir (Massif Central, France), together with temperature, chlorophyll, bacteria (abundance and production), and heterotrophic nanoflagellates (abundance and potential bacterivory). Virus abundance (VA) ranged from 0.6 to 13 x 10(10) viruses l(-1), exceeding bacterial abundance (BA) approximately sixfold on average. In terms of carbon, viruses corresponded to up to 25% of bacterial biomass. A multiple regression model indicated that BA was the best predictor for VA (R(2) = 0.75). The frequency of infected bacteria (estimated from the percentage of visibly infected cells) varied from 1% to 32% and was best explained by a combination of temperature (R(2) = 0.20) and bacterial production (R(2) = 0.25). Viruses and flagellates contributed about equally to bacterial mortality. Both factors destroyed 55% of bacterial production, with a shift from phage bacteriolysis in early spring to protistan bacterivory in late summer. The vertical differences in most of the biological variables were not significant, contrasting with the seasonal differences (i.e., spring vs. summer-autumn). All biological variables under study were indeed significantly coupled to temperature. We regarded this to be the consequence of the enhanced discharge of the reservoir in 2003 (compared to previous years). This substantially weakened the stability and the thermal inertia of the water column, thereby establishing temperature as a stronger forcing factor in setting the conditions for optimal metabolic activity of microbial communities. [TOP OF PAGE]

  414. A diversity of bacteriophage forms and genomes can be isolated from the surface sands of the Sahara Desert. Prigent,M., Leroy,M., Confalonieri,F., Dutertre,M., DuBow,M.S. (2005). Extremophiles 9:289-296. The surface sands of the Sahara Desert are exposed to extremes of ultraviolet light irradiation, desiccation and temperature variation. Nonetheless, the presence of bacteria has recently been demonstrated in this environment by cultivation methods and by 16S rDNA analyses from total DNA isolated from surface sands. To discern the presence of bacteriophages in this harsh environment, we searched for extracellular phages and intracellularly located phages present as prophages or within pseudolysogens. Mild sonication of the sand, in different liquid culture media, incubated with and without Mitomycin-C, was followed by differential centrifugation to enrich for dsDNA phages. The resulting preparations, examined by electron microscopy, revealed the presence of virus-like particles with a diversity of morphotypes representative of all three major double-stranded DNA bacteriophage families (Myoviridae, Siphoviridae and Podoviridae). Moreover, pulsed-field gel electrophoresis of DNA, extracted from the enriched bacteriophage preparations, revealed the presence of distinct bands suggesting the presence of putative dsDNA phage genomes ranging in size from 45 kb to 270 kb. Characterization of the bacteriophages present in the surface sands of the Sahara Desert extends the range of environments from which bacteriophages can be isolated, and provides an important point of departure for the study of phages in extreme terrestrial environments. [TOP OF PAGE]

  415. [Bacteriophages use in the treatment of pyoseptic complications in a female patient with renal allotransplant]. Prokopenko,E.I., Shcherbakova,E.O., Vatazin,A.V., Budnikova,N.E., Iankovoi,A.G., Pasov,S.A., Agafonova,S.G. (2005). Urologiia 43-46. [TOP OF PAGE]

  416. Detection of enteric viruses and bacterial indicators in German environmental waters. Pusch,D., Oh,D.Y., Wolf,S., Dumke,R., Schroter-Bobsin,U., Hohne,M., Roske,I., Schreier,E. (2005). Arch. Virol. 150:929-947. A German mining lake and the supplying surface waters, which are located downstream of a sewage plant, were examined regarding their microbiological and virological quality. Between October 2002 and September 2003, specific PCR methods were used to determine the occurrence of enteric viruses in 123 water specimens drawn at different sites downstream of the waste water treatment plant and in 9 samples from the sewage plant influent. Detection rates in sewage plant effluents and surface water samples depended on sampling sites and were: 29-76% for enterovirus (EntV), 24-42% (astrovirus, AstV), 15-53% (norovirus, NV), 3-24% (rotavirus, RoV), 5-20% (hepatitis A virus, HAV) and 20% (adenovirus, AdV). AstV genome load of selected samples was between 3.7 x 10(3) to 1.2 x 10(8) genome equivalents per liter (gen.equ./l), depending on sampling location; NV average genome load ranged from 1.8 x 10(4) to 9.7 x 10(5) gen.equ./l. Cell culture methods showed that three out of 18 PCR positive samples contained infectious EntV. Even though microbiological parameters such as Escherichia coli, enterococci and coliphages indicated acceptable microbiological water quality, the virological data of this study suggest the possibility that surface waters may be a source for enteric viral infections. [TOP OF PAGE]

  417. Isolation of lactococcal prolate phage-phage recombinants by an enrichment strategy reveals two novel host range determinants. Rakonjac,J., O'Toole,P.W., Lubbers,M. (2005). J. Bacteriol. 187:3110-3121. Virulent lactococcal prolate (or c2-like) phages are the second most common phage group that causes fermentation failure in the dairy industry. We have mapped two host range determinants in two lactococcal prolate phages, c2 and 923, for the host strains MG1363 and 112. Each phage replicates on only one of the two host strains: c2 on MG1363 and 923 on 112. Phage-phage recombinants that replicated on both strains were isolated by a new method that does not require direct selection but rather employs an enrichment protocol. After initial mixed infection of strain 112, two rotations, the first of which was carried out on strain MG1363 and the second on 112, permitted continuous amplification of double-plating recombinants while rendering one of the parent phages unamplified in each of the two rotations. Mapping of the recombination endpoints showed that the presence of the N-terminal two-thirds of the tail protein L10 of phage c2 and a 1,562-bp cosR-terminal fragment of phage 923 genome overcame blocks of infection in strains MG1363 and 112, respectively. Both infection inhibition mechanisms act at the stage of DNA entry; in strain MG1363, the infection block acts early, before phage DNA enters the cytoplasm, and in strain 112, it acts late, after most of the DNA has entered the cell but before it undergoes cos-end ligation. These are the first reported host range determinants in bacteriophage of lactic acid bacteria required for overcoming inhibition of infection at the stage of DNA entry and cos-end ligation. [TOP OF PAGE]

  418. Control of bacteriophage mu lysogenic repression. Ranquet,C., Toussaint,A., de Jong,H., Maenhaut-Michel,G., Geiselmann,J. (2005). J. Mol. Biol. 353:186-195. The transposable and temperate phage Mu infects Escherichia coli where it can enter the lytic life-cycle or reside as a repressed and integrated prophage. The repressor protein Rep is the key element in the lysis-lysogeny decision. We have analyzed the fate of Rep in different mutants by Western blotting under two conditions that can induce a lysogen: high temperature and stationary phase. We show that, unexpectedly, Rep accumulates under all conditions where the prophage is completely derepressed, and that this accumulation is ClpX-dependent. An analysis of the degradation kinetics shows that Rep is a target of two protease systems: inactivation of either the clpP or lon gene results in a stabilization of Rep. Such a reaction scheme explains the counterintuitive observation that derepression is correlated with high repressor concentration. We conclude that under all conditions of phage induction the repressor is sequestered in a non-active form. A quantitative simulation accounts for our experimental data. It provides a model that captures the essential features of Mu induction and explains some of the mechanisms by which the physiological signals affecting the lysis-lysogeny decision converge onto Rep. [TOP OF PAGE]

  419. A transgenic mouse model for studying the clearance of blood-borne pathogens via human complement receptor 1 (CR1). Repik,A., Pincus,S.E., Ghiran,I., Nicholson-Weller,A., Asher,D.R., Cerny,A.M., Casey,L.S., Jones,S.M., Jones,S.N., Mohamed,N., Klickstein,L.B., Spitalny,G., Finberg,R.W. (2005). Clinical and experimental immunology 140:230-240. Complement receptor 1 (CR1) on the surface of human erythrocytes facilitates intravascular clearance of complement-opsonized pathogens. The need for complement activation can be circumvented by directly coupling the organism to CR1 using a bispecific monoclonal antibody heteropolymer (HP). Lack of a functional homologue to CR1 on mouse erythrocytes has made it difficult to study HP-dependent clearance of pathogens in small animals. We have developed a transgenic mouse that expresses human CR1 on erythrocytes. CR1 antigen is of appropriate size and in a clustered distribution as confirmed by immunoblotting and fluorescence microscopy, respectively. HP that immobilized bacteriophage fX174 prototype pathogen to erythrocyte CR1 of the transgenic mice increased the rate of clearance of the virus compared with HP that bound bacteriophage, but not CR1. This transgenic mouse model will allow evaluation of different HPs for their in vivo efficacy and potential as human therapeutics. [TOP OF PAGE]

  420. Phage release from biofilm and planktonic Staphylococcus aureus cells. Resch,A., Fehrenbacher,B., Eisele,K., Schaller,M., Gotz,F. (2005). FEMS Microbiol. Lett. 252:89-96. The ability of pathogenic staphylococci to form biofilms facilitates colonization and the development of chronic infections. Therapy is hampered by the high tolerance of biofilms towards antibiotic treatment and the immune system. We found evidence that lysogenic Staphylococcus aureus cells in a biofilm and in planktonic cultures spontaneously release phages into their surroundings. Phages were detected over a much longer period in biofilm cultures than in planktonic supernatants because the latter were degraded by secreted proteases. Phage release in planktonic and biofilm cultures was artificially increased by adding mitomycin C. Two morphologically distinct phages in the S. aureus strain used in this work were observed by electron microscopy. We postulate that phage-release is a frequent event in biofilms. The resulting lysis of cells in a biofilm might promote the persistence and survival of the remaining cells, as they gain a nutrient reservoir from their dead and lysed neighboring cells. This might therefore be an early differentiation and apoptotic mechanism. [TOP OF PAGE]

  421. A relative-entropy algorithm for genomic fingerprinting captures host-phage similarities. Robins,H., Krasnitz,M., Barak,H., Levine,A.J. (2005). J. Bacteriol. 187:8370-8374. The degeneracy of codons allows a multitude of possible sequences to code for the same protein. Hidden within the particular choice of sequence for each organism are over 100 previously undiscovered biologically significant, short oligonucleotides (length, 2 to 7 nucleotides). We present an information-theoretic algorithm that finds these novel signals. Applying this algorithm to the 209 sequenced bacterial genomes in the NCBI database, we determine a set of oligonucleotides for each bacterium which uniquely characterizes the organism. Some of these signals have known biological functions, like restriction enzyme binding sites, but most are new. An accompanying scoring algorithm is introduced that accurately (92%) places sequences of 100 kb with their correct species among the choice of hundreds. This algorithm also does far better than previous methods at relating phage genomes to their bacterial hosts, suggesting that the lists of oligonucleotides are "genomic fingerprints" that encode information about the effects of the cellular environment on DNA sequence. Our approach provides a novel basis for phylogeny and is potentially ideally suited for classifying the short DNA fragments obtained by environmental shotgun sequencing. The methods developed here can be readily extended to other problems in bioinformatics. [TOP OF PAGE]

  422. Experimental evolution of conflict mediation between genomes. Sachs,J.L., Bull,J.J. (2005). Proc. Natl. Acad. Sci. USA 102:390-395. Transitions to new levels of biological complexity often require cooperation among component individuals, but individual selection among those components may favor a selfishness that thwarts the evolution of cooperation. Biological systems with elements of cooperation and conflict are especially challenging to understand because the very direction of evolution is indeterminate and cannot be predicted without knowing which types of selfish mutations and interactions can arise. Here, we investigated the evolution of two bacteriophages (f1 and IKe) experimentally forced to obey a life cycle with elements of cooperation and conflict, whose outcome could have ranged from extinction of the population (due to selection of selfish elements) to extreme cooperation. Our results show the de novo evolution of a conflict mediation system that facilitates cooperation. Specifically, the two phages evolved to copackage their genomes into one protein coat, ensuring cotransmission with each other and virtually eliminating conflict. Thereafter, IKe evolved such extreme genome reduction that it lost the ability to make its own virions independent of f1. Our results parallel a variety of conflict mediation mechanisms existing in nature: evolution of reduced genomes in symbionts, cotransmission of partners, and obligate coexistence between cooperating species. [TOP OF PAGE]

  423. A snapshot of viral evolution from genome analysis of the tectiviridae family. Saren,A.M., Ravantti,J.J., Benson,S.D., Burnett,R.M., Paulin,L., Bamford,D.H., Bamford,J.K.H. (2005). J. Mol. Biol. 350:427-440. The origin, evolution and relationships of viruses are all fascinating topics. Current thinking in these areas is strongly influenced by the tailed double-stranded (ds) DNA bacteriophages. These viruses have mosaic genomes produced by genetic exchange and so new natural isolates are quite dissimilar to each other, and to laboratory strains. Consequently, they are not amenable to study by current tools for phylogenetic analysis. Less attention has been paid to the Tectiviridae family, which embraces icosahedral dsDNA bacterial viruses with an internal lipid membrane. It includes viruses, such as PRD1, that infect Gram-negative bacteria, as well as viruses like Bam35 with Gram-positive hosts. Although PRD1 and Bam35 have closely related virion morphology and genome organization, they have no detectable sequence similarity. There is strong evidence that the Bam35 coat protein has the "double-barrel trimer" arrangement of PRD1 that was first observed in adenovirus and is predicted to occur in other viruses with large facets. It is very likely that a single ancestral virus gave rise to this very large group of viruses. The unprecedented degree of conservation recently observed for two Bam35-like tectiviruses made it important to investigate those infecting Gram-negative bacteria. The DNA sequences for six PRD1-like isolates (PRD1, PR3, PR4, PR5, L17, PR772) have now been determined. Remarkably, these bacteriophages, isolated at distinctly different dates and global locations, have almost identical genomes. The discovery of almost invariant genomes for the two main Tectiviridae groups contrasts sharply with the situation in the tailed dsDNA bacteriophages. Notably, it permits a sequence analysis of the isolates revealing that the tectiviral proteins can be dissected into a slowly evolving group descended from the ancestor, the viral self, and a more rapidly changing group reflecting interactions with the host. [TOP OF PAGE]

  424. Growth of cyanophage N-1 under the influence of heavy metal ions. Sarma,T.A., Kaur,S.P. (2005). Acta virologica. English ed 49:23-28. The growth of cyanophage N-1 in the cyanobacterium Nostoc muscorum under the influence of heavy metal ions, namely Co2+, Cr6+, Cu2+, Mn2+ and Ni2+ has been studied. One-step growth experiments revealed that heavy metal ions extended the latent period by 1-2 hrs with a concomitant decrease in the phage burst size. The latter was reduced in the order Cu2/Mn2+, Ni2+, Co2+ and Cr6+. The treatment of the phage-infected bacteria with heavy metal ions did not induce mutations affecting either the phage plaque morphology or burst size. The final phage titer after such a treatments was lowest with Co2+, Cu2+ and Cr6+. The inhibition of the phage growth under the influence of heavy metal ions is discussed in context with the interaction of cyanophage N-1 with the photosynthetic reactions in the host bacteria. [TOP OF PAGE]

  425. Synonymous codon usage bias in 16 Staphylococcus aureus phages: implication in phage therapy. Sau,K., Gupta,S.K., Sau,S., Ghosh,T.C. (2005). Virus Res. 113:123-131. To reveal the factors influencing architecture of protein-coding genes in staphylococcal phages, relative synonymous codon usage variation has been investigated in 920 protein-coding genes of 16 staphylococcal phages. As expected for AT rich genomes, there are predominantly A and T ending codons in all 16 phages. Both Nc plot and correspondence analysis on relative synonymous codon usage indicates that mutation bias influences codon usage variation in the 16 phages. Correspondence analysis also suggests that translational selection and gene length also influence the codon usage variation in the phages to some extent and codon usage in staphylococcal phages is phage-specific but not S. aureus-specific. Further analysis indicates that among 16 staphylococcal phages, 44AHJD, P68 and K may be extremely virulent in nature as most of their genes have high translation efficiency. If this is true, then above three phages may be useful for curing staphylococcal infections. [TOP OF PAGE]

  426. Escherichia coli K1's capsule is a barrier to bacteriophage T7. Scholl,D., Adhya,S., Merril,C. (2005). Appl. Environ. Microbiol. 71:4872-4874. Escherichia coli strains that produce the K1 polysaccharide capsule have long been associated with pathogenesis. This capsule is believed to increase the cell's invasiveness, allowing the bacteria to avoid phagocytosis and inactivation by complement. It is also recognized as a receptor by some phages, such as K1F and K1-5, which have virion-associated enzymes that degrade the polysaccharide. In this report we show that expression of the K1 capsule in E. coli physically blocks infection by T7, a phage that recognizes lipopolysaccharide as the primary receptor. Enzymatic removal of the K1 antigen from the cell allows T7 to adsorb and replicate. This observation suggests that the capsule plays an important role as a defense against some phages that recognize structures beneath it and that the K1-specific phages evolved to counter this physical barrier. [TOP OF PAGE]

  427. The genome of bacteriophage K1F, a T7-like phage that has acquired the ability to replicate on K1 strains of Escherichia coli. Scholl,D., Merril,C. (2005). J. Bacteriol. 187:8499-8503. Bacteriophage K1F specifically infects Escherichia coli strains that produce the K1 polysaccharide capsule. Like several other K1 capsule-specific phages, K1F encodes an endo-neuraminidase (endosialidase) that is part of the tail structure which allows the phage to recognize and degrade the polysaccharide capsule. The complete nucleotide sequence of the K1F genome reveals that it is closely related to bacteriophage T7 in both genome organization and sequence similarity. The most striking difference between the two phages is that K1F encodes the endosialidase in the analogous position to the T7 tail fiber gene. This is in contrast with bacteriophage K1-5, another K1-specific phage, which encodes a very similar endosialidase which is part of a tail gene "module" at the end of the phage genome. It appears that diverse phages have acquired endosialidase genes by horizontal gene transfer and that these genes or gene products have adapted to different genome and virion architectures. [TOP OF PAGE]

  428. Isolation and characterization of bacteriophages of the Burkholderia cepacia complex. Seed,K.D., Dennis,J.J. (2005). FEMS Microbiol. Lett. 251:273-280. The Burkholderia cepacia complex consists of nine phenotypically similar but genotypically distinct beta-proteobacteria that are metabolically diverse and highly antibiotic resistant. Because of this exceptional intrinsic antibiotic resistance, infections with B. cepacia complex members are difficult to treat clinically and new alternative therapies are required. One strategy that holds some promise is the use of naturally occurring antibacterial bacteriophages that could potentially bind to and lyse B. cepacia complex cells in vivo. Towards that end, we used enrichment techniques to isolate lytic and lysogenic bacteriophages specific to the B. cepacia complex. The newly isolated bacteriophages were characterized by host range analysis, electron microscopy, genome restriction analysis, and partial DNA sequencing. These isolates include a bacteriophage with one of the broadest host ranges yet identified for any bacteriophage specific to the B. cepacia complex, and the first description of bacteriophages capable of lysing B. ambifaria. [TOP OF PAGE]

  429. Bacteriophage MS-2 removal by submerged membrane bioreactor. Shang,C., Wong,H.M., Chen,G. (2005). Water Res. 39:4211-4219. A membrane bioreactor (MBR) may serve as a pre-disinfection or disinfection unit, in addition to its solid/liquid separation and biological conversion functions, to produce sewage effluent of high quality. This bench-scale pilot study focuses on investigating the performance of a submerged MBR in pathogen removal and the factors affecting the removal, using a 0.4-microm hollow-fiber membrane module submerged in an aeration tank and bacteriophage MS-2 as the indicator organism. Removal of the MS-2 phage was found to be contributed by physical filtration by the membrane itself, biomass activity in the aeration tank and bio-filtration achieved by the biofilm developed on the membrane surface. The membrane alone gave poor virus removal (0.4+/-0.1 log) but the overall removal increased substantially with the presence of biomass and the membrane-surface-attached biofilm. The contributions of the suspended biomass and attached biofilm to the phage removal are dependent on the inter-related parameters including the concentration of mixed liquor suspended solids (MLSS), the sludge retention time (SRT) and the food to mass (F/M) ratio. The correlations between effluent flux/trans-membrane pressure and virus removal give evidence that phage removal in the MBR is most likely susceptible to both biological and physical factors including the quantity and property of the biomass and the biofilm and the membrane pore size reduction. [TOP OF PAGE]

  430. Inactivation of Escherichia coli O157:H7 in biofilm on stainless steel by treatment with an alkaline cleaner and a bacteriophage. Sharma,M., Ryu,J.H., Beuchat,L.R. (2005). J. Appl. Microbiol. 99:449-459. AIMS: To determine the effectiveness of an alkaline cleaner used in food-processing plants and a lytic bacteriophage specific for Escherichia coli O157:H7 in killing wild type and rpoS-deficient cells of the pathogen in a biofilm. METHODS AND RESULTS: Wild type and rpoS-deficient cells were attached to stainless steel coupons (c. 7-8 log CFU per coupon) on which biofilms were developed during incubation at 22 degrees C for 96 h in M9 minimal salts media (MSM) with one transfer to fresh medium. Coupons were treated with 100 and 25% working concentrations of a commercial alkaline cleaner (pH 11.9, with 100 microg ml(-1) free chlorine) used in the food industry, chlorine solutions (50 and 100 microg ml(-1) free chlorine), or sterile deionized water (control) at 4 degrees C for 1 and 3 min. Treatment with 100% alkaline cleaners reduced populations by 5-6 log CFU per coupon, a significant (P < or = 0.05) reduction compared with treatment with water. Initial populations (2.6 log CFU per coupon) of attached cells of both strains were reduced by 1.2 log CFU per coupon when treated with bacteriophage KH1 (7.7 log PFU ml(-1)) for up to 4 days at 4 degrees C. Biofilms containing low populations (2.7-2.8 log CFU per coupon) of wild type and rpoS-deficient cells that had developed for 24 h at 22 degrees C were not decreased by more than 1 log CFU per coupon when treated with KH1 (7.5 log PFU ml(-1)) at 4 degrees C. CONCLUSIONS: Higher numbers of cells of E. coli O157:H7 in biofilms are killed by treatment with an alkaline cleaner than with hypochlorite alone, possibly through a synergistic mechanism of alkaline pH and hypochlorite. Populations of cells attached on coupons were reduced by treating with bacteriophage but cells enmeshed in biofilms were protected. SIGNIFICANCE AND IMPACT OF THE STUDY: The alkaline pH, in combination with hypochlorite, in a commercial cleaner is responsible for killing E. coli O157:H7 in biofilms. Treatment with bacteriophage KH1 reduces populations of cells attached to coupon surfaces but not cells in biofilms. [TOP OF PAGE]

  431. Nearly identical bacteriophage structural gene sequences are widely distributed in both marine and freshwater environments. Short,C.M., Suttle,C.A. (2005). Appl. Environ. Microbiol. 71:480-486. Primers were designed to amplify a 592-bp region within a conserved structural gene (g20) found in some cyanophages. The goal was to use this gene as a proxy to infer genetic richness in natural cyanophage communities and to determine if sequences were more similar in similar environments. Gene products were amplified from samples from the Gulf of Mexico, the Arctic, Southern, and Northeast and Southeast Pacific Oceans, an Arctic cyanobacterial mat, a catfish production pond, lakes in Canada and Germany, and a depth of ca. 3,246 m in the Chuckchi Sea. Amplicons were separated by denaturing gradient gel electrophoresis, and selected bands were sequenced. Phylogenetic analysis revealed four previously unknown groups of g20 clusters, two of which were entirely found in freshwater. Also, sequences with >99% identities were recovered from environments that differed greatly in temperature and salinity. For example, nearly identical sequences were recovered from the Gulf of Mexico, the Southern Pacific Ocean, an Arctic freshwater cyanobacterial mat, and Lake Constance, Germany. These results imply that closely related hosts and the viruses infecting them are distributed widely across environments or that horizontal gene exchange occurs among phage communities from very different environments. Moreover, the amplification of g20 products from deep in the cyanobacterium-sparse Chuckchi Sea suggests that this primer set targets bacteriophages other than those infecting cyanobacteria. [TOP OF PAGE]

  432. Comparative efficacy of hand hygiene agents in the reduction of bacteria and viruses. Sickbert-Bennett,E.E., Weber,D.J., Gergen-Teague,M.F., Sobsey,M.D., Samsa,G.P., Rutala,W.A. (2005). Am. J. Infect. Contr. 33:67-77. BACKGROUND: Health care-associated infections most commonly result from person-to-person transmission via the hands of health care workers. METHODS: We studied the efficacy of hand hygiene agents (n = 14) following 10-second applications to reduce the level of challenge organisms (Serratia marcescens and MS2 bacteriophage) from the hands of healthy volunteers using the ASTM-E-1174-94 test method. RESULTS: The highest log 10 reductions of S marcescens were achieved with agents containing chlorhexidine gluconate (CHG), triclosan, benzethonium chloride, and the controls, tap water alone and nonantimicrobial soap and water (episode 1 of hand hygiene, 1.60-2.01; episode 10, 1.60-3.63). Handwipes but not alcohol-based handrubs were significantly inferior from these agents after a single episode of hand hygiene, but both groups were significantly inferior after 10 episodes. After a single episode of hand hygiene, alcohol/silver iodide, CHG, triclosan, and benzethonium chloride were similar to the controls in reduction of MS2, but, in general, handwipes and alcohol-based handrubs showed significantly lower efficacy. After 10 episodes, only benzethonium chloride (1.33) performed as well as the controls (1.59-1.89) in the reduction of MS2. CONCLUSIONS: Antimicrobial handwashing agents were the most efficacious in bacterial removal, whereas waterless agents showed variable efficacy. Alcohol-based handrubs compared with other products demonstrated better efficacy after a single episode of hand hygiene than after 10 episodes. Effective hand hygiene for high levels of viral contamination with a nonenveloped virus was best achieved by physical removal with a nonantimicrobial soap or tap water alone. [TOP OF PAGE]

  433. [Permeability to phi chi 174 bacteriophages in polyolephin membrane condoms]. Sierra,O.E., Gaona de Hernandez,M.A., Rey,G.J. (2005). Biomedica : revista del Instituto Nacional de Salud 25:603-608. Membranes used for the manufacture of condoms eventually can develop tiny pores, thereby decreasing dramatically their effectiveness as a physical barrier against the transmission of infectious agents. A technique was designed that was based on the ability of bacteriophage viruses to trespass membranes and to infect certain bacteria species, and then developing lysis plaques in the colonies of the host bacteria. The effectiveness of 60 polyolefin condoms in preventing the diffusion of the bacteriophage phi chi 174(ATCC13706-B1), 27 nm diameter, was compared to 20 latex condoms. Physiological conditions such as pressure, pH, superficial tension, length, time of exposure and viral titre were simulated. A pressurization system was designed, in which compressed air was injected simultaneously to ten condoms. Four of the 60 polyolefin condoms and one of the 20 latex condoms were permeable to the virus. Therefore, at least 93% of the condoms evaluated were able to contain the virus. The difference in permeability between the two types of membranes was not statistically significant (P = 0.79). [TOP OF PAGE]

  434. Widespread genetic exchange among terrestrial bacteriophages. Silander,O.K., Weinreich,D.M., Wright,K.M., O'Keefe,K.J., Rang,C.U., Turner,P.E., Chao.L. (2005). Proc. Natl. Acad. Sci. USA 102:19009-19014. Bacteriophages are the most numerous entities in the biosphere. Despite this numerical dominance, the genetic structure of bacteriophage populations is poorly understood. Here, we present a biogeography study involving 25 previously undescribed bacteriophages from the Cystoviridae clade, a group characterized by a dsRNA genome divided into three segments. Previous laboratory manipulation has shown that, when multiple Cystoviruses infect a single host cell, they undergo (i) rare intrasegment recombination events and (ii) frequent genetic reassortment between segments. Analyzing linkage disequilibrium (LD) within segments, we find no significant evidence of intrasegment recombination in wild populations, consistent with (i). An extensive analysis of LD between segments supports frequent reassortment, on a time scale similar to the genomic mutation rate. The absence of LD within this group of phages is consistent with expectations for a completely sexual population, despite the fact that some segments have >50% nucleotide divergence at 4-fold degenerate sites. This extraordinary rate of genetic exchange between highly unrelated individuals is unprecedented in any taxa. We discuss our results in light of the biological species concept applied to viruses. [TOP OF PAGE]

  435. In-house phage amplification assay is a sound alternative for detecting rifampin-resistant Mycobacterium tuberculosis in low-resource settings. Simboli,N., Takiff,H., McNerney,R., Lopez,B., Martin,A., Palomino,J.C., Barrera,L., Ritacco,V. (2005). Antimicrob. Agents Chemother. 49:425-427. An in-house mycobacteriophage amplification assay for detecting rifampin-resistant Mycobacterium tuberculosis showed 100% sensitivity, 97.7% specificity, and 95.2% predictive value for resistance in a test of 129 isolates from a hot spot area of multidrug-resistant M. tuberculosis. The applicability of the test was demonstrated in the routine work flow of a low-resource reference laboratory. [TOP OF PAGE]

  436. [Effects of the recombinant plasmid carrying the genes of cholera prophages CTX and RS1 on the expression of virulence and immunogenicity genes in the cholera pathogen]. Smirnov,N.I., Zadnova,S.P., Toporkov,A.V. (2005). Molekuliarnaia genetika, mikrobiologiia i virusologiia 3-8. Using toxin-coregulated adhesion pili (TCP), the etiologic agent of cholera is able to colonize human small intestine, where this pathogen proceeds with the production of the secreted cholera toxin (CT), inducing the development of severe diarrhea. At the same time, TCP and CT are not only the major factors of pathogenicity but also form a part of the group of key protective antigens. Immunoenzyme, immunoblotting, self-agglutination investigations, electron-microscopic studies, and electrophoretic assay of the outer membrane proteins showed that the recombinant plasmid carrying a number of cloned genes of two prophages, CTX and RS1, introduced into model Vibrio cholerae strains classical biovariant, resulted in the formation of strains with an enhanced rate of synthesis of three protective antigens: CT, TCP, and an outer membrane protein, OmpU. A simultaneous increase in the level of biosynthesis of the three antigens in V. cholerae was demonstrated to be specified by alterations in the expression of the toxR regulatory gene. Information was obtained suggesting that the transcriptional activity of toxR gene was dependent on the activity of rstC antirepressor gene derived from RS1 pro-phage and localized in the cloned fragment. Strains hyperproducing the three protective antigens can be used to construct more efficient non-living cholera vaccines, and to isolate the indicated proteins applicable to the development of diagnostic test-systems. [TOP OF PAGE]

  437. Effects of temperature and moisture on coliphage PRD-1 survival in soil. Song,I., Choi,C.Y., O'Shaughnessy,S., Gerba,C.P. (2005). J. Food Prot. 68:2118-2122. The goal of this study was to quantitatively assess the effects of temperature and soil moisture on the survival of coliphage PRD-1 in soil. PRD-1 was added to sandy loam soil at five different soil moisture levels. The soil seeded with PRD-1 was packed into sterile polyethylene jars and exposed to eight different temperatures in an oven. Samples were collected over 14 to 25 days depending on the temperature. The inactivation rate of PRD-1 increased linearly with increased temperature. The inactivation rate gradually decreased when the soil moisture level decreased from 20.9 to 8.9%. However, the inactivation rate increased when the soil moisture content reached 5.1%, suggesting the existence of an optimal soil moisture condition for PRD-1 survival. It is also possible that there is a threshold soil moisture level below which the inactivation of PRD-1 suddenly increases. Marked reductions in recoveries were observed as the soil moisture approached or fell below 5.0% as a result of evaporation. The increased inactivation of PRD-1 due to strong association with soil particles may have caused rapid reductions in recoveries. The evaporation process appeared to affect PRD-1 survival substantially at higher temperatures whereas little effect was observed at lower temperatures. A model developed from this study predicted PRD-1 survival in subsurface soil in field conditions with an average error of 11.0%. [TOP OF PAGE]

  438. Gene order constrains adaptation in bacteriophage T7. Springman,R., Badgett,M.R., Molineux,I.J., Bull,J.J. (2005). Virology 341:141-152. The order of genes in the genome is commonly thought to have functional significance for gene regulation and fitness but has not heretofore been tested experimentally. We adapted a bacteriophage T7 variant harboring an ectopically positioned RNA polymerase gene to determine whether it could regain the fitness of the wild type. Two replicate lines maintained the starting gene order and showed only modest recovery of fitness, despite the accumulation of over a dozen mutations. In both lines, a mutation in the early terminator signal is responsible for the majority of the fitness recovery. In a third line, the phage evolved a new gene order, restoring the wild-type position of the RNA polymerase gene but also displacing several other genes to ectopic locations. Due to the recombination, the fitness of this replicate was the highest obtained but it falls short of the wild type adapted to the same growth conditions. The large benefits afforded by the terminator mutation and the recombination are explicable in terms of T7 biology, whereas several mutations with lesser benefits are not easily accounted for. These results support the premise that gene order is important to fitness and that wild-type fitness is not rapidly re-evolved in reorganized genomes. [TOP OF PAGE]

  439. Virus detection using filament-coupled antibodies. Stone,G.P., Mernaugh,R., Haselton,F.R. (2005). Biotech. Bioeng. 91:699-706. Two attractive features of ELISA are the specificity of antibody-antigen recognition and the sensitivity achieved by enzymatic amplification. This report describes the development of a non-enzymatic molecular recognition platform adaptable to point-of-care clinical settings and field detection of biohazardous materials. This filament-antibody recognition assay (FARA) is based on circumferential bands of antibody probes coupled to a 120 microm diameter polyester filament. One advantage of this design is that automated processing is achieved by sequential positioning of filament-coupled probes through a series of 25-60 mL liquid filled microcapillary chambers. This approach was evaluated by testing for the presence of M13KO7 bacterial virus using anti-M13KO7 IgG(1) monoclonal antibody coupled to a filament. Filament motion first positioned the antibodies within a microcapillary tube containing a solution of M13KO7 virus before moving the probes through subsequent chambers, where the filament-coupled probes were washed, exposed to a fluorescently labeled anti-M13K07 antibody, and washed again. Filament fluorescence was then measured using a flatbed microarray scanner. The presence of virus in solution produced a characteristic increase in filament fluorescence only in regions containing coupled antibody probes. Even without the enzymatic amplification of a typical ELISA, the presence of 8.3 x 10(8) virus particles produced a 30-fold increase in fluorescence over an immobilized negative control antibody. In an ELISA comparison study, the filament-based approach had a similar lower limit of sensitivity of approximately 1.7 x 10(7) virus particles. This platform may prove attractive for point-of-care settings, the detection of biohazardous materials, or other applications where sensitive, rapid, and automated molecular recognition is desired. [TOP OF PAGE]

  440. Phage therapy in animals and agribusiness. Sulakvelidze,A., Barrow,P. (2005). pp. 335-380. In In Kutter,E. and Sulakvelidze,A. (eds.), Bacteriophages: Biology and Application. CRC Press, Boca Raton, Florida. [first paragarph (but may not be final version)] Since the discovery of bacteriophages in 1915-1917, they have been used to prevent and treat various bacterial infections. Although "phage therapy" has been historically associated with the use of bacteriophages in human medicine, phages also have been extensively used in veterinary medicine and in various agricultural settings. The history and various aspects of phage therapy in humans are reviewed in Chapter 14 of this book. In this Chapter, we review the past and current use of phages to prevent and treat naturally-occurring and experimentally-induced infections of animals. In addition, we discuss the potential applications of phage therapy in various agricultural settings, including the potential value of bacteriophages for improving the safety of foods and preventing foodborne diseases of bacterial etiology, and their potential to reduce the use of antibiotics in livestock. [TOP OF PAGE]

  441. Bacteriophage therapy in humans. Sulakvelidze,A., Kutter,E. (2005). pp. 381-436. In In Kutter,E. and Sulakvelidze,A. (eds.), Bacteriophages: Biology and Application. CRC Press, Boca Raton, Florida. [first two paragarphs (but may not be final version)] The discovery of bacteriophages is one of the most important milestones in the history of biomedical research -- one which has led to many fundamental discoveries and breakthroughs in the life sciences. In the pre-antibiotic era of the early 20th century, the potential of bacteriophages to be a powerful tool in dealing with infectious diseases of bacterial etiology also captured the imagination of the scientific and non-scientific communities, and it inspired many writers and journalists to write about bacteriophages and their possible therapeutic applications (Ho 2001). Bacteriophage therapy also served as a leitmotif of Arrowsmith, a well-known, Pulitzer Prize-winning novel by Sinclair Lewis (1925). The novel, published in 1925 during the early days of phage research, told the story of Martin Arrowsmith, a physician who used bacteriophages to deal with an outbreak of bubonic plague on a Caribbean island. However, for a variety of reasons (some discussed later in this chapter), the initially strong interest in phage therapy was short-lived and phage therapy was all but forgotten, in the Western world, shortly after antibiotics became widely available. The recent and rapidly increasing emergence of multiantibiotic-resistant mutants of pathogenic bacteria has rekindled the interest of the Western scientific community, industry, and general public in this almost century-old approach - as documented by the appearance of a number of phage therapy-related publications in the Western peer-reviewed literature (Alisky, et al. 1998; Sulakvelidze, et al. 2001; Biswas, et al. 2002; Cerveny, et al. 2002; Duckworth and Gulig 2002) and media (Radetsky 1996; Kuchment 2001; Martin 2003), and the formation of new biotechnology firms commercializing phage-based technology in the West (Sivitz 2002; Thacker 2003). Phages also recently resurfaced as the "saviors of humankind" in a best-selling novel - Prey by Michael Crichton (2002) - in which phages are used to destroy laboratory-escaped "bacterial nanoparticles" threatening life on earth. ¶ Biomedical technology today is very different from what it was in the early days of phage therapy research, and our understanding of biological properties of phages and the basic mechanisms of phage-bacterial host interaction has improved dramatically since the days of early therapeutic uses of bacteriophages. These advances can have a profound impact on the development of safe therapeutic phage preparations having optimal efficacy against their specific bacterial hosts, and on designing science-based strategies for integrating phage therapy into our arsenal of tools for preventing and treating bacterial infections. In this context, although bacteriophage therapy can probably be used to prevent and/or treat most bacterial infections, it is likely that antibiotic-resistant bacteria will become the first "clinical" targets of this re-emerging technology. It is also likely that, despite phage therapy's long history in Eastern Europe and the former Soviet Union (and, in the pre-antibiotic era, in the United States, France and other countries), therapeutic phage preparations will have to undergo a rigorous approval process by various regulatory agencies (e.g., the Food and Drug Administration in the United States) before they can be commercialized and used to treat humans in the Western world. On the other hand, therapeutic phages may find an easier acceptance in non-clinical settings, such as agribusiness and veterinary medicine. For example, they may be used to improve the safety of food products, or to help reduce the use of antibiotics in poultry and other livestock industries. This chapter primarily focuses on therapeutic applications of bacteriophages in humans, and the uses of phages in veterinary medicine and agricultural settings are described in more detail in chapter 13. [TOP OF PAGE]

  442. Phage therapy: an attractive option for dealing with antibiotic-resistant bacterial infections. Sulakvelidze,A. (2005). Drug Discov. Today 10:807-809. [first paragraph] The discovery of 'compound 606' (trade named 'Salvarsan') - the first 'magic bullet' for fighting bacterial infections - by Paul Erlich in 1909 is considered the birth of chemotherapy. The search for new 'magic bullets' increased in subsequent years, and it focused on identifying novel and safe compounds effective against bacterial infections. The discovery of antibiotics during the 1940s was the pinnacle of this process, and it revolutionized medicine from that point forward. Hundreds of antibiotics have been developed by various pharmaceutical companies since Eli Lilly pioneered the production of penicillin more than half a century ago, and many of them are currently available for clinical use. Antibiotics have saved more lives than any other drugs in the history of humankind, and their phenomenal success led the USA's Surgeon General to declare, during the late 1960s, that it was time to close the book on bacterial diseases. Indeed, partially triggered by this type of sentiment and, more importantly, by financial considerations, many large pharmaceutical companies have recently been 'closing the book' on developing new antibiotics, and they have been redirecting much of their R&D activities to more lucrative targets, such as drugs for treating chronic conditions. This trend and the increasing emergence of antibioticresistant bacterial pathogens could have very serious public health ramifications. In that regard, a recent report by a special Task Force co-chaired by the CDC, FDA and NIH stated that 'The world may soon be faced with previously treatable diseases that have again become untreatable, as in the pre-antibiotic era' [1]. [TOP OF PAGE]

  443. Three Prochlorococcus cyanophage genomes: Signature features and ecological interpretations. Sullivan,M.B., Coleman,M., Weigele,P., Rohwer,F., Chisholm,S.W. (2005). PLoS Biol. 3:e144 The oceanic cyanobacteria Prochlorococcus are globally important, ecologically diverse primary producers. It is thought that their viruses (phages) mediate population sizes and affect the evolutionary trajectories of their hosts. Here we present an analysis of genomes from three Prochlorococcus phages: a podovirus and two myoviruses. The morphology, overall genome features, and gene content of these phages suggest that they are quite similar to T7-like (P-SSP7) and T4-like (P-SSM2 and P-SSM4) phages. Using the existing phage taxonomic framework as a guideline, we examined genome sequences to establish ''core'' genes for each phage group. We found the podovirus contained 15 of 26 core T7-like genes and the two myoviruses contained 43 and 42 of 75 core T4-like genes. In addition to these core genes, each genome contains a significant number of ''cyanobacterial'' genes, i.e., genes with significant best BLAST hits to genes found in cyanobacteria. Some of these, we speculate, represent ''signature'' cyanophage genes. For example, all three phage genomes contain photosynthetic genes (psbA, hliP) that are thought to help maintain host photosynthetic activity during infection, as well as an aldolase family gene (talC) that could facilitate alternative routes of carbon metabolism during infection. The podovirus genome also contains an integrase gene (int) and other features that suggest it is capable of integrating into its host. If indeed it is, this would be unprecedented among cultured T7-like phages or marine cyanophages and would have significant evolutionary and ecological implications for phage and host. Further, both myoviruses contain phosphate-inducible genes (phoH and pstS) that are likely to be important for phage and host responses to phosphate stress, a commonly limiting nutrient in marine systems. Thus, these marine cyanophages appear to be variations of two well-known phages—T7 and T4—but contain genes that, if functional, reflect adaptations for infection of photosynthetic hosts in low-nutrient oceanic environments. [TOP OF PAGE]

  444. Therapeutic use of phage cocktail for controlling Escherichia coli O157:H7 in gastrointestinal tract of mice. Tanji,Y., Shimada,T., Fukudomi,H., Miyanaga,K., Nakai,Y., Unno,H. (2005). Journal of Bioscience and Bioengineering 100:280-287. To investigate the therapeutical use of phage mixture for controlling gastrointestinal Escherichia coli O157:H7 cells, in vitro and in vivo experiments were conducted. Three phages, SP15, SP21, and SP22 were selected from 26 phage stock screened from feces of stock animals and sewage influent. Addition of single or binary phage to the E. coli cell batch-culture reduced the turbidity of the culture. However, reascend of the turbidity due to the appearance of phage resistance cell was observed. On the other hand, addition of three phage mixture (SP15-21-22) did not produce reascend of culture turbidity under aerobic condition. Under anaerobic condition, slight reascend of culture turbidity was observed after SP15-21-22 addition. Chemostat continuous culture was operated under anaerobic condition to optimize the titer of phage cocktail and frequency of the addition for controlling E. coli cells. Five-log decrease of E. coli cell concentration after addition of phage cocktail of 10(9) Plaque forming unit (PFU)/ml was observed. However, reascend of cell concentration was observed after 1 d incubation. Repeated addition of phage cocktail was effective to reduce the cell concentration. Suspension of phage cocktail in the buffer containing 0.25% CaCO3 neutralized 9 times much more buffer of pH 2. Based on this in vitro experiment, phage cocktail (SP15-21-22) suspended in the buffer containing 0.25% CaCO3 was orally administrated to the mice in which E. coli O157:H7 cells was administrated in 2-d advance. E. coli and phage concentration in the feces was monitored for 9 d after phage addition. High titer of phage was detected in the feces when the phage cocktail administrated daily. E. coli O157:H7 concentration in the feces has been reduced according to the time period. However, difference of E. coli concentration in the feces of mice administrates with phage and in the control mice without phage addition became slight after 9-d test period. High titer of the phage settled down in the gastrointestinal tracts and reduced the concentration of E. coli cell. Repeated oral administration of SP15-21-22 was effective for rapid evacuation of E. coli O157:H7 from the feces and gastrointestinal tract of mice. [TOP OF PAGE]

  445. Bioaerosol emission rate and plume characteristics during land application of liquid class B biosolids. Tanner,B.D., Brooks,J.P., Haas,C.N., Gerba,C.P., Pepper,I.L. (2005). Environ. Sci. Technol. 39:1584-1590. This study investigated bioaerosol emission rates and plume characteristics of bioaerosols generated during land application of liquid Class B biosolids. In addition, it compared the rate of aerosolization of coliphages and total coliform bacteria during land application of liquid Class B biosolids to the rate of aerosolization during land application of groundwater inoculated with similar concentrations of Escherichia coli and coliphage MS2. Air samples were taken immediately downwind of a spray applicator as it applied liquid (approximately 8% solids) biosolids to farmland near Tucson, Arizona. Air samples were also collected immediately downwind of groundwater seeded with MS2 and E. coli applied to land in an identical manner. Air samples, collected with liquid impingers, were taken in horizontal and vertical alignment with respect to the passing spray applicator. Vertical and horizontal sample arrays made it possible to calculate the flux of microorganisms through a virtual plane of air samplers, located 2 m downwind of the passing spray applicator. Neither coliphages nor coliform bacteria were detected in air downwind of spray application of liquid Class B biosolids. Based on limits of detection for the methodology, the rate of aerosolization during land application of liquid biosolids was calculated to be less than 33 plaque forming units (PFU) of coliphage and 10 colony forming units (CFU) of coliform bacteria per meter traveled by the spray applicator. The rate of aerosolization during land application of seeded groundwater was found to be, on average, 2.02 x 10(3) CFU E. coli and 3.86 x 10(3) PFU MS2 aerosolized per meter traveled by the spray applicator. This is greater aerosolization than was observed during land application of biosolids. Because concentrations of coliphages and coliforms were similar in the liquid biosolids and the seeded water, it was concluded that some property of biosolids reduces aerosolization of microorganisms relative to groundwater. Additional experiments utilizing a novel air sampling protocol showed that the duration of bioaerosol exposure immediately (2 m) downwind of biosolids spray application is brief and the plume of bioaerosols generated is discrete. Additional air samples showed that aerosolization of coliphages and coliform bacteria after liquid biosolids have been applied to land does not occur at detectable levels. [TOP OF PAGE]

  446. Phages and bacterial vaginosis. Tao,L., Pavlova,S.I., Kiliç,A.O. (2005). pp. 256-279. In In Waldor,M.K., Friedman,D.I., and Adhya,S.L. (eds.), Phages: Their Role in Pathogenesis and Biotechnology. ASM Press, Washington DC. [concluding paragraph] Undoubtably, phages are natural factors that suppress vaginal lactobacilli, and we have obtained extensive data to support this claim. However, a direct link between phages and BV has yet to be established. Of course, the most convincing evidence would be obtained by directly observing phage infections of vaginal lactobacilli in women at the early stage of BV. Unfortunately, this is a rather difficult task. when a woman realizes that she has developed BV and comes to a gynocologic clinic, her vaginal lactobacilli have already decreased in number by being depleted by an unknown factor, possible phage. To overcome this problem, future clinical studies should include a longitudinal sampling of healthy women with a high risk of developing BV or of women suffering from recurrent BV. An animal model is also important for testing Koch's postulate. Phages should be tested in animals to determine if they initiate BV and transmit the disease among different animals. Moreover, fundamental knowledge of Lactobacillus phages should be further advanced. Future studies should include establishing phage taxonomy, characterizing phage diversity, determining phage genomic sequences, and studying interactions bteween phages and lactobacilli. These advancements in basic sciences will help us to understand the role of Lactobacillus phages in the etiology of BV. [TOP OF PAGE]

  447. Towards rational treatment of bacterial infections during extended space travel. Taylor,P.W., Sommer,A.P. (2005). International Journal of Antimicrobial Agents 26:183-187. In the next 15-30 years, manned space flight to Mars, our planetary neighbour, will become a reality and astronauts are likely to spend at least 2-3 years away from Earth. Time spent in such extreme environments will result in a diminution of immune status and profound changes in the human bacterial microflora. In microgravity, the efficacy of antibiotics is reduced and microbial mutation rates increase dramatically. These factors will impinge on the capacity to treat effectively the infections that will doubtless arise during such long and stressful endeavour. We highlight new rationales for the treatment of infectious disease that may be applicable to therapy in extreme environments such as deep space. [TOP OF PAGE]

  448. Inactivation of particle-associated viral surrogates by ultraviolet light. Templeton,M.R., Andrews,R.C., Hofmann,R. (2005). Water Res. 39:3487-3500. This study investigated whether colloid-sized particles can enmesh and protect viruses from 254-nm ultraviolet (UV) light and sought to determine the particle characteristics (e.g. size, chemical composition) that are most relevant in causing a protective effect. Two viral surrogates (MS2 coliphage and bacteriophage T4), three types of particles (kaolin clay, humic acid powder, and activated sludge), two coagulants (alum and ferric chloride), two filtration conditions (none and 0.45 microm), and two UV doses (40 and 80 mJ/cm2 for MS2 coliphage; 2 and 7 mJ/cm2 for bacteriophage T4) were considered in a series of bench-scale UV collimated beam experiments. Transmission electron microscopy was used to qualitatively confirm the phage particle-association after coagulation. Humic acid and activated sludge floc particles shielded both viral surrogates to a statistically significant degree (with >99% confidence) relative to particle-free control conditions, while the kaolin clay particles provided no significant protection. The results of the study suggest that particles <2 microm in diameter are large enough to protect viruses from UV light and that particulate chemical composition (e.g. UV-absorbing organic content) may be a critical factor in the survival of particle-associated viruses during UV disinfection. [TOP OF PAGE]

  449. Microbial quality of runoff following land application of cattle manure and swine slurry. Thurston-Enriquez,J.A., Gilley,J.E., Eghball,B. (2005). J. Water Health 3:157-171. Concentrations of human health-related microorganisms in runoff from agricultural plots (0.75 m x 2 m) treated with fresh and aged cattle manure, swine slurry and no manure (control) were determined. Three consecutive simulated rainfall events, producing 35 mm rainfall and separated by 24 h, were carried out for each plot. Fecal indicator (Escherichia coli, enterococci, Clostridium perfringens and coliphage) loads released in rainfall runoff from plots treated with fresh cattle manure, aged cattle manure and swine slurry treatments ranged from 5.52 x 10(5) to 4.36 x 10(9), 3.92 x 10(4) to 4.86 x 10(8), and 9.63 x 10(5) to 3.05 x 10(8), respectively. Plot runoff concentrations of protozoa (Cryptosporidium oocysts and Giardia cysts) ranged from 1.65 x 10(5) to 1.04 x 10(6), 2.93 x 10(3) to 2.75 x 10(5), and 9.12 x 10(4) to 3.58 x 10(6) for fresh cattle manure, aged cattle manure and swine slurry plot treatments, respectively. These results suggest that large microbial loads could be released via heavy precipitation events that produce runoff from livestock manure-applied agricultural fields, of even modest size, and could have a significant impact on water bodies within the watershed. Because of the lack of multiplication in the environment, highly elevated concentrations in manured land runoff, and correlation to protozoan parasite presence, Clostridium may be an alternative indicator for livestock manure contamination. [TOP OF PAGE]

  450. Use of bacteriophages in combination with competitive exclusion to reduce Salmonella from infected chickens. Toro,H., Price,S.B., McKee,A.S., Hoerr,F.J., Krehling,J., Perdue,M., Bauermeister,L. (2005). Avian Diseases 49:118-124. Salmonella-spedfic bacteriophages (BP) and competitive exclusion (CE) were used to reduce Salmonella colonization in experimentally infected chickens. A "cocktail" of distinct phage (i.e., phage showing different host ranges and inducing different types of plaques on Salmonella Typhimurium [ST] cultures) was developed. The killing activity of the selected BPs on ST cultures differed significantly, as determined in in vitro killing assays. BPs were administered orally to the chickens several days prior and after ST challenge but not simultaneously. BPs were readily isolated from the feces of the BP-treated chickens approximately 48 hr after administration. A CE product consisting of a defined culture of seven different microbial species was used either alone or in combination with BP treatment. CE was administered orally at hatch. Salmonella counts in intestine, ceca, and a pool of liver/spleen were evaluated in Salmonella-challenged chickens treated with BP or with BP and CE. In both trials 1 and 2, a beneficial effect of the phage treatment on weight gain performance was evident. A reduction in Salmonella counts was detected in cecum and ileum of BP-, CE-, and BP+CE-treated chickens as compared with nontreated birds. In trial 1, BP treatment reduced ST counts to marginal levels in the ileum and reduced counts sixfold in the ceca. A reduction of Salmonella counts with BP, CE, and BP+CE treatments was evident in chickens from trial 2. Both CE and BP treatments showed differences in the reduction of Salmonella counts after challenge between spedmens obtained at days 4 and 14 postchallenge in ceca, liver/spleen, and ileum. The preliminary data presented in this report show that isolation and characterization of Salmonella-specific BP is uncomplicated and feasible on a larger scale. Results indicate a protective effect of both Salmonella-specific BPs and a defined competitive exclusion product against Salmonella colonization of experimentally infected chickens. These results are encouraging for further work on the use of BP as an effective alternative to antibiotics to reduce Salmonella infections in poultry. [TOP OF PAGE]

  451. Identification of cyanophage Ma-LBP and infection of the cyanobacterium Microcystis aeruginosa from an Australian subtropical lake by the virus. Tucker,S., Pollard,P. (2005). Appl. Environ. Microbiol. 71:629-635. Viruses can control the structure of bacterial communities in aquatic environments. The aim of this project was to determine if cyanophages (viruses specific to cyanobacteria) could exert a controlling influence on the abundance of the potentially toxic cyanobacterium Microcystis aeruginosa (host). M. aeruginosa was isolated, cultured, and characterized from a subtropical monomictic lake-Lake Baroon, Sunshine Coast, Queensland, Australia. The viral communities in the lake were separated from cyanobacterial grazers by filtration and chloroform washing. The natural lake viral cocktail was incubated with the M. aeruginosa host growing under optimal light and nutrient conditions. The specific growth rate of the host was 0.023 h-1; generation time, 30.2 h. Within 6 days, the host abundance decreased by 95%. The density of the cyanophage was positively correlated with the rate of M. aeruginosa cell lysis (r2 = 0.95). The cyanophage replication time was 11.2 h, with an average burst size of 28 viral particles per host cell. However, in 3 weeks, the cultured host community recovered, possibly because the host developed resistance (immunity) to the cyanophage. The multiplicity of infection was determined to be 2,890 virus-like particles/cultured host cell, using an undiluted lake viral population. Transmission electron microscopy showed that two types of virus were likely controlling the host cyanobacterial abundance. Both viruses displayed T7-like morphology and belonged to the Podoviridiae group (short tails) of viruses that we called cyanophage Ma-LBP. In Lake Baroon, the number of the cyanophage Ma-LBP was 5.6 x 104 cyanophage · ml-1, representing 0.23% of the natural viral population of 2.46 x 107 · ml-1. Our results showed that this cyanophage could be a major natural control mechanism of M. aeruginosa abundance in aquatic ecosystems like Lake Baroon. Future studies of potentially toxic cyanobacterial blooms need to consider factors that influence cyanophage attachment, infectivity, and lysis of their host alongside the physical and chemical parameters that drive cyanobacterial growth and production. [TOP OF PAGE]

  452. Lambdoid phages and shiga toxin. Tyler,J.S., Livny,J., Friedman,D.I. (2005). pp. 131-164. In In Waldor,M.K., Friedman,D.I., and Adhya,S.L. (eds.), Phages: Their Role in Bacterial Pathogenesis and Biotechnology. ASM Press, Washington DC. [TOP OF PAGE]

  453. [Meningococci infection by a bacteriophage with a virulence factor]. van den Broek,P.J. (2005). Nederlands tijdschrift voor geneeskunde 149:2600-2602. Meningococci are bacteria dreaded for their ability to kill young people. However, meningococci and humans usually live together peacefully. In a minority of cases, the co-existence results in disease. Recently, whole genome comparisons between hyperinvasive clones and clones not associated with disease revealed that a chromosomally integrated bacteriophage was related to invasiveness. Many examples of bacteriophage-encoded virulence factors are known--as such, this finding is not remarkable. However, the way this virulence factor was found is a nice example of unravelling the pathogenesis of infectious diseases in the genomic era. [TOP OF PAGE]

  454. Evaluation of microbiological quality of coastal waters in Greece. Vantarakis,A.C., Tsibouxi,A., Venieri,D., Komninou,G., Athanassiadou,A., Papapetropoulou,M. (2005). J. Water Health 3:371-380. To evaluate the microbiological water quality of bathing sites along the Achaia coastline (south western Greece), a survey was conducted to determine the concentration of faecal bacterial and phage indicators as well as the presence of human viruses. Seawater samples (234) were collected from nine bathing sites on the Achaia coastline and were analysed for the presence of: total coliforms, faecal coliforms, faecal streptococci, Escherichia coli, somatic coliphages, F-RNA bacteriophages, bacteriophages infecting Bacteroides fragilis, enteroviruses, adenoviruses and hepatitis A viruses. Most of the bacteriological analysis results were in accordance with the European Union standards. In all sites, bacteriophages were detected occasionally. Enteroviruses and adenoviruses were detected in 24 samples (10.26%) and 37 samples (15.81%) respectively. No samples were positive for the presence of hepatitis A virus. The overall data indicates that bathing sites are impacted by human faecal material. Both bacterial indicators and phages have low predictive capability for the presence of human viruses in coastal waters. None of the environmental parameters analysed was strongly related to the presence of the indicator organisms and viruses. Appropriate and effective administrative measures that should be taken into account may be considered in order to improve water quality and reduce public health risk. [TOP OF PAGE]

  455. Variability of virus attachment patterns to butterhead lettuce. Vega,E., Smith,J., Garland,J., Matos,A., Pillaii,S.D. (2005). J. Food Prot. 68:2112-2117. Enteric viruses account for most foodborne illness in the United States. The objective of this study was to determine whether the isoelectric point (pI) of viruses such as feline calicivirus (FCV), echovirus 11, and bacteriophages fX174 and MS2 had any effect on their attachment to butterhead lettuce. The adsorption of virus particles to the lettuce was variable. Bacteriophage MS2 was the only virus that fit the current Derjaguin-Landau-Verway-Overbeek model of virus attachment. Echovirus 11 had the highest affinity to lettuce surface. Echovirus 11 appeared to exhibit reversible attachment above its pI, whereas below its pI strong adsorption was observed. Adsorption of FCV was at its maximum above its pI. Bacteriophage fX174 exhibited the most complex adsorption pattern, with attachment occurring only at the pH extremes (pH 3.0 and 8.0). These results suggest the current model for virus adsorption to sediment does not adequately explain the attachment of virus to lettuce. Importantly, the results indirectly suggest that current sample processing methods to recover viruses from lettuce may differentially select for the recovery of only certain virus types. [TOP OF PAGE]

  456. Bacteriophage in the treatment of experimental septicemic mice from a clinical isolate of multidrug resistant Klebsiella pneumoniae. Vinodkumar,C.S., Neelagund,Y.F., Kalsurmath,S. (2005). J. Commun. Dis. 37:18-29. Drug resistance is the major cause of increase in morbidity and mortality in neonates. The emergence of antibiotic-resistant bacterial strains requires the exploration of alternative antibacterial therapies and the concern that human kind in re-entering the 'pre-antibiotic era' has become very real and the development of alternative anti-infection modalities has become one of the highest priorities of modern medicine and biotechnology. This has spurred biomedical researchers to expand their efforts to identify new technologies and products that employ novel mechanism of action against the "super-bugs". One of such alternatives stems up from an old idea is the bacteriophage therapy, which led our group to study the ability of bacterial viruses (bacteriophages or phages) to rescue septicemic mice with multidrug resistant (MDR) Klebsiella pneumoniae isolated from neonatal septicemia. The phage strain used in this study had lytic activity against a wide range of clinical isolates of MDR Klebsiella pneumoniae. One of these MDR Klebsiella strain was used to induce septicemia in mice by intraperitoneal (i.p.) injection of 10(9) CFU. The resulting bacteremia was fatal within 48 h. A single i.p. injection of 3x10(8) PFU of the phage strain administered 45 min after the bacterial challenge, was sufficient to rescue 100% of the animals. Even when treatment was delayed to the point where all animals were moribund, approximately 50% of them were rescued by a single injection of this phage preparation. The ability of this phage to rescue septicemic mice was demonstrated to be due to the functional capabilities of the phage and not to a nonspecific immune effect. The rescue of septicemic mice could be affected only by phage strains able to grow in vitro on the bacterial host used to infect the animals and when such strains are heat inactivated they lose their ability to rescue the infected mice. [TOP OF PAGE]

  457. An amino acid substitution in a capsid protein enhances phage survival in mouse circulatory system more than a 1000-fold. Vitiello,C.L., Merril,C.R., Adhya,S. (2005). Virus Res. 114:101-103. In experiments with germ free mice, free from adaptive antibodies to the bacterial virus lambda phage, titers of the virus in the circulatory system have been reported to decrease by more than 10(9)pfu within 48 h of intraperitoneal intravenous or oral administration. Based on these observations, serial passage techniques have been used to select lambda phage mutants, with 13,000-16,000-fold greater capacity to remain in the mouse circulatory system 24h after intraperitoneal injection. In these prior studies the "long-circulating" phage, designated lambdaArgo phage, had at least three mutations including one in the major phage capsid (E) protein, which resulted in the change of glutamic acid to a lysine at residue 158. In the current study, we demonstrate that this single specific substitution in the E protein is sufficient to confer the "long-circulating" phenotype. The isogenic pair of phage developed in this study consisting of the long-circulating marker-rescued lambdaArgo phage, and the parental wild type phage can be used for studies of viral recognition mechanisms of the innate immune system and for the development of more effective antibacterial therapeutic phage strains. [TOP OF PAGE]

  458. Phage therapy reduces Campylobacter jejuni colonization in broilers. Wagenaar,J.A., Van Bergen,M.A.P., Mueller,M.A., Wassenaar,T.M., Carlton,R.M. (2005). Vet Microbiol 109:275-283. The effect of phage therapy in the control of Campylobacter jejuni colonization in young broilers, either as a preventive or a therapeutic measure, was tested. A prevention group was infected with C. jejuni at day 4 of a 10-day phage treatment. A therapeutic group was phage treated for 6 days, starting 5 days after C. jejuni colonization of the broilers had been established. Treatment was monitored by enumerating Campylobacter colony forming units (CFU) and phage plaque forming units (PFU) from caecal content. Counts were compared with control birds not receiving phage therapy. A clear 3 log decline in C. jejuni counts was initially observed in the therapeutic group, however, after 5 days bacterial counts stabilized at a level 1 log lower than that of the control group. Colonization of C. jejuni in the prevention group was delayed by the treatment and after an initial 2 log reduction, colonization stabilized within a week at levels comparable to the therapeutic group. The CFU and PFU counts displayed opposing highs and lows over time, indicative of alternating shifts in amplification of bacteria and phages. There were no adverse health effects from the phage treatment. Two different phages were combined as therapeutic treatment of Campylobacter positive chickens challenged at the age approaching broiler harvest. This again resulted in a significant decrease in Campylobacter colonization. We conclude that phage treatment is a promising alternative for reducing C. jejuni colonization in broilers. [TOP OF PAGE]

  459. Phages: Their Role in Bacterial Pathogenesis and Biotechnology. Waldor,M.K., Friedman,D., Adhya,S. (2005). ASM Press, Washington, DC.[TOP OF PAGE]

  460. Riverbank filtration for control of microorganisms: results from field monitoring. Weiss,W.J., Bouwer,E.J., Aboytes,R., Lechevallier,M.W., O'Melia,C.R., Le,B.T., SCHWAB,K.J. (2005). Water Res. 39:1990-2001. Microbial monitoring was conducted over a period of more than 1 year at three full-scale riverbank filtration (RBF) facilities, located in the United States along the Ohio, Missouri, and Wabash Rivers. Results of this study demonstrated the potential for RBF to provide substantial reductions in microorganism concentrations relative to the raw water sources. Cryptosporidium and Giardia were detected occasionally in the river waters but never in any of the well waters. Average concentrations and log reductions of Cryptosporidium and Giardia could not be accurately determined due to the low and variable concentrations in the river waters and the lack of detectable concentrations in the well waters. Average concentrations of aerobic and anaerobic spore-forming bacteria, which have both been proposed as potential surrogates for the protozoans, were reduced at the three facilities by 0.8 to > 3.1 logs and 0.4 to > 4.9 logs, respectively. Average concentrations of male-specific and somatic bacteriophage were reduced by > 2.1 logs and 3.2 logs, respectively. Total coliforms were rarely detected in the well waters, with 5.5 and 6.1 log reductions in average concentrations at the two wells at one of the sites relative to the river water. Average turbidity reductions upon RBF at the three sites were between 2.2 and 3.3 logs. Turbidity and microbial concentrations in the river waters generally tracked the river discharge; a similar relationship between the well water concentrations and river discharge was not observed, due to the low, relatively constant well water turbidities and lack of a significant number of detections of microorganisms in the well waters. Further research is needed to better understand the relationships among transport of pathogens (e.g., Cryptosporidium, Giardia, viruses) and potential surrogate parameters (including bacterial spores and bacteriophage) during RBF and the effects of water and sediment characteristics on removal efficiency. [TOP OF PAGE]

  461. Coevolutionary arms races between bacteria and bacteriophage. Weitz,J.S., Hartman,H., Levin,S.A. (2005). Proc. Natl. Acad. Sci. USA 102:9535-9540. We propose a computational and theoretical framework for analyzing rapid coevolutionary dynamics of bacteriophage and bacteria in their ecological context. Bacteriophage enter host cells via membrane-bound surface receptors often responsible for nutrient uptake. As such, a selective pressure will exist for the bacteria to modify its receptor configuration and, in turn, for the phage to modify its tail fiber. A mathematical model of these trait adaptations is developed by using the framework of adaptive dynamics. Host strains differ in their efficiency of resource uptake and resistance to phage, whereas phage strains differ in their host preference for adsorption. We solve the evolutionary ecology model and find the conditions for coevolutionary branching and relevant dimensionless parameters leading to distinct quasispecies. We confirm these calculations using stochastic Monte Carlo simulations of populations evolving in a chemostat with fixed washout rate and inflow resource density. We find that multiple quasispecies of bacteria and phage can coexist in a homogeneous medium with a single resource. When diversification occurs, quasispecies of phage adsorb effectively to only a limited subset of the total number of quasispecies of bacteria, i.e., functional differences between quasispecies arise endogenously within the evolutionary ecology framework. Finally, we discuss means to relate predictions of this model to experimental studies in the chemostat, using the model organisms Escherichia coli and the virulent strain of l phage. [TOP OF PAGE]

  462. Evolution of genomic content in the stepwise emergence of Escherichia coli O157:H7. Wick,L.M., Qi,W., Lacher,D.W., Whittam,T.S. (2005). J. Bacteriol. 187:1783-1791. Genome comparisons have demonstrated that dramatic genetic change often underlies the emergence of new bacterial pathogens. Evolutionary analysis of Escherichia coli O157:H7, a pathogen that has emerged as a worldwide public health threat in the past two decades, has posited that this toxin-producing pathogen evolved in a series of steps from O55:H7, a recent ancestor of a nontoxigenic pathogenic clone associated with infantile diarrhea. We used comparative genomic hybridization with 50-mer oligonucleotide microarrays containing probes from both pathogenic and nonpathogenic genomes to infer when genes were acquired and lost. Many ancillary virulence genes identified in the O157 genome were already present in an O55:H7-like progenitor, with 27 of 33 genomic islands of >5 kb and specific for O157:H7 (O islands) that were acquired intact before the split from this immediate ancestor. Most (85%) of variably absent or present genes are part of prophages or phage-like elements. Divergence in gene content among these closely related strains was approximately 140 times greater than divergence at the nucleotide sequence level. A >100-kb region around the O-antigen gene cluster contained highly divergent sequences and also appears to be duplicated in its entirety in one lineage, suggesting that the whole region was cotransferred in the antigenic shift from O55 to O157. The beta-glucuronidase-positive O157 variants, although phylogenetically closest to the Sakai strain, were divergent for multiple adherence factors. These observations suggest that, in addition to gains and losses of phage elements, O157:H7 genomes are rapidly diverging and radiating into new niches as the pathogen disseminates. [TOP OF PAGE]

  463. Abundance and diversity of viruses in six Delaware soils. Williamson,K.E., Radosevich,M., Wommack,K.E. (2005). Appl. Environ. Microbiol. 71:3119-3125. The importance of viruses in marine microbial ecology has been established over the past decade. Specifically, viruses influence bacterial abundance and community composition through lysis and alter bacterial genetic diversity through transduction and lysogenic conversion. By contrast, the abundance and distribution of viruses in soils are almost completely unknown. This study describes the abundance and diversity of autochthonous viruses in six Delaware soils: two agricultural soils, two coastal plain forest soils, and two piedmont forest soils. Viral abundance was measured using epifluorescence microscopy, while viral diversity was assessed from morphological data obtained through transmission electron microscopy. Extracted soil virus communities were dominated by bacteriophages that demonstrated a wide range of capsid diameters (20 nm to 160 nm) and morphologies, including filamentous forms and phages with elongated capsids. The reciprocal Simpson's index suggests that forest soils harbor more diverse assemblages of viruses, particularly in terms of morphological distribution. Repeated extractions of virus-like particles (VLPs) from soils indicated that the initial round of extraction removes approximately 70% of extractable viruses. Higher VLP abundances were observed in forest soils (1.31 x 10(9) to 4.17 x 10(9) g(-1) dry weight) than in agricultural soils (8.7 x 10(8) to 1.1 x 10(9) g(-1) dry weight). Soil VLP abundance was significantly correlated to moisture content (r = 0.988) but not to soil texture. Land use (agricultural or forested) was significantly correlated to both bacterial (r = 0.885) and viral (r = 0.812) abundances, as were soil organic matter and water content. Thus, land use is a significant factor influencing viral abundance and diversity in soils. [TOP OF PAGE]

  464. Experimental bacteriophage protection against Staphylococcus aureus abscesses in a rabbit model. Wills,Q.F., Kerrigan,C., Soothill,J.S. (2005). Antimicrob. Agents Chemother. 49:1220-1221. In a rabbit model of wound infection caused by Staphylococcus aureus, 2 x 10(9) PFU of staphylococcal phage prevented abscess formation in rabbits when it was injected simultaneously with S. aureus (8 x 10(7) CFU) into the same subcutaneous site. Phage multiplied in the tissues. Phages might be a valuable prophylaxis against staphylococcal infection. [TOP OF PAGE]

  465. Bacteriophages—potential for application in wastewater treatment processes. Withey,S., Cartmell,E., Avery,L.M., Stephenson,T. (2005). Science of Total Environment 339:1-18. Bacteriophages are viruses that infect and lyse bacteria. Interest in the ability of phages to control bacterial populations has extended from medical applications into the fields of agriculture, aquaculture and the food industry. Here, the potential application of phage techniques in wastewater treatment systems to improve effluent and sludge emissions into the environment is discussed. Phage-mediated bacterial mortality has the potential to influence treatment performance by controlling the abundance of key functional groups. Phage treatments have the potential to control environmental wastewater process problems such as: foaming in activated sludge plants; sludge dewaterability and digestibility; pathogenic bacteria; and to reduce competition between nuisance bacteria and functionally important microbial populations. Successful application of phage therapy to wastewater treatment does though require a fuller understanding of wastewater microbial community dynamics and interactions. Strategies to counter host specificity and host cell resistance must also be developed, as should safety considerations regarding pathogen emergence through transduction. [TOP OF PAGE]

  466. Diversity in times of adversity: probabilistic strategies in microbial survival games. Wolf,D.M., Vazirani,V.V., Arkin,A.P. (2005). J. Theor. Biol. 234:227-253. Population diversification strategies are ubiquitous among microbes, encompassing random phase-variation (RPV) of pathogenic bacteria, viral latency as observed in some bacteriophage and HIV, and the non-genetic diversity of bacterial stress responses. Precise conditions under which these diversification strategies confer an advantage have not been well defined. We develop a model of population growth conditioned on dynamical environmental and cellular states. Transitions among cellular states, in turn, may be biased by possibly noisy readings of the environment from cellular sensors. For various types of environmental dynamics and cellular sensor capability, we apply game-theoretic analysis to derive the evolutionarily stable strategy (ESS) for an organism and determine when that strategy is diversification. We find that: (1) RPV, effecting a sort of Parrondo paradox wherein random alternations between losing strategies produce a winning strategy, is selected when transitions between different selective environments cannot be sensed, (2) optimal RPV cell switching rates are a function of environmental lifecycle asymmetries and environmental autocorrelation, (3) probabilistic diversification upon entering a new environment is selected when sensors can detect environmental transitions but have poor precision in identifying new environments, and (4) in the presence of excess additive noise, low-pass filtering is required for evolutionary stability. We show that even when RPV is not the ESS, it may minimize growth rate variance and the risk of extinction due to 'unlucky' environmental dynamics. [TOP OF PAGE]

  467. Bacteriophage Esc-A is an efficient therapy for Escherichia coli 3-1 caused diarrhea in chickens. Xie,H., Zhuang,X., Kong,J., Ma,G., Zhang,H. (2005). J. Gen. Appl. Microbiol. 51:159-163. The bacteriophage Esc-A was isolated from sewage by using the intestinal pathogenic Escherichia coli 3-1 as the host. Toxicity in chickens showed its safety as a bio-product. Phage therapy against diarrhea in chickens indicated that Esc-A could decrease the death rate more efficiently compared with antibiotic treatments. [TOP OF PAGE]

  468. Alteration of tail fiber protein gp38 enables T2 phage to infect Escherichia coli O157:H7. Yoichi,M., Abe,M., Miyanaga,K., Unno,H., Tanji,Y. (2005). J. Biotechnol. 115:101-107. Artificial control of phage specificity may contribute to practical applications, such as the therapeutic use of phages and the detection of bacteria by their specific phages. To change the specificity of phage infection, gene products (gp) 37 and 38, expressed at the tip of the long tail fiber of T2 phage, were exchanged with those of PP01 phage, an Escherichia coli O157:H7 specific phage. Homologous recombination between the T2 phage genome and a plasmid encoding the region around genes 37-38 of PP01 occurred in transformant E. coli K12 cells. The recombinant T2 phage, named T2ppD1, carried PP01 gp37 and 38 and infected the heterogeneous host cell E. coli O157:H7 and related species. On the other hand, T2ppD1 could not infect E. coli K12, the original host of T2, or its derivatives. The host range of T2ppD1 was the same as that of PP01. Infection of T2ppD1 produced turbid plaques on a lawn of E. coli O157:H7 cells. The binding affinity of T2ppD1 to E. coli O157:H7 was weaker than that of PP01. The adsorption rate constant (ka) of T2ppD1 (0.17 x 10(-9)(ml CFU(-1) min(-1)) was almost 1/6 that of PP01 (1.10 x 10(-9)(ml CFU(-1) min(-1))). In addition to the tip of the long tail fiber, exchange of gene products expressed in the short tail fiber may be necessary for tight binding of recombinant phage. [TOP OF PAGE]

  469. Virus removal by iron coagulation-microfiltration. Zhu,B., Clifford,D.A., Chellam,S. (2005). Water Res. 39:5153-5161. This study was undertaken to determine virus removal efficiency by iron coagulation followed by microfiltration (MF) in water treatment using the MS2 bacteriophage (approximately 25 nm diameter) as a tracer virus. Results from these bench-scale studies were used to propose a mechanism for virus removal by iron coagulation-MF. Ferric chloride was used as coagulant, and the dosages were 0, 2, 5, and 10 mg/L as Fe(III) with pH adjusted during mixing to 6.3, 7.3 and 8.3. In the absence of iron-coagulation and with less than 2 mg/L Fe, MF alone achieved less than a 0.5 log removal of MS2 virus. However, iron-coagulation pretreatment dramatically improved virus removal, especially in the 5-10 mg/L Fe dose range, ultimately achieving more than 4-log removal at pH 6.3 with 10-mg/L Fe dose. For the 5 and 10 mg/L Fe dosages, decreasing pH in the 8.3-6.3 range resulted in significantly greater virus removal. For 0 and 2 mg/L iron dosages, decreasing pH in the 8.3-6.3 range also improved virus removal, but to a lesser extent. The experimental data indicates negatively charged MS2 viruses first adsorbed onto the positively charged iron oxyhydroxide floc particles before being removed by MF. MS2 viruses were not inactivated in iron or aluminum coagulation as evidenced by the fact that their concentrations before and after coagulation, settling, and re-suspension of the coagulated sludge were not statistically different. [TOP OF PAGE]

  470. Renaissance phage. Anonymous (2004). Nat. Rev. Microbiol. 2:922 In today's culture of spin, 'renaissance' is a term that is often applied undeservingly to particular areas of science. In the case of current phage research, however, its use is easily justified. [TOP OF PAGE]

  471. Virus particles monitored by fluorescence spectroscopy: a potential detection assay for macromolecular assembly. Alimova,A., Katz,A., Podder,R., Minko,G., Wei,H., Alfano,R.R., Gottlieb,P. (2004). Photochem Photobiol 80:41-46. Native fluorescence spectroscopy was used for in situ investigations of two lipid-containing bacteriophages from the cystovirus family as well as their Pseudomonad host cells. Both the viruses f6 and f12 and their bacterial host proteins contain the amino acid tryptophan (trp), which is the predominant fluorophore in UV. Within proteins, trp's structural environment differs, and the differences are reflected in their spectroscopic signatures. It was observed that the peak of the trp emission from both viruses was at 330 nm, a significantly shorter wavelength than trp in either the Pseudomonad host cells or the amino acid's chemical form. This allowed us to monitor the viral attachment process and subsequent lytic release of progeny virus particles by measurement of the trp emission spectra during the infection process. This work demonstrates that fluorescence may offer a novel tool to detect viruses and monitor viral infection of cells and may be part of a biodefense application. [TOP OF PAGE]

  472. Occurrence of Escherichia coli, noroviruses, and F-specific coliphages in fresh market-ready produce. Allwood,P.B., Malik,Y.S., Maherchandani,S., Vought,K., Johnson,L.A., Braymen,C., Hedberg,C.W., Goyal,S.M. (2004). J. Food Prot. 67:2387-2390. Forty samples of fresh produce collected from retail food establishments were examined to determine the occurrence of Escherichia coli, F-specific coliphages, and noroviruses. An additional six samples were collected from a restaurant undergoing investigation for a norovirus outbreak. Nineteen (48%) of the retail samples and all outbreak samples were preprocessed (cut, shredded, chopped, or peeled) at or before the point of purchase. Reverse transcription-PCR, with the use of primers JV 12 and JV 13, failed to detect norovirus RNA in any of the samples. All six outbreak samples and 13 (33%) retail samples were positive for F-specific coliphages (odds ratio undefined, P = 0.003). Processed retail samples appeared more likely to contain F-specific coliphages than unprocessed samples (odds ratio 3.8; 95% confidence interval 0.8 to 20.0). Only two (5.0%) retail samples were positive for E. coli; outbreak samples were not tested for E. coli. The results of this preliminary survey suggest that F-specific coliphages could be useful conservative indicators of fecal contamination of produce and its associated virological risks. Large-scale surveys should be conducted to confirm these findings. [TOP OF PAGE]

  473. Effect of temperature and sanitizers on the survival of feline calicivirus, Escherichia coli, and F-specific coliphage MS2 on leafy salad vegetables. Allwood,P.B., Malik,Y.S., Hedberg,C.W., Goyal,S.M. (2004). J. Food Prot. 67:1451-1456. We conducted a series of experiments to compare the survival of Escherichia coli, feline calicivirus, and F-specific coliphage MS2 on lettuce and cabbage with and without disinfection. Inoculated produce was held at 4, 25, or 37ºC for 21 days or was treated with different concentrations of sodium bicarbonate, chlorine bleach, peroxyacetic acid, or hydrogen peroxide. Survival was measured by the decimal reduction value (time to 90% reduction in titer) and the change in log titers of the test organisms. A stronger correlation of survival measures was observed between feline calicivirus and MS2 than between E. coli and either of the viral agents at 25 and 37ºC. The maximum time to detection limit for MS2 at all temperatures was 9 days, whereas feline calicivirus was detected for a maximum of 14 days at 4ºC. In contrast, E. coli was detectable for 21 days at 4 and 25ºC and for 14 days at 37ºC. Significant increases in E. coli titer occurred within the first 5 days, but virus titers decreased steadily throughout the experiments. E. coli was also highly susceptible to all disinfectants except 1% sodium bicarbonate and 50 ppm chlorine bleach, whereas the viruses were resistant to all four disinfectants. [TOP OF PAGE]

  474. Micro-organism re-growth in wastewater disinfected by UV radiation and ozone: a micro-biological study. Alonso,E., Santos,A., Riesco,P. (2004). Environ. Technol. 25:433-441. A series of disinfection experiments using UV radiation and ozone was performed on the secondary effluent from a wastewater treatment plant at a pilot plant scale. The microbial population in the inflowing wastewater and the treated outflow water were quantified for each of the treatment modules (fecal coliforms, fecal streptococci, Salmonella spp. (presence/absence), Clostridium Sulphite-reducers, Pseudomonas aeruginosa, Staphylococcus aureus, coliphages, nematodes, intestinal nematodes and pathogenic fungi). Treated water was stored in opaque tanks at a temperature between 20 and 22°C, after which, a one-month study of the regrowth of the bacterial flora, nematodes and fungi was carried out. Clostridium Sulphite-reducers, pathogenic fungi and nematodes were the micro-organisms showing a greatest degree of resistence to UV- and Ozone-treatment. It was only concerning Clostridium and Pseudomonas abatement that significant elimination results were achieved with both technologies. [TOP OF PAGE]

  475. Cyanophage infection and photoinhibition in marine cyanobacteria. Bailey,S., Clokie,M.R.J., Millard,A., Mann,N.H. (2004). Res. Microbiol. 155:720-725. Members of two cyanobacterial genera, Synechococcus and Prochlorococcus, are dominant within the prokaryotic component of the picophytoplankton and contribute significantly to global photosynthetic productivity. These organisms are known to be susceptible to infection by bacteriophages (viruses that infect bacteria) and it is believed that phage infection in the oceans has exerted selective pressures on the evolution of both phage and host and continues to influence community structure. Understanding of the processes of host-phage interaction within the marine environment is limited; however, new insights have arisen from sequence analysis of the genome of the bacteriophage S-PM2, which infects Synechococcus strains. The phage was found to encode homologs of the key photosystem II reaction center core polypeptides, D1 and D2. These reaction center polypeptides are known to be rapidly turned over in uninfected cells in a repair cycle that helps to protect oxygenic phototrophs against photoinhibition. This finding suggests that bacteriophages infecting marine cyanobacteria may play an active role in protecting their hosts against photoinhibition, thereby ensuring an energy supply for replication by preventing the deleterious effects on host cell integrity seen during acute photoinhibition. [TOP OF PAGE]

  476. Viral activity in two contrasting lake ecosystems. Bettarel,Y., Sime-Ngando,T., Amblard,C., Dolan,J. (2004). Appl. Environ. Microbiol. 70:2941-2951. For aquatic systems, especially freshwaters, there is little data on the long-term (i.e., >6-month period) and depth-related variability of viruses. In this study, we examined virus-induced mortality of heterotrophic bacteria over a 10-month period and throughout the water column in two lakes of the French Massif Central, the oligomesotrophic Lake Pavin and the eutrophic Lake Aydat. Concurrently, we estimated nonviral mortality through heterotrophic nanoflagellate and ciliate bacterivory. Overall, viral infection parameters were much less variable than bacterial production. We found that the frequency of visibly infected cells (FVIC), estimated using transmission electron microscopy, peaked in both lakes at the end of spring (May to June) and in early autumn (September to October). FVIC values were significantly higher in Lake Pavin (mean [M] = 1.6%) than in Lake Aydat (M = 1.1%), whereas the opposite trend was observed for burst sizes, which averaged 25.7 and 30.2 virus particles bacterium(-1), respectively. We detected no significant depth-related differences in FVIC or burst size. We found that in both lakes the removal of bacterial production by flagellate grazing (M(Pavin) = 37.7%, M(Aydat) = 18.5%) was nearly always more than the production removed by viral lysis (M(Pavin) = 16.2%, M(Aydat) = 19%) or ciliate grazing (M(Pavin) = 2.7%, M(Aydat) = 8.8%). However, at specific times and locations, viral lysis prevailed over protistan grazing, for example, in the anoxic hypolimnion of Lake Aydat. In addition, viral mortality represented a relatively constant mortality source in a bacterial community showing large variations in growth rate and subject to large variations in loss rates from grazers. Finally, although viruses did not represent the main agent of bacterial mortality, our data seem to show that their relative importance was higher in the less productive system. [TOP OF PAGE]

  477. Smarter than the average phage. Blakely,G.W. (2004). Mol. Microbiol. 54:851-854. The seventh cholera pandemic emerged in the poorer nations of the world towards the end of the 20th century and continues to kill thousands of people per year. The causative agent of cholera, the Gram-negative bacterium Vibrio cholera, is only pathogenic when it contains a lysogenic bacteriophage, CTXphi, that encodes the toxin responsible for inducing massive fluid loss from the human host. Site-specific integration of CTXphi into chromosome I of V. cholera occurs at a site, dif, that is normally required for resolution of chromosome dimers generated by homologous recombination. An article in this issue of Molecular Microbiology reports the analysis of interactions between two host encoded recombinases, XerC and XerD, and the recombination sites involved in lysogeny. Surprisingly, recombination between the CTXphi attP site and the chromosomal dif site requires additional recombinase binding sites, downstream from the positions of strand exchange, which might play an architectural role. The positions of strand cleavage also differ significantly between the two sites, suggesting a novel recombination mechanism that implicates additional host factors in resolution of the Holliday junction intermediate. [TOP OF PAGE]

  478. Tracking the origin of faecal pollution in surface water: an ongoing project within the European Union research programme. Blanch,A.R., Belanche-Munoz,L., Bonjoch,X., Ebdon,J., Gantzer,C., Lucena,F., Ottoson,J., Kourtis,C., Iversen,A., Kuhn,I., Moce,L., Muniesa,M., Schwartzbrod,J., Skraber,S., Papageorgiou,G., Taylor,H.D., Wallis,J., Jofre,J. (2004). Water Hlth. 2:249-260. The objectives of this study are to generate knowledge about methods to track the sources of faecal pollution in surface waters, with the aim of having one or a few easy procedures applicable to different geographic areas in Europe. For this, a first field study using already proposed methods (genotypes of F-specific RNA bacteriophages, bacteriophages infecting Bacteroides fragilis, phenotypes of faecal coliforms and enterococci, and sterols) has been done in five areas representing a wide array of conditions in Europe. The present faecal indicators (faecal coliforms, enterococci, sulfite reducing clostridia and somatic coliphages) have also been included in this first field study. At the same time some emerging methods have been settled or adapted to water samples and assayed in a limited number of samples. The results of this first field study indicate that no single parameter alone is able to discriminate the sources, human or non-human, of faecal pollution, but that a 'basket' of 4 or 5 parameters, which includes one of the present faecal indicators, will do so. In addition, numerical analysis of the data shows that this 'basket' will allow the successful building of predictive models. Both the statistical analyses and the studied predictive models indicate that genotype II of F-specific RNA bacteriophages, the coprostanol and the ratio coprostanol: coprostanol+epicoprostanol are, out of the studied parameters, those with a greater discriminating power. Either because unsuccessful adaptation of the methods to water samples or because the preliminary assays in water samples indicated low discriminating capability, only three (sorbitol-fermenting bifidobacteria, some species of bifidobacteria detected by PCR with specific primers and phages infecting Bacteroides tethaiotaomicron) of the newly assayed methods have been considered for a second field study, which is currently underway. Expectations are that these new tools will minimize the number of parameters in the 'basket', or at least minimize the difficulty in assaying them. [TOP OF PAGE]

  479. A is for adaptation. Boeke,J.D. (2004). Nature 431:408-409. Studies of a bacterial virus have revealed an unexpected weapon that helps it to overcome its host's rapidly changing defences. A look at
    other organisms hints that the mechanism might be widespread. [TOP OF PAGE]

  480. Preparation of endotoxin-free bacteriophages. Boratynski,J., Syper,D., Weber-Dabrowska,B., Lusiak-Szelachowska,M., Pozniak,G., Gorski,A. (2004). Cellular & molecular biology letters 9:253-259. Bacteriophages (phages) are bacterial viruses that interact with bacterial walls and invade bacterial cells. Moreover, they disturb bacterial metabolism and lead to bacteria lysis. In the case of Gram-negative bacteria crude phage cultures, apart from the phages themselves, the bacterial debris, bacterial proteins and nucleic acids contain endotoxins. These endotoxins (lipopolysaccharides) posses a high degree of toxicity in vitro and in vivo, and their removal is essential for safety in antibacterial bacteriophage therapy. An effective, scaleable purification of bacteriophages from endotoxins was accomplished by sequential ultrafiltration through polysulfone membrane (30 nm) followed by chromatography on sepharose 4B and Matrex Cellulofine Sulfate. The phage fraction after gel filtration chromatography routinely contained endotoxins in the 150-2500 EU/ml range. The procedure yielded bacteriophages contaminated with as little as 0.4-7 EU/ml (Limulus assay). This value lies within the permitted level for intravenous applications (5 EU/kg/h by European Pharmacopoeia, 1997). [TOP OF PAGE]

  481. Vulnerability of drinking-water wells in La Crosse, Wisconsin, to enteric-virus contamination from surface water contributions. Borchardt,M.A., Haas,N.L., Hunt,R.J. (2004). Appl. Environ. Microbiol. 70:5937-5946. Human enteric viruses can contaminate municipal drinking-water wells, but few studies have examined the routes by which viruses enter these wells. In the present study, the objective was to monitor the municipal wells of La Crosse, Wisconsin, for enteric viruses and determine whether the amount of Mississippi River water infiltrating the wells was related to the frequency of virus detection. From March 2001 to February 2002, one river water site and four wells predicted by hydrogeological modeling to have variable degrees of surface water contributions were sampled monthly for enteric viruses, microbial indicators of sanitary quality, and oxygen and hydrogen isotopes. (18)O/(16)O and (2)H/(1)H ratios were used to determine the level of surface water contributions. All samples were collected prior to chlorination at the wellhead. By reverse transcription-PCR (RT-PCR), 24 of 48 municipal well water samples (50%) were positive for enteric viruses, including enteroviruses, rotavirus, hepatitis A virus (HAV), and noroviruses. Of 12 river water samples, 10 (83%) were virus positive by RT-PCR. Viable enteroviruses were not detected by cell culture in the well samples, although three well samples were positive for culturable HAV. Enteroviruses detected in the wells by RT-PCR were identified as several serotypes of echoviruses and group A and group B coxsackieviruses. None of the well water samples was positive for indicators of sanitary quality, namely male-specific and somatic coliphages, total coliform bacteria, Escherichia coli, and fecal enterococci. Contrary to expectations, viruses were found in all wells regardless of the level of surface water contributions. This result suggests that there were other unidentified sources, in addition to surface water, responsible for the contamination. [TOP OF PAGE]

  482. Bacteriophage Flux in Endosymbionts (Wolbachia): Infection Frequency, Lateral Transfer, and Recombination Rates. Bordenstein,S.R., Wernegreen,J.J. (2004). Mol. Biol. Evol. 21:1981-1991. The highly specialized genomes of bacterial endosymbionts typically lack one of the major contributors of genomic flux in the free-living microbial world-bacteriophages. This study yields three results that show bacteriophages have, to the contrary, been influential in the genome evolution of the most prevalent bacterial endosymbiont of invertebrates, Wolbachia. First, we show that bacteriophage WO is more widespread in Wolbachia than previously recognized, ccurring in at least 89% (35/39) of the sampled genomes. Second, we show through several phylogenetic approaches that bacteriophage WO underwent recent lateral transfers between Wolbachia bacteria that coinfect host cells in the dipteran Drosophila simulans and the hymenopteran Nasonia vitripennis. These two cases, along with a previous report in the lepidopteran Ephestia cautella, support a general mechanism for genetic exchange in endosymbionts-the ''intracellular arena'' hypothesis-in which genetic material moves horizontally between bacteria that coinfect the same intracellular environment. Third, we show recombination in this bacteriophage; in the region encoding a putative capsid protein, the recombination rate is faster than that of any known recombining genes in the endosymbiont genome. The combination of these three lines of genetic evidence indicates that this bacteriophage is a widespread source of genomic instability in the intracellular bacterium Wolbachia and potentially the invertebrate host. More generally, it is the first bacteriophage implicated in frequent lateral transfer between the genomes of bacterial endosymbionts. Gene transfer by bacteriophages could drive significant evolutionary change in the genomes of intracellular bacteria that are typically considered highly stable and prone to genomic degradation. [TOP OF PAGE]

  483. "My enemy's enemy is my friend." Using phages to fight bacteria. Bradbury,J. (2004). Lancet 363:624-625. [first paragraph] Bacteriophages, viruses that prey upon bacteria, typically attack only a single bacterial strain. This specificity, together with the killing capacity of "phages", says phage researcher Martin Loessner, makes them the "natural enemies" of bacteria. "We are now endeavouring to make this enemy our friend", says Loessner, a professor of food microbiology at the Swiss Federal Institute of Technology in Zurich, turning phages into potentially important allies in our battle against bacteria. [TOP OF PAGE]

  484. Phage community dynamics in hot springs. Breitbart,M., Wegley,L., Leeds,S., Schoenfeld,T., Rohwer,F. (2004). Appl. Environ. Microbiol. 70:1633-1640. In extreme thermal environments such as hot springs, phages are the only known microbial predators. Here we present the first study of prokaryotic and phage community dynamics in these environments. Phages were abundant in hot springs, reaching concentrations of a million viruses per milliliter. Hot spring phage particles were resistant to shifts to lower temperatures, possibly facilitating DNA transfer out of these extreme environments. The phages were actively produced, with a population turnover time of 1 to 2 days. Phage-mediated microbial mortality was significant, making phage lysis an important component of hot spring microbial food webs. Together, these results show that phages exert an important influence on microbial community structure and energy flow in extreme thermal environments. [TOP OF PAGE]

  485. Global distribution of nearly identical phage-encoded DNA sequences. Breitbart,M., Miyake,J.H., Rohwer,F. (2004). FEMS Microbiol. Lett. 236:249-256. Phages, the most abundant biological entities on the planet, play important roles in biogeochemical cycling, horizontal gene transfer, and defining microbial community composition. However, very little is known about phage diversity or biogeography, and there has not yet been a systematic effort to compare the phages found in different ecosystems. Here, we report that T7-like Podophage DNA polymerase sequences occur in every major biome investigated, including marine, freshwater, sediment, terrestrial, extreme, and metazoan-associated. The majority of these sequences belong to a unique clade that is only distantly related to cultured isolates. Some identical T7-like phage-encoded DNA polymerase genes from this clade were >99% conserved at the nucleotide level in multiple different environments, suggesting that these phages are moving between biomes in recent evolutionary time and that the global genomic pool for T7-like phages may be smaller than previously hypothesized. [TOP OF PAGE]

  486. Diversity and population structure of a near-shore marine sediment viral community. Breitbart,M., Felts,B., Kelley,S., Mahaffy,J.M., Nulton,J., Salamon,P., Rohwer,F. (2004). Proc. R. Soc. Lond. B Biol. Sci. 271:565-574. Viruses, most of which are phage, are extremely abundant in marine sediments, yet almost nothing is known about their identity or diversity. We present the metagenomic analysis of an uncultured near-shore marine-sediment viral community. Three-quarters of the sequences in the sample were not related to anything previously reported. Among the sequences that could be identified, the majority belonged to double-stranded DNA phage. Temperate phage were more common than lytic phage, suggesting that lysogeny may be an important lifestyle for sediment viruses. Comparisons between the sediment sample and previously sequenced seawater viral communities showed that certain phage phylogenetic groups were abundant in all marine viral communities, while other phage groups were under-represented or absent. This 'marineness' suggests that marine phage are derived from a common set of ancestors. Several independent mathematical models, based on the distribution of overlapping shotgun sequence fragments from the library, were used to show that the diversity of the viral community was extremely high, with at least 10(4) viral genotypes per kilogram of sediment and a Shannon index greater than 9 nats. Based on these observations we propose that marine-sediment viral communities are one of the largest unexplored reservoirs of sequence space on the planet. [TOP OF PAGE]

  487. Comparison of bacteriophages for use in iodine inactivation: batch and continuous flow studies. Brion,G.M., O'Banion,N.B., Marchin,G.L. (2004). Water Hlth. 2:261-266. Inactivation rates in batch studies for four commonly used surrogate bacteriophages were measured in stable aqueous iodine solutions for the purpose of determining which was the most suited to evaluate iodine disinfection efficacy in batch and continuous flow conditions. Two types of group Leviviridae bacteriophages were used, Type I (MS2) and Type II (GA), along with group Microviridae, Phi-X174, and group Tectiviridae, PRD1. Inactivation was compared at iodine doses of 1.0-1.5 mg l2/l. MS2 was the most susceptible to iodine inactivation of the four phages tested. Inactivation of naked, icosahedral bacteriophages, MS2 and Phi-X174 demonstrated removals to below detection limits (>99.99%) in less than 10 min. Lipid-containing PRD1 and F+ssRNA GA bacteriophages demonstrated the greatest iodine resistance in batch experiments with an average of 1.82 logs of inactivation (98.5%) after 60 min and 1.05 logs of inactivation (91.1%) after 30 min respectively. Similarly, in continuous flow studies through pentaiodide quaternary ammonium strong base resin, MS2, GA and Phi-X174 were more strongly inactivated than PRD1. The lipid component of PRD1 is thought to enhance resistance to iodine over non-lipid-containing bacteriophages by protecting easily oxidized groups on the protein capsid, but further research is needed before proving this hypothesis. The results from this research may provide a surrogate standard for more rigorous and developed research into the mode of iodine disinfection and its inactivation kinetics. [TOP OF PAGE]

  488. The effect of spatial heterogeneity and parasites on the evolution of host diversity. Brockhurst,M.A., Rainey,P.B., Buckling,A. (2004). Proc. R. Soc. Lond. B Biol. Sci. 271:107-111. Both spatial heterogeneity and exploiters (parasites and predators) have been implicated as key ecological factors driving population diversification. However, it is unclear how these factors interact. We addressed this question using the common plant-colonizing bacterium Pseudomonas fluorescens, which has been shown to diversify rapidly into spatial niche-specialist genotypes when propagated in laboratory microcosms. Replicate populations were evolved in spatially homogeneous and heterogeneous environments (shaken and static microcosms, respectively) with and without viral parasites (bacteriophage) for approximately 60 bacterial generations. Consistent with previous findings, exploiters reduced diversity in heterogeneous environments by relaxing the intensity of resource competition. By contrast, exploiters increased diversity in homogeneous environments where there was little diversification through resource competition. Competition experiments revealed this increase in diversity to be the result of fitness trade-offs between exploiter resistance and competitive ability. In both environments, exploiters increased allopatric diversity, presumably as a result of divergent selection for resistance between populations. Phage increased total diversity in homogeneous environments, but had no net effect in heterogeneous environments. Such interactions between key ecological variables need to be considered when addressing diversification and coexistence in future studies. [TOP OF PAGE]

  489. Bioaerosols from the land application of biosolids in the desert southwest USA. Brooks,J.P., Tanner,B.D., Josephson,K.L., Gerba,C.P., Pepper,I.L. (2004). Water Sci. Technol. 50:7-12. This study evaluated bioaerosol emissions during land application of Class B biosolids in and around Tucson, Arizona, to aid in developing models of the fate and transport of bioaerosols generated from the land application of biosolids. Samples were collected for 20 min at distances between 2 m and 20 m downwind of point sources, using an SKC BioSampler impinger. A total of six samples were collected per sampling event, which consisted of a biosolid spray applicator applying liquid biosolids to a cotton field. Each application represented one exposure. Samples were collected in deionised water amended with peptone and antifoam agent. Ambient weather conditions were also monitored every 10 min following initiation of sampling. Concurrently with downwind samples, background (ambient) air samples were collected to compensate for any ambient airborne microorganisms. In addition, biosolids samples were collected for analysis of target indicator and pathogenic organisms. Soil samples were also collected and analysed. Significant numbers of heterotrophic plate count (HPC) bacteria were found in air samples collected during the biosolid application process. These could have arisen from soil particles being aerosolised during the land application process. Aerosolised soil may contribute significantly to the amount of aerosolised microorganisms. Soil particles may be able to more readily aerosolise, due to their low density, small particle size and low mass. Aerosolised HPC bacteria found during biosolids land application were similar to those found during normal tractor operation on non-biosolids applied fields. Coliforms and coliphages were not routinely detected even though they were found to be present in the biosolids at relatively high concentrations, 10(6) and 10(4)/g (dry weight) of biosolids respectively. This could be due to the die-off rate of aerosolised Gram-negative bacteria or sorption to the solid portion of the biosolids. Low numbers of aerosolised coliphages may likewise be due to sorption phenomena. We theorise that only organisms in the aqueous phase of the biosolids were available to desorb and be aerosolised. Animal viruses, which were not detected in the biosolids, were likewise not detected in the aerosol samples. Clostridium perfringens was detected in only a small percent of aerosol samples although it was detected during all weather conditions; other microorganisms were detected during more favourable environmental conditions (relative humidity >10%). Despite the fact that many of these organisms were present in the biosolids at significant concentrations, their presence in bioaerosols generated during the land application of biosolids was limited to only a small percentage of samples. Bacteria as well as viruses may sorb to biosolids, which contain a high percentage of organic matter, and desorption during land application of biosolids may not readily take place; therefore, these microorganisms may not be readily aerosolised. [TOP OF PAGE]

  490. The discovery of a novel algal virus type infection of the phytoplankter Micromonas pusilla by a double stranded RNA reovirus. Brussard,C.P.D., Sandaa,R.-A., Bratbak,G. (2004). Virology 319:280-291. We report the isolation of the first double-stranded (ds) RNA virus in the family Reoviridae that infects a protist (microalga Micromonas pusilla, Prasinophyceae). The dsRNA genome was composed of 11 segments ranging between 0.8 and 5.8 kb, with a total size of approximately 25.5 kb. The virus (MpRNAV-01B) could not be assigned to the genus level because host type, genome size, and number of segments smaller than 2 kb did not correspond to either of the two existing 11-segmented dsRNA genera Rotavirus and Aquareovirus. MpRNAV-01B has a particle size of 65-80 nm, a narrow host range, a latent period of 36 h, and contains five major proteins (120, 95, 67, 53, and 32 kDa). MpRNAV-01B was stable to freeze-thawing, resistant to chloroform, ether, nonionic detergents, chelating and reducing agents. The virus was inactivated at temperatures above 35ºC and by ionic detergent, ethanol, acetone, and acidic conditions (pH 2-5). [TOP OF PAGE]

  491. Phages and the evolution of bacterial pathogens: from genomic rearrangements to lysogenic conversion. Brüssow,H., Canchaya,C., Hardt,W.D. (2004). Microbiol. Mol. Biol. Rev. 68:560-602, table. Comparative genomics demonstrated that the chromosomes from bacteria and their viruses (bacteriophages) are coevolving. This process is most evident for bacterial pathogens where the majority contain prophages or phage remnants integrated into the bacterial DNA. Many prophages from bacterial pathogens encode virulence factors. Two situations can be distinguished: Vibrio cholerae, Shiga toxin-producing Escherichia coli, Corynebacterium diphtheriae, and Clostridium botulinum depend on a specific prophage-encoded toxin for causing a specific disease, whereas Staphylococcus aureus, Streptococcus pyogenes, and Salmonella enterica serovar Typhimurium harbor a multitude of prophages and each phage-encoded virulence or fitness factor makes an incremental contribution to the fitness of the lysogen. These prophages behave like "swarms" of related prophages. Prophage diversification seems to be fueled by the frequent transfer of phage material by recombination with superinfecting phages, resident prophages, or occasional acquisition of other mobile DNA elements or bacterial chromosomal genes. Prophages also contribute to the diversification of the bacterial genome architecture. In many cases, they actually represent a large fraction of the strain-specific DNA sequences. In addition, they can serve as anchoring points for genome inversions. The current review presents the available genomics and biological data on prophages from bacterial pathogens in an evolutionary framework. [TOP OF PAGE]

  492. Isolation and characterization of a generalized transducing phage for Pseudomonas aeruginosa strains PAO1 and PA14. Budzik,J.M., Rosche,W.A., Rietsch,A., O'Toole,G.A. (2004). J. Bacteriol. 186:3270-3273. A temperate, type IV pilus-dependent, double-stranded DNA bacteriophage named DMS3 was isolated from a clinical strain of Pseudomonas aeruginosa. A clear-plaque variant of this bacteriophage was isolated. DMS3 is capable of mediating generalized transduction within and between P. aeruginosa strains PA14 and PAO1, thus providing a useful tool for the genetic analysis of P. aeruginosa. [TOP OF PAGE]

  493. Genetic details, optimization, and phage life histories. Bull,J.J., Pfening,D.W., Wang,I.-W. (2004). Trends Ecol. Evol. 19:76-82. Optimality models assume that phenotypes evolve by natural selection largely independently of underlying genetic mechanisms. This neglect of genetic mechanisms is considered an advantage by some evolutionary biologists but a fatal flaw by others. The controversy has gone unresolved, in part, from a lack of complex phenotypes that meet optimality criteria and for which the underlying genetic mechanisms are known. Here, we look at both perspectives for lysis time in bacteriophages. We find that the basic assumptions of the optimality model are compatible with the genetic details, but the optimality model is limited in its ability to accommodate lysis time plasticity because the mechanistic underpinnings of plasticity are poorly known. [TOP OF PAGE]

  494. Genome properties and the limits of adaptation in bacteriophages. Bull,J.J., Badgett,M.R., Springman,R., Molineux,I.J. (2004). Evolution 58:692-701. Eight bacteriophages were adapted for rapid growth under similar conditions to compare their evolved, endpoint fitnesses. Four pairs of related phages were used, including two RNA phages with small genomes (MS2 and Qb) two single-stranded DNA phages with small genomes (fX174 and G4), two T-odd phages with medium-sized, double-stranded DNA genomes (T7 and T3), and two T-even phages with large, double-stranded DNA genomes (T6 and RB69). Fitness was measured as absolute growth rate per hour under the same conditions used for adaptation. T7 and T3 achieved the highest fitnesses, able to increase by 13 billionfold and three-quarters billionfold per hour, respectively. In contrast, the RNA phages achieved low fitness maxima, with growth rates approximately 400-fold and 4000-fold per hour. The highest fitness limits were not attributable to high mutation rates or small genome size, even though both traits are expected to enhance adaptation for fast growth. We suggest that major differences in fitness limits stem from different "global" constraints, determined by the organization and composition of the phage genome affecting whether and how it overcomes potentially rate-limiting host processes, such as transcription, translation, and replication. Adsorption rates were also measured on the evolved phages. No consistent pattern of adsorption rate and fitness was observed across the four different types of phages, but within each pair of related phages, higher adsorption was associated with higher fitness. Different adsorption rate limits within pairs may stem from "local" constraints-sequence differences leading to different local optima in the sequence space. [TOP OF PAGE]

  495. Epistasis and Its relationship to canalization in the RNA virus f6. Burch,C.L., Chao,L. (2004). Genetics 167:559-567. Although deleterious mutations are believed to play a critical role in evolution, assessing their realized effect has been difficult. A key parameter governing the effect of deleterious mutations is the nature of epistasis, the interaction between the mutations. RNA viruses should provide one of the best systems for investigating the nature of epistasis because the high mutation rate allows a thorough investigation of mutational effects and interactions. Nonetheless, previous investigations of RNA viruses by S. Crotty and co-workers and by S. F. Elena have been unable to detect a significant effect of epistasis. Here we provide evidence that positive epistasis is characteristic of deleterious mutations in the RNA bacteriophage 6. We estimated the effects of deleterious mutations by performing mutation-accumulation experiments on five viral genotypes of decreasing fitness. We inferred positive epistasis because viral genotypes with low fitness were found to be less sensitive to deleterious mutations. We further examined environmental sensitivity in these genotypes and found that low-fitness genotypes were also less sensitive to environmental perturbations. Our results suggest that even random mutations impact the degree of canalization, the buffering of a phenotype against genetic and environmental perturbations. In addition, our results suggest that genetic and environmental canalization have the same developmental basis and finally that an understanding of the nature of epistasis may first require an understanding of the nature of canalization. [TOP OF PAGE]

  496. Use of aqueous silver to enhance inactivation of coliphage MS-2 by UV disinfection. Butkus,M.A., Labare,M.P., Starke,J.A., Moon,K., Talbot,M. (2004). Appl. Environ. Microbiol. 70:2848-2853. A synergistic effect between silver and UV radiation has been observed that can appreciably enhance the effectiveness of UV radiation for inactivation of viruses. At a fluence of ca. 40 mJ/cm(2), the synergistic effect between silver and UV was observed at silver concentrations as low as 10 microg/liter (P < 0.0615). At the same fluence, an MS-2 inactivation of ca. 3.5 logs (99.97%) was achieved at a silver concentration of 0.1 mg/liter, a significant improvement (P < 0.0001) over the ca. 1.8-log (98.42%) inactivation of MS-2 at ca. 40 mJ/cm(2) in the absence of silver. Modified Chick-Watson kinetics were used to model the synergistic effect of silver and UV radiation. For an MS-2 inactivation of 4 logs (99.99%), the coefficient of dilution (n) was determined to be 0.31, which suggests that changes in fluence have a greater influence on inactivation than does a proportionate change in silver concentration. [TOP OF PAGE]

  497. Rapid diagnosis of pulmonary tuberculosis by mycobacteriophage assay. Butt,T., Ahmad,R.N., Kazmi,S.Y., Mahmood,A. (2004). Int. J. Tuberc. Lung Dis. 8:899-902. We evaluated FASTPlaqueTB, a recently introduced bacteriophage assay for rapid detection of Mycobacterium tuberculosis complex in sputum specimens, using 169 non-duplicate sputum specimens from patients suspected of pulmonary tuberculosis. The results of 160 specimens were analysed. FASTPlaqueTB assay detected tuberculosis in 77% (46/60) of culture-positive cases. Among the AFB smear-positive cases (n = 47) it had a sensitivity of 76% and specificity of 60% while among AFB smear-negative cases (n = 113) its sensitivity and specificity were 78% and 98%, respectively. The overall sensitivity and specificity of the technique were 77% and 96%, respectively, and the positive and negative predictive values were respectively 92% and 87%. The overall efficiency of the test was 89%. Test results were available in 48 h. [TOP OF PAGE]

  498. Recent pre-harvest supplementation strategies to reduce carriage and shedding of zoonotic enteric bacterial pathogens in food animals. Callaway,T.R., Anderson,R.C., Edrington,T.S., Genovese,K.J., Harvey,R.B., Poole,T.L., Nisbet,D.J. (2004). An. Hlth. Res. Rev. 5:35-47. Food-borne bacterial illnesses strike more than 76 million North Americans each year. Many of these illnesses are caused by animal-derived foodstuffs. Slaughter and processing plants do an outstanding job in reducing bacterial contamination after slaughter and during further processing, yet food-borne illnesses still occur at an unacceptable frequency. Thus, it is imperative to widen the window of action against pathogenic bacteria. Attacking pathogens on the farm or in the feedlot will improve food safety all the way to the consumer's fork. Because of the potential improvement in overall food safety that pre-harvest intervention strategies can provide, a broad range of preslaughter intervention strategies are currently under investigation. Potential interventions include direct anti-pathogen strategies, competitive enhancement strategies and animal management strategies. Included in these strategies are competitive exclusion, probiotics, prebiotics, antibiotics, antibacterial proteins, vaccination, bacteriophage, diet, and water trough interventions. The parallel and simultaneous application of one or more preslaughter strategies has the potential to synergistically reduce the incidence of human food-borne illnesses by erecting multiple hurdles, thus preventing entry of pathogens into the food chain. This review emphasizes work with Escherichia coli O157:H7 to illustrate the various strategies. [TOP OF PAGE]

  499. What are we doing about Escherichia coli O157:H7 in cattle? Callaway,T.R., Anderson,R.C., Edrington,T.S., Genovese,K.J., Bischoff,K.M., Poole,T.L., Jung,Y.S., Harvey,R.B., Nisbet,D.J. (2004). Journal of Animal Science 82 E-Suppl:E93-E99 Many human foodborne illnesses can be caused by consumption of foodstuffs (including meat products) contaminated with pathogenic bacteria from animal intestinal contents or hides. Steps that have been taken in the slaughter plant to decrease the spread of foodborne pathogenic bacteria (e.g., hazard analysis and critical control point methods) have been very effective; however, meat products are still the source of foodborne bacterial human illnesses. Increasing numbers of human Escherichia coli O157:H7 illnesses have also been related to contact with animals or to water supplies contaminated by run-off from cattle farms. Thus, strategies that specifically target foodborne pathogenic bacteria in the animal at the farm or feedlot level have great potential to improve food safety and decrease human illnesses. In this review, we describe a broad range of live-animal intervention strategies, both probiotic and antipathogen. Additionally, we examine some of the effects of diet and management strategies on foodborne pathogenic bacterial populations. The use of antibiotics in food animals to decrease foodborne pathogens also will be briefly examined. Overall, the concurrent use of several of these preslaughter intervention strategies could synergistically decrease human illnesses by providing for additional barriers in a multiple-hurdle approach to improving food safety. [TOP OF PAGE]

  500. The impact of prophages on bacterial chromosomes. Canchaya,C., Fournous,G., Brüssow,H. (2004). Mol. Microbiol. 53:9-18. Prophages were automatically localized in sequenced bacterial genomes by a simple semantic script leading to the identification of 190 prophages in 115 investigated genomes. The distribution of prophages with respect to presence or absence in a given bacterial species, the location and orientation of the prophages on the replichore was not homogeneous. In bacterial pathogens, prophages are particularly prominent. They frequently encoded virulence genes and were major contributors to the genetic individuality of the strains. However, some commensal and free-living bacteria also showed prominent prophage contributions to the bacterial genomes. Lysogens containing multiple sequence-related prophages can experience rearrangements of the bacterial genome across prophages, leading to prophages with new gene constellations. Transfer RNA genes are the preferred chromosomal integration sites, and a number of prophages also carry tRNA genes. Prophage integration into protein coding sequences can lead to either gene disruption or new proteins. The phage repressor, immunity and lysogenic conversion genes are frequently transcribed from the prophage. The expression of the latter is sometimes integrated into control circuits linking prophages, the lysogenic bacterium and its animal host. Prophages are apparently as easily acquired as they are lost from the bacterial chromosome. Fixation of prophage genes seems to be restricted to those with functions that have been co-opted by the bacterial host. [TOP OF PAGE]

  501. Thermal and chemical resistance of Lactobacillus casei and Lactobacillus paracasei bacteriophages. Capra,M.L., Quiberoni,A., Reinheimer,J.A. (2004). Lett. Appl. Microbiol. 38:499-504. AIMS: The survival of two collection Lactobacillus casei and L. paracasei bacteriophages when subjected to thermal and chemical treatments was investigated. METHODS AND RESULTS: Thermal resistance was evaluated by heating phage suspensions at 63, 72 and 90°C in three different media [Tris-magnesium gelatin (TMG) buffer: 10 mmol l(-1) Tris-Cl, 10 mmol l(-1) MgSO(4) and 0.1% w/v gelatin; Man Rogosa Sharpe (MRS) broth and reconstituted nonfat dry skim milk (RSM)]. A marked heat sensitivity was evident in both phages, as 15 min at 72°C was enough to completely inactivate (6 log(10) reduction) them. No clear influence was demonstrated by the suspension media. The phages also showed similar resistance to biocides. Peracetic acid and sodium hypochlorite (800 ppm) were the most effective ones, destroying the phages within 5 min. Concentrations of 75 and 100% ethanol were not suitable to inactivate phage particles even after 45 min. Isopropanol did not show an effect on phage viability. CONCLUSIONS: The data obtained in this work are important to design more effective control procedures in order to inactivate phages in dairy plants and laboratories. SIGNIFICANCE AND IMPACT OF THE STUDY: This work will contribute to enhance the background knowledge about phages of probiotic bacteria. [TOP OF PAGE]

  502. Treating cocaine addiction with viruses. Carrera,M.R., Kaufmann,G.F., Mee,J.M., Meijler,M.M., Koob,G.F., Janda,K.D. (2004). Proc. Natl. Acad. Sci. USA 101:10416-10421. Cocaine addiction continues to be a major health and social problem in the United States and other countries. Currently used pharmacological agents for treating cocaine abuse have proved inadequate, leaving few treatment options. An alternative is to use protein-based therapeutics that can eliminate the load of cocaine, thereby attenuating its effects. This approach is especially attractive because the therapeutic agents exert no pharmacodynamic action of their own and therefore have little potential for side effects. The effectiveness of these agents, however, is limited by their inability to act directly within the CNS. Bacteriophage have the capacity to penetrate the CNS when administered intranasally. Here, a method is presented for engineering filamentous bacteriophage to display cocaine-binding proteins on its surface that sequester cocaine in the brain. These antibody-displaying constructs were examined by using a locomotor activity rodent model to assess the ability of the phage-displayed proteins to block the psychoactive effects of cocaine. Results presented demonstrate a strategy in the continuing efforts to find effective treatments for cocaine addiction and suggest the application of this protein-based treatment for other drug abuse syndromes. [TOP OF PAGE]

  503. The chromosome of Shigella flexneri bacteriophage Sf6: complete nucleotide sequence, genetic mosaicism, and DNA packaging. Casjens,S., Winn-Stapley,D.A., Gilcrease,E.B., Morona,R., Kuhlewein,C., Chua,J.E.H., Manning,P.A., Inwood,W., Clark,A.J. (2004). J. Mol. Biol. 339:379-394. Shigella flexneri temperate bacteriophage Sf6 is of interest in part because its prophage expresses the oac gene that alters the antigenic properties of the surface O-antigen polysaccharide of its host bacterium. We have determined the complete sequence of its 39,044 bp genome. The sequence shows that Sf6 is a member of the canonical lambdoid phage group, and like other phages of this type has a highly mosaic genome. It has chromosomal regions that encode proteins >80% identical with at least 15 different previously characterized lambdoid phages and prophages, but 43% of the genome, including the virion assembly genes, is homologous to the genome of one phage, HK620. An analysis of the nucleotide differences between Sf6 and HK620 indicates that even these similar regions are highly mosaic. This mosaicism suggests ways in which the virion structural proteins might interact with each other. The Sf6 early operons are arranged like a typical lambdoid phage, with "boundary sequences" often found between functional modules in the "metabolic" genome domain. By virtue of high degree of similarity in the encoding genes and their DNA target sites, we predict that the integrase, early transcription anti-terminator, CI and Cro repressors, and CII protein of Sf6 have DNA binding specificities very similar to the homologous proteins encoded by phages HK620, lambda, 434 and P22, respectively. The late operon contains two tRNA genes. The Sf6 terminase genes are unusual. Analysis of in vivo initiation of the DNA packaging series showed that the Sf6 apparatus that recognizes DNA for packaging appears to cleave DNA for initiation of packaging series at many sites within a large region of about 1800 bp that includes a possible pac site. This is unlike previously characterized phage packaging mechanisms. [TOP OF PAGE]

  504. Pressure inactivation kinetics of phage l cI 857. Chen,H., Joerger,R.D., Kingsley,D.H., Hoover,D.G. (2004). J. Food Prot. 67:505-511. Inactivation curves of phage lambda cI 857 inactivated by high hydrostatic pressure were obtained at three pressure levels (300, 350, and 400 MPa) in buffered media and ultrahigh-temperature 2% reduced fat milk. Pressurization of phage lambda in buffered media at 300 MPa for 300 min, 350 MPa for 36 min, and 400 MPa for 8 min reduced the titer of phage lambda by 7.5, 6.7, and 7.7 log, respectively. Pressurization of phage lambda in milk at 300 MPa for 400 min, 350 MPa for 80 min, and 400 MPa for 20 min reduced the titer of phage lambda by 5.4, 6.4, and 7.1 log, respectively. Tailing was observed in all inactivation curves, indicating that the linear model was not adequate for describing these curves. Among the three nonlinear models studied, the Weibull and log-logistic models consistently produced best fits to all inactivation curves, and the modified Gompertz model the poorest. Because there were no significant differences in the values of shape factor (n) for suspension medium buffer, we reduced the number of parameters in the Weibull model from two to one by setting n at the mean value. The simplified Weibull model produced a fit comparable to the full model. Additionally, the simplified Weibull model allowed predictions to be made at pressures different from the experimental pressures. Menstruum was found to significantly affect the pressure resistance of phage lambda. Comparison of pressure inactivation of hepatitis A virus and phage lambda indicated that phage lambda is more sensitive to pressure than hepatitis A virus in Dulbecco's modified Eagle medium with 10% fetal bovine sera. [TOP OF PAGE]

  505. Phage-host interaction: an ecological perspective. Chibani-Chennoufi,S., Bruttin,A., Dillmann,M.L., Brüssow,H. (2004). J. Bacteriol. 186:3677-3686. [first three paragraphs] Nearly 100 years ago, Felix d'Herelle, the codiscoverer of bacteriophages, used bacteria to control insect pests and used phages against bacterial disease. His approaches reflected ecological insights before this branch of biology became an established scientific discipline. In fact, one might have predicted that phage research would become the springboard for biotechnology and ecology. However, d'Herelle was ahead of his time, and the zeitgeist in the 1930s pushed physicists into the question "What is life?" Phages as the simplest biological systems were the logical choice for this question, and phage research became the cradle of molecular biology. ¶ Now many researchers speak of a "new age of phage research." It is now realized that phages play an important role in ecology (e.g., phage impact on the cycling of organic matter in the biosphere at a global level) (27), that phages influence the evolution of bacterial genomes (most obviously in the development of bacterial pathogenicity) (7), and that phages might provide potential tools to face the antibiotic resistance crisis in medicine (59). With this new trend, we now see a clear shift from the reductionist approach, focusing on a handful of phages in carefully controlled laboratory conditions, towards the study of many different phages in the complexity of real-life situations. ¶ In contrast to the molecular biology-oriented phage research where the interaction of molecules took center stage, ecology focuses on the interactions between organisms and their physical environment. Much of ecology is therefore about the evolution of biological diversity in space and time. In contrast to many branches of biology, ecology attributes a great importance to quantitative relationships and numbers and aims at a mathematical formulation of its observations. It is thus appropriate to start this review with an overview of phage titers encountered in the biosphere. Next, we ask how a parasite targets its host if the latter is scarce or not in an appropriate physiological state. Finally, we report on research that tries to bridge phage ecology and genomics and cell biology approaches. It is concluded that the integration of phages into complex networks of interacting biological systems, and analysis by molecular techniques, could give phage research a model character in biology again. [TOP OF PAGE]

  506. Lactobacillus plantarum Bacteriophage LP65: a New Member of the SPO1-Like Genus of the Family Myoviridae. Chibani-Chennoufi,S., Dillmann,M.L., Marvin-Guy,L., Rami-Shojaei,S., Brüssow,H. (2004). The Journal of Bacteriology 186:7069-7083. The virulent Lactobacillus plantarum myophage LP65 was isolated from industrial meat fermentation. Tail contraction led to reorganization of the tail sheath and the baseplate; a tail tube was extruded. In ultrathin section the phage adsorbed via its baseplate to the exterior of the cell, while the tail tube tunneled through the thick bacterial cell wall. Convoluted membrane structures were induced in the infected cell. Progeny phage was detected 100 min postinfection, and lysis occurred after extensive digestion of the cell wall. Sequence analysis revealed a genome of 131,573 bp of nonredundant DNA. Four major genome regions and a large tRNA gene cluster were observed. One module corresponded to DNA replication genes. Helicase/primase and two replication/recombination enzymes represented the only links to T4-like Myoviridae from gram-negative bacteria. Another module corresponded to the structural genes. Sequence relatedness identified links with Listeria phage A511, Staphylococcus phage K, and Bacillus phage SPO1. LP65 structural proteins were identified by two-dimensional proteome analysis and mass spectrometry. The putative tail sheath protein showed a shear-induced change in electrophoretic migration behavior. The genome organization of the structural module in LP65 resembled that of Siphoviridae from the lambda supergroup. [TOP OF PAGE]

  507. Isolation of Escherichia coli bacteriophages from the stool of pediatric diarrhea patients in Bangladesh. Chibani-Chennoufi,S., Sidoti,J., Bruttin,A., Dillmann,M.L., Kutter,E., Qadri,F., Sarker,S.A., Brüssow,H. (2004). J. Bacteriol. 186:8287-8294. A 3-week coliphage survey was conducted in stool samples from 140 Bangladeshi children hospitalized with severe diarrhea. On the Escherichia coli indicator strain K803, all but one phage isolate had 170-kb genomes and the morphology of T4 phage. In spot tests, the individual T4-like phages infected up to 27 out of 40 diarrhea-associated E. coli, representing 22 O serotypes and various virulence factors; only five of them were not infected by any of these new phages. A combination of diagnostic PCR based on g32 (DNA binding) and g23 (major capsid protein) and Southern hybridization revealed that half were T-even phages sensu strictu, while the other half were pseudo-T-even or even more distantly related T4-like phages that failed to cross-hybridize with T4 or between each other. Nineteen percent of the acute stool samples yielded T4-like phages, and the prevalence was lower in convalescent stool samples. T4-like phages were also isolated from environmental and sewage water, but with low frequency and low titers. On the enteropathogenic E. coli strain O127:K63, 14% of the patients yielded phage, all of which were members of the phage family Siphoviridae with 50-kb genomes, showing the morphology of Jersey- and beta-4 like phages and narrow lytic patterns on E. coli O serotypes. Three siphovirus types could be differentiated by lack of cross-hybridization. Only a few stool samples were positive on both indicator strains. Phages with closely related restriction patterns and, in the case of T4-like phages, identical g23 gene sequences were isolated from different patients, suggesting epidemiological links between the patients. [TOP OF PAGE]

  508. Comparative genomics of the T4-Like Escherichia coli phage JS98: implications for the evolution of T4 phages. Chibani-Chennoufi,S., Canchaya,C., Bruttin,A., Brüssow,H. (2004). J. Bacteriol. 186:8276-8286. About 130 kb of sequence information was obtained from the coliphage JS98 isolated from the stool of a pediatric diarrhea patient in Bangladesh. The DNA shared up to 81% base pair identity with phage T4. The most conserved regions between JS98 and T4 were the structural genes, but their degree of conservation was not uniform. The head genes showed the highest sequence conservation, followed by the tail, baseplate, and tail fiber genes. Many tail fiber genes shared only protein sequence identity. Except for the insertion of endonuclease genes in T4 and gene 24 duplication in JS98, the structural gene maps of the two phages were colinear. The receptor-recognizing tail fiber proteins gp37 and gp38 were only distantly related to T4, but shared up to 83% amino acid identity to other T6-like phages, suggesting lateral gene transfer. A greater degree of variability was seen between JS98 and T4 over DNA replication and DNA transaction genes. While most of these genes came in the same order and shared up to 76% protein sequence identity, a few rearrangements, insertions, and replacements of genes were observed. Many putative gene insertions in the DNA replication module of T4 were flanked by intron-related endonuclease genes, suggesting mobile DNA elements. A hotspot of genome diversification was located downstream of the DNA polymerase gene 43 and the DNA binding gene 32. Comparative genomics of 100-kb genome sequence revealed that T4-like phages diversify more by the accumulation of point mutations and occasional gene duplication events than by modular exchanges. [TOP OF PAGE]

  509. In vitro and in vivo bacteriolytic activities of Escherichia coli phages: implications for phage therapy. Chibani-Chennoufi,S., Sidoti,J., Bruttin,A., Kutter,E., Sarker,S., Brüssow,H. (2004). Antimicrob. Agents Chemother. 48:2558-2569. Four T4-like coliphages with broad host ranges for diarrhea-associated Escherichia coli serotypes were isolated from stool specimens from pediatric diarrhea patients and from environmental water samples. All four phages showed a highly efficient gastrointestinal passage in adult mice when added to drinking water. Viable phages were recovered from the feces in a dose-dependent way. The minimal oral dose for consistent fecal recovery was as low as 10(3) PFU of phage per ml of drinking water. In conventional mice, the orally applied phage remained restricted to the gut lumen, and as expected for a noninvasive phage, no histopathological changes of the gut mucosa were detected in the phage-exposed animals. E. coli strains recently introduced into the intestines of conventional mice and traced as ampicillin-resistant colonies were efficiently lysed in vivo by phage added to the drinking water. Likewise, an in vitro phage-susceptible E. coli strain freshly inoculated into axenic mice was lysed in vivo by an orally applied phage, while an in vitro-resistant E. coli strain was not lysed. In contrast, the normal E. coli gut flora of conventional mice was only minimally affected by oral phage application despite the fact that in vitro the majority of the murine intestinal E. coli colonies were susceptible to the given phage cocktail. Apparently, the resident E. coli gut flora is physically or physiologically protected against phage infection. [TOP OF PAGE]

  510. Role of irrigation and wastewater reuse: comparison of subsurface irrigation and furrow irrigation. Choi,C., Song,I., Stine,S., Pimentel,J., Gerba,C. (2004). Water Sci. Technol. 50:61-68. Two different irrigation systems, subsurface drip irrigation and furrow irrigation, are tested to investigate the level of viral contamination and survival when tertiary effluent is used in arid and semi-arid regions. The effluent was injected with bacteriophages of PRD1 and MS2. A greater number of PRD1 and MS2 were recovered from the lettuce in the subsurface drip-irrigated plots as compared to those in the furrow-irrigated plots. Shallow drip tape installation and preferential water paths through cracks on the soil surface appeared to be the main causes of high viral contamination in subsurface drip irrigation plots, which led to the direct contact of the lettuce stems with the irrigation water which penetrated the soil surface. The water use efficiency of the subsurface drip irrigation system was higher than that of the furrow irrigation system. Thus, subsurface drip irrigation is an efficient irrigation method for vegetable crops in arid and semi-arid regions if viral contamination can be reduced. Deeper installation of drip tapes, frequent irrigations, and timely harvests based on cumulative heat units may further reduce health risks by ensuring viral die-off under various field conditions. [TOP OF PAGE]

  511. Bacteriophage MAV1 is not associated with virulence of Mycoplasma arthritidis. Clapper,B., Tu,A.H., Simmons,W.L., Dybvig,K. (2004). Infect. Immun. 72:7322-7325. Previous studies demonstrated that Mycoplasma arthritidis strain 158 acquired a high degree of virulence upon lysogenization with bacteriophage MAV1. In the present study, the association between MAV1 and virulence was reexamined by creating new lysogens of 158 and of a relatively avirulent mutant, strain 158-1. In the absence of lysogenization, 158 was more virulent than expected. The virulence of 158 and 158-1 did not increase upon lysogenization. A major antigenic difference between 158 and 158-1 was identified that is unrelated to MAV1 and could account for the difference in virulence. [TOP OF PAGE]

  512. The vir gene of bacteriophage MAV1 confers resistance to phage infection on Mycoplasma arthritidis. Clapper,B., Tu,A.H., Elgavish,A., Dybvig,K. (2004). J. Bacteriol. 186:5715-5720. Lysogenization of Mycoplasma arthritidis with the MAV1 bacteriophage increases the virulence of the mycoplasma in rats. The MAV1 vir gene is one of only two constitutively transcribed phage genes in the lysogen. We show here that Vir is a lipoprotein and is located on the outer surface of the cell membrane. To investigate whether Vir is a virulence factor, the vir gene was cloned into the transposon vector Tn4001T and inserted in the genome of the nonlysogen strain 158. The virulence of the resulting transformants was no different from that of the parent strain. Interestingly, all vir-containing transformants were resistant to infection by MAV1. Vir had no effect on MAV1 adsorption. We conclude that Vir is not a virulence factor but functions to exclude superinfecting phage, possibly by blocking the injection of phage DNA into the bacterial cytoplasm. [TOP OF PAGE]

  513. Bacterial viruses as human vaccines? Clark,J.R., March,J.B. (2004). Expert review of vaccines 3:463-476. Bacteriophages (or phages) are viruses of bacteria, consisting of nucleic acid packaged within a protein coat. In eukaryotic hosts, phages are unable to replicate and in the absence of a suitable prokaryotic host, behave as inert particulate antigens. In recent years, work has shown that whole phage particles can be used to deliver vaccines in the form of immunogenic peptides attached to modified phage coat proteins or as delivery vehicles for DNA vaccines, by incorporating a eukaryotic promoter-driven vaccine gene within their genome. While both approaches are promising by themselves, in future there is also the exciting possibility of creating a hybrid phage combining both components to create phage that are cheap, easy and rapid to produce and that deliver both protein and DNA vaccines via the oral route in the same construct. [TOP OF PAGE]

  514. Encapsidation of host DNA by bacteriophages infecting marine Synechococcus strains. Clokie,M.R., Millard,A.D., Wilson,W.H., Mann,N.H. (2004). FEMS Microbiol. Ecol. 46:349-352. It has been speculated that horizontal gene transfer might be important in the evolution of strains of the marine cyanobacterium Synechococcus and that phages might mediate this process, but until now there has been no direct evidence to support this idea. We have rigorously purified bacteriophages (cyanomyoviruses) from their Synechococcus host and performed a series of experiments on phage encapsidated DNA to reveal the presence of chromosomal Synechococcus DNA. Quantitative polymerase chain reaction has shown that V1 in 105 Synechococcus phage particles contain a host marker gene in their capsids. This is the first study that has shown that phages infecting marine Synechococcus strains can package host DNA and this provides evidence for the potential importance of these phage in horizontal gene transfer. [TOP OF PAGE]

  515. Genetic exchange in biofilms. Cvikovitch,D.G. (2004). pp. 192-205. In In Ghannoum,M. and O'Toole,G.A. (eds.), Microbial Biofilms. ASM Press, Washington, DC. [TOP OF PAGE]

  516. Anticancer activity of bacteriophage T4 and its mutant HAP1 in mouse experimental tumour models. Dabrowska,K., Opolski,A., Wietrzyk,J., Switala-Jelen,K., Godlewska,J., Boratynski,J., Syper,D., Weber-Dabrowska,B., Gorski,A. (2004). Anticancer research 24:3991-3995. BACKGROUND: Previously, we have shown the ability of the bacteriophage T4 and its substrain HAP1 (selected for a higher affinity to melanoma cells) to reveal antimetastatic activity in a mouse melanoma model. Here, we investigated the potential phage anticancer activity in primary tumour models. MATERIALS AND METHODS: Mice were inoculated subcutaneously with B16 or LLC cells (collected from in vitro culture). Bacteriophages T4 and HAP1 were injected intraperitoneally daily (8 x 10(8)pfu/mouse, except the experiment concerning the dose-dependence). RESULTS: Treatment with purified preparations of bacteriophage T4 resulted in significant reduction of tumour size, the effect being dose-dependent. HAP1 was more effective than T4 and its activity was also dose-dependent. Parallel experiments with non-purified bacteriophage lysates resulted in significant stimulation of tumour growth. CONCLUSION: These data suggest that purified bacteriophages may inhibit tumour growth, a phenomenon with potentially important clinical implications in oncology. [TOP OF PAGE]

  517. Antitumor activity of bacteriophages in murine experimental cancer models caused possibly by inhibition of b3 integrin signaling pathway. Dabrowska,K., Opolski,A., Wietrzyk,J., Switala-Jelen,K., Boratynski,J., Nasulewicz,A., Lipinska,L., Chybicka,A., Kujawa,M., Zabel,M., Dolinska-Krajewska,B., Piasecki,E., Weber-Dabrowska,B., Rybka,J., Salwa,J., Wojdat,E., Nowaczyk,M., Gorski,A. (2004). Acta virologica. English ed 48:241-248. Bacteriophages (phages) as bacterial viruses are generally believed to have no intrinsic tropism for mammalian cells. In this study the interactions between phages and various eukaryotic cells were investigated. Binding of phages to the membranes of cancer and normal blood cells was observed. Moreover, it was shown that the wild-type phage T4 (wtT4) and its substrain HAP1 with enhanced affinity for melanoma cells inhibit markedly and significantly experimental lung metastasis of murine B16 melanoma cells by 47% and 80%, respectively. A possible molecular mechanism of these effects, namely a specific interaction between the Lys-Gly-Asp motif of the phage protein 24 and b3-integrin receptors on target cells is proposed. It was also shown that anti-b3 antibodies and synthetic peptides mimicking natural b3 ligands inhibit the phage binding to cancer cells. This is in line with the well-described b3 integrin-dependent mechanism of tumor metastasis. It is concluded that the blocking of b3 integrins by phage preparations results in a significant decrease in tumor invasiveness. [TOP OF PAGE]

  518. Start-up entities in the origin of new genes. Daubin,V., Ochman,H. (2004). Cur. Opin. Gen. Devel. 14:616-619. The remarkable diversity in the contents of genomes raises questions about how new genes and new functions originate. Recent evidence indicates that parasitism, particularly the molecular interactions between phage and their bacterial hosts, is a likely mechanism for generating new genes. This invention of such novel functions seems to be founded on a strategy that secures the short-term survival of parasitic elements and thereby contributes to the renovation of gene repertoires in their host. [TOP OF PAGE]

  519. Bacterial sensitivity to bacteriophage in the aquatic environment. Day,M. (2004). Sci. Prog. 87:179-191. There are several unusual features about phage when you first encounter them as a biologist. They are small, but conform to one of a few morphological types. Next their genomes can be composed of DNA or RNA and be single or double stranded. Finally they are numerically more abundant than prokaryotes and a significant proportion of them form an association in their microbial host populations termed lysogeny. The latter findings indicate that they are numerically significant in microbial populations. Since bacterial and phage abundance or lack of it is related in environments, this implies that the phage populations 'titrate' their hosts, and more probably the host's physiological status. Microbial populations wax and wane with nutritional inputs and there is a dynamic relationship between phage population sizes and host numbers and physiology. Overlay this with the different phage life cycle strategies, exemplified at the extremes by phage lambda (temperate) and phage T4 (virulent), then it becomes apparent that phage are a component in nutrient cycling in ecology. But their contribution does not stop there. Many are capable of transduction, moving DNA from one cell into another. So they can also aid the evolutionary progress of microbial populations by allowing them to share genes, just as gene exchange via plasmids and transformation does. Our perception of bacteria has been derived from pure culture studies and we are just being able to appreciate how subtle their ecological interactions are. This is no less true of the studies on bacteriophage, which are almost all based on laboratory experimentation, where the hosts are physiologically stressed by growing in 'high nutritional and optimum conditions'. The natural environment is naturally discontinuous and life evolved in this. Thus our perceptions of bacteriophage and their life cycle patterns derived from laboratory experimentation may be a little off the mark when we come to understand how they and their hosts interact in the niches available to them. It is worth just considering this as you read the article, as I suspect phage behaviours are more intimately involved in, and moderated by the physiological stresses in the life cycle of bacteria than we currently believe. [TOP OF PAGE]

  520. Phage therapy: where East meets West. Debattista,J. (2004). Exp. Rev. Anti-Infect. Ther. 2:815-819. [first paragraph (may not be same as published)] The emergence of vancomycin-resistant enterococcus (VRE) has once more reminded health authorities and the general public of the narrow lead antibiotic therapy has afforded medical science over bacterial infection. Therefore, it is opportune at this time to recall that alternate nonchemical therapies have, in the past, been subject to considerable investigation. They are still currently being utilized in some countries, and are receiving renewed interest in others. Biological control through the application of host-specific bacteriophages as a means of treating bacterial infection predates the introduction of penicillin by almost 20 years, and has continued to attract interest as a potential alternative, or at least adjunct to conventional chemical bactericides. [TOP OF PAGE]

  521. Reduced fecundity is the cost of cheating in RNA virus f6. Dennehy,J.J., Turner,P.E. (2004). Proc. R. Soc. Lond. B Biol. Sci. 271:2275-2282. Co-infection by multiple viruses affords opportunities for the evolution of cheating strategies to use intracellular resources. Cheating may be costly, however, when viruses infect cells alone. We previously allowed the RNA bacteriophage f6 to evolve for 250 generations in replicated environments allowing coinfection of Pseudomonas phaseolicola bacteria. Derived genotypes showed great capacity to compete during co-infection, but suffered reduced performance in solo infections. Thus, the evolved viruses appear to be cheaters that sacrifice between-host fitness for within-host fitness. It is unknown, however, which stage of the lytic growth cycle is linked to the cost of cheating. Here, we examine the cost through burst assays, where lytic infection can be separated into three discrete phases (analogous to phage life history): dispersal stage, latent period (juvenile stage), and burst (adult stage). We compared growth of a representative cheater and its ancestor in environments where the cost occurs. The cost of cheating was shown to be reduced fecundity, because cheaters feature a significantly smaller burst size (progeny produced per infected cell) when infecting on their own. Interestingly, latent period (average burst time) of the evolved virus was much longer than that of the ancestor, indicating the cost does not follow a life history trade-off between timing of reproduction and lifetime fecundity. Our data suggest that interference competition allows high fitness of derived cheaters in mixed infections, and we discuss preferential encapsidation as one possible mechanism. [TOP OF PAGE]

  522. New dawn for phage therapy. Dixon,B. (2004). The Lancet infectious diseases 4:186 [first two paragraphs] Perhaps Antony Twort was 10 years too early in publishing his father Frederick's biography. A marvellous portrait of the eccentric co-discoverer of the bacteriophage, whose work helped to usher in the era of molecular biology, the book appeared only after numerous rejections from publishers (Lancet Infect Dis 2003; 3: 58). It also received little review attention, because literary editors are largely unaware of the role of science and scientists in shaping the modern world. ¶ However, the decade since publication of In Focus, Out of Step (Stroud, UK: Alan Sutton) has seen increasing interest in phages, especially in administering them therapeutically. Most recently there have been promising advances towards real applications. Now, thanks to work in Vienna, Austria, the major obstacle to phage therapy seems well on the way to being removed. At a time when antibiotic resistance is provoking real concern even in the most sober quarters, this is excellent news. [TOP OF PAGE]

  523. Contribution of the colmation layer to the elimination of coliphages by slow sand filtration. Dizer,H., Grutzmacher,G., Bartel,H., Wiese,H.B., Szewzyk,R., Lopez-Pila,J.M. (2004). Water Sci. Technol. 50:211-214. River bank or slow sand filtration is a major procedure for processing surface water to drinking water in central europe. In order to model the performance of river bank and slow sand filtration plants, we are studying the different mechanisms by which the elimination of pathogens is realized. An important question concerning the mode of action of slow sand filters and river bank filtration units is the role of the colmation layer or "schmutzdecke" on the elimination of human pathogens. The schmutzdecke is an organic layer which develops at the surface of the sand filter short after the onset of operation. We have inoculated a pilot plant for slow sand filtration with coliphages and determined their rate of breakthrough and their final elimination. In the first experiment, with a colmation layer still missing, the breakthrough of the coliphages in the 80 cm mighty sandy bed amounted to ca. 40 %. In contrast, less than 1 % of coliphages escaped from the filter as the same experiment was repeated two months later, when a substantial colmation layer had developed. Our preliminary conclusions are that the colmation layer is extremely efficient in eliminating of viruses. [TOP OF PAGE]

  524. Cyanophage diversity, inferred from g20 gene analyses, in the largest natural lake in France, Lake Bourget. Dorigo,U., Jacquet,S., Humbert,J.F. (2004). Appl. Environ. Microbiol. 70:1017-1022. The genetic diversity of the natural freshwater community of cyanophages and its variations over time have been investigated for the first time in the surface waters of the largest natural lake in France. This was done by random screening of clone libraries for the g20 gene and by denaturing gradient gel electrophoresis (DGGE). Nucleotide sequence analysis revealed 35 distinct cyanomyovirus g20 genotypes among the 47 sequences analyzed. Phylogenetic analyses showed that these sequences fell into seven genetically distinct operational taxonomic units (OTUs). The distances between these OTUs were comparable to those reported between marine clusters. Moreover, some of these freshwater cyanophage sequences were genetically more closely related to marine cyanophage sequences than to other freshwater sequences. Both approaches for the g20 gene (sequencing and DGGE analysis) showed that there was a clear seasonal pattern of variation in the composition of the cyanophage community that could reflect changes in its biological, chemical, and/or physical environment. [TOP OF PAGE]

  525. Tropism switching in Bordetella bacteriophage defines a family of diversity-generating retroelements. Doulatov,S., Hodes,A., Dai,L., Mandhana,N., Liu,M., Deora,R., Simons,R.W., Zimmerly,S., Miller,J.F. (2004). Nature 431:476-481. Bordetella bacteriophages generate diversity in a gene that specifies host tropism. This microevolutionary adaptation is produced by a genetic element that combines the basic retroelement life cycle of transcription, reverse transcription and integration with site-directed, adenine-specific mutagenesis. Central to this process is a reverse transcriptase-mediated exchange between two repeats; one serving as a donor template (TR) and the other as a recipient of variable sequence information (VR). Here we describe the genetic basis for diversity generation. The directionality of information transfer is determined by a 21-base-pair sequence present at the 3' end of VR. On the basis of patterns of marker transfer in response to variant selective pressures, we propose that a TR reverse transcript is mutagenized, integrated into VR as a single non-coding strand, and then partially converted to the parental VR sequence. This allows the diversity-generating system to minimize variability to the subset of bases under selection. Using the Bordetella phage cassette as a signature, we have identified numerous related elements in diverse bacteria. These elements constitute a new family of retroelements with the potential to confer selective advantages to their host genomes. [TOP OF PAGE]

  526. La vraie vie de Félix d Herelle avant la découverte du bactériophage. Dublanchet,A. (2004). Association de Anciens Elèves de l Institut Pasteur 45:80-82. [TOP OF PAGE]

  527. Identification of peptide sequences that induce the transport of phage across the gastrointestinal mucosal barrier. Duerr,D.M., White,S.J., Schluesener,H.J. (2004). J. Virol. Meth. 116:177-180. To investigate whether specific peptide sequences could induce virion transport across the intestinal barrier, we used phage display to both identify signalling peptides capable of inducing trans-intestinal transport and also provide a suitable model of virion translocation. We utilised simple, single-round high input in vivo biopanning protocol using a 7-mer random amino acid phage display library. Phage were applied by gavage and translocation across the intestinal barrier assessed by phage recovery from the spleen 2h later. Following isolation, a number of phage were sequenced and several homologies with HIV gp120 were identified. Immunocytochemical analysis of phage translocation across the intestinal barrier by a phage bearing the peptide YPRLLTP demonstrated that phage were actively transported along specific channels. It is concluded that utilisation of in vivo phage display (IVPD) has provided evidence for a specific peptide-guided transport of undegraded cargo across the intestinal barrier, modelled by M13 phage. [TOP OF PAGE]

  528. An evaluation of mycophage therapy, chemotherapy and vaccination for control of Mycobacterium avium subsp. paratuberculosis infection. Emery,D.L., Whittington,R.J. (2004). Vet Microbiol 104:143-155. The control of ovine Johne's disease (OJD) is important for domestic trade, the viability of farming units and possibly also for public health. Current strategies in Australia have included quarantine and pasture spelling to decrease prevalence and shedding rates and reduce numbers of Mycobacterium paratuberculosis (Mptb) ingested by susceptible sheep. However, alternative procedures are needed and vaccination with Gudair has recently commenced. This review examines prospects for the control of OJD by chemotherapy, vaccination and mycophages. Current chemotherapeutic regimes for treatment of M. paratuberculosis in ruminants are prohibitively expensive and of dubious efficacy, and apart from environmental concerns, mycophage therapy lacks a track record of success against intracellular bacteria. There is substantial evidence that live and killed mycobacterial vaccines reduce the incidence of clinical disease and shedding rates in OJD. An appraisal of recent experimental results suggests that neonatal vaccination with a defined dose of M. paratuberculosis offers the best prospects for the induction of protective Th1-type immunity. [TOP OF PAGE]

  529. Polylysogeny and prophage induction by secondary infection in Vibrio cholerae. Espeland,E.M., Lipp,E.K., Huq,A., Colwell,R.R. (2004). Environ. Microbiol. 6:760-763. Strains of Vibrio cholerae O1, biotypes El Tor and classical, were infected with a known temperate phage (PhiP15) and monitored over a 15-day period for prophage induction. Over the course of the experiment two morphologically and three genomically distinct virus-like particles were observed from the phage-infected El Tor strain by transmission electron microscopy and field inversion gel electrophoresis, respectively, whereas only one phage, PhiP15, was observed from the infected classical strain. In the uninfected El Tor culture one prophage was spontaneously induced after 6 days. No induction in either strain was observed after treatment with mitomycin C. Data indicate that El Tor biotypes of V. cholerae may be polylysogenic and that secondary infection can promote multiple prophage induction. These traits may be important in the transfer of genetic material among V. cholerae by providing an environmentally relevant route for multiple prophage propagation and transmission. [TOP OF PAGE]

  530. Removal of coliphages in secondary effluent by microfiltration-mechanisms of removal and impact of operating parameters. Farahbakhsh,K., Smith,D.W. (2004). Water Res. 38:585-592. The efficacy of a microfiltration (MF) pilot plant in removing somatic coliphages (referred hereafter as coliphages) present in the secondary effluent was evaluated during this study. The impact of operating parameters such as feed coliphage concentrations, permeate flux and membrane fouling on the removal of coliphages by the MF plant was investigated. The study showed that membrane fouling was beneficial for removing coliphages by MF. It was also shown that the removal of coliphages by MF was initially governed by adsorption on membrane surface or in membrane pores. As the membrane fouled, however, the removal of coliphages was primarily governed by direct interception on the cake layer formed on the surface of the membrane. Increases in feed coliphage concentrations resulted in the passage of larger numbers of coliphages when the MF was clean but had little impact on the passage of coliphages when the membrane became fouled. Increasing permeate flux lowered log-removal values (LRVs) for the clean membrane but resulted in an initial increase in LRVs for the fouled membrane followed by a drop in LRVs with further increases in permeate flux. [TOP OF PAGE]

  531. Resuscitation of a defective prophage in Salmonella cocultures. Figueroa-Bossi,N., Bossi,L. (2004). J. Bacteriol. 186:4038-4041. Widely studied Salmonella enterica serovar Typhimurium strains ATCC 14028s and SL1344 harbor a cryptic ST64B prophage unable to produce infectious virions. We found that coculturing either strain with an isogenic sibling lacking the prophage leads to the appearance of active forms of the virus. Active phage originates from reversion of a +1 frameshift mutation at a monotonous G:C run in a presumptive tail assembly pseudogene. [TOP OF PAGE]

  532. Adaptation varies through space and time in a coevolving host–parasitoid interaction. Forde,S.E., Thompson,J.N., Bohannan,B.J.M. (2004). Nature 431:841-844. One of the central challenges of evolutionary biology is to understand how coevolution organizes biodiversity over complex geographic landscapes. Most species are collections of genetically differentiated populations, and these populations have the potential to become adapted to their local environments in different ways. The geographic mosaic theory of coevolution incorporates this idea by proposing that spatial variation in natural selection and gene flow across a landscape can shape local coevolutionary dynamics(1, 2, 3, 4, 5, 6, 7). These effects may be particularly strong when populations differ across productivity gradients, where gene flow will often be asymmetric among populations(8). Conclusive empirical tests of this theory have been particularly difficult to perform because they require knowledge of patterns of gene flow, historical population relationships and local selection pressures(2). We have tested these predictions empirically using a model community of bacteria and bacteriophage (viral parasitoids of bacteria). We show that gene flow across a spatially structured landscape alters coevolution of parasitoids and their hosts and that the resulting patterns of adaptation can fluctuate in both space and time. [TOP OF PAGE]

  533. Co-infection weakens selection against epistatic mutations in RNA viruses. Froissart,R., Wilke,C.O., Montville,R., Remold,S.K., Chao,L., Turner,P.E. (2004). Genetics 168:9-19. Co-infection may be beneficial in large populations of viruses because it permits sexual exchange between viruses that is useful in combating the mutational load. This advantage of sex should be especially substantial when mutations interact through negative epistasis. In contrast, co-infection may be detrimental because it allows virus complementation, where inferior genotypes profit from superior virus products available within the cell. The RNA bacteriophage phi6 features a genome divided into three segments. Co-infection by multiple phi6 genotypes produces hybrids containing reassorted mixtures of the parental segments. We imposed a mutational load on phi6 populations by mixing the wild-type virus with three single mutants, each harboring a deleterious mutation on a different one of the three virus segments. We then contrasted the speed at which these epistatic mutations were removed from virus populations in the presence and absence of co-infection. If sex is a stronger force, we predicted that the load should be purged faster in the presence of co-infection. In contrast, if complementation is more important we hypothesized that mutations would be eliminated faster in the absence of co-infection. We found that the load was purged faster in the absence of co-infection, which suggests that the disadvantages of complementation can outweigh the benefits of sex, even in the presence of negative epistasis. We discuss our results in light of virus disease management and the evolutionary advantage of haploidy in biological populations. [TOP OF PAGE]

  534. Isolation and characterization of the bacteriophage WO from Wolbachia, an arthropod endosymbiont. Fujii,Y., Kubo,T., Ishikawa,H., Sasaki,T. (2004). Biochem. Biophys. Res. Com. 317:1183-1188. Wolbachia is a group of obligate symbiotic bacteria found in many insects and other arthropods. The presence of Wolbachia alters reproduction in the host, but the mechanisms are unknown. Molecular biological studies of Wolbachia have delayed significantly, and one of the reasons is the lack of transformation techniques of this bacterium. In the present study, bacteriophage particles were isolated from Wolbachia for the first time. The purified phage had an isometric head that was approximately 40 nm in diameter and contained linear double-stranded DNA of approximately 20 kbp. Partial sequence information (total of 20,484 bp) revealed that there were 24 open reading frames including a structural gene module, and genes for replication and lysogenic conversion. This bacteriophage is the only known mobile genetic element potentially used for transformation of Wolbachia. [TOP OF PAGE]

  535. Detection of bacteriophage infection and prophage induction in bacterial cultures by means of electric DNA chips. Gabig-Ciminska,M., Los,M., Holmgren,A., Albers,J., Czyz,A., Hintsche,R., Wegrzyn,G., Enfors,S.O. (2004). Analytical biochemistry 324:84-91. Infections of bacterial cultures by bacteriophages are common and serious problems in many biotechnological laboratories and factories. A method for specific, quantitative, and quick detection of phage contamination, based on the use of electric DNA chip is described here. Different phages of Escherichia coli and Bacillus subtilis were analyzed. Phage DNA was isolated from bacterial culture samples and detected by combination of bead-based sandwich hybridization with enzyme-labeled probes and detection of the enzymatic product using silicon chips. The assay resulted in specific signals from all four tested phages without significant background. Although high sensitivity was achieved in 4h assay time, a useful level of sensitivity (10(7)-10(8) phages) is achievable within 25 min. A multiplex DNA chip technique involving a mixture of probes allows for detection of various types of phages in one sample. These analyses confirmed the specificity of the assay. [TOP OF PAGE]

  536. [Sensitivity of nosocomial microflora circulating in a transplantation clinic to medicinal bacteriophages]. Gabrielian,N.I., Gorskaia,E.M., Spirina,T.S., Darbeeva,O.S., Maiskaia,L.M. (2004). Zhurnal mikrobiologii, epidemiologii, i immunobiologii 6-10. The sensitivity of 239 isolates obtained from patients with postoperative infectious complications to phagolysis was determined. Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli were found to have the highest sensitivity to phages. Variations in the sensitivity of the same cultures to phages from different producers and even from the same producer were established. The sensitivity of cultures to phages may serve as an additional criterion of the biological properties of strains and their marker. [TOP OF PAGE]

  537. Diversity and host range of Shiga toxin-encoding phage. Gamage,S.D., Patton,A.K., Hanson,J.F., Weiss,A.A. (2004). Infect. Immun. 72:7131-7139. Shiga toxin 2 (Stx2) from the foodborne pathogen Escherichia coli O157:H7 is encoded on a temperate bacteriophage. Toxin-encoding phages from C600::933W and from six clinical E. coli O157:H7 isolates were characterized for PCR polymorphisms, phage morphology, toxin production, and lytic and lysogenic infection profiles on O157 and non-O157 serotype E. coli. The phages were found to be highly variable, and even phages isolated from strains with identical pulsed-field gel electrophoresis profiles differed. Examination of cross-plaquing and lysogeny profiles further substantiated that each phage is distinct; reciprocal patterns of susceptibility and resistance were not observed and it was not possible to define immunity groups. The interaction between Shiga toxin-encoding phage and intestinal E. coli was examined. Lytic infection was assessed by examining Shiga toxin production following overnight incubation with phage. While not common, lytic infection was observed, with a more-than-1,000-fold increase in Stx2 seen in one case, demonstrating that commensal E. coli cells can amplify Shiga toxin if they are susceptible to infection by the Shiga toxin-encoding phages. Antibiotic-resistant derivatives of the Stx2-encoding phages were used to examine lysogeny. Different phages were found to lysogenize different strains of intestinal E. coli. Lysogeny was found to occur more commonly than lytic infection. The presence of a diverse population of Shiga toxin-encoding phages may increase the pathogenic fitness of E. coli O157:H7. [TOP OF PAGE]

  538. Rapid response of marginal zone B cells to viral particles. Gatto,D., Ruedl,C., Odermatt,B., Bachmann,M.F. (2004). J. Immunol. 173:4308-4316. Marginal zone (MZ) B cells are thought to be responsible for the first wave of Abs against bacterial Ags. In this study, we assessed the in vivo response of MZ B cells in mice immunized with viral particles derived from the RNA phage Qb. We found that both follicular (FO) and MZ B cells responded to immunization with viral particles. MZ B cells responded with slightly faster kinetics, but numerically, FO B cells dominated the response. B1 B cells responded similarly to MZ B cells. Both MZ and FO B cells underwent isotype switching, with MZ B cells again exhibiting faster kinetics. In fact, almost all Qb-specific MZ B cells expressed surface IgG by day 5. Histological analysis demonstrated that a population of activated B cells remain associated with the MZ, probably due to the elevated integrin levels expressed by these cells. Thus, both MZ and FO B cells respond with rapid proliferation to viral infection and both populations undergo isotype switching, but MZ B cells remain in the MZ and may be responsible for local Ab production, opsonizing pathogens entering the spleen. [TOP OF PAGE]

  539. Diversity, distribution and specificity of WO phage infection in Wolbachia of four insect species. Gavotte,L., Vavre,F., Henri,H., Ravallec,M., Stouthamer,R., Bouletreau,M. (2004). Insect Mol. Biol. 13:147-153. The bacteriophage WO was recently characterized in Wolbachia, a strictly intracellular bacterium that causes several reproductive alterations in its arthropod hosts. To gain insights into the phage-Wolbachia relationships, we studied the phage presence among Wolbachia infecting four insect species sharing several Wolbachia strains, two Drosophila and two of their parasitoid wasps. Based on the phage sequence of ORF7, we identified five different phages in six Wolbachia strains. Among these five bacteriophages, some are specific for a given bacterial strain whereas others are not, but globally phage infection appears stable on a large geographical scale and across insect generations. Their specificity contrasts with the absence of congruence between Wolbachia and phage phylogenies, suggesting phage exchanges between different Wolbachia lineages. [TOP OF PAGE]

  540. Drastically lowering the titer of waterborne bacteriophage PRD1 by exposure to immobilized hydrophobic polycations. Gelman,F., Lewis,K., Klibanov,A.M. (2004). Biotechnology Letters 26:1695-1700. Decrease in the titer of bacteriophage PRD1 (a model of animal adenoviruses) in aqueous solutions caused by the presence of systematically chemically derivatized surfaces was kinetically investigated. The greatest loss of infectivity--up to a 4-log reduction in the titer--was observed with immobilized hydrophobic polyethylenimine-based and dendrimer-based polycations. [TOP OF PAGE]

  541. Virus and bacteria dynamics of a coastal sediment: Implication for benthic carbon cycling. Glud,R.N., Middelboe,M. (2004). Limnol. Oceanogr. 49:2073-2081. We measured microbial heterotrophic activity, bacteria, and virus-like particle (VLP) abundance in homogenized, undiluted, and anoxic enclosures of sediment collected at a coastal station. The bacterial growth rate and VLP net production increased along with the respiratory activity in response to temperature. This suggests that VLPs represent a dynamic component of benthic microbial communities and that the net production of viriobenthos is regulated by the metabolic activity of bacteria. The abundance, net production, and decay rate of VLPs were significantly higher than those encountered in most pelagic systems. However, the rates were lower than the very few available potential rates (three studies) of viriobenthic activity, which all were obtained applying different slurry approaches. Our measurements support the general observation that virus abundance and production correlate with the trophic status of the environment and show that microbial activity can regulate the viriobenthic production in undiluted, homogenized marine sediments. The virus-induced bacterial mortality corresponded to similar to 20% of bacterial net production and similar to 2% h(-1) of the total bacterial population. This is moderate compared with the results of most pelagic studies, and the associated leakage of lysates (dissolved organic carbon) only amounted to 4-8% of the produced dissolved inorganic carbon. Despite high standing stocks and relatively high turnover rates, VLP-induced bacterial lysis represented only a minor shunt in the benthic carbon cycle at the investigated site. [TOP OF PAGE]

  542. Bacteriophage biocontrol of plant pathogens: fact or fiction? Goodridge,L.D. (2004). Trends Biotechnol. 22:384-385. Bacterial resistance due to the misuse of antibiotics has become a global issue and alternative methods are being developed that might decrease the use of antimicrobials in agricultural settings. Bacteriophage therapy represents a novel way to control the growth of plant-based bacterial pathogens. Although this method shows promise, a recent paper by Gill and Abedon has shown that the complex bacteriophage-host interactions in the plant environment must be investigated further. [TOP OF PAGE]

  543. Childhood tuberculosis and its early diagnosis. Gray,J.W. (2004). Clinical Biochemistryy 37:450-455. Traditional methods for laboratory diagnosis of tuberculosis are unsatisfactory, especially for children, in whose specimens mycobacteria are usually sparse. Recent changes in tuberculosis epidemiology in developed countries, including a large increase in incidence in children from certain ethnic minorities, have prompted interest in newer diagnostic methods. Liquid-based culture detection systems offer improved sensitivity and speed of diagnosis, although the time taken for detection of growth is still upwards of 1 week. Nucleic acid amplification techniques offer more rapid results, but perform best on smear-positive samples; sensitivities may be as low as 50% in smear-negative specimens. Although these newer techniques are widely used in some developed countries, in others, they are not perceived as offering sufficient benefit to justify their routine use. The diagnostic accuracy of mycobacteriophage and serologic methods is insufficient to justify their wide use even in developing countries. Despite recent developments, there is still no panacea for diagnosis of childhood tuberculosis. [TOP OF PAGE]

  544. Variation in the Shiga toxin region of 20th-century epidemic and endemic Shigella dysenteriae 1 strains. Greco,K.M., McDonough,M.A., Butterton,J.R. (2004). J. Infect. Dis. 190:330-334. The Shiga toxin (Stx) region of Shigella dysenteriae 1 lies on a defective prophage homologous to lambdoid bacteriophages in Stx-producing Escherichia coli. S. dysenteriae 1 strains obtained in locations throughout the world over the course of the past 60 years were assessed for variations in the Stx region by use of polymerase chain reaction and sequence analysis. The defective prophage was present in all strains examined, suggesting that all S. dysenteriae 1 isolates derive from a clone that resulted from a single phage-integration event. All western-hemisphere strains have an additional iso-IS1 insertion element upstream of stxAB, implying that there has been minimal exchange of strains between hemispheres in recent decades. [TOP OF PAGE]

  545. Microorganisms resistant to free-living amoebae. Greub,G., Raoult,D. (2004). Clin. Microbiol. Rev. 17:413-433. Free-living amoebae feed on bacteria, fungi, and algae. However, some microorganisms have evolved to become resistant to these protists. These amoeba-resistant microorganisms include established pathogens, such as Cryptococcus neoformans, Legionella spp., Chlamydophila pneumoniae, Mycobacterium avium, Listeria monocytogenes, Pseudomonas aeruginosa, and Francisella tularensis, and emerging pathogens, such as Bosea spp., Simkania negevensis, Parachlamydia acanthamoebae, and Legionella-like amoebal pathogens. Some of these amoeba-resistant bacteria (ARB) are lytic for their amoebal host, while others are considered endosymbionts, since a stable host-parasite ratio is maintained. Free-living amoebae represent an important reservoir of ARB and may, while encysted, protect the internalized bacteria from chlorine and other biocides. Free-living amoebae may act as a Trojan horse, bringing hidden ARB within the human "Troy," and may produce vesicles filled with ARB, increasing their transmission potential. Free-living amoebae may also play a role in the selection of virulence traits and in adaptation to survival in macrophages. Thus, intra-amoebal growth was found to enhance virulence, and similar mechanisms seem to be implicated in the survival of ARB in response to both amoebae and macrophages. Moreover, free-living amoebae represent a useful tool for the culture of some intracellular bacteria and new bacterial species that might be potential emerging pathogens. [TOP OF PAGE]

  546. Therapy of experimental pseudomonas infections with a nonreplicating genetically modified phage. Hagens,S., Habel,A., von Ahsen,U., von Gabain,A., Bläsi,U. (2004). Antimicrob. Agents Chemother. 48:3817-3822. Bacteriophage therapy of bacterial infections has received renewed attention owing to the increasing prevalence of antibiotic-resistant pathogens. A side effect of many antibiotics as well as of phage therapy with lytic phage is the release of cell wall components, e.g., endotoxins of gram-negative bacteria, which mediate the general pathological aspects of septicemia. Here we explored an alternative strategy by using genetically engineered nonreplicating, nonlytic phage to combat an experimental Pseudomonas aeruginosa infection. An export protein gene of the P. aeruginosa filamentous phage Pf3 was replaced with a restriction endonuclease gene. This rendered the Pf3 variant (Pf3R) nonreplicative and concomitantly prevented the release of the therapeutic agent from the target cell. The Pf3R phage efficiently killed a wild-type host in vitro, while endotoxin release was kept to a minimum. Treatment of P. aeruginosa infections of mice with Pf3R or with a replicating lytic phage resulted in comparable survival rates upon challenge with a minimal lethal dose of 3. However, the survival rate after phage therapy with Pf3R was significantly higher than that with the lytic phage upon challenge with a minimal lethal dose of 5. This higher survival rate correlated with a reduced inflammatory response elicited by Pf3R treatment relative to that with the lytic phage. Therefore, this study suggests that the increased survival rate of Pf3R-treated mice could result from reduced endotoxin release. Thus, the use of a nonreplicating modified phage for the delivery of genes encoding proteins toxic to bacterial pathogens may open up a new avenue in antimicrobial therapy. [TOP OF PAGE]

  547. Escherichia coli mazEF-mediated cell death as a defense mechanism that inhibits the spread of phage P1. Hazan,R., Engelberg-Kulka,H. (2004). Mol. Gen. Genom. 272:227-234. The Escherichia coli gene pair mazEF is a regulatable chromosomal toxin-antitoxin module: mazF encodes a stable toxin and mazE encodes for a labile antitoxin that overcomes the lethal effect of MazF. Because MazE is labile, inhibition of mazE expression results in cell death. We studied the effect of mazEF on the development of bacteriophage P1 upon thermoinduction of the prophage P1CM c1ts and upon infection with virulent phage particles (P1vir). In several E. coli strains, we showed that the Delta mazEF derivative strains produced significantly more phages than did the parent strain. In addition, upon induction of K38(P1CM c1ts), nearly all of the Delta mazEF mutant cells lysed; in contrast, very few of the parental mazEF + K38 cells underwent lysis. However, most of these cells did not remain viable. Thus, while the Delta mazEF cells die as a result of the lytic action of the phage, most of the mazEF+ cells are killed by a different mechanism, apparently through the action of the chromosomal mazEF system itself. Furthermore, the introduction of lysogens into a growing non-lysogenic culture is lethal to Delta mazEF but not for mazEF+ cultures. Thus, although mazEF action causes individual cells to die, upon phage growth this is generally beneficial to the bacterial culture because it causes P1 phage exclusion from the bacterial population. These results provide additional support for the view that bacterial cultures may share some of the characteristics of multicellular organisms. [TOP OF PAGE]

  548. Hot new virus, deep connections. Hendrix,R.W. (2004). Proc. Natl. Acad. Sci. USA 101:7495-7496. [first two paragraphs] Biologists of the 17th, 18th, and 19th centuries-call them natural historians-somehow got along without PCR, chip technology, mass spectrometry, or highspeed computers. From our vantage point in the 21st century we may wonder how our scientific ancestors, lacking our sophisticated tools, could have accomplished anything that we would recognize as forward progress in understanding how biology works. Yet they were in the enviable position that much of their biological world was completely unexplored; all they had to do to make a name for themselves was step out of their home habitat, usually Europe, and with a little luck they might find a new and wonderful organism, unlike anything previously known to science. Many examples of such discoveries can be cited (cycads, duck-billed platypuses, giant tortoises, Komodo dragons, and animalcules among them) and, in the most successful cases (think of Mr. Darwin), the new creature(s) dramatically enhanced our understanding of the structure and history of the biological world as a whole. Fortunately for the excitement quotient of modern-day natural historians, Mother Nature's reservoir of undiscovered bizarre and wonderful organisms is not yet empty, and a new one makes the transition from unknown to known with the report by Rice et al. (1) in this issue of PNAS. ¶ The new entry is a virus plucked from the near-boiling water of a thermal pool in Yellowstone National Park, and it is every bit as interesting to 21st century science as something like the Galapagos marine iguana (Fig. 1A) was to European science when it first came on the stage a few centuries ago. The new virus's host is the hyperthermophilic archaeon Sulfolobus sulfataricus, which grows happily at temperatures above 80°C and a pH of 2. Very few viruses of Archaea have been described to date [they amount to <1% of the viruses enumerated by the International Committee on the Taxonomy of Viruses (2)] but these early indications suggest that archaeal viruses likely are just as diverse as the more extensively characterized viruses of Bacteria and Eukarya. The viruses that infect the archaeal halophiles are so far confined to ones that have the same virion morphology and even occasional sequence similarity with the familiar tailed bacteriophages, but the viruses of the hyperthermophiles are a strange and diverse group with virion morphologies including filaments as well as shapes resembling food items such as lemons and corndogs. In this context, perhaps it is not surprising that the new virus would not look quite like anything described before. It is a spherical or, more properly, an icosahedrally symmetric virus (Fig. 1B), and, like most such viruses, the surface morphological features follow the rules enunciated by Caspar and Klug (3), although it has a previously undescribed triangulation number of 31. The most dramatic morphological feature of the virion is the protruding ''turrets'' that extend 13 nm above the capsid surface at the 12 fivefold symmetrical positions of the icosahedron. The function of the turrets is not known, but a plausible guess is that they have a role in attaching the virus to the cell and initiating infection. The morphological features are the basis for the authors' name for the virus: STIV, for Sulfolobus turreted icosahedral virus (1). [TOP OF PAGE]

  549. Shiga toxin-encoding bacteriophages--genomes in motion. Herold,S., Karch,H., Schmidt,H. (2004). Int. J. Med. Microbiol. 294:115-121. Shiga toxins (Stx) represent a group of bacterial toxins that are involved in human and animal disease. Stx are mainly produced by Escherichia coli isolated from human and non-human sources, Shigella dysenteriae type 1, and sporadically, by Citrobacter freundii, Enterobacter cloacae and Shigella flexneri. The genes encoding Stx are encoded in the genome of heterogeneous lambdoid prophages (Stx-converting bacteriophages; Stx-phages). They are located in a similar position in the late region of the prophage genome and stx is under control of phage genes. Therefore, induction of Stx-converting prophages triggers increased production of Stx. Following induction, Stx-phages can infect other bacteria in vivo and in vitro. Stx-phages may be considered to represent highly mobile genetic elements that play an important role in the expression of Stx, in horizontal gene transfer, and hence in genome diversification. [TOP OF PAGE]

  550. Evidence of Trichodesmium viral lysis and potential significance for biogeochemical cycling in the oligotrophic ocean. Hewson,I., Govil,S.R., Capone,D.G., Carpenter,E.J., Fuhrman,J.A. (2004). Aquat. Microb. Ecol. 36:1-8. The planktonic cyanobacterium Trichodesmium spp. is a globally important diazotroph, fixing at least 80 Tg N yr(-1) in tropical waters. Despite its biogeochemical importance, the mechanisms of Trichodesmium mortality, and the means by which its fixed N enters upper levels of the food web, are poorly understood. Potential virus-like particle (VLP) production by Trichodesmium spp. was observed in both culture (IMS101) and field samples from the tropical North Pacific Ocean, in oceanic waters around the Hawaiian Islands. VLP observed by TEM in IMS101 lysate were approximately 56 nm wide and untailed, and VLP with similar morphology were observed in tissue treated with mitomycin C. A most-probable number cultivation technique (utilizing bacterized cultures of Trichodesmium) detected moderate abundances of Trichodesmium-infecting cyanophage (605 to 9750 ml(-1) infecting only 1 cultured strain) in 0.2 mum-filtered seawater samples from surface, subsurface and deep chlorophyll maximum samples. Estimation of mortality from virus production was not possible due to rapid VLP release in the first 6 h of dilution incubations. Rather, an indirect approach using burst size (determined from mitomycin C treatment of washed trichomes resuspended in virus-free seawater), decay rate of VLP (from latter part of virus production incubations) and average cyanophage titer was used to estimate mortality. These conservative calculations suggested that 0.3 to 6.5 % d(-1) (mean = 1.65 +/- 0.99 % d(-1)) of trichomes could potentially be lysed by viruses, representing the release of approximately 3 to 64 % fixed N d(-1). These estimates are based upon a steady-state maintenance of observed VLP abundance, which in nature could be from lysogeny, pseudolysogeny, carrier state, or successive lytic infection. Viral lysis therefore may represent a significant novel mechanism of N release from Trichodesmium spp., even in non-bloom conditions. [TOP OF PAGE]

  551. Elimination of viruses, bacteria and protozoan oocysts by slow sand filtration. Hijnen,W.A.M., Schijven,J.F., Bonne,P., Visser,A., Medema,G.J. (2004). Water Sci. Technol. 50:147-154. The decimal elimination capacity (DEC) of slow sand filters (SSF) for viruses, bacteria and oocysts of Cryptosporidium has been assessed from full-scale data and pilot plant and laboratory experiments. DEC for viruses calculated from experimental data with MS2-bacteriophages in the pilot plant filters was 1.5-2 log10. E. coli and thermotolerant coliforms (Coli44) were removed at full-scale and in the pilot plant with 2-3 log10. At full-scale, Campylobacter bacteria removal was 1 log10 more than removal of Coli44, which indicated that Coli44 was a conservative surrogate for these pathogenic bacteria. Laboratory experiments with sand columns showed 2-3 and >5-6 log10 removal of spiked spores of sulphite-reducing clostridia (SSRC; C. perfringens) and oocysts of Cryptosporidium respectively. Consequently, SSRC was not a good surrogate to quantify oocyst removal by SSF. Removal of indigenous SSRC by full-scale filters was less efficient than observed in the laboratory columns, probably due to continuous loading of these filter beds with spores, accumulation and retarded transport. It remains to be investigated if this also applies to oocyst removal by SSF. The results additionally showed that the schmutzdecke and accumulation of (in)organic charged compounds in the sand increased the elimination of microorganisms. Removal of the schmutzdecke reduced DEC for bacteria by +/-2 log10, but did not affect removal of phages. This clearly indicated that, besides biological activity, both straining and adsorption were important removal mechanisms in the filter bed for microorganisms larger than viruses. [TOP OF PAGE]

  552. Isolation of bacteriophages from the oral cavity. Hitch,G., Pratten,J., Taylor,P.W. (2004). Lett. Appl. Microbiol. 39:215-219. AIMS: To isolate bacteriophages lytic for oral pathogens from human saliva, dental plaque and mature biofilms constituted from saliva-derived bacteria. METHODS AND RESULTS: Saliva and dental plaque samples from healthy volunteers and from patients with gingivitis and periodontitis were examined for the presence of lytic bacteriophage using a panel of oral pathogens and bacteria isolated from the samples. Samples were also enriched for bacteriophage using static culture techniques and mature biofilms. A limited number of samples contained bacteriophage particles that were visualized using electron microscopy. Cultures yielded phage infecting non-oral bacteria (Proteus mirabilis) but no bacteriophage specific for recognized oral pathogens were found. Some micro-organisms from the oral microflora elaborated antibacterial substances that inhibited growth of other residents of the oral cavity. CONCLUSIONS: Unlike other ecosystems, the composition of the oral cavity does not appear to be heavily influenced by interactions between bacteriophages and their hosts. SIGNIFICANCE AND IMPACT OF THE STUDY: Bacteriophage for control of oral infections may need to be obtained from other sources. Antibacterial substances derived from some members of the oral microflora warrant investigation as potential antibiotics. [TOP OF PAGE]

  553. The use of microbial tracers to monitor seasonal variations in effluent retention in a constructed wetland. Hodgson,C.J., Perkins,J., Labadz,J.C. (2004). Water Res. 38:3833-3844. Effluent retention in a constructed wetland was determined using both microbial and chemical tracers. Seasonal variation in effluent retention was the main focus of the study. The biotracers used in the study were the coliphage MS2, a bacteriophage of Enterobacter cloacae and antibiotic resistant endospores of Bacillus globigii. Two separate tracer runs were conducted, Winter high flow (January 2002) and Summer low flow (June 2002). The three biotracers were evaluated simultaneously on both occasions, with the commonly used chemical tracer, rhodamine WT, a bright red fluorescent dye, being evaluated during the final experiment. The Winter tracer run was conducted during a typical Winter storm, with a mean effluent discharge of 4.1 ls(-1). Tracer recovery was 98% MS2, 91% Ent. cloacae phage and 2% endospore. Effluent retention was estimated at between 2 and 4 h at 90% phage tracer recovery. The Summer tracer run was conducted at a typical site operating discharge rate of 0.8 ls(-1). Tracer recovery was 23% MS2, 36% Ent. cloacae phage, 8% rhodamine and 14% for the endospores. Effluent retention was estimated at between 11 and 18 h at 90% of phage tracer recovery. Initial results are encouraging and indicate bacteriophage to have further potential as tracing agents in wetlands. [TOP OF PAGE]

  554. Evaluation of the purification capacity of nine portable, small-scale water purification devices. Horman,A., Rimhanen-Finne,R., Maunula,L., von Bonsdorff,C.H., Rapala,J., Lahti,K., Hanninen,M.L. (2004). Water Sci. Technol. 50:179-183. A test was performed to evaluate the microbial and chemical purification capacity of nine portable, small-scale water purification filter devices with production capacity less than 100 L/h. The devices were tested for simultaneous removal capacity of bacteria (cultured Escherichia coli, Clostridium perfringens, Klebsiella pneumoniae and Enterobacter cloacae), enteric protozoans (formalin-stored Cryptosporidium parvum oocysts), viral markers (F-RNA bacteriophages) and microcystins produced by toxic cyanobacterial cultures. In general, the devices tested were able to remove bacterial contaminants by 3.6-6.9 log10 units from raw water. Those devices based only on filtration through pores 0.2-0.4 microm or larger failed in viral and chemical purification. Only one device, based on reverse osmosis, was capable of removing F-RNA phages at concentrations under the detection limit and microcystins by 2.5 log10. The present study emphasised the need for evaluation tests of water purification devices from the public safety and HACCP (Hazard Analysis and Critical Control Point) points of view. Simultaneous testing for various pathogenic/indicator microbes and microcystins was shown to be a useful and practical way to obtain essential data on actual purification capacity of commercial small-scale drinking-water filters. [TOP OF PAGE]

  555. Therapeutic efficacy of bacteriophage and Baytril (enrofloxacin) individually and in combination to treat colibacillosis in broilers. Huff,W.E., Huff,G.R., Rath,N.C., Balog,J.M., Donoghue,A.M. (2004). Poult. Sci. 83:1944-1947. A study was conducted to evaluate the therapeutic efficacy of bacteriophage and the antibiotic enrofloxacin individually and in combination to treat colibacillosis. The experimental design was a 2 x 2 x 2 factorial with 8 treatments and 4 replicate pens of 10 birds. The treatments were 1) control, 2) unchallenged birds treated with bacteriophage, 3) enrofloxacin, or 4) the combination; 5) birds challenged with Escherichia coli, and birds challenged with E. coli and treated with 6) bacteriophage, 7) enrofloxacin, or 8) the combination of bacteriophage and enrofloxacin. Birds in the E. coli challenged treatments were challenged at 7 d of age by injecting 10(4) cfu of E. coli into the thoracic air sac. The antibiotic treatment was initiated immediately after the birds were challenged and consisted of 50 ppm enrofloxacin in the drinking water for 7 consecutive days. The bacteriophage treatment consisted of a single intramuscular injection of 2 different bacteriophage (10(9) pfu) administered immediately after the E. coli challenge. Mortality in the birds challenged with E. coli and untreated was 68%, and the bacteriophage and enrofloxacin treatments significantly decreased mortality to 15 and 3%, respectively. There was total protection in birds that received both the bacteriophage and enrofloxacin representing a significant synergy. The decrease in mortality with enrofloxacin (3%) was significantly better than the decrease in mortality with bacteriophage (15%). Airsacculitis lesion scores and lesion incidence in surviving birds were significantly less in the enrofloxacin treatment compared with the bacteriophage treatment. Both bacteriophage and enrofloxacin provided effective treatments of colibacillosis, and the synergy between these 2 treatments suggests that bacteriophage combined with antibiotic treatment has significant value. [TOP OF PAGE]

  556. Cloning Serratia entomophila antifeeding genes--a putative defective prophage active against the grass grub Costelytra zealandica. Hurst,M.R.H., Glare,T.R., Jackson,T.A. (2004). J. Bacteriol. 186:5116-5128. Serratia entomophila and Serratia proteamaculans (Enterobacteriaceae) cause amber disease in the grass grub Costelytra zealandica (Coleoptera: Scarabaeidae), an important pasture pest in New Zealand. Larval disease symptoms include cessation of feeding, clearance of the gut, amber coloration, and eventual death. A 155-kb plasmid, pADAP, carries the genes sepA, sepB, and sepC, which are essential for production of amber disease symptoms. Transposon insertions in any of the sep genes in pADAP abolish gut clearance but not cessation of feeding, indicating the presence of an antifeeding gene(s) elsewhere on pADAP. Based on deletion analysis of pADAP and subsequent sequence data, a 47-kb clone was constructed, which when placed in either an Escherichia coli or a Serratia background exerted strong antifeeding activity and often led to rapid death of the infected grass grub larvae. Sequence data show that the antifeeding component is part of a large gene cluster that may form a defective prophage and that six potential members of this prophage are present in Photorhabdus luminescens subsp. laumondii TTO1, a species which also has sep gene homologues. [TOP OF PAGE]

  557. Isolation of phages infecting Actinoplanes SN223 and characterization of two of these viruses. Jarling,M., Bartkowiak,K., Robenek,H., Pape,H., Meinhardt,F. (2004). Applied Microbiology and Biotechnology 64:250-254. Phages infecting the industrially important Actinoplanes strain SN223 were isolated from soil samples collected at the shores of inland waters in Germany. The genome sizes range from 53 kb to 58 kb. Preliminary analyses revealed G+C contents comparable with the G/C bias of the host. Electron microscopy of three selected viruses displayed no obvious morphological differences, the phage heads being icosahedral and their tails non-contractible. Two of the phages (FAsp2, FAsp3.1) characterized in more detail are capable of provoking putative pseudolysogenic growth of the host bacterium. The carrier state for FAsp2, in which cells are tightly packed with viruses, was demonstrated by electron microscopy. The latter phage is apparently widely distributed, as it was isolated from regions which are distantly located, i.e. more than 600 km apart from each other. [TOP OF PAGE]

  558. Bacteriophage lambda is a highly stable DNA vaccine delivery vehicle. Jepson,C.D., March,J.B. (2004). Vaccine 22:2413-2419. The stability of whole bacteriophage lambda particles, used as a DNA vaccine delivery system has been examined. Phage were found to be highly stable under normal storage conditions. In liquid suspension, no decrease in titre was observed over a 6-month period at 4 and -70°C, and phage stability was unaffected by freeze/thawing. The measured half life of phage in suspension was 36 days at 20°C, 3.4 days at 37°C and 2.3 days at 42°C. Freeze drying of a phage suspension (with or without the stabilizers dry skim milk or trehalose) resulted in 5-20% residual viability. Following desiccation (with or without stabilizers), measured half lives ranged from 20 to 100 days at 20°C, 2.6 to 38 days at 37°C, 2.1 to 26 days at 42°C, 7 to 33 h at 70°C, and 1.3 to 6m at 100°C. In all cases the addition of trehalose significantly increased the stability of the desiccated phage. When stored at -70°C, desiccated phage appeared to be stable in the absence of stabilizers. When phage lambda was diluted into water, a marginal loss in titre was observed over a 2-week period. Over a 24 h period, liquid phage suspensions were stable within the pH range pH 3-11, therefore oral administration of bacteriophage DNA vaccines via drinking water may be possible. [TOP OF PAGE]

  559. Big questions, small worlds: microbial model systems in ecology. Jessep,C.M., Kassen,R., Forde,S.E., Kerr,B., Buckling,A., Rainey,P.B., Bohannan,B.J.M. (2004). Trends Ecol. Evol. 19:189-197. Although many biologists have embraced microbial model systems as tools to address genetic and physiological questions, the explicit use of microbial communities as model systems in ecology has traditionally been more restricted. Here, we highlight recent studies that use laboratory-based microbial model systems to address ecological questions. Such studies have significantly advanced our understanding of processes that have proven difficult to study in field systems, including the genetic and biochemical underpinnings of traits involved in ecological interactions, and the ecological differences driving evolutionary change. The use of microbial model systems is not without criticism, however. Many ecologists have voiced concern that microbial microcosm experiments are too simplified, contrived, and small in spatial and temporal scale to adequately address ecological questions. We argue that these concerns reflect a misunderstanding of the purpose of microcosm studies. It is the simplicity of microbial model systems that makes them such powerful tools for the study of ecology; such simplicity allows the high degrees of experimental control and replication necessary to address many questions that are inaccessible through field observation or experimentation. Furthermore, the tractability of the microbial model systems also allows ecologists to bridge ecological and evolutionary questions, and to analyze experiments post hoc to better understand the mechanisms underlying particular results. [TOP OF PAGE]

  560. Abundance, distribution, and diversity of viruses in alkaline, hypersaline Mono Lake, California. Jiang,S., Steward,G., Jellison,R., Chu,W., Choi,S. (2004). Microb. Ecol. 47:9-17. Mono Lake is a large (180 km(2)), alkaline (pH approximately 10), moderately hypersaline (70-85 g kg(-1)) lake lying at the western edge of the Great Basin. An episode of persistent chemical stratification (meromixis) was initiated in 1995 and has resulted in depletion of oxygen and accumulation of ammonia and sulfide beneath the chemocline. Although previous studies have documented high bacterial abundances and marked seasonal changes in phytoplankton abundance and community composition, there have been no previous reports on the occurrence of viruses in this unique lake. Based on the high concentrations and diversity of microbial life in this lake, we hypothesized that planktonic viruses are also abundant and diverse. To examine the abundance and distribution of viruses and bacteria, water samples were collected from four stations along 5 to 15 vertical depths at each station. Viral abundance ranged from 1 x 10(8) to 1 x 10(9) mL(-1), among the highest observed in any natural aquatic system examined so far. Increases (p < 0.1) in viral densities were observed in the anoxic bottom water at multiple stations. However, regression analysis indicated that viral abundance could not be predicted by any single environmental parameter. Pulsed field gel electrophoresis revealed a diverse viral community in Mono Lake with genome sizes ranging from approximately 14 to >400 kb with most of the DNA in the 30 to 60 kb size range. Cluster analysis grouped the anoxic bottom-water viral community into a unique cluster differentiating it from surface and mid-water viral communities. A hybridization study using an indigenous viral isolate as a probe revealed an episodic pattern of temporal phage distribution with strong niche stratification between oxic and anoxic waters. [TOP OF PAGE]

  561. PCR detection of pathogenic viruses in southern California urban rivers. Jiang,S.C., Chu,W. (2004). J. Appl. Microbiol. 97:17-28. AIMS: To investigate human viral contamination in urban rivers and its impact on coastal waters of southern California, USA. METHODS AND RESULTS: Three types of human viruses (adeno, entero and hepatitis A) were detected using nested- and RT-PCR from 11 rivers and creeks. Faecal indicator bacteria as well as somatic and F-specific coliphage were also tested. Approximately 50% of the sites were positive for human adenoviruses. However, there was no clear relationship between detection of human viruses and the concentration of indicator bacteria and coliphage. Both faecal indicator bacteria and human viral input at beaches near river mouths were associated with storm events. The first storm of the wet season seemed to have the greatest impact on the quality of coastal water than following storm events. CONCLUSIONS: This study provides the first direct evidence that human viruses are prevalent in southern California urban rivers. Urban run-off impacts coastal water quality most significantly during the storm season. SIGNIFICANCE AND IMPACT OF THE STUDY: To protect human health during water recreational activities, it is necessary to develop effective strategies to manage urban run-off during storm events. [TOP OF PAGE]

  562. Shiga toxin phages in water. Jofre,J., Muniesa,M. (2004). pp. 79-89. In In Spencer,J.F.T. and Ragout de Spencer,A.L. (eds.), Methods in Molecular Biology. Totowa,N.J., Humana Press. [TOP OF PAGE]

  563. The persistence and removal of enteric pathogens in constructed wetlands. Karim,M.R., Manshadi,F.D., Karpiscak,M.M., Gerba,C.P. (2004). Water Res. 38:1831-1837. Sedimentation is thought to be one of the mechanisms of microbial reduction from wetlands used for wastewater treatment. This study compared the occurrence and survival of enteric indicator microorganisms and pathogens in the water column and sediments of two constructed surface flow wetlands in Arizona. On a volume/wet weight basis the concentration of fecal coliforms and coliphage in the water column and sediment was similar. However, on a volume/dry weight basis the numbers were one to two orders of magnitude higher in the sediment. Giardia cyst and Cryptosporidium oocyst concentrations were one to three orders of magnitude greater in the sediment compared to the water column. The die-off rates of all the bacteria and coliphage were greater in the water column than the sediment. The die-off rates of fecal coliforms in the water and sediment were 0.256log(10)day(-1) and 0.151log(10)day(-1), respectively. The die-off rates of Salmonella typhimurium in the water and sediment were 0.345log(10)day(-1) and 0.312log(10)day(-1), respectively. The die-off rates of naturally occurring coliphage in water column and sediment were 0.397log(10)day(-1) and 0.107log(10)day(-1), respectively, and the die-off rates of and PRD-1 in water and sediment were 0.198log(10)day(-1) and 0.054log(10)day(-1), respectively. In contrast Giardia die-off in the sediment was greater compared to the water column. The die-off rates of Giardia in water and sediment were 0.029log(10)day(-1) and 0.37log(10)day(-1), respectively. Coliphage survived the longest of any group of organisms in the sediment and the least in the water column. In contrast Giardia survived best in the water column and least in the sediment. [TOP OF PAGE]

  564. [To the expediency of determination of coliphages in water]. Kashkarova,G.P., Blagova,O.E., Boitsov,A.G., Lastovka,O.N., Evel'son,E.A., Akhapkina,E.N., Kontorovich,V.B., Dorodnikov,A.I. (2004). Gigiena i Sanitariia 75-77. [TOP OF PAGE]

  565. Pathogen removal efficiency from UASB + BF effluent using conventional and UV post-treatment systems. Keller,R., Passamani-Franca,R.F., Passamani,F., Vaz,L., Cassini,S.T., Sherrer,N., Rubim,K., Sant'Ana,T.D., Goncalves,R.F. (2004). Water Sci. Technol. 50:1-6. The aim of this study was to verify the efficiency of removal of microorganisms in effluents of a Wastewater Treatment Plant (WWTP) comprising an association of a UASB reactor followed by three submerged aerated biofilters (BAF) and one tertiary filter. The WWTP designed to treat domestic wastewater from a population of 1,000 inhabitants showed high removal efficiency for organic matter and suspended solids. Helminth eggs were also efficiently removed from the tertiary effluent and were found in the sludge from the UASB reactor; however, removal of bacteria in this system was very low. To enhance the efficiency of the system, the effluent from tertiary filters was submitted to UV disinfection in a real scale reactor. Our results showed that UV irradiation was very effective at lowering the concentrations of E. coli, thermotolerant coliforms and coliphages to acceptable levels for agricultural reuse. Salmonella spp. and helminth eggs were seeded into the tertiary effluent before passing through the UV reactor. Salmonella was not found in the final effluent, but helminth eggs were not completely inactivated by UV irradiation and viable eggs were detected after 28 d of incubation. [TOP OF PAGE]

  566. [Spread of viruses through the food chain]. Klein,G. (2004). Deutsche Tier. Wchschr. 111:312-314. Food associated viruses are responsible for a high number of infectious diseases in man, mainly gastroenteritis and hepatitis. The three most important viral agents are noroviruses (NV) (formerly known as Norwalk-like viruses), Rotavirus (RV) and Hepatitis A-Virus (HAV). The numbers of infections in man were in 2002 according to the Robert Koch-Institut for NV and RV 50,000, respectively, and for HAV 1,500, slightly decreasing in 2003. The rate of foodborne infections caused by viruses can only be estimated (appr. 20% of total cases). On the other hand only a very small part of viral gastroenteritis can be diagnosed and notified. Besides the direct infection through contaminated food the human to human infection is the most important source, also responsible for outbreaks. There is at the moment no routine diagnostic tool available for the detection of viruses in food because of the lack of standardized methods. For NV, one of the most important foodborne (live bivalve molluscs) viral pathogens, indicator organisms are in use. There is a scientific evaluation in different member states concerning the value of bacterial indicators vs. bacteriophages. In addition to foodborne viruses (via faecal contamination present in the food chain) there are emerging zoonotic viral agents. Food may be a vector for this agents depending on the production structures (e.g. SARS or influenca). [TOP OF PAGE]

  567. Bacteriophages that infect the cellulolytic ruminal bacterium Ruminococcus albus AR67. Klieve,A.V., Bain,P.A., Yokoyama,M.T., Ouwerkerk,D., Forster,R.J., Turner,A.F. (2004). Lett. Appl. Microbiol. 38:333-338. AIM: To isolate bacterial viruses that infect the ruminal cellulolytic bacterium Ruminococcus albus. METHODS: Four phages infecting R. albus AR67 were isolated under anaerobic conditions using the soft-agar overlay technique. The phages were characterized on morphology, solvent stability, nucleic acid type and digestion characteristics. Two phages, fRa02 and fRa04 comprised icosahedral virions with linear double-stranded DNA and appeared to belong to the family Tectiviridae. The other two phages are most likely filamentous phages with circular single-stranded DNA of the family Inoviridae. SIGNIFICANCE OF THE STUDY: Viruses of the families Tectiviridae and Inoviridae have not previously been isolated from rumen bacteria. The phages isolated in this study are the first phages shown to infect the cellulolytic bacteria of the rumen. This suggests that the cellulolytic populations of the rumen are subject to lytic events that may impact on the ability of these bacteria to degrade plant fibre and on the nutrition of the animal. [TOP OF PAGE]

  568. Treatment and prevention of enterococcal infections--alternative and experimental approaches. Koch,S., Hufnagel,M., Huebner,J. (2004). Expert Opinion on Biological Therapy 4:1519-1531. Enterococci are one of the leading types of organisms isolated from infections of hospitalised patients and the third most common cause of nosocomial bloodstream infections. They contribute significantly to patient mortality and morbidity, as well as healthcare costs. The emergence of resistance against virtually all clinically available antibiotics and the ability to transfer these resistance determinants to other pathogens demonstrates the urgency for an improved understanding of enterococcal virulence mechanisms, and the development of alternative treatment and prevention options. This article reviews new antimicrobials, vaccine targets, bacteriophage therapy, as well as treatments targeting virulence factors and biofilm, for their potential to treat and/or prevent enterococcal infections. Although clinical isolates often cause serious infections, so-called 'non-pathogenic' strains are used as therapeutics in the form of probiotics. Understanding the differences between true pathogens and beneficial commensals may help to evaluate future treatment and prophylactic options. [TOP OF PAGE]

  569. Group I intron homing in Bacillus phages SPO1 and SP82: a gene conversion event initiated by a nicking homing endonuclease. Landthaler,M., Lau,N.C., Shub,D.A. (2004). J. Bacteriol. 186:4307-4314. Many group I introns encode endonucleases that promote intron homing by initiating a double-stranded break-mediated homologous recombination event. In this work we describe intron homing in Bacillus subtilis phages SPO1 and SP82. The introns encode the DNA endonucleases I-HmuI and I-HmuII, respectively, which belong to the H-N-H endonuclease family and possess nicking activity in vitro. Coinfections of B. subtilis with intron-minus and intron-plus phages indicate that I-HmuI and I-HmuII are required for homing of the SPO1 and SP82 introns, respectively. The homing process is a gene conversion event that does not require the major B. subtilis recombination pathways, suggesting that the necessary functions are provided by phage-encoded factors. Our results provide the first examples of H-N-H endonuclease-mediated intron homing and the first demonstration of intron homing initiated by a nicking endonuclease. [TOP OF PAGE]

  570. Spring phytoplankton bloom in Norwegian coastal waters: Microbial community dynamics, succession and diversity. Larsen,A., Fonnes,G.A., Sandaa,R.A., Castberg,T., Thyrhaug,R., Erga,S.E., Jacquet,S., Bratbak,G. (2004). Limnol. Oceanogr. 49:180-190. Most studies of spring bloom succession in Norwegian waters have employed light microscopy and accounted for species composition of phyto- and zooplankton. Flow cytometry and molecular tools enable us to extend such investigations to include smaller organisms like bacterio- and virioplankton. Here, we describe succession and diversity of algae, bacteria, and viruses in relation to environmental changes from 15 February to 27 April. The spring succession started with an increase in autotrophic picoeukaryotes and Synechococcus sp. The diatoms bloomed around the middle of March and caused nutrient depletion in the upper part of the water column. Upwelling in the beginning of April gave rise to a second bloom, consisting of diatoms and Phaeocystis pouchetii. Numerically, autotrophic picoeukaryotes and Synechococcus sp. dominated the periods between and after these two major blooms. Heterotrophic bacterial abundance increased throughout the experimental period and reached peak values during and after phytoplankton blooms. These bacteria were succeeded by viruses having low DNA fluorescence, whereas viruses with medium DNA fluorescence bloomed during or after blooms of autotrophic picoeukaryotes. High-DNA fluorescence viruses reached maximum concentrations during and after the diatom and Phaeocystis blooms. The diversity of the bacterial community remained relatively stable, whereas viral diversity varied more and increased after major phytoplankton blooms. Our investigation thus demonstrates how virioplankton are important elements of the total microbial diversity and how they are intimately linked to the rest of the microbial community and possibly act as an internal driving force in spring bloom successions. [TOP OF PAGE]

  571. Bxz1, a new generalized transducing phage for mycobacteria. Lee,S., Kriakov,J., Vilcheze,C., Dai,Z., Hatfull,G.F., Jacobs,W.R.J. (2004). FEMS Microbiol. Lett. 241:271-276. We have isolated and characterized a new generalized transducing phage, Bxz1, from soil sampling at a neighboring Wildlife Preservation Park. The hosts of the phage, measured by the formation of plaques, include fast growing Mycobacterium smegmatis and Mycobacterium vaccae. Bxz1 is capable of transducing chromosomal markers, point mutations, and plasmids at frequencies ranging from 10(-8) to 10(-6) per plaque forming unit between strains of M. smegmatis. We also demonstrated cotransduction of a transposon insertion linked to a point mutation of the ndh gene. [TOP OF PAGE]

  572. Optimizing concentration and timing of a phage spray application to reduce Listeria monocytogenes on honeydew melon tissue. Leverentz,B., Conway,W.S., Janisiewicz,W., Camp,M.J. (2004). J. Food Prot. 67:1682-1686. A phage cocktail was applied to honeydew melon pieces 1, 0.5, and 0 h before contamination with Listeria monocytogenes strain LCDC 81-861 and 0.5, 1, 2, and 4 h after contamination. The phage application was most effective when applied 1, 0.5, or 0 h before contamination with L. monocytogenes, reducing pathogen populations by up to 6.8 log units after 7 days of storage. This indicates that under commercial conditions, if contamination occurs at the time of cutting, phage would have to be applied as soon as possible after cutting the produce. However, all phage applications from 1 h before to 4 h after contamination and all phage concentrations ranging from 10(4) to 10(8) PFU/ml reduced bacterial populations on honeydew melon pieces. Higher phage concentrations were more effective in reducing pathogen populations. A phage concentration of approximately 10(8) PFU/ml was necessary to reduce the pathogen populations to nondetectable levels immediately after treatment, and pathogen growth was suppressed by phage concentrations of 10(6) through 10(8) throughout the storage period of 7 days at 10ºC. In an attempt to enhance the effectiveness of the phage cocktail on low pH fruit, such as apples, the phage was applied in combination with MnCl(2). This combination, however, did not enhance the effectiveness of the phage on apple tissue. The results from this study indicate that the effectiveness of the phage application on honeydew melon pieces can be optimized by using a phage concentration of at least 10(8) PFU/ml applied up to 1 h after processing of the honeydew melons. [TOP OF PAGE]

  573. Population and evolutionary dynamics of phage therapy. Levin,B.R., Bull,J.J. (2004). Nat. Rev. Microbiol. 2:166-173. Following a sixty-year hiatus in western medicine, bacteriophages (phages) are again being advocated for treating and preventing bacterial infections. Are attempts to use phages for clinical and environmental applications more likely to succeed now than in the past? Will phage therapy and prophylaxis suffer the same fates as antibiotics--treatment failure due to acquired resistance and ever-increasing frequencies of resistant pathogens? Here, the population and evolutionary dynamics of bacterial-phage interactions that are relevant to phage therapy and prophylaxis are reviewed and illustrated with computer simulations. [TOP OF PAGE]

  574. Involvement of colicin in the limited protection of the colicin producing cells against bacteriophage. Lin,Y.H., Liao,C.C., Liang,P.H., Yuan,H.S., Chak,K.F. (2004). Biochem. Biophys. Res. Com. 318:81-87. The restriction/modification system is considered to be the most common machinery of microorganisms for protection against bacteriophage infection. However, we found that mitomycin C induced Escherichia coli containing ColE7-K317 can confer limited protection against bacteriophage M13K07 and l infection. Our study showed that degree of protection is correlated with the expression level of the ColE7 operon, indicating that colicin E7 alone or the colicin E7-immunity protein complex is directly involved in this protection mechanism. It was also noted that the degree of protection is greater against the single-strand DNA bacteriophage M13K07 than the double-strand bacteriophagel. Coincidently, the K(A) value of ColE7-Im either interacting with single-strand DNA (2.94x10(5)M(-1)) or double-strand DNA (1.75x10(5)M(-1)) reveals that the binding affinity of ColE7-Im with ssDNA is 1.68-fold stronger than that of the protein complex interacting with dsDNA. Interaction between colicin and the DNA may play a central role in this limited protection of the colicin-producing cell against bacteriophages. Based on these observations, we suggest that the colicin exporting pathway may interact to some extent with the bacteriophage infection pathway leading to a limited selective advantage for and limited protection of colicin-producing cells against different bacteriophages. [TOP OF PAGE]

  575. Transfer of photosynthesis genes to and from Prochlorococcus viruses. Lindell,D., Sullivan,M.B., Johnson,Z.I., Tolonen,A.C., Rohwer,F., Chisholm,S.W. (2004). Proc. Natl. Acad. Sci. USA 101:11013-11018. Comparative genomics gives us a new window into phage-host interactions and their evolutionary implications. Here we report the presence of genes central to oxygenic photosynthesis in the genomes of three phages from two viral families (Myoviridae and Podoviridae) that infect the marine cyanobacterium Prochlorococcus. The genes that encode the photosystem II core reaction center protein D1 (psbA), and a high-light-inducible protein (HLIP) (hli) are present in all three genomes. Both myoviruses contain additional hli gene types, and one of them encodes the second photosystem II core reaction center protein D2 (psbD), whereas the other encodes the photosynthetic electron transport proteins plastocyanin (petE) and ferredoxin (petF). These uninterrupted, full-length genes are conserved in their amino acid sequence, suggesting that they encode functional proteins that may help maintain photosynthetic activity during infection. Phylogenetic analyses show that phage D1, D2, and HLIP proteins cluster with those from Prochlorococcus, indicating that they are of cyanobacterial origin. Their distribution among several Prochlorococcus clades further suggests that the genes encoding these proteins were transferred from host to phage multiple times. Phage HLIPs cluster with multicopy types found exclusively in Prochlorocococus, suggesting that phage may be mediating the expansion of the hli gene family by transferring these genes back to their hosts after a period of evolution in the phage. These gene transfers are likely to play a role in the fitness landscape of hosts and phages in the surface oceans. [TOP OF PAGE]

  576. Occurrence of microbial indicators and Clostridium perfringens in wastewater, water column samples, sediments, drinking water, and Weddell seal feces collected at McMurdo Station, Antarctica. Lisle,J.T., Smith,J.J., Edwards,D.D., McFeters,G.A. (2004). Appl. Environ. Microbiol. 70:7269-7276. McMurdo Station, Antarctica, has discharged untreated sewage into McMurdo Sound for decades. Previous studies delineated the impacted area, which included the drinking water intake, by using total coliform and Clostridium perfringens concentrations. The estimation of risk to humans in contact with the impacted and potable waters may be greater than presumed, as these microbial indicators may not be the most appropriate for this environment. To address these concerns, concentrations of these and additional indicators (fecal coliforms, Escherichia coli, enterococci, coliphage, and enteroviruses) in the untreated wastewater, water column, and sediments of the impacted area and drinking water treatment facility and distribution system at McMurdo Station were determined. Fecal samples from Weddell seals in this area were also collected and analyzed for indicators. All drinking water samples were negative for indicators except for a single total coliform-positive sample. Total coliforms were present in water column samples at higher concentrations than other indicators. Fecal coliform and enterococcus concentrations were similar to each other and greater than those of other indicators in sediment samples closer to the discharge site. C. perfringens concentrations were higher in sediments at greater distances from the discharge site. Seal fecal samples contained concentrations of fecal coliforms, E. coli, enterococci, and C. perfringens similar to those found in untreated sewage. All samples were negative for enteroviruses. A wastewater treatment facility at McMurdo Station has started operation, and these data provide a baseline data set for monitoring the recovery of the impacted area. The contribution of seal feces to indicator concentrations in this area should be considered. [TOP OF PAGE]

  577. The occurrence of lysogenic bacteria and microbial aggregates in the lakes of the McMurdo Dry Valleys, Antarctica. Lisle,J.T., Priscu,J.C. (2004). Microb. Ecol. 47:427-439. The McMurdo Dry Valleys of Antarctica form the coldest and driest ecosystem on Earth. Within this region there are a number of perennially ice-covered (3-6 m thick) lakes that support active microbial assemblages and have a paucity of metazoans. These lakes receive limited allochthonous input of carbon and nutrients, and primary productivity is limited to only 6 months per year owing to an absence of sunlight during the austral winters. In an effort to establish the role that bacteria and their associated viruses play in carbon and nutrient cycling in these lakes, indigenous bacteria, free bacteriophage, and lysogen abundances were determined. Total bacterial abundances (TDC) ranged from 3.80 x 10(4) to 2.58 x 10(7) cells mL(-1) and virus-like particle (VLP) abundances ranged from 2.26 x 10(5) to 5.56 x 10(7) VLP mL(-1). VLP abundances were significantly correlated (P < 0.05) with TDC, bacterial productivity (TdR), chlorophyll a (Chl a), and soluble reactive phosphorus (SRP). Lysogenic bacteria, determined by induction with mitomycin C, made up between 2.0% and 62.5% of the total population of bacteria when using significant decreases and increases in TDC and VLP abundances, respectively, and 89.5% when using increases in VLP abundances as the sole criterion for a successful induction event. The contribution of viruses released from induced lysogens contributed <0.015% to the total viral production rate. Carbohydrate and protein based organic aggregates were abundant within the water column of the lakes and were heavily colonized by bacteria and VLPs. Alkaline phosphatase activity was detected within the matrix of the aggregates, implying phosphorus deficiency and consortial nutrient exchanges among microorganisms. [TOP OF PAGE]

  578. Genomic and genetic analysis of Bordetella bacteriophages encoding reverse transcriptase-mediated tropism-switching cassettes. Liu,M., Gingery,M., Doulatov,S.R., Liu,Y., Hodes,A., Baker,S., Davis,P., Simmonds,M., Churcher,C., Mungall,K., Quail,M.A., Preston,A., Harvill,E.T., Maskell,D.J., Eiserling,F.A., Parkhill,J., Miller,J.F. (2004). J. Bacteriol. 186:1503-1517. Liu et al. recently described a group of related temperate bacteriophages that infect Bordetella subspecies and undergo a unique template-dependent, reverse transcriptase-mediated tropism switching phenomenon (Liu et al., Science 295: 2091-2094, 2002). Tropism switching results from the introduction of single nucleotide substitutions at defined locations in the VR1 (variable region 1) segment of the mtd (major tropism determinant) gene, which determines specificity for receptors on host bacteria. In this report, we describe the complete nucleotide sequences of the 42.5- to 42.7-kb double-stranded DNA genomes of three related phage isolates and characterize two additional regions of variability. Forty-nine coding sequences were identified. Of these coding sequences, bbp36 contained VR2 (variable region 2), which is highly dynamic and consists of a variable number of identical 19-bp repeats separated by one of three 5-bp spacers, and bpm encodes a DNA adenine methylase with unusual site specificity and a homopolymer tract that functions as a hotspot for frameshift mutations. Morphological and sequence analysis suggests that these Bordetella phage are genetic hybrids of P22 and T7 family genomes, lending further support to the idea that regions encoding protein domains, single genes, or blocks of genes are readily exchanged between bacterial and phage genomes. Bordetella bacteriophages are capable of transducing genetic markers in vitro, and by using animal models, we demonstrated that lysogenic conversion can take place in the mouse respiratory tract during infection. [TOP OF PAGE]

  579. Characterizing spontaneous induction of Stx encoding phages using a selectable reporter system. Livny,J., Friedman,D.I. (2004). Mol. Microbiol. 51:1691-1704. Shiga toxin (Stx) genes in Stx producing Escherichia coli (STEC) are encoded in prophages of the lambda family, such as H-19B. The subpopulation of STEC lysogens with induced prophages has been postulated to contribute significantly to Stx production and release. To study induced STEC, we developed a selectable in vivo expression technology, SIVET, a reporter system adapted from the RIVET system. The SIVET lysogen has a defective H-19B prophage encoding the TnpR resolvase gene downstream of the phage P<sub>R</sub> promoter and a cat gene with an inserted tet gene flanked by targets for the TnpR resolvase. Expression of resolvase results in excision of tet , restoring a functional cat gene; induced lysogens survive and are chloramphenicol resistant. Using SIVET we show that: (i) approximately 0.005% of the H-19B lysogens are spontaneously induced per generation during growth in LB. (ii) Variations in cellular physiology (e.g. RecA protein) rather than in levels of expressed repressor explain why members of a lysogen population are spontaneously induced. (iii) A greater fraction of lysogens with stx encoding prophages are induced compared to lysogens with non-Stx encoding prophages, suggesting increased sensitivity to inducing signal(s) has been selected in Stx encoding prophages. (iv) Only a small fraction of the lysogens in a culture spontaneously induce and when the lysogen carries two lambdoid prophages with different repressor/ operators, 933W and H-19B, usually both prophages in the same cell are induced. [TOP OF PAGE]

  580. A comparison of the survival of F+RNA and F+DNA coliphages in lake water microcosms. Long,S.C., Sobsey,M.D. (2004). Water Hlth. 2:15-22. The survival of seven F+RNA phages (MS2 Group I ATCC type strain, two Group I environmental isolates, a Group II environmental isolate, a Group III environmental isolate, and two Group IV environmental isolates) and six F+DNA phages (M13, fd, f1, and ZJ/2 ATCC type strains, and two environmental isolates) were examined in microcosms using a surface drinking water source. Phages were spiked into replicate aliquots of a source water at about 20,000 pfu/ml. Replicate spikes were incubated at 4 and 20ºC and monitored for 110 days. At 4 degrees C, Groups I and II F+ RNA phages were detectable through 110 days, with reductions of about 1 and 3 log10, respectively. The Group III F+RNA phage demonstrated 5 log10 reduction after 3 weeks, and the Group IV F+RNA phages were reduced to detection limits (5 log10 reduction) within 10 days. Of the F+DNA phages, all four type strains were detectable with about 2.5 log10 reduction after 110 days at 4 degrees C. The F+DNA environmental isolates were detectable with about a 4 log10 reduction after 110 days at 4ºC. All phages demonstrated faster decay at 20ºC. These results suggest that differences in F+ phage survival may influence their prevalence in environmental waters and the ability to attribute their prevalence to specific human and animal sources of faecal contamination. [TOP OF PAGE]

  581. Streptococcus pneumoniae and its bacteriophages: one long argument. Lopez,R. (2004). Int. Microbiol. 7:163-171. Infectious diseases currently kill more than 15 million people annually, and the WHO estimates that every year 1.6 million people die from pneumococcal diseases. Streptococcus pneumoniae (pneumococcus), a bacterium with a long biological pedigree, best illustrates the rapid evolution of antibiotic resistance, which has led to major public health concern. This article discusses the molecular basis of the two main virulence factors of pneumococcus, the capsule and cell-wall hydrolases, as well as new approaches to developing medicinal weapons for preventing pneumococcal infections. In addition, current knowledge regarding pneumococcal phages as potential contributors to virulence and the use of lytic enzymes encoded by these phages as therapeutic tools is reviewed. [TOP OF PAGE]

  582. Bacteriophage contamination: is there a simple method to reduce its deleterious effects in laboratory cultures and biotechnological factories? Los,M., Czyz,A., Sell,E., Wegrzyn,A., Neubauer,P., Wegrzyn,G. (2004). Journal of Applied Genetics 45:111-120. Infection of bacterial cultures by bacteriophages as well as prophage induction in the host cells are serious problems in both research and biotechnological laboratories. Generally, prevention strategies (like good laboratory/factory hygiene, sterilisation, decontamination and disinfection) are necessary to avoid bacteriophage contamination. However, it is well known that no matter how good the laboratory/factory practice and hygiene are, bacteriophage infections occur from time to time. The use of immunised or resistant bacterial strains against specific phages may be helpful, but properties of the genetically modified strains resistant to phages are often worse (from the point of view of a researcher or a biotechnological company) than those of the parental, phage-sensitive strains. In this article we review recent results that may provide a simple way to minimise deleterious effects of bacteriophage infection and prophage induction. It appears that low bacterial growth rates result in a significant inhibition of lytic development of various bacteriophages. Moreover, spontaneous prophage induction is less frequent in slowly growing bacteria. [TOP OF PAGE]

  583. [Inactivation of T4 phage in water environment using proteinase]. Lu,W.z., Yang,Q.x., Zhang,Y., Yang,M., Zhu,C.f. (2004). Huan Jing Ke Xue 25:93-96. The inactivation effectiveness of proteinase to viruses was investigated by using T4 phage as a model virus. The results showed that the inactivation effectiveness of proteinase to T4 phage was obvious. In the optimum conditions and 67.5 u/mL concentration, the inactivation rate of proteinase K to T4 phage in sterilized water and in sewage achieved 99.4% and 49.4% respectively in an hour, and achieved >99.9% and 81.1% in three hours. The inactivation rate of the industrial proteinase 1398 to T4 phage in sterilized water achieved 74.4% in an hour. The effects of pH and temperature on the inactivation effectiveness was not evident. [TOP OF PAGE]

  584. Reduction of bacterial indicators and bacteriophages infecting faecal bacteria in primary and secondary wastewater treatments. Lucena,F., Duran,A.E., Moron,A., Calderon,E., Campos,C., Gantzer,C., Skraber,S., Jofre,J. (2004). J. Appl. Microbiol. 97:1069-1076. AIMS: To compare the suitability of various bacterial and viral indicators to assess the removal of faecal micro-organisms by primary and secondary wastewater treatment processes. METHODS AND RESULTS: The numbers of several bacterial indicators [faecal coliforms (FC), enterococci (ENT) and sulphite-reducing clostridia (SRC)] and bacteriophages (somatic coliphages, F-specific RNA phages and bacteriophages infecting Bacteroides fragilis strain RYC2056) were determined in incoming raw sewage and effluents from various primary and secondary wastewater treatment processes in several geographical areas. Reductions in the numbers of indicators were calculated as log10 reductions. Processes based on removal and mild disinfection, showed no significant differences in the elimination of any of the indicators tested or between geographical areas. In contrast, treatment processes that include strong microbial inactivation, such as lime-aided flocculation and lagooning, showed significant differences between the log10 reductions of the various micro-organisms studied, FC showing the highest reduction and spores of SRC and phages infecting B. fragilis the lowest. CONCLUSIONS: The microbial elimination performance of treatment processes based principally on removal and mild disinfection can be evaluated with a single indicator. In contrast, processes with additional disinfecting capabilities require more than one indicator for accurate evaluation of the treatment; bacteriophages are good candidates for use as second indicators. SIGNIFICANCE AND IMPACT OF THE STUDY: Bacteriophages provide additional information for the evaluation of microbial elimination in some treatment plants. The easy, fast and cheap methods available for phage determination are feasible both in industrialized and developing countries. [TOP OF PAGE]

  585. Usefulness of monitoring tropical streams for male-specific RNA coliphages. Luther,K., Fujioka,R. (2004). Water Hlth. 2:171-181. The objective of this study was to evaluate the usefulness of monitoring streams in Hawaii for FRNA coliphages as a reliable indicator of sewage contamination. This study was undertaken as a result of our previous findings that monitoring streams in Hawaii for traditional faecal indicator bacteria (faecal coliform, Escherichia coli, enterococci) was not useful in determining when streams are contaminated with sewage, because environmental (soil) sources rather than sewage accounted for the high concentrations of faecal bacteria in streams. Two perennial streams, sewage and soil samples were monitored for traditional faecal indicator bacteria (faecal coliform, E. coli, enterococci) and FRNA coliphages. The results showed that sewage treatment processes and disinfection drastically reduced the concentrations of traditional faecal indicator bacteria but FRNA coliphages were still present in significant concentrations in the treated sewage effluents. These results indicate that monitoring sewage effluents and environmental waters for only traditional faecal indicator bacteria may not be adequately protective of human health effects. Ambient concentrations of traditional faecal indicator bacteria in soil and streams of Hawaii were consistently high but consistently low for FRNA coliphages, indicating that monitoring streams of Hawaii for FRNA coliphages can be used to determine when streams are contaminated with sewage. [TOP OF PAGE]

  586. Milk contamination and resistance to processing conditions determine the fate of Lactococcus lactis bacteriophages in dairies. Madera,C., Monjardin,C., Suarez,J.E. (2004). Appl. Environ. Microbiol. 70:7365-7371. Milk contamination by phages, the susceptibility of the phages to pasteurization, and the high levels of resistance to phage infection of starter strains condition the evolution dynamics of phage populations in dairy environments. Approximately 10% (83 of 900) of raw milk samples contained phages of the quasi-species c2 (72%), 936 (24%), and P335 (4%). However, 936 phages were isolated from 20 of 24 (85%) whey samples, while c2 was detected in only one (4%) of these samples. This switch may have been due to the higher susceptibility of c2 to pasteurization (936-like phages were found to be approximately 35 times more resistant than c2 strains to treatment of contaminated milk in a plate heat exchanger at 72 degrees C for 15 s). The restriction patterns of 936-like phages isolated from milk and whey were different, indicating that survival to pasteurization does not result in direct contamination of the dairy environment. The main alternative source of phages (commercial bacterial starters) does not appear to significantly contribute to phage contamination. Twenty-four strains isolated from nine starter formulations were generally resistant to phage infection, and very small progeny were generated upon induction of the lytic cycle of resident prophages. Thus, we postulate that a continuous supply of contaminated milk, followed by pasteurization, creates a factory environment rich in diverse 936 phage strains. This equilibrium would be broken if a particular starter strain turned out to be susceptible to infection by one of these 936-like phages, which, as a consequence, became prevalent. [TOP OF PAGE]

  587. Evolutionary potential of an RNA virus. Makeyev,E.V., Bamford,D.H. (2004). J. Virol. 78:2114-2120. RNA viruses are remarkably adaptable to changing environments. This is medically important because it enables pathogenic viruses to escape the immune response and chemotherapy and is of considerable theoretical interest since it allows the investigation of evolutionary processes within convenient time scales. A number of earlier studies have addressed the dynamics of adapting RNA virus populations. However, it has been difficult to monitor the trajectory of molecular changes in RNA genomes in response to selective pressures. To address the problem, we developed a novel in vitro evolution system based on a recombinant double-stranded RNA bacteriophage, phi 6, containing a beta-lactamase (bla) gene marker. Carrier-state bacterial cells are resistant to ampicillin, and after several passages, they become resistant to high concentrations of another beta-lactam antibiotic, cefotaxime, due to mutations in the virus-borne bla gene. We monitored the changes in bla cDNAs induced by cefotaxime selection and observed an initial explosion in sequence variants with multiple mutations throughout the gene. After four passages, a stable, homogeneous population of bla sequences containing three specific nonsynonymous mutations was established. Of these, two mutations (E104K and G238S) have been previously reported for beta-lactamases from cefotaxime-resistant bacterial isolates. These results extend our understanding of the molecular mechanisms of viral adaptation and also demonstrate the possibility of using an RNA virus as a vehicle for directed evolution of heterologous proteins. [TOP OF PAGE]

  588. [Bacteriophage of Bacillus polymyxa BC153-29]. Matseliukh,D.I., Burova,L.M. (2004). ??? 66:22-27. A virulent bacteriophage has been isolated from culture liquid of the sowing and production fermenter in the process of biosynthesis of polymyxin B; the bacteriophage lyses a sensitive producer's culture of antibiotic Bacillus polymyxa BC153-29. The bacteriophage has been purified by ultracentrifugation in the density gradient of cesium chloride, and its morphology has been investigated by the election microscopy. The hexagonal phage head is 72 +/- 1.8 nm in diameter, with the length and width of the elastic noncontractile appendix--300 +/- 2.3 and 14.2 +/- 0.5 nm, respectively. The phage was related to Siphoviridae (B1) family. The phage inactivation dynamics has been studied using specific antiserum, the phage neutralization velocity constant (595 min(-1)) and phage adsorption velocity constant on sensitive cells (9.39 x 10(-8) ml/min) have been determined. Exogenic origin of the isolated virulent bacteriophage of Bacillus polymyxa BC153-29 has been supposed. [TOP OF PAGE]

  589. Use of bacteriophages as surrogates for mammalian viruses. McAlister,M., Aranha,H., Larson,R. (2004). Developments in biologicals 118:89-98. The threat of viral contamination is common to all processes using biological products of animal or human origin. Therefore, demonstration of virus clearance (i.e. validation of virus removal and/or inactivation steps) is of utmost importance to the biopharmaceutical industries. Ultimately, virus clearance studies should show that any virus removal/inactivation stage incorporated into the manufacturing process not only removes or inactivates known viruses that may be conceivably present (e.g. from cell banks and source materials), but also other viruses that may be introduced adventitiously (e.g. by addition of supplements downstream of the manufacturing process). In this paper, we outline the shared properties of mammalian viruses and similar sized bacteriophages, and factors that may influence the virus clearance process. We also present test data from filtration studies, showing similar titre reductions for both types of virus. We propose that well-characterised bacteriophage, such as PP7 and PR772 can be used as models for mammalian viruses if the virus removal mechanism is based on size exclusion. [TOP OF PAGE]

  590. The impact of bacteriophage genomics. McGrath,S., Fitzgerald,G.F., van Sinderen,D. (2004). Curr. Opin. Biotechnol. 15:94-99. The discovery of (bacterio)phages revolutionised microbiology and genetics, while phage research has been integral to answering some of the most fundamental biological questions of the twentieth century. The susceptibility of bacteria to bacteriophage attack can be undesirable in some cases, especially in the dairy industry, but can be desirable in others, for example, the use of bacteriophage therapy to eliminate pathogenic bacteria. The relative ease with which entire bacteriophage genome sequences can now be elucidated has had a profound impact on the study of these bacterial parasites. [TOP OF PAGE]

  591. Development of a bacteriophage phage replication assay for diagnosis of pulmonary tuberculosis. McNerney,R., Kambashi,B.S., Kinkese,J., Tembwe,R., Godfrey-Faussett,P. (2004). J. Clin. Microbiol. 42:2115-2120. Successful infection and replication of bacteriophages is indicative of the presence of viable bacteria. We describe here the development of a bacteriophage replication assay for the detection of Mycobacterium tuberculosis by using mycobacteriophage D29. Optimization of phage inoculate and incubation times allowed highly sensitive detection of M. bovis BCG. Fewer than 10 CFU (100 CFU/ml) were detected. No false-positive results were observed in negative samples. Application of the assay to 496 sputum specimens in the National Reference Laboratory of Zambia produced sensitivity, specificity, and positive and negative predictive values of 44.1, 92.6, 82.2, and 67.5%, respectively, compared to culture on Lowenstein-Jensen medium. The equivalent corresponding results for direct fluorescent smear microscopy were 42.3, 96.8, 91.2, and 67.6%. The small increase in sensitivity over that of direct microscopy does not justify the introduction of this technique for routine diagnosis of pulmonary tuberculosis at this time. [TOP OF PAGE]

  592. Assessment of drinking water quality using indicator bacteria and bacteriophages. Mendez,J., Audicana,A., Cancer,M., Isern,A., Llaneza,J., Moreno,B., Navarro,M., Tarancon,M.L., Valero,F., Ribas,F., Jofre,J., Lucena,F. (2004). Journal of Water Health 2:201-214. Bacterial indicators and bacteriophages suggested as potential indicators of water quality were determined by public laboratories in water from springs, household water wells, and rural and metropolitan water supplies in north-eastern Spain. Indicator bacteria were detected more frequently than bacteriophages in springs, household water wells and rural water supplies. In contrast, positive bacteriophage detections were more numerous than those of bacteria in metropolitan water supplies. Most of the metropolitan water supply samples containing indicators had concentrations of chlorine below 0.1 mg l(-1), their indicator loads resembling more closely those of rural water supplies than any other samples taken from metropolitan water supplies. The number of samples from metropolitan water supplies containing more than 0.1 mg l(-1) of chlorine that contained phages clearly outnumbered those containing indicator bacteria. Some association was observed between rainfall and the presence of indicators. Sediments from service reservoirs and water from dead ends in the distribution network of one of the metropolitan water supplies were also tested. Bacterial indicators and phages were detected in a higher percentage than in samples of tap water from the same network. Additionally, indicator bacteria were detected more frequently than bacteriophages in sediments of service reservoirs and water from dead end samples. We conclude that naturally occurring indicator bacteria and bacteriophages respond differently to chlorination and behave differently in drinking water distribution networks. Moreover, this study has shown that testing for the three groups of phages in routine laboratories is easy to implement and feasible without the requirement for additional material resources for the laboratories. [TOP OF PAGE]

  593. Standardised evaluation of the performance of a simple membrane filtration-elution method to concentrate bacteriophages from drinking water. Mendez,J., Audicana,A., Isern,A., Llaneza,J., Moreno,B., Tarancon,M.L., Jofre,J., Lucena,F. (2004). J. Virol. Meth. 117:19-25. The bacteriophage elution procedure described further after adsorption to acetate-nitrate cellulose membrane filters allows better recovery of phages concentrated from 1l of water than elution procedures used previously. The improvement is due to the combined effect of the eluent (3% (w/v) beef extract, 3% (v/v) Tween 80, 0.5M NaCl, pH 9.0) and the application of ultrasound instead of agitation or swirling. Average recovery of somatic coliphages, 82 +/- 7%, was the greatest, and that of phages infecting Bacteroides fragilis, 56 +/- 8%, the lowest, with intermediate values for F-specific and F-specific RNA bacteriophages. Thus, the method allowed recovery of over 56% for all the phages suggested as surrogate indicators. The method was then validated according to an International Standardisation Organisation validation standard procedure and implemented in routine laboratories, which obtained reproducible results. [TOP OF PAGE]

  594. Removal of biological and non-biological viral surrogates by spiral-wound reverse osmosis membrane elements with intact and compromised integrity. Mi,B., Eaton,C., Kim,J.H., Colvin,C.K., Lozier,J.C., Marinas,B.J. (2004). Water Res. 38:3821-3832. The removal of bacteriophage MS2 and fluorescent-dyed polystyrene microspheres with intact and purposely compromised spiral-wound RO membrane elements was investigated. MS2 rejection with intact membrane elements was >99.9995%. A model developed for data evaluation revealed that the advective passage of MS2 through imperfections of intact membrane elements was <2 x 10(-5)% of the overall product water flow produced. The advective passage of MS2 and microspheres through a pinhole induced in one of the elements was 0.05-0.1% of the overall product water flow. Prolonged testing of both intact and compromised elements resulted in increased MS2 rejection corresponding to advective MS2 passage through membrane imperfections of <3 x 10(-7)% of the overall product water flow. The permeate flow rate obtained with an element with a larger pinhole was 5-13% greater than that of the intact element, and the corresponding rejection of MS2 and microspheres was similar to that observed for sodium chloride. The use of a cracked o-ring in the connection of the permeate tube to the element vessel end-cup resulted in advective passage of MS2 through the crack of <0.0001% of the overall permeate flow. [TOP OF PAGE]

  595. Genetic organization of the psbAD region in phages infecting marine Synechococcus strains. Millard,A., Clokie,M., Shub,D.A., Mann,N.H. (2004). Proc. Natl. Acad. Sci. USA 101:11007-11012. The discovery of the genes psbA and psbD, encoding the D1 and D2 core components of the photosynthetic reaction center PSII (photosystem II), in the genome of the bacteriophage S-PM2 (a cyanomyovirus) that infects marine cyanobacteria begs the question as to how these genes were acquired. In an attempt to answer this question, it was established that the occurrence of the genes is widespread among marine cyanomyovirus isolates and may even extend to podoviruses. The phage psbA genes fall into a clade that includes the psbA genes from their potential Synechococcus and Prochlorococcus hosts, and thus, this phylogenetic analysis provides evidence to support the idea of the acquisition of these genes by horizontal gene transfer from their cyanobacterial hosts. However, the phage psbA genes form distinct subclades within this lineage, which suggests that their acquisition was not very recent. The psbA genes of two phages contain identical 212-bp insertions that exhibit all of the canonical structural features of a group I self-splicing intron. The different patterns of genetic organization of the psbAD region are consistent with the idea that the psbA and psbD genes were acquired more than once by cyanomyoviruses and that their horizontal transfer between phages via a common phage gene pool, as part of mobile genetic modules, may be a continuing process. In addition, genes were discovered encoding a high-light inducible protein and a putative key enzyme of dark metabolism, transaldolase, extending the areas of host-cell metabolism that may be affected by phage infection. [TOP OF PAGE]

  596. Bacteriophage-mediated transduction: an engine for change and evolution. Miller,R.V. (2004). pp. 144-157. In In Miller,R.V. and Day,M.J. (eds.), Microbial Evolution: Gene Establishment, Survival, and Exchange. ASM Press, Washington DC. [TOP OF PAGE]

  597. Nodulation competitiveness between contrasting phage phenotypes of pigeonpea rhizobial strains. Mishra,A., Dhar,B., Singh,R.M. (2004). Indian J. Exp. Biol. 42:611-615. Competitiveness between (I) lysogenic vs. phage-indicator strains, (II) phage-resistant vs phage-sensitive strains, and (III) large plaque vs. small plaque developing strains was examined under laboratory and field conditions in order to study the involvement of these crucial phage sensitivity patterns in the competition for nodule occupancy of pigeonpea rhizobia. The phage-indicator strain (A039) exhibited higher competitiveness over the lysogenic strain (A025 Sm(r)); the phage sensitive strain (IHP-195) over the phage resistant strain (IHP 195 Sm(r)V(r)); and the large plaque developing strain (A059) over the small plaque developing strain (IHP195 Sm(r)) in association with pigeonpea cv. bahar both under laboratory and field conditions. Dual inoculation of A025 Sm(r) + A039 and A059 + IHP195 Sm(r) (mixed in equal proportion just before treatment) improved the nodule occupancy by inoculant strains against native rhizobia and resulted into higher plant dry weight and yield as compared to their application as single inoculum. The phage-resistant mutant IHP195 Sm(r)V(r) showed reduced competitiveness against native rhizobia, compared to its parental strain. The dual inoculation of parental strain and phage-resistant mutant gave the same result as the inoculation of parental strain alone. [TOP OF PAGE]

  598. Parasites mediate the relationship between host diversity and disturbance frequency. Morgan,A.D., Buckling,A. (2004). Ecol. Lett. 7:1029-1034. Patterns of community and population diversity are likely to be dependent on interactions between ecological variables. Here we address how two important ecological variables - extrinsic periodic mortality events (disturbances) and the presence of obligate-killing parasites - interact to affect the diversity of niche-specialist genotypes in laboratory populations of the bacterium Pseudomonas fluorescens. Consistent with previous studies, diversity was maximized at intermediate frequencies of disturbance in the absence of parasitic bacteriophages (phages). By contrast, no relationship was found between diversity and disturbance frequency in the presence of phage. The results can be explained in part by differential effects of phage on bacterial densities, and hence resource competition, under different disturbance regimes. [TOP OF PAGE]

  599. Assessment of the microbial integrity, sensu G.S. Wilson, of piped and bottled drinking water in the condition as ingested. Mossel,D.A.A., Struijk,C.B. (2004). Int. J. Food Microbiol. 92:375-390. The second half of the 20th century witnessed substantial progress in the assurance and verification of microbiological integrity, i.e., safety and sensory quality, of drinking water. Enteropathogenic agents, such as particular viruses and protozoa, not previously identified as transmitted by industrially provided water supplies, were demonstrated to cause disease outbreaks, when ingested with piped water. The potential harm posed by carry-over of orally toxic metabolites of organisms, producing 'algal' (cyanophytic) blooms, was considered. In addition, earlier observations on the colonization of attenuated drinking water bodies by a variety of oligotrophic Gram-negative bacteria were confirmed and extended. This new evidence called for updating both water purification technologies and analytical methodology, serving to verify that goals had been attained. For the former purpose, the hazard analysis empowering control of critical practices (HACCP) strategy, introduced about 1960 in industrial food processing, was successfully adopted. Elimination, devitalization or barrier technologies for the more recently identified water-borne pathogens were elaborated, taking account of the hazard of production of chlorinated compounds with alleged adverse health effects. Biofilm formation throughout water distribution networks was brought under control by strict limitation of concentrations of compounds, assimilable by oligotrophic bacteria. Upon acknowledging that direct detection tests for pathogens were futile, because of their most sporadic and erratic distribution, Schardinger's marker organism concept was anew embraced, rigorously revised and substantially enlarged. Misleading designations, like searches for 'faecal coliforms' were replaced by boundary testing for Escherichia coli and appropriate Enterococcus spp. In addition, though still to be perfected, detection protocols for relevant bacteriophages or index viruses and, to a certain extent, also for spores of aerobic and anaerobic sporing rods were also elaborated. In all monitoring account was taken of sublethally injured target organisms, surviving purification technologies, though not deprived of their ecological significance. A need remains for a rigorously standardized operating procedure (SOP) for colony counts of psychrotrophic, oligotrophic Gram-negative rod-shaped bacteria ('heterotrophic plate count'), which constitute a useful criterion of indicator value. As in the contemporary HACCP approach to food safety, guidelines for assessing success or failure in control of integrity (Water Safety Objectives) were empirically elaborated. These rely on surveys on water samples, originating from drinking water supplies, previously verified as complying with longitudinally integrated HACCP-based purification technologies. Structured Academic dissemination of these innovations, through professional microbiologists to operator and executive levels, is recommended. Web based Distance Learning MSc Programmes, like the one, since the academic year 2003-2004, offered by the University of Hertfordshire, Hatfield, UK, may contribute to such endeavours. Though the complete Course is centered around Food Safety, the Modules in-Residence Practicals and Science and Technology of Drinking Water can be studied as an entity while being employed. [TOP OF PAGE]

  600. Free Shiga toxin bacteriophages isolated from sewage showed diversity although the stx genes appeared conserved. Muniesa,M., Serra-Moreno,R., Jofre,J. (2004). Environ. Microbiol. 6:716-725. Phages carrying the stx2 gene were detected in a range of sewage samples using a plaque hybridization-based method. After detection, phages were isolated and propagated with a laboratory strain of Escherichia coli as host for characterization purposes. Although it was not possible to conduct propagation or transduction experiments on most of the phages, 11 reached a sufficiently high titre for studies of host infectivity, electron microscopy and sequencing of the stx2 flanking regions to be performed. These phages showed a wide range of host infectivity and morphology. The genetic structure of the 5' stx flanking region appeared conserved whereas the 3' region differed from that of previously described phages. This is the first description of infectious stx-phages isolated as free particles in the environment, and as such constitutes a new contribution to the study of the ecology of these phages. [TOP OF PAGE]

  601. Factors influencing the replication of somatic coliphages in the water environment. Muniesa,M., Jofre,J. (2004). Antonie van Leeuwenhoek J. Microbiol. 86:65-76. The potential replication of somatic coliphages in the environment has been considered a drawback for their use as viral indicators, although the extent to which this affects their numbers in environmental samples has not been assessed. In this study, the replication of somatic coliphages in various conditions was assayed using suspensions containing naturally occurring somatic coliphages and Escherichia coli WG5, which is a host strain recommended for detecting somatic coliphages. The effects on phage replication of exposing strain WG5 and phages to a range of physiological conditions and the effects of the presence of suspended particles or other bacteria were also assayed. Phage replication was further tested using a strain of Klebsiella terrigena and naturally occurring E. coli cells as hosts. Our results indicate that threshold densities of both host bacterium and phages should occur simultaneously to ensure appreciable phage replication. Host cells originating from a culture in the exponential growth phase and incubation at 37 °C were the best conditions for phage replication in E. coli WG5. In these conditions the threshold densities required to ensure phage replication were about 10(4) host cells/ml and 10(3) phages/ml, or 10(3) host cells/ml and 10(4) phages/ml, or intermediate values of both. The threshold densities needed for phage replication were higher when the cells proceeded from a culture in the stationary growth phase or when suspended particles or other bacteria were present. Furthermore E. coli WG5 was more efficient in supporting phage replication than either K. terrigenae or E. coli cells naturally occurring in sewage. Our results indicate that the phage and bacterium densities and the bacterial physiological conditions needed for phage replication are rarely expected to be found in the natural water environments. [TOP OF PAGE]

  602. Bacteriophages and diffusion of b-lactamase genes. Muniesa,M., Garcia,A., Miro,E., Mirelis,B., Prats,G., Jofre,J., Navarro,F. (2004). Emerg. Infect. Dis. 10:1134-1137. We evaluated the presence of various b-lactamase genes within the bacteriophages in sewage. Results showed the occurrence of phage particles carrying sequences of bla(OXA-2), bla(PSE-1) or bla(PSE-4) and bla(PSE)-type genes. Phages may contribute to the spread of some b-lactamase genes. [TOP OF PAGE]

  603. Diversity of stx2 converting bacteriophages induced from Shiga-toxin-producing Escherichia coli strains isolated from cattle. Muniesa,M., Blanco,J.E., de Simón,M., Serra-Moreno,R., Blanch,A.R., Jofre,J. (2004). Microbiology 150:2959-2971. The presence of bacteriophages encoding Shiga toxin 2 (stx<sub>2</sub> phages) was analysed in 168 strains of Shiga-toxin-producing Escherichia coli (STEC) isolated from cattle. Following mitomycin C induction, strains carrying stx<sub>2</sub> phages were screened by plaque blot and hybridization with an stx<sub>2</sub>A-probe. In the stx<sub>2</sub>-phage-carrying strains, the amounts of phage production, phage DNA extracted and Stx<sub>2</sub> produced after induction were assessed. The induced stx<sub>2</sub> phages were characterized morphologically and genetically. Assays to obtain lysogens from different strains were also carried out and phages induced from the lysogens were compared with those induced from the STEC isolates. Results indicated that 18% of the strains carried an inducible stx<sub>2</sub> phage. Most of them showed a direct relationship between phage induction and toxin production. Each strain carried only one inducible stx<sub>2</sub> phage, although a few strains had two copies of the stx<sub>2</sub> in the chromosome. The stx<sub>2</sub> phages showed diverse morphology and a wide variability in their genome. Assays to obtain lysogens showed that not all the phages were transduced with the same frequency and only six lysogens were obtained. Phages in the lysogens were the same as those induced from their respective initial STEC host strains, although the induction and relative toxin production of the lysogens varied. Most phages carried the stx<sub>2</sub> gene, while a few carried stx<sub>2</sub> variants. Infectivity of the phages depended on the different hosts, although O157 :H7 was preferentially infected by phages induced from O157 strains. The results show that inducible stx<sub>2</sub> phages are common among STEC of animal origin and that they may enhance the spread of stx<sub>2</sub>. [TOP OF PAGE]

  604. Abundance in sewage of bacteriophages infecting Escherichia coli O157:H7. Muniesa,M., Jofre,J. (2004). Methods in Molecular Biology 268:79-88. Bacterial virulence factors such as toxins are often encoded by bacteriophages. Among other examples, factors encoded by phages have been described in some of the emerging or re-emerging pathogens, including the pyrogenic exotoxin A production in group A streptococci, the cholera toxin in Vibrio cholerae, or enterotoxin production in enterohemorrhagic (EHEC) strains of E. coli. Most described virulence factors in Shiga toxin (Stx)-producing E. coli strains are located in mobile genetic elements such as plasmids and bacteriophages. Stx, which are one of the most important virulence elements in Shiga toxin-producing E. coli (STEC), are encoded in the genome of temperate bacteriophages infecting E. coli and other Enterobacteriaceae. Studies on Stx phages indicate that they are transmitted between different bacteria in vivo and in vitro. Phages could also be transmitted extraintestinally, hence the observed presence of infectious Shiga toxin phages in sewage and in fecally contaminated rivers. Stx phages also show a higher persistence under natural inactivation and disinfectant treatments in aquatic environments.This background shows that phages or lysogenic strains carrying Stx2 phages might be the natural reservoir of Stx2 genes and that lysogenization could be the main cause of the emergence of STEC strains, as suggested by several authors. It has also been suggested that lysogenization/conversion processes could take place in food and water and probably inside the human and animal gut. Ingestion of Stx2 phages could produce conversion of non-Stx2-E. coli strains, present inside the gut and producing new pathogenic strains. To control these phenomena, it is first necessary to gain more information about the distribution of Stx phages in the environment.For this purpose, a method of detecting Stx2 phages present in environmental water samples has been developed. The particularity of this method is that it allows detection of all (infectious and noninfectious) Stx2 phages in a water sample; in a second stage, the method allows detection of those phages able to infect and replicate on E. coli O157:H7. Although this method has been applied to Stx2 phages able to infect E. coli O157:H7, it is also applicable to detection in the natural environment of other genes carried by other bacteriophages and other bacteria. [TOP OF PAGE]

  605. Detection of enteric viruses in shellfish from the Norwegian coast. Myrmel,M., Berg,E.M.M., Rimstad,E., Grinde,B. (2004). Appl. Environ. Microbiol. 70:2678-2684. Common blue mussels (Mytilus edulis), horse mussels (Modiolus modiolus), and flat oysters (Ostrea edulis) obtained from various harvesting and commercial production sites along the Norwegian coast were screened for the presence of norovirus by a real-time reverse transcription (RT)-nested PCR assay and for possible indicators of fecal contamination, i.e., for F-specific RNA bacteriophages (F-RNA phages) by plaque assay and for human adenoviruses and human circoviruses by nested PCR assay. The aims were to obtain relevant information for assessing the risk of transmission of enteric viruses by shellfish and to investigate the potential of various indicator viruses in routine screening. Noroviruses were detected in 6.8% of the samples, and the indicators were detected in 23.8% (F-RNA phages), 18.6% (adenoviruses), and 8.0% (circoviruses) of the samples. A seasonal variation was observed, with the exception of circoviruses, with more positive samples in the winter. A positive correlation was found between F-RNA phages and noroviruses. However, F-RNA phages were present in only 43% of the norovirus-positive samples. The results show that mussels from the Norwegian coast can constitute a risk of infection with enteric viruses and that routine testing of samples may be justified. Advantages and disadvantages of various options for screening are discussed. [TOP OF PAGE]

  606. Phage taxonomy: we agree to disagree. Nelson,D. (2004). J. Bacteriol. 186:7029-7031. [first paragraph] Bacteriophage (phage) researchers are in universal agreement that phage biology has undergone a renaissance in recent years. No longer just tools of molecular biology, phage are now recognized to play critical roles in bacterial pathogenesis (3, 29) and bacterial population dynamics (5, 10). Likewise, phage therapy is enjoying renewed interest in Western medicine (24). Phage-derived proteins are currently being used as molecular machines (23), diagnostic agents (22), and therapeutic agents (15, 17, 22) and for drug discovery (14). However, the resurgence in phage popularity has only fanned the flames of an ongoing debate, namely, that of phage taxonomy. Many phage biologists feel that the current taxonomic classification scheme, devised over 3 decades ago by the International Committee on Taxonomy of Viruses (ICTV), is outdated and in need of revision in light of the postgenomic age. However, choosing one methodology on the basis of which to classify all known and yet to be discovered phage is an area where phage researchers agree to disagree. In this issue of the Journal of Bacteriology, Chibani-Chennoufi et al. provide support for a comparative genomics-based alternative classification scheme (6). [TOP OF PAGE]

  607. Presence of bacterial phage-like DNA sequences in commercial Taq DNA polymerase reagents. Newsome,T., Li,B.J., Zou,N., Lo,S.C. (2004). J. Clin. Microbiol. 42:2264-2267. Many studies have reported the presence of bacterial DNA contamination in commercial Taq DNA polymerase reagents. This is the first report of the presence of phage-like DNA sequences in certain commercial Taq DNA polymerase reagents. Precautions are needed when using amplification reagents with exogenous DNAs. [TOP OF PAGE]

  608. Removal of F-specific RNA bacteriophages in artificial recharge of groundwater--a field study. Niemi,R.M., Kytovaara,A., Paakkonen,J., Lahti,K. (2004). Water Sci. Technol. 50:155-158. Artificial recharge of groundwater offers a semi-natural means to produce raw water for drinking-water plants. Surface water works are increasingly being replaced by artificial groundwater works in Finland. Two municipalities, one serving 30,000 and the other 170,000 inhabitants, have considered filtering river water through eskers for the production of potable water. In this study the removal of bacteriophages during infiltration of river water was estimated, for the evaluation of treatment adequacy in a field study. A 5-m-deep column of sand was constructed and used to mimic the percolating phase in infiltration. An artificial esker was constructed on the riverbank by isolating a 2-m-wide, 2-m-deep and 18-m-long bed of coarse sand with plastic. The sand bed represented the saturated zone. River water was pumped at a rate of 40 L/h to the sand column. The river water was spiked with F+ specific RNA phage MS2 by adding phage suspension during one week at an average concentration of 4.3 x 10(9) PFU/mL. Samples for phage assays were taken during one month, from four sampling sites, on the basis of detention time as estimated by a tracer experiment with sodium chloride. The median count of MS2 for percolated water was 2.4 x 10(5) PFU/mL, representing a 96.7% reduction. During the passage of 6 m in the saturated zone, a further reduction of 98.5% occurred. During the passage from 6 m to 12 m the additional reduction was 99.97%. The overall reduction was between 6 and 7 log10 units. The removal of MS2 phages was rather efficient, although the esker material was coarse, mainly sandy, gravel. [TOP OF PAGE]

  609. Site-specific recombination links the evolution of P2-like coliphages and pathogenic enterobacteria. Nilsson,A.S., Karlsson,J.L., Haggard-Ljungquist,E. (2004). Mol. Biol. Evol. 21:1-13. The genome of the tailed temperate coliphage P2 (Myoviridae) contains some genes that probably are horizontally transferred additions to the genome. One of these genes, the Z/fun gene, was recently found intact in the genome of Neisseria meningitidis. We have investigated the presence of P2-like phages, and the genetic variation at the position corresponding to the phage P2 Z/fun locus, in the Escherichia coli reference collection (ECOR). P2-like phages are common in E. coli since they are present in about 30% of the ECOR strains. Hybridizations and PCR amplifications indicate that the overall variation among these phages is small. Amplification of the region corresponding to the phage P2 Z/fun locus in 11 prophages revealed that this is a multivariable locus. Sequencing of the region resulted in 10 completely different sequences but with a similar high AT-content as the Z/fun gene. All sequences contained at least one open reading frame with good transcription and translation signals. All sequences were also surrounded by a highly similar, previously undiscovered, inverted repeat (IR). We also found this IR in genetically unstable regions in pathogenic enterobacteria. This demonstrates that P2-like phages are important factors in the evolution of bacteria, not only because they carry a diversity of lysogenic conversion genes but also because they can act as vectors for single genes. The genes found between the IRs have unknown functions, and only a few clearly similar genes have been found in other bacteria. [TOP OF PAGE]

  610. Inactivation of indicator micro-organisms from various sources of faecal contamination in seawater and freshwater. Noble,R.T., Lee,I.M., Schiff,K.C. (2004). J. Appl. Microbiol. 96:464-472. AIM: The survival of indicator micro-organisms in aquatic systems is affected by both biotic and abiotic factors. Much of the past research on this topic has been conducted using laboratory-generated cultures of indicator bacteria. For this study, we used natural sources of faecal contamination as inoculants into environmental water samples, thereby representing the wide diversity of organisms likely to be found in faecal contamination. METHODS AND RESULTS: Rates of inactivation of water quality indicators, total coliforms (TC), Escherichia coli, enterococci (EC) and F+-specific coliphage were studied in three experiments using inoculants of sewage influent, sewage effluent and urban storm drain run-off. Effects of temperature, nutrients, total suspended solids, bacterial load and solar irradiation were studied in fresh and seawater matrices. Results demonstrated that temperature and solar irradiation had significant effects upon rates of inactivation (anova, P < 0.001). Inactivation rates were similar, regardless of the inoculant type. EC degraded the slowest in the dark with T90s of 115-121 and 144-177 h at 20 and 14°C, respectively. When incubated in sunlight, EC was inactivated significantly more rapidly than either E. coli or F+-specific coliphage (P < 0.001). CONCLUSIONS: Inactivation of indicator bacteria is not dependent upon the original source of contamination. Inactivation rates of indicator bacteria were similar in fresh and seawater matrices. However, EC degraded more rapidly in sunlight than E. coli. SIGNIFICANCE AND IMPACT OF THE STUDY: This study suggests that the source of faecal contamination is not an important factor to inactivation rates of indicator bacteria. However, rates of inactivation of indicator bacteria are likely system specific. [TOP OF PAGE]

  611. Testing for viral penetration of non-latex surgical and examination gloves: a comparison of three methods. O'Connell,K.P., El Masri,M., Broyles,J.B., Korniewicz,D.M. (2004). Clin. Microbiol. Infect. 10:322-326. Currently, there are no international standards based on microbiological methodology for testing the ability of medical examination or surgical gloves to prevent the passage of viruses. Three protocols for the direct examination of the viral barrier properties of non-latex gloves were compared with 1080 gloves (270 gloves from each of two surgical brands and two medical examination brands). In two of the methods, gloves were filled with and suspended in a nutrient broth solution, and bacteriophage fX174 was placed either inside or outside the glove, while the entire test vessel was agitated. Gloves tested using the third method were filled with a suspension of bacteriophage and allowed to rest in a vessel containing nutrient broth. Gloves were tested directly from the manufacturer's packaging, or after being punctured intentionally or subjected to a stress protocol. The passage of bacteriophage was detected with plaque assays. Significant differences in failure rates between glove brands were apparent only among gloves that had been subjected to the stress protocol. Overall, the two methods in which bacteriophage were placed inside the gloves provided more sensitivity than the method in which bacteriophage was spiked into broth outside the gloves. Thus the placement of bacteriophage inside test gloves (or the use of pressure across the glove barrier during testing), and the use of a standardised stress protocol, will improve significantly the ability of a glove test protocol to determine the relative quality of the barrier offered by medical examination and surgical gloves. Further research is needed to provide test methods that can incorporate reproducibly both the use of bacteriophage and simulated glove use in an industrial quality control setting. [TOP OF PAGE]

  612. Genome of staphylococcal phage K: a new lineage of myoviridae infecting Gram-positive bacteria with a low G-C content. O'Flaherty,S., Coffey,A., Edwards,R., Meaney,W., Fitzgerald,G.F., Ross,R.P. (2004). J. Bacteriol. 186:2862-2871. Phage K is a polyvalent phage of the Myoviridae family which is active against a wide range of staphylococci. Phage genome sequencing revealed a linear DNA genome of 127,395 bp, which carries 118 putative open reading frames. The genome is organized in a modular form, encoding modules for lysis, structural proteins, DNA replication, and transcription. Interestingly, the structural module shows high homology to the structural module from Listeria phage A511, suggesting intergenus horizontal transfer. In addition, phage K exhibits the potential to encode proteins necessary for its own replisome, including DNA ligase, primase, helicase, polymerase, RNase H, and DNA binding proteins. Phage K has a complete absence of GATC sites, making it insensitive to restriction enzymes which cleave this sequence. Three introns (lys-I1, pol-I2, and pol-I3) encoding putative endonucleases were located in the genome. Two of these (pol-I2 and pol-I3) were found to interrupt the DNA polymerase gene, while the other (lys-I1) interrupts the lysin gene. Two of the introns encode putative proteins with homology to HNH endonucleases, whereas the other encodes a 270-amino-acid protein which contains two zinc fingers (CX2CX22CX2C and CX2CX23CX2C). The availability of the genome of this highly virulent phage, which is active against infective staphylococci, should provide new insights into the biology and evolution of large broad-spectrum polyvalent phages. [TOP OF PAGE]

  613. Evaluation of a cocktail of three bacteriophages for biocontrol of Escherichia coli O157:H7. O'Flynn,G., Ross,R.P., Fitzgerald,G.F., Coffey,A. (2004). Appl. Environ. Microbiol. 70:3417-3424. Escherichia coli O157:H7 is an endemic pathogen causing a variety of human diseases including mild diarrhea, hemorrhagic colitis, hemolytic-uremic syndrome, and thrombotic thrombocytopenic purpura. This study concerns the exploitation of bacteriophages as biocontrol agents to eliminate the pathogen E. coli O157:H7. Two distinct lytic phages (e11/2 and e4/1c) isolated against a human strain of E. coli O157:H7, a previously isolated lytic phage (pp01), and a cocktail of all three phages were evaluated for their ability to lyse the bacterium in vivo and in vitro. Phage e11/2, pp01, and the cocktail of all three virulent phages resulted in a 5-log-unit reduction of pathogen numbers in 1 h at 37°C. However, bacteriophage-insensitive mutants (BIMs) emerged following the challenge. All tested BIMs had a growth rate which approximated that of the parental O157 strain, although many of these BIMs had a smaller, more coccoid cellular morphology. The frequency of BIM formation (10(-6) CFU) was similar for e11/2, pp01, and the phage cocktail, while BIMs insensitive to e4/1c occurred at the higher frequency (10(-4) CFU). In addition, BIMs commonly reverted to phage sensitivity within 50 generations. In an initial meat trial experiment, the phage cocktail completely eliminated E. coli O157:H7 from the beef meat surface in seven of nine cases. Given that the frequency of BIM formation is low (10(-6) CFU) for two of the phages, allied to the propensity of these mutants to revert to phage sensitivity, we expect that BIM formation should not hinder the use of these phages as biocontrol agents, particularly since low levels of the pathogen are typically encountered in the environment. [TOP OF PAGE]

  614. Management of tomato bacterial spot in the field by foliar applications of bacteriophages and SAR inducers. Obradovic,A., Jones,J.B., Momol,M.T., Balogh,B., Olson,S.M. (2004). Plant Dis. 88:736-740. Various combinations of the harpin protein, acibenzolar-S-methyl, and bacteriophages were compared for controlling tomato bacterial spot in field experiments. Harpin protein and acibenzolar- S-methyl were applied every 14 days beginning twice before transplanting and then an additional four applications throughout the season. Formulated bacteriophages were applied prior to inoculation followed by twice a week at dusk. A standard bactericide treatment, consisting of copper hydroxide plus mancozeb, was applied once prior to inoculation and then every 7 days, while untreated plants served as an untreated control. Experiments were conducted in north and central Florida fields during fall 2001, spring 2002, and fall 2002. In three consecutive seasons, acibenzolar-S-methyl applied in combination with bacteriophage or bacteriophage and harpin significantly reduced bacterial spot compared with the other treatments. However, it did not significantly affect the total yield compared with the standard or untreated control. Application of host-specific bacteriophages was effective against the bacterial spot pathogen in all three experiments, providing better disease control than copper-mancozeb or untreated control. When results of the disease severity assessments or harvested yield from the bacteriophage-treated plots were grouped and compared with the results of the corresponding nonbacteriophage group, the former provided significantly better disease control and yield of total marketable fruit. [TOP OF PAGE]

  615. Rapid detection of Escherichia coli O157:H7 by using green fluorescent protein-labeled PP01 bacteriophage. Oda,M., Morita,M., Unno,H., Tanji,Y. (2004). Appl. Environ. Microbiol. 70:527-534. A previously isolated T-even-type PP01 bacteriophage was used to detect its host cell, Escherichia coli O157:H7. The phage small outer capsid (SOC) protein was used as a platform to present a marker protein, green fluorescent protein (GFP), on the phage capsid. The DNA fragment around soc was amplified by PCR and sequenced. The gene alignment of soc and its upstream region was g56-soc.2-soc.1-soc, which is the same as that for T2 phage. GFP was introduced into the C- and N-terminal regions of SOC to produce recombinant phages PP01-GFP/SOC and PP01-SOC/GFP, respectively. Fusion of GFP to SOC did not change the host range of PP01. On the contrary, the binding affinity of the recombinant phages to the host cell increased. However, the stability of the recombinant phages in alkaline solution decreased. Adsorption of the GFP-labeled PP01 phages to the E. coli cell surface enabled visualization of cells under a fluorescence microscope. GFP-labeled PP01 phage was not only adsorbed on culturable E. coli cells but also on viable but nonculturable or pasteurized cells. The coexistence of insensitive E. coli K-12 (W3110) cells did not influence the specificity and affinity of GFP-labeled PP01 adsorption on E. coli O157:H7. After a 10-min incubation with GFP-labeled PP01 phage at a multiplicity of infection of 1,000 at 4ºC, E. coli O157:H7 cells could be visualized by fluorescence microscopy. The GFP-labeled PP01 phage could be a rapid and sensitive tool for E. coli O157:H7 detection. [TOP OF PAGE]

  616. Effects of freezing and storage temperature on MS2 viability. Olson,M.R., Axler,R.P., Hicks,R.E. (2004). J. Virol. Meth. 122:147-152. Monitoring human enteric virus levels in domestic wastewater effluent is crucial to protecting human health. Occasionally, during intensive sampling, wastewater samples must be stored for later viral analysis. Little data exist regarding how enteric viruses survive during storage at different temperatures in secondary treated wastewater. During a field-scale study assessing pathogen removal performance by various onsite treatment technologies, the MS2 bacteriophage, an indicator of enteric viruses, was inoculated into septic tank (STE), sand filter, peat filter and constructed wetland (CW) effluents to determine virus decay at various storage temperatures. Virus stored at temperatures > or =10°C and at -20°C decayed nearly twice as fast as those stored at 4°C or -80°C. Decreased water quality decreased viral decay rates at 4°C and -80°C, with slowest decay occurring in STE and the fastest in sterile PBS and low pH peat effluent. In CW effluent after 8 days, less MS2 was inactivated when stored at 4°C (20%) compared to -80°C (58%); however, during extended storage (approximately 300 days), less MS2 was inactivated at -80°C (75%) compared to 4°C (93%). We recommend that viruses in wastewater be stored in the dark at 4°C unless storage for >40 days is necessary. [TOP OF PAGE]

  617. Approximate solution to the speed of spreading viruses. Ortega-Cejas,V., Fort,J., Méndez,V., Campos,D. (2004). Phys. Rev. E 69:031909-1-031909-4. Recently, it has been shown that the speed of virus infections can be explained by time-delayed reactiondiffusion [J. Fort and V. Méndez, Phys. Rev. Lett. 89, 178101 (2002)], but no analytical solutions were found. Here we derive formulas for the front speed, valid in appropriate limits. We also integrate numerically the evolution equations of the system. There is good agreement with both numerical and experimental speeds. [TOP OF PAGE]

  618. Estimation of septic tank setback distances based on transport of E. coli and F-RNA phages. Pang,L., Close,M., Goltz,M., Sinton,L., Davies,H., Hall,C., Stanton,G. (2004). Environment International 29:907-921. Setback distances between septic tank systems and the shorelines of Lake Okareka, New Zealand were determined from model simulations for a worst-case scenario, using the highest hydraulic conductivity and gradient measured in the field, removal rates of the microbial indicators (Escherichia coli and F-RNA phages) determined from a column experiment, and maximum values of the design criteria for the disposal system, and assuming an absence of an unsaturated zone, a continuous discharge of the raw effluent from a failed or non-complying treatment system (both indicators at concentrations of 1x10(7) counts/100 ml) into the groundwater and no sorption of pathogens in the aquifer. Modelling results suggest that the minimal setback distances were 16 m to satisfy the New Zealand Recreational Water Quality Guidelines for E. coli <126 per 100 ml (Ministry for the Environment, 1999) and 48 m to meet the Drinking-Water Standards for New Zealand 2000 for enteric virus <1 per 100 l (Ministry of Health, 2000). These distances may be applicable for other lakeshores in pumice sand aquifers with groundwater velocities <7 m/day. Findings of laboratory column and batch experiments provided an insight into the microbial attenuation and transport processes in pumice sand aquifers. Bacterial removal was predominately through filtration (87-88%) and partially by die-off (12-13%), while viral removal was by both die-off (45%) and filtration (55%). In addition, microbial die-off in groundwater without aquifer material (i.e., free microbes) was much lower than die-off in groundwater with aquifer material (i.e., sorbed microbes) and contributed only 2-6% to the total removal. This implies that the setback distances estimated from die-off rates for the free microbes, determined in the laboratory without considering aquifer media and other removal processes, which are often reported in the literature, could be larger than necessary. [TOP OF PAGE]

  619. Identification of bacteriophage types and their carriage in Staphylococcus aureus. Pantucek,R., Doskar,J., Ruzicková,V., Kaspárek,P., Orácová,E., Kvardová,V., Rosypal,S. (2004). Archives in Virology 149:1689-1703. Conserved genomic sequences distinctive of Staphylococcus aureus phage types 3A, 11, 77, 187 and Twort, representative of phage serogroups A, B, F, L and D, were identified and characterized. PCR primers designed for the above sequences were used for development of a multiplex PCR assay which enabled us not only to classify all phages of the International Typing Set plus 16 additional phages, but also to detect prophages in S. aureus genomes. One to four different prophages were unambiguously detected in experimentally lysogenized S. aureus strains, and substantial variation in prophage content was found in 176 S. aureus clinical strains of different provenance. In addition, by using a comparative genomics approach, all the prophages in the S. aureus genomes sequenced to date could be revealed and classified. [TOP OF PAGE]

  620. The ability of two different Vibrio spp. bacteriophages to infect Vibrio harveyi, Vibrio cholerae and Vibrio mimicus. Payne,M., Oakey,J., Owens,L. (2004). J. Appl. Microbiol. 97:663-672. AIMS: To determine the host range of the Vibrio harveyi myovirus-like bacteriophage (VHML) and the cholera toxin conversion bacteriophage (CTX Phi) within a range of Vibrio cholerae and V. mimicus and V. harveyi, V. cholerae and V. mimicus isolates respectively. METHODS AND RESULTS: Three V. harveyi, eight V. cholerae and five V. mimicus isolates were incubated with VHML and CTX Phi. Polymerase chain reaction (PCR) was used to determine the presence of VHML and CTX Phi in infected isolates. We demonstrated that it was possible to infect one isolate of V. cholerae (isolate ACM #2773/ATCC #14035) with VHML. This isolate successfully incorporated VHML into its genome as evident by positive PCR amplification of the sequence coding part of the tail sheath of VHML. Attempts to infect all other V. cholerae and V. mimicus isolates with VHML were unsuccessful. Attempts to infect V. cholerae non-01, V. harveyi and V. mimicus isolates with CTX Phi were unsuccessful. CONCLUSIONS: Bacteriophage infection is limited by bacteriophage-exclusion systems operating within bacterial strains and these systems appear to be highly selective. One system may allow the co-existence of one bacteriophage while excluding another. VHML appears to have a narrow host range which may be related to a common receptor protein in such strains. The lack of the vibrio pathogenicity island bacteriophage (VPI Phi) in the isolates used in this study may explain why infections with CTX Phi were unsuccessful. SIGNIFICANCE AND IMPACT OF THE STUDY: The current study has demonstrated that Vibrio spp. bacteriophages may infect other Vibrio spp. [TOP OF PAGE]

  621. Bacteria and viruses in the water column of tropical freshwater reservoirs. Peduzzi,P., Schiemer,F. (2004). Environ. Microbiol. 6:707-715. In tropical freshwater reservoirs of Sri Lanka, which are linked in an aquatic network, bacterial abundance and production as well as virus abundance, frequency of viral infection and virus production were investigated together with a set of nutrient species (Kjeldahl-N, NO3-N, total P, soluble P, PO4-P). At two characteristic seasons (wet season, dry season), samples were taken from two types of reservoirs (new upland impoundment and ancient, shallow lowland reservoir), each during 4 days at various depths of the entire water columns. Kjeldahl-N and total P were greatly elevated in the wind-mixed water body of the shallow impoundment during the dry season, whereas the deeper reservoir type exhibited no obvious seasonality. In SYBR green trade mark -stained samples, bacterial abundance showed no seasonal pattern in either reservoir type. Bacterial secondary production, however, was significantly elevated in the entire water column of the shallow impoundment under wind-mixed conditions in the dry season. Highest abundance of virus particles and elevated frequency of bacteria containing mature phages were also observed in the shallow reservoir during the dry season indicating favourable conditions for virus propagation. Data from this aquatic network show that most virus parameters, such as abundance or frequency of visibly infected cells, were positively linked to bacterial abundance and production, but also to organic nitrogen or some phosphorus species. We calculated that between 13.2% and 46.1% of the bacterial standing stocks would be subjected to virus-mediated mortality. Estimates of bacteriophage production revealed that from 10 x 10(9) up to 98 x 10(9) phages were produced per litre and day. Bacteria and viruses in the studied tropical freshwater system appear to be linked to various environmental conditions and may affect processes at the ecosystem scale. [TOP OF PAGE]

  622. Sewage impact on shellfish microbial contamination. Pommepuy,M., Dumas,F., Caprais,M.P., Camus,P., Le Mennec,C., Parnaudeau,S., Haugarreau,L., Sarrette,B., Vilagines,P., Pothier,P., Kholi,E., Le Guyader,F. (2004). Water Sci. Technol. 50:117-124. Coastal areas are frequently contaminated by microorganisms of human origin, due to high population density and low seawater renewal. To evaluate the impact of wastewater input on shellfish quality, a study was conducted in Brittany (France) over a period of 20 months. A hydrodynamic model was used to simulate wastewater impact on microbial water quality. To validate the model, wastewater from the three main sewage treatment plants and shellfish from three sites were sampled monthly. Bacterial indicators (E. coli), F-RNA phages were searched for by culture and noroviruses by RT-PCR and hybridisation. These microorganisms were detected in the three effluents and clams, with no marked seasonal variation. The microbial concentrations in the two oyster beds, distant from the effluent outfall, were low, and only three of the samples were positive for norovirus. For simulation, the winter wastewater inputs of E. coli and phages were calculated and an estimation for norovirus flux was made from the epidemic situation in the population. The microbial behaviour was included in the model by a decay-rate factor. Results from the model calculations were found to be very similar to E. coli and phage concentrations observed in shellfish. For noroviruses, the model indicated that shellfish distant from the wastewater input were under the detection limit of the RT-PCR method. This study demonstrated the use of modelisation to interpret norovirus contamination in various areas. [TOP OF PAGE]

  623. Drift increases the advantage of sex in RNA bacteriophage F6. Poon,A., Chao,L. (2004). Genetics 166:19-24. The pervasiveness of sex and recombination remains one of the most enigmatic problems in evolutionary biology. According to many theoretical models, recombination can increase the rate of adaptation by restoring genetic variation. However, the potential for genetic drift to generate conditions that produce this outcome has yet to be studied experimentally. We have designed and performed an experiment that reveals the effects of drift on existing genetic variation by minimizing the influence of variation on beneficial mutation rate. Our experiment was conducted in populations of RNA bacteriophage F6 initiated from a common source population at varying bottleneck sizes. The segmented genome of this virus results in genetic exchange between viruses that co-infect the same host cell. In response to selection for growth in a high-temperature environment, sexual lines outperformed their asexual counterparts on average. The advantage of sex attenuated with increasing effective population size, implying that the rate of adaptation was limited by clonal interference among segments caused by drift. This is the first empirical evidence that the advantage of sex during adaptation increases with the intensity of drift. [TOP OF PAGE]

  624. The Pasteurella multocida toxin is encoded within a lysogenic bacteriophage. Pullinger,G.D., Bevir,T., Lax,A.J. (2004). Mol. Microbiol. 51:255-269. Toxigenic strains of Pasteurella multocida produce a 146 kDa toxin (PMT) that acts as a potent mitogen. Sequence analysis of the structural gene for PMT, toxA, previously suggested it was horizontally acquired, because it had a low G + C content relative to the P. multocida genome. To address this, the sequence of DNA flanking toxA was determined. The sequence analysis showed the presence of homologues to bacteriophage tail protein genes and a bacteriophage antirepressor, suggesting that the toxin gene resides within a prophage. In addition to phage genes, the toxA flanking DNA contained a homologue of a restriction/modification system that was shown to be functional. The presence of a bacteriophage was demonstrated in spent medium from toxigenic P. multocida isolates. Its production was increased by mitomycin C addition, a treatment that is known to induce the lytic cycle of many temperate bacteriophages. The genomes of bacteriophages from three different toxigenic P. multocida strains had similar but not identical restriction profiles, and were approximately 45-50 kb in length. The prophages from two of these had integrated at the same site in the chromosome, in a tRNA gene. Southern blot analysis confirmed that these bacteriophages contained the toxA gene. [TOP OF PAGE]

  625. Characterization of three Lactobacillus delbrueckii subsp. bulgaricus phages and the physicochemical analysis of phage adsorption. Quiberoni,A., Guglielmotti,D., Binetti,A., Reinheimer,J. (2004). J. Appl. Microbiol. 96:340-351. AIMS: Three indigenous Lactobacillus delbrueckii subsp. bulgaricus bacteriophages and their adsorption process were characterized. METHODS AND RESULTS: Phages belonged to Bradley's group B or the Siphoviridae family (morphotype B1). They showed low burst size and short latent periods. A remarkably high sensitivity to pH was also demonstrated. Indigenous phage genomes were linear and double-stranded DNA molecules of approx. 31-34 kbp, with distinctive restriction patterns. Only one phage genome appeared to contain cohesive ends. Calcium ions did not influence phage adsorption, but it was necessary to accelerate cell lysis and improve plaque formation. The adsorption kinetics were similar on viable and nonviable cells, and the adsorption rates were high between 0 and 50 degrees C. SDS and proteinase K treatments did not influence the phage adsorption but mutanolysin and TCA reduced it appreciably. No significant inhibitory effect on phage adsorption was observed for the saccharides tested. This study also revealed the irreversibility of phage adsorption to their hosts. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: The study increases the knowledge on phages of thermophilic lactic acid bacteria. [TOP OF PAGE]

  626. [Combined use of active chlorine and coagulants for drinking water purification and disinfection]. Rakhmanin,I.A., Zholdakova,Z.I., Poliakova,E.E., Kir'ianova,L.F., Miasnikov,I.N., Tul'skaia,E.A., Artemova,T.Z., Ivanova,L.V., Dmitrieva,R.A., Doskina,T.V. (2004). Gigiena i Sanitariia 6-9. The authors made an experimental study of the efficiency of water purification procedures based on the combined use of active chlorine and coagulants and hygienically evaluated the procedures. The study included the evaluation of water disinfection with various coagulants and active chlorine; the investigation of the processes of production of deleterious organic chlorine compounds; the assessment of the quality of water after its treatment. The coagulants representing aluminum polyoxychloride: RAX-10 (AQUA-AURATE 10) and RAX-18 (AQUA-AURATE 18), and aluminum sulfate, technically pure grade were tested. The treatment of river water with the coagulants RAX-10 and RAX-18, followed by precipitation, filtration, and chlorination under laboratory conditions, was shown to result in water disinfection to the levels complying with the requirements described in SanPiN 2.1.4.1074-01. RAX-18 showed the best disinfecting activity against total and heat-tolerant coliform bacteria, but also to the highly chlorine-resistant microrganisms--the spores of sulfite-reducing Clostridia, phages, and viruses. Since the coagulants have an increased sorptive capacity relative to humus and other organic substances, substitution of primary chlorination for coagulant treatment may induce a reduction in the risk of formation of oncogenically and mutagenically hazardous chlorinated hydrocarbons. [TOP OF PAGE]

  627. The structure of a thermophilic archaeal virus shows a double-stranded DNA viral capsid type that spans all domains of life. Rice,G., Tang,L., Stedman,K., Roberto,F., Spuhler,J., Gillitzer,E., Johnson,J.E., Douglas,T., Young,M. (2004). Proc. Natl. Acad. Sci. USA 101:7716-7720. Of the three domains of life (Eukarya, Bacteria, and Archaea), the least understood is Archaea and its associated viruses. Many Archaea are extremophiles, with species that are capable of growth at some of the highest temperatures and extremes of pH of all known organisms. Phylogenetic rRNA-encoding DNA analysis places many of the hyperthermophilic Archaea (species with an optimum growth 80°C) at the base of the universal tree of life, suggesting that thermophiles were among the first forms of life on earth. Very few viruses have been identified from Archaea as compared to Bacteria and Eukarya. We report here the structure of a hyperthermophilic virus isolated from an archaeal host found in hot springs in Yellowstone National Park. The sequence of the circular double-stranded DNA viral genome shows that it shares little similarity to other known genes in viruses or other organisms. By comparing the tertiary and quaternary structures of the coat protein of this virus with those of a bacterial and an animal virus, we find conformational relationships among all three, suggesting that some viruses may have a common ancestor that precedes the division into three domains of life >3 billion years ago. [TOP OF PAGE]

  628. The genome and proteome of coliphage T1. Roberts,M.D., Martin,N.L., Kropinski,A.M. (2004). Virology 318:245-266. The genome of enterobacterial phage T1 has been sequenced, revealing that its 50.7-kb terminally redundant, circularly permuted sequence contains 48,836 bp of nonredundant nucleotides. Seventy-seven open reading frames (ORFs) were identified, with a high percentage of small genes located at the termini of the genomes displaying no homology to existing phage or prophage proteins. Of the genes showing homologs (47%), we identified those involved in host DNA degradation (three endonucleases) and T1 replication (DNA helicase, primase, and single-stranded DNA-binding proteins) and recombination (RecE and Erf homologs). While the tail genes showed homology to those from temperate coliphage N15, the capsid biosynthetic genes were unique. Phage proteins were resolved by 2D gel electrophoresis, and mass spectrometry was used to identify several of the spots including the major head, portal, and tail proteins, thus verifying the annotation. [TOP OF PAGE]

  629. Conjugative and mobilizable transposons. Salyers,A.A., Whittle,G., Shoemaker,N.B. (2004). pp. 125-143. In In Miller,R.V. and Day,M.J. (eds.), Mirobial Evolution: Gene Establishment, Survival, and Exchange. ASM Press, Washington, DC. [TOP OF PAGE]

  630. Distribution, sequence homology, and homing of group I introns among T-even-like bacteriophages: evidence for recent transfer of old introns. Sandegren,L., Sjoberg,B.M. (2004). The Journal of Biological Chemistry 279:22218-22227. Self-splicing group I introns are being found in an increasing number of bacteriophages. Most introns contain an open reading frame coding for a homing endo-nuclease that confers mobility to both the intron and the homing endonuclease gene (HEG). The frequent occurrence of intron/HEG has raised questions whether group I introns are spread via horizontal transfer between phage populations. We have determined complete sequences for the known group I introns among T-even-like bacteriophages together with sequences of the intron-containing genes td, nrdB, and nrdD from phages with and without introns. A previously uncharacterized phage isolate, U5, is shown to contain all three introns, the only phage besides T4 found with a "full set" of these introns. Sequence analysis of td and nrdB genes from intron-containing and intronless phages provides evidence that recent horizontal transmission of introns has occurred among the phages. The fact that several of the HEGs have suffered deletions rendering them non-functional implies that the homing endonucleases are of no selective advantage to the phage and are rapidly degenerating and probably dependent upon frequent horizontal transmissions for maintenance within the phage populations. Several of the introns can home to closely related intronless phages during mixed infections. However, the efficiency of homing varies and is dependent on homology in regions flanking the intron insertion site. The occurrence of optional genes flanking the respective intron-containing gene can strongly affect the efficiency of homing. These findings give further insight into the mechanisms of propagation and evolution of group I introns among the T-even-like bacteriophages. [TOP OF PAGE]

  631. Movement of viruses between biomes. Sano,E., Carlson,S., Wegley,L., Rohwer,F. (2004). Appl. Environ. Microbiol. 70:5842-5846. Viruses are abundant in all known ecosystems. In the present study, we tested the possibility that viruses from one biome can successfully propagate in another. Viral concentrates were prepared from different near-shore marine sites, lake water, marine sediments, and soil. The concentrates were added to microcosms containing dissolved organic matter as a food source (after filtration to allow 100-kDa particles to pass through) and a 3% (vol/vol) microbial inoculum from a marine water sample (after filtration through a 0.45-mm-pore-size filter). Virus-like particle abundances were then monitored using direct counting. Viral populations from lake water, marine sediments, and soil were able to replicate when they were incubated with the marine microbes, showing that viruses can move between different ecosystems and propagate. These results imply that viruses can laterally transfer DNA between microbes in different biomes. [TOP OF PAGE]

  632. Newly isolated Vibrio cholerae non-O1, non-O139 phages. Sarkar,B.L., Ghosh,A.N., Sen,A., Rodrigues,D.P. (2004). Emerg. Infect. Dis. 10:754-756. To the Editor: The epidemic cholera caused by Vibrio cholerae O1 appeared in Latin America in 1991 after a 100-year absence. Following its explosive appearance in Peru, travelers on the Amazon River brought cholera to Brazil by April 1991. It spread southward along the Atlantic Coast of Brazil, reaching Rio de Janeiro in February 1993. ¶ Phage typing is a useful tool for studying the source or origin of V. cholerae for epidemiologic importance. Because of limitations of the Basu and Mukerjee scheme, a new phage-typing scheme for V. cholerae O1 was developed at the National Institute of Cholera and Enteric Diseases, India (1-3). During the course of a comprehensive study on the phage typing of V. cholerae O1, most strains isolated in Brazil were found to be sensitive with a set of 10 El Tor phages (ATCC 51352-B1-B10) (4). This finding prompted us to explore or ascertain the natural habitat of V. cholerae and cholera phages, if any, in an environmental reservoir in Brazil, particularly in Rio de Janeiro. [TOP OF PAGE]

  633. Characterization of nutrient-induced dispersion in Pseudomonas aeruginosa PAO1 biofilm. Sauer,K., Cullen,M.C., Rickard,A.H., Zeef,L.A.H., Davies,D.G., Gilbert,P. (2004). J. Bacteriol. 186:7312-7326. The processes associated with early events in biofilm formation have become a major research focus over the past several years. Events associated with dispersion of cells from late stage biofilms have, however, received little attention. We demonstrate here that dispersal of Pseudomonas aeruginosa PAO1 from biofilms is inducible by a sudden increase in carbon substrate availability. Most efficient at inducing dispersal were sudden increases in availability of succinate > glutamate > glucose that led to approximately 80% reductions in surface-associated biofilm biomass. Nutrient-induced biofilm dispersion was associated with increased expression of flagella (fliC) and correspondingly decreased expression of pilus (pilA) genes in dispersed cells. Changes in gene expression associated with dispersion of P. aeruginosa biofilms were studied by using DNA microarray technology. Results corroborated proteomic data that showed gene expression to be markedly different between biofilms and newly dispersed cells. Gene families that were upregulated in dispersed cells included those for flagellar and ribosomal proteins, kinases, and phage PF1. Within the biofilm, genes encoding a number of denitrification pathways and pilus biosynthesis were also upregulated. Interestingly, nutrient-induced dispersion was associated with an increase in the number of Ser/Thr-phosphorylated proteins within the newly dispersed cells, and inhibition of dephosphorylation reduced the extent of nutrient-induced dispersion. This study is the first to demonstrate that dispersal of P. aeruginosa from biofilms can be induced by the addition of simple carbon sources. This study is also the first to demonstrate that dispersal of P. aeruginosa correlates with a specific dispersal phenotype. [TOP OF PAGE]

  634. Phage offer a real alternative. Schoolnik,G.K., Summers,W.C., Watson,J.D. (2004). Nat. Biotech. 22:505-506. A News and Views piece by Steven Projan in your February issue offers a gratuitous, pessimistic assessment for the prospects of phage therapy per se (Nat. Biotechnol. 22, 167-168, 2004). We believe Projan's criticisms are overly broad and fail to consider the published literature and the impact that contemporary phage biology is having on the development of phage therapeutics. We would not have been moved to respond to his comments were it not for our view that the pharmaceutical industry's capacity to develop truly novel chemical antibiotics or antibacterials is being outstripped through the evolution of antimicrobial resistance by a broad array of infectious agents. Thus, in the spirit of a constructive dialogue, we—participants at a Cold Spring Harbor Banbury Conference—offer the following rejoinder. [TOP OF PAGE]

  635. Viral effects on bacterial community composition in marine plankton microcosms. Schwalbach,M.S., Hewson,I., Fuhrman,J.A. (2004). Aquat. Microb. Ecol. 34:117-127. Recent theory suggests that viruses influence bacterial community composition by killing the winners of resource competition. We tested aspects of this theory by growing natural marine bacteria communities in seawater microcosms that had either significantly reduced or increased virus abundances and monitoring changes in the bacterial communities. Bacterial community composition was assayed by 2 whole-community fingerprinting techniques, terminal restriction fragment length polymorphisms (TRFLP) and automated ribosomal intergenic spacer analysis (ARISA), at the beginning and end of experiments to examine the effect of changes in viral abundance on bacterial community composition. Our results suggest that changes in viral abundances have mixed effects on microbial community fingerprints. Modest, but statistically significant, changes in community fingerprints were seen when most viruses were removed by filtration and bacteria subsequently grown over 5 d compared to growth at normal virus density. There were no significant differences between community fingerprints from microcosms grown with a normal versus 3-fold density of viruses over 2 d (possibly because of slow growth rates); however, significant changes occurred over time, regardless of virus abundance, suggesting that manipulation and containment alone had a strong influence on community fingerprints, which may have masked some virus effects. Also, moderate natural variation in composition between replicate microcosms made it difficult to distinguish statistically significant virus effects. Given that relatively few significant changes were apparent in community fingerprints between virus treatments at the end of our experiments, it is possible that current models of virus infection and the possible roles of viruses in controlling community composition need re-evaluation. The persistence of high viral abundance and apparent high turnover, in concert with our results, suggests that viruses and their hosts may have a more stable coexistence than is now currently thought. However, it is possible that the modest effects of viral infection observed in this short-term study could be significantly amplified over longer time-scales of weeks or months, resulting in viruses having a more substantial influence on bacterial community composition. [TOP OF PAGE]

  636. Improved isolation of undersampled bacteriophages: finding of distant terminase genes. Serwer,P., Hayes,S.J., Zaman,S., Lieman,K., Rolando,M., Hardies,S.C. (2004). Virology 329:412-424. Isolation and characterization of new environmental bacteriophages are performed for (1) analyzing microbial evolution and ecology and (2) delivering biological therapy. The sampling of environmental bacteriophages appears, however, to be limited by the procedure (usually liquid enrichment culture) used to propagate them. An alternative, less competitive procedure is developed here for the purpose of isolating new bacteriophages. This procedure involves extraction directly into and then propagation in a dilute agarose gel. Adaptations of this procedure are used to avoid repeated isolation of the same bacteriophage. Some newly isolated bacteriophages grow so poorly that they appear inaccessible to liquid enrichment culture. Four comparatively high titer bacteriophages were isolated and characterized by a genomic sequence survey. Some had genomes with extremely distant relationships to those of other bacteriophages, based on a tree built from the large terminase genes. These methods find novel genomes by rapidly isolating and screening diverse bacteriophages. [TOP OF PAGE]

  637. [Bacteriophages as antibacterial agents]. Shasha,S.M., Sharon,N., Inbar,M. (2004). Harefuah 143:121-5, 166. Bacteriophages are viruses that only infect bacteria. They have played an important role in the development of molecular biology and have been used as anti-bacterial agents. Since their independent discovery by Twort and d'Herelle, they have been extensively used to prevent and treat bacterial infections, mainly in Eastern Europe and the former Soviet Union. In western countries this method has been sporadically employed on humans and domesticated animals. However, the discovery and widespread use of antibiotics, coupled with doubts about the efficacy of phage therapy, led to an eclipse in the use of phage in medicine. The emergence of antibiotic resistant bacteria, especially strains that are multiply resistant, has resulted in a renewed interest in alternatives to conventional drugs. One of the possible replacements for antibiotics is the use of bacteriophages as antimicrobial agents. This brief review aims to describe the history of bacteriophage and early clinical studies on their use in bacterial disease prophylaxis and therapy, and discuss the advantages and disadvantages of bacteriophage in this regard. [TOP OF PAGE]

  638. Pseudomonas fluorescens infection by bacteriophage PhiS1: the influence of temperature, host growth phase and media. Sillankorva,S., Oliveira,R., Vieira,M.J., Sutherland,I., Azeredo,J. (2004). FEMS Microbiol. Lett. 241:13-20. The influence of host growth temperature, phase and media, together with the effect of infection temperature on bacteriophage PhiS1 infection of Pseudomonas fluorescens were examined. The rates of cell lysis and phage release were determined and showed that the efficacy of phage infection was optimal with host cells grown and infected at 26°C. The host physiological state also affected these rates. Infection was dependent on the presence of cell wall proteins with molecular weights of 17.5+/-1 and 99+/-5 kDa. [TOP OF PAGE]

  639. Bacteriophage F S1 infection of Pseudomonas fluorescens planktonic cells versus biofilms. Sillankorva,S., Oliveira,R., Vieira,M.J., Sutherland,I.W., Azeredo,J. (2004). Biofouling 20:133-138. This communication focuses on the efficacy of a specific lytic phage, phage F S1, as a control agent of Pseudomonas fluorescens biofilms. The effect of phage infection temperature and the host growth temperature were evaluated. The results obtained showed that the phage infection process was temperature dependent and that the optimum temperature of infection of planktonic cells and biofilms was 26 degrees C. At this temperature, bacteriophage F S1, at a multiplicity of infection (MOI) of 0.5 infected both planktonic cells and biofilms causing a biomass reduction of about 85% in both cases. [TOP OF PAGE]

  640. Comparison of coliforms and coliphages as tools for assessment of viral contamination in river water. Skraber,S., Gassilloud,B., Gantzer,C. (2004). Appl. Environ. Microbiol. 70:3644-3649. The aim of the study was to evaluate the presence of pathogenic viruses in the Moselle River and to compare the usefulness of thermotolerant coliforms and somatic coliphages as tools for river water quality assessment in terms of viral contamination. Thermotolerant coliforms and somatic coliphages were enumerated by standardized methods in 170 samples of river water drawn from five sampling sites along the Moselle River (eastern France). BGM cell culture and integrated cell culture-reverse transcription-PCR DNA enzyme immunoassay were used to determine the presence of pathogenic viral genome (Enterovirus and Norovirus genogroup II [GGII]) and infectious Enterovirus spp. in 90 1-liter samples. No infectious Enterovirus spp. were isolated, but Enterovirus and Norovirus GGII genomes were detected in 38% of the samples. Norovirus GGII genome was mostly detected in winter, whereas Enterovirus genome was mostly detected in summer and fall. Somatic coliphages appeared to be less sensitive to higher river water temperature than thermotolerant coliforms. Furthermore, the number of river water samples positive for pathogenic viral genome increased with increasing concentration of somatic coliphages, whereas coliform concentration was unrelated to viral genome contamination. Consequently somatic coliphages, which are less sensitive to environmental factors than thermotolerant coliforms in river water, would provide a promising tool for assessment of river water quality in terms of fecal and viral pollution. [TOP OF PAGE]

  641. Ten new temperate bacteriophages of Citrobacter youngae. Smarda,J., Slovackova,H. (2004). Folia Microbiol (Praha) 49:671-678. In a cross-test, we examined 55 strains of Citrobacter youngae against each other as potential producers of temperate bacteriophages and as potential sensitive indicators for them. Ten strains (18.2 %) showed the production of phages. Seven different strain-specific spectra of activity (from 1 to 11 strains each) were found. Phage production by 6 strains was inducible with mitomycin C, in 4 strains it was not inducible. The plaques of the phages were more or less turbid, without a lytic halo, tiny to small, 0.2-1.3 mm in diameter. Using a polyclonal, specific anti-lambda serum, all 10 phages were found to be clearly distinct from E. coli lambda phage, the phage 31/47 showing the highest neutralization titre of all. Interspecific tests with 15 strains of 8 species of Enterobacteriaceae revealed not a single case of activity of Citrobacter phages towards any of them. Five phage-immune clones lysogenized with 5 of the phages kept their remaining phage sensitivity spectra, though extended by sensitivity to 1-3 phages; 2 of these strains acquired also sensitivity to phage lambda. The phages belong to the morphotypes of Myoviridae (6 phages) and Siphoviridae (4 phages), with head diameters of 51-58 nm and tail length of 97-173 nm. Three strains produced corpuscular bacteriocins. [TOP OF PAGE]

  642. Effects of culturing on the population structure of a hyperthermophilic virus. Snyder,J.C., Spuhler,J., Wiedenheft,B., Roberto,F.F., Douglas,T., Young,M.J. (2004). Microb. Ecol. 48:561-566. The existence of a culturing bias has long been known when sampling organisms from the environment. This bias underestimates microbial diversity and does not accurately reflect the most ecologically relevant species. Until now no study has examined the effects of culture bias on viral populations. We have employed culture independent methods to assess the diversity of Sulfolobus spindle-shaped viruses (SSVs) from extremely hyperthermal environments. This diversity is then compared to the viral diversity of cultured samples. We detected a clear culturing bias between environmental samples and cultured isolates. This is the first study identifying a culture bias in a viral population. [TOP OF PAGE]

  643. Development and evaluation of methods to detect coliphages in large volumes of water. Sobsey,M.D., Yates,M.V., Hsu,F.C., Lovelace,G., Battigelli,D., Margolin,A., Pillai,S.D., Nwachuku,N. (2004). Water Sci. Technol. 50:211-217. New and improved methods have been developed to detect somatic and male-specific coliphages in large volumes of water by single agar layer (SAL), enrichment and membrane filter methods. Somatic coliphages were detected efficiently on E. coli hosts C and CN13, male-specific coliphages were detected more efficiently on E. coli Famp than on Salmonella typhimurium WG49 and both types of coliphages were detected simultaneously on E. coli C3000. For water volumes of up to 100 ml, the SAL method was efficient and reliable. For water volumes of <1 L and as many as 10 multiple 1 L volumes, the enrichment method was efficient in detecting very low numbers of coliphages. Membrane filter methods, in which coliphages were adsorbed to and eluted from filters, also were relatively efficient, but they were less efficient than SAL and enrichment methods and were considered to be more cumbersome. For filter adsorption-elution methods, coliphage recoveries were most efficient for cellulose ester filters, less efficient for electropositive 1 MDS filters and least efficient for a direct membrane filter method. Overall, the enrichment method was preferred because of its ability to easily and rapidly detect low levels of coliphages in large sample volumes by either presence-absence or most probable number quantification. [TOP OF PAGE]

  644. Bdellovibrio as therapeutic agents: a predatory renaissance? Sockett,R.E., Lambert,C. (2004). Nat. Rev. Microbiol. 2:669-675. Bdellovibrio are predatory bacteria that invade the periplasm of other Gram negative bacteria where they undergo a complex developmental cycle that culminates in killing of the prey cell. Their intracellular niche allows Bdellovibrio to feed without competition and their lytic action can rapidly reduce bacterial populations, including pathogens, making these predatory bacteria interesting potential candidates for therapeutic applications. With the complete genome sequence for one Bdellovibrio strain now available, researchers now have an opportunity to evaluate the therapeutic potential of these predatory bacteria. [TOP OF PAGE]

  645. Microbicidal efficacy of an advanced oxidation process using ozone/hydrogen peroxide in water treatment. Sommer,R., Pribil,W., Pfleger,S., Haider,T., Werderitsch,M., Gehringer,P. (2004). Water Sci. Technol. 50:159-164. The combined application of ozone and hydrogen peroxide represents a kind of advanced oxidation for water treatment. The radicals that are generated during the process are used for the degradation of organic pollutants from groundwater and industrial effluents. The aim of our study was to evaluate the possible microbicidal, and particularly virucidal, efficacy of such a process, since no substantial data were available. The investigations were performed at a pilot plant installed for the elimination of perchloroethylene from polluted groundwater (reduction efficacy for perchloroethylene from 26 mg/L to 5 mg/L). To enable a reliable evaluation of the microbicidal effect, a set of alternate test organisms was used. As model viruses we chose bacteriophages MS2 (F+ specific, single-stranded RNA), fX174 (single-stranded DNA) and PRD-1 (coated, double-stranded DNA). Furthermore, spores of Bacillus subtilis were included as possible surrogates for protozoa and Escherichia coli as representative for traditional indicator bacteria used in water analysis. The microbicidal efficiency was compared to the inactivation by means of ozone under two standard conditions (20ºC): (a) 0.4 mg/L residual after 4 min and (b) 0.1 mg/L residual after 10 min. Surprisingly, a good microbicidal effect of the ozone/hydrogen peroxide process was found. This was somewhat unexpected, because we had assumed that the disinfection potential of ozone would have been interfered with by the presence of hydrogen peroxide. Escherichia coli and the three test viruses revealed a reduction of about 6-log. In contrast, spores of Bacillus subtilis showed after the total process a reduction of 0.4-log. These results matched the effect of the ozone treatment (a) with a residual of 0.4 mg/L after 4 min contact time (20ºC). The test condition (b) with a residual of 0.1 mg/L ozone after a contact time of 10 min at 20ºC gave a higher reduction of the B. subtilis spores (1.5-log). The presented study revealed a satisfying microbicidal efficacy of the ozone/hydrogen peroxide process with respect to vegetative bacteria and viruses (bacteriophages). However, it has to be emphasised that intense mixing and sufficient contact time have to be optimised and tested for each individual installation. [TOP OF PAGE]

  646. Therapeutic use of bacteriophages. Soothill,J., Hawkins,C., Anggard,E., Harper,D. (2004). The Lancet infectious diseases 4:544-545. [first two paragraphs] We respond to two articles, published in The Lancet and The Lancet Infectious Diseases on the use of bacteriophages as therapeutic agents. Jane Bradbury gave much attention to uncontrolled work from eastern Europe, but did not include the extensive, carefully controlled, and positive work of Smith and colleagues. The findings of our recent research challenge Bernard Dixon's discussion of the effects of bacteriophage-induced lysis and the usefulness of inhibiting bacteriophage replication. ¶ We have done the first regulatory approved clinical study of the efficacy of bacteriophage therapy, addressing chronic, antibiotic resistant Pseudomonas aeruginosa ear infections in pet dogs that have not responded to conventional therapy. Dixon proposed that endotoxins released as a result of bacterial lysis lead to side effects, particularly circulatory shock, and that this is a problem with bacteriophage medicine for human beings. We do not know of any published evidence that bacteriophage multiplication or lysis of bacteria resulting from the use of bacteriophages has been associated with circulatory shock in patients. In carefully conducted animal experiments, including those of Smith and colleagues such effects have not been noted. In our research we minimised any such theoretical issue by focusing on local rather than systemic infections. [TOP OF PAGE]

  647. Immunological factors that affect the in vivo fate of T7 phage in the mouse. Srivastava,A.S., Kaido,T., Carrier,E. (2004). J. Virol. Meth. 115:99-104. Phage display is a powerful method to study organ and tissue specific addresses. As part of our studies on the in vivo panning of tissue-homing peptide libraries, we examined the survival of T7 phage in the blood of C57BL/6J mice to estimate the half-life of T7 phage and the factors responsible for its inactivation. Amplified and purified T7 phage particles with or without random peptide library inserts were injected intravenously into the tail vein of wild-type (C57BL/6J) and immunocompromized (C57BL/6J) female mice. In wild-type mice, both the parent phage as well as phage carrying a peptide library were eliminated quickly from the blood, with only approximately 1% survival of detectable infectious phage after 60 min of injection. In SCID (C57BL/6J-Prkdc(scid)) mice, phage titers were stable over the same period of time with or without peptide library, suggesting a role for either B- or T cells or both in phage inactivation. The presumed role of B cell was indicated by demonstration of stable phage in the B-cell deficient mouse (C57BL/10-Igh-6(tm1Cgn)). In other immunocompromized mice, the phage titers were unstable, similar to that found in wild-type mice. In no case, was there a difference between phage with or without random peptide library. These data indicate that the presence of random C-X7-C peptides on the T7 phage coat protein does not affect the clearance of the phage in murine blood. Most likely, host immune factors play a role in the neutralization of T7 phage in blood by reacting with B-cell dependent immunoglobin. [TOP OF PAGE]

  648. In utero detection of T7 phage after systemic administration to pregnant mice. Srivastava,A.S., Chauhan,D.P., Carrier,E. (2004). BioTechniques 37:81-83. The phage is used as a scaffold to display recombinant libraries of peptides, which provides the means to rescue and amplify peptides that bind target macromolecules. Many reports showed that the T7 phage display method can be used to obtain a ligand-binding peptidefor tissue-targeted therapies in adult animals. In utero tissue targeting of fetal tissues may help in the correction of many genetic and metabolic diseases. Here we demonstrate the distribution and detection of T7 phage displaying the C-X7-C peptide library in mouse fetal tissues after systemic injection of T7 phage into pregnant mouse tail vein. T7 phage was recovered from fetal tissues 15 min after injection of T7 phage. Our results suggest that T7 phage may be a useful tool in selecting the tissue-specific ligand-binding peptide for fetal tissues. This approach may be helpful in designing in utero tissue-targeted therapies. [TOP OF PAGE]

  649. Viral abundance and a high proportion of lysogens suggest that viruses are important members of the microbial community in the Gulf of Trieste. Stopar,D., Cerne,A., Zigman,M., Poljsak-Prijatelj,M., Turk,V. (2004). Microb. Ecol. 47:1-8. Epifluorescence microscopy and transmission electron microscopy were applied to study virioplankton community in the Gulf of Trieste (northern Adriatic Sea). The total viral abundance was in a range between 2.5 x 10(9)/L and 2.9 x 10(10)/L and was positively correlated with trophic status of the environment. Viruslike particles were significantly correlated with bacterial abundance in all samples studied. Correlations with other physicochemical or biological parameters were not significant. The data suggest that, because of the substantial fraction of tailed viruses present (26%), bacteriophages are an important component of the virioplankton community in the Gulf of Trieste. The abundance of viruslike particles in the seawater changed at hour intervals in a range from 1.3 x 10(9)/L to 5.1 x 10(9)/L. A significant fraction (71%) of the bacterial isolates was inducible in vitro by mitomycin C, and a high occurrence (51%) of lysogenic isolates with more than one phage morphotype present in the lysate was detected. The presence of lysogenic bacteria in the seawater was confirmed in situ with a mitomycin C induction experiment on the natural bacterial population. Results suggest that virioplankton is an abundant component of the microbial community in the Gulf of Trieste. [TOP OF PAGE]

  650. First-time isolation and characterization of a bacteriophage encoding the Shiga toxin 2c variant, which Is globally spread in strains of Escherichia coli O157 . Strauch,E., Schaudinn,C., Beutin,L. (2004). Infect. Immun. 72:7030-7039. A bacteriophage encoding the Shiga toxin 2c variant (Stx2c) was isolated from the human Escherichia coli O157 strain CB2851 and shown to form lysogens on the E. coli K-12 laboratory strains C600 and MG1655. Production of Stx2c was found in the wild-type E. coli O157 strain and the K-12 lysogens and was inducible by growing bacteria in the presence of ciprofloxacin. Phage 2851 is the first reported viable bacteriophage which carries an stx2c gene. Electron micrographs of phage 2851 showed particles with elongated hexagonal heads and long flexible tails resembling phage lambda. Sequence analysis of an 8.4-kb region flanking the stx2c gene and other genetic elements revealed a mosaic gene structure, as found in other Stx phages. Phage 2851 showed lysis of E. coli K-12 strains lysogenic for Stx phages encoding Stx1 (H19), Stx2 (933W), Stx (7888), and Stx1c (6220) but showed superinfection immunity with phage lambda, presumably originating from the similarity of the cI repressor proteins of both phages. Apparently, phage 2851 integrates at a different chromosomal locus than Stx2 phage 933W and Stx1 phage H19 in E. coli, explaining why Stx2c is often found in combination with Stx1 or Stx2 in E. coli O157 strains. Diagnostic PCR was performed to determine gene sequences specific for phage 2851 in wild-type E. coli O157 strains producing Stx2c. The phage 2851 q and o genes were frequently detected in Stx2c-producing E. coli O157 strains, indicating that phages related to 2851 are associated with Stx2c production in strains of E. coli O157 that were isolated in different locations and time periods. [TOP OF PAGE]

  651. Bacteriophage defense systems and strategies for lactic acid bacteria. Sturino,J.M., Klaenhammer,T.R. (2004). Adv. Appl. Microbiol. 56:331-378. Bacteriophages (phages) have the potential to interfere with any industry that produces bacteria as an end product or uses them as biocatalysts in the production of fermented products or bioactive molecules. Using microorganisms that drive food bioprocesses as an example, this review will describe a set of genetic tools that are useful in the engineering of customized phage-defence systems. Special focus will be given to the power of comparative genomics as a means of streamlining target selection, providing more widespread phage protection, and increasing the longevity of these industrially important bacteria in the bioprocessing environment. [TOP OF PAGE]

  652. Burkholderia cenocepacia phage BcepMu and a family of Mu-like phages encoding potential pathogenesis factors. Summer,E.J., Gonzalez,C.F., Carlisle,T., Mebane,L.M., Cass,A.M., Savva,C.G., LiPuma,J.J., Young,R. (2004). J. Mol. Biol. 340:49-65. We have isolated BcepMu, a Mu-like bacteriophage whose host range includes human pathogenic Burkholderia cenocepacia (formally B. cepacia genomovar III) isolates, and determined its complete 36,748 bp genomic sequence. Like enteric bacteriophage Mu, the BcepMu genomic DNA is flanked by variable host sequences, a result of transposon-mediated replication. The BcepMu genome encodes 53 proteins, including capsid assembly components related to those of Mu, and tail sheath and tube proteins related to those of bacteriophage P2. Seventeen of the BcepMu genes were demonstrated to encode homotypic interacting domains by using a cI fusion system. Most BcepMu genes have close homologs to prophage elements present in the two published Salmonella typhi genomes, and in the database sequences of Photorhabdus luminescens, and Chromobacterium violaceum. These prophage elements, designated SalMu, PhotoMu and ChromoMu, respectively, are collinear with BcepMu through nearly their entire lengths and show only limited mosaicism, despite the divergent characters of their hosts. The BcepMu family of Mu-like phages has a number of notable differences from Mu. Most significantly, the critical left end region of BcepMu is inverted with respect to Mu, and the BcepMu family of transposases is clearly of a distinct lineage with different molecular requirements at the transposon ends. Interestingly, a survey of 33 B.cepacia complex strains indicated that the BcepMu prophage is widespread in human pathogenic B.cenocepacia ET12 lineage isolates, but not in isolates from the PHDC or Midwest lineages. Identified members of the BcepMu family all contain a gene possibly involved in bacterial pathogenicity, a homolog of the type-two-secretion component exeA, but only BcepMu also carries a lipopolysaccharide modification acyltransferase which may also contribute a pathogenicity factor. [TOP OF PAGE]

  653. The interaction of phage and biofilms. Sutherland,I.W., Hughes,K.A., Skillman,L.C., Tait,K. (2004). FEMS Microbiol. Lett. 232:1-6. Biofilms present complex assemblies of micro-organisms attached to surfaces. they are dynamic structures in which various metabolic activities and interactions between the component cells occur. When phage come in contact with biofilms, further interactions occur dependent on the susceptibility of the biofilm bacteria to phage and to the availability of receptor sites. If the phage also possess polysaccharide-degrading enzymes, or if considerable cell lysis is effected by the phage, the integrity of the biofilm may rapidly be destroyed. Alternatively, coexistence between phage and host bacteria within the biofilm may develop. Although phage have been proposed as a means of destroying or controlling biofilms, the technology for this has not yet been successfully developed. [TOP OF PAGE]

  654. Toward rational control of Escherichia coli O157:H7 by a phage cocktail. Tanji,Y., Shimada,T., Yoichi,M., Miyanaga,K., Hori,K., Unno,H. (2004). Applied Microbiology and Biotechnology 64:270-274. Twenty six phages infected with Escherichia coli O157:H7 were screened from various sources. Among them, nine caused visible lysis of E. coli O157:H7 cells in LB liquid medium. However, prolonged incubation of E. coli cells and phage allowed the emergence of phage-resistant cells. The susceptibility of the phage-resistant cells to the nine phages was diverse. A rational procedure for selecting an effective cocktail of phage for controlling bacteria was investigated based on the mechanism of phage-resistant cell conversion. Deletion of OmpC from the E. coli cells facilitated the emergence of cells resistant to SP21 phage. After 8 h of incubation, SP21-resistant cells appeared. By contrast, alteration of the lipopolysaccharide (LPS) profile facilitated cell resistance to SP22 phage, which was observed following a 6-h incubation. When a cocktail of phages SP21 and SP22 was used to infect E. coli O157:H7 cells, 30 h was required for the emergence of cells (R-C) resistant to both phages. The R-C cells carried almost the same outer membrane and LPS components as the wild-type cells. However, the reduced binding ability of both phages to R-C cells suggested disturbance of phage adsorption to the R-C surface. Even though R-C cells resistant to both phages appeared, this work shows that rational selection of phages has the potential to at least delay the emergence of phage resistance. [TOP OF PAGE]

  655. Evaluation of electrochemically generated ozone for the disinfection of water and wastewater. Tanner,B.D., Kuwahara,S., Gerba,C.P., Reynolds,K.A. (2004). Water Sci. Technol. 50:19-25. Effective wastewater treatment is critical to public health and well-being. This is especially true in developing countries, where disinfection of wastewater is frequently inadequate. People who live in these areas may benefit from wastewater disinfection using ozone. This study evaluated the ability of a new electrochemical process of ozone generation, which produced ozone continuously at high pressure and concentration by the electrolysis of water, to disinfect tap water and secondarily treated wastewater. Inactivation of Klebsiella terrigena, Escherichia coli, MS2 bacteriophage and poliovirus 1 was evaluated first in reverse osmosis (RO) treated water. Inactivation of K. terrigena (6-log), E. coli (6-log), MS2 (6-log) and poliovirus 1 (>3-log) was observed after 1 min of ozonation in a 1 L batch reactor. Experiments were then performed to assess the microbiological impact of disinfection using ozone on secondarily treated municipal wastewater. The effect of ozonation on wastewater was determined for total and faecal coliforms, bacteriophages and heterotrophic plate count (HPC) bacteria. Electrochemical ozone generators provided an effective, rapid and low-cost method of wastewater disinfection. Based on the results of this research, electrochemically generated ozone would be well suited to remote, small-scale, disinfection operations and may provide a feasible means of wastewater disinfection in developing countries. [TOP OF PAGE]

  656. Old dogma, new tricks--21st Century phage therapy. Thiel,K. (2004). Nat. Biotech. 22:31-36. As antibiotic resistant bacteria threaten a public health crisis, biotechnology is turning to bacteriophages, nature's tiniest viruses. But can phage therapy overcome its historical baggage? [TOP OF PAGE]

  657. Bead-based electrochemical immunoassay for bacteriophage MS2. Thomas,J.H., Kim,S.K., Hesketh,P.J., Halsall,H.B., Heineman,W.R. (2004). Analyt. Chem. 76:2700-2707. Viruses are one of four classes of biothreat agents, and bacteriophage MS2 has been used as a simulant for biothreat viruses, such as smallpox. A paramagnetic bead-based electrochemical immunoassay has been developed for detecting bacteriophage MS2. The immunoassay sandwich was made by attaching a biotinylated rabbit anti-MS2 IgG to a streptavidin-coated bead, capturing the virus, and then attaching a rabbit anti-MS2 IgG-beta-galactosidase conjugate to another site on the virus. beta-Galactosidase converts p-aminophenyl galactopyranoside (PAPG) to p-aminophenol (PAP). PAPG is electroinactive at the potential at which PAP is oxidized to p-quinone imine (PQI), so the current resulting from the oxidation of PAP to PQI is directly proportional to the concentration of antigen in the sample. The immunoassay was detected with rotating disk electrode (RDE) amperometry and an interdigitated array (IDA) electrode. With an applied potential of +290 mV vs Ag/AgCl and a rotation rate of 3000 rpm, the detection limit was 200 ng/mL MS2 or 3.2 x 10(10) viral particles/mL with RDE amperometry. A trench IDA electrode was incorporated into a poly(dimethyl siloxane) channel, within which beads were collected, incubated with PAPG, and PAP generation was detected. The two working electrodes were held at +290 and -300 mV vs Ag/AgCl, and electrochemical recycling of the PAP/PQI couple by the IDA electrode lowered the limit of detection to 90 ng/mL MS2, or 1.5 x 10(10) MS2 particles/mL. [TOP OF PAGE]

  658. The role of prophage-like elements in the diversity of Salmonella enterica serovars. Thomson,N., Baker,S., Pickard,D., Fookes,M., Anjum,M., Hamlin,N., Wain,J., House,D., Bhutta,Z., Chan,K., Falkow,S., Parkhill,J., Woodward,M., Ivens,A., Dougan,G. (2004). J. Mol. Biol. 339:279-300. The Salmonella enterica serovar Typhi CT18 (S.Typhi) chromosome harbours seven distinct prophage-like elements, some of which may encode functional bacteriophages. In silico analyses were used to investigate these regions in S.Typhi CT18, and ultimately compare these integrated bacteriophages against 40 other Salmonella isolates using DNA microarray technology. S.Typhi CT18 contains prophages that show similarity to the lambda, Mu, P2 and P4 bacteriophage families. When compared to other S.Typhi isolates, these elements were generally conserved, supporting a clonal origin of this serovar. However, distinct variation was detected within a broad range of Salmonella serovars; many of the prophage regions are predicted to be specific to S.Typhi. Some of the P2 family prophage analysed have the potential to carry non-essential "cargo" genes within the hyper-variable tail region, an observation that suggests that these bacteriophage may confer a level of specialisation on their host. Lysogenic bacteriophages therefore play a crucial role in the generation of genetic diversity within S.enterica. [TOP OF PAGE]

  659. Automatic identification of bacterial types using statistical imaging methods. Trattner,S., Greenspan,H., Tepper,G., Abboud,S. (2004). IEEE Transactions on Medical Imaging 23:807-820. The objective of the current study is to develop an automatic tool to identify microbiological data types using computer-vision and statistical modeling techniques. Bacteriophage (phage) typing methods are used to identify and extract representative profiles of bacterial types out of species such as the Staphylococcus aureus. Current systems rely on the subjective reading of profiles by a human expert. This process is time-consuming and prone to errors, especially as technology is enabling the increase in the number of phages used for typing. The statistical methodology presented in this work, provides for an automated, objective and robust analysis of visual data, along with the ability to cope with increasing data volumes. [TOP OF PAGE]

  660. ST64B is a defective bacteriophage in Salmonella enterica serovar Typhimurium DT64 that encodes a functional immunity region capable of mediating phage-type conversion. Tucker,C.P., Heuzenroeder,M.W. (2004). Int. J. Med. Microbiol. 294:59-63. The Salmonella enterica subsp. enterica serovar Typhimurium (S. Typhimurium) defective bacteriophage ST64B has a putative immunity (immC) region consisting of cI, cro and cII-like genes. Since ST64B is widespread in S. Typhimurium, studies were undertaken to determine whether this region might be functional and influence phage typing results. Cloning of ST64B immC-like genes and their subsequent expression in S. Typhimurium DTs showed that this region is able to mediate phage-type conversion in DTs 41 and 44. This confirms the functionality of the immC region and the patterns of lysis produced by phage typing are consistent with the predicted mechanism of action of the encoded protein products. [TOP OF PAGE]

  661. Evaluating microbial purification during soil treatment of wastewater with multicomponent tracer and surrogate tests. Van Cuyk,S., Siegrist,R.L., Lowe,K., Harvey,R.W. (2004). J. Environ. Qual. 33:316-329. Soil treatment of wastewater has the potential to achieve high purification efficiency, yet the understanding and predictability of purification with respect to removal of viruses and other pathogens is limited. Research has been completed to quantify the removal of virus and bacteria through the use of microbial surrogates and conservative tracers during controlled experiments with three-dimensional pilot-scale soil treatment systems in the laboratory and during the testing of full-scale systems under field conditions. The surrogates and tracers employed included two viruses (MS-2 and PRD-1 bacteriophages), one bacterium (ice-nucleating active Pseudomonas), and one conservative tracer (bromide ion). Efforts have also been made to determine the relationship between viruses and fecal coliform bacteria in soil samples below the wastewater infiltrative surface, and the correlation between Escherichia coli concentrations measured in percolating soil solution as compared with those estimated from analyses of soil solids. The results suggest episodic breakthrough of virus and bacteria during soil treatment of wastewater and a 2 to 3 log (99-99.9%) removal of virus and near complete removal of fecal coliform bacteria during unsaturated flow through 60 to 90 cm of sandy medium. Results also suggest that the fate of fecal coliform bacteria may be indicative of that of viruses in soil media near the infiltrative surface receiving wastewater effluent. Concentrations of fecal coliform in percolating soil solution may be conservatively estimated from analysis of extracted soil solids. [TOP OF PAGE]

  662. Inactivation of enteric microbes in water by electro-chemical oxidant from brine (NaCl) and free chlorine. Venczel,L.V., Likirdopulos,C.A., Robinson,C.E., Sobsey,M.D. (2004). Water Sci. Technol. 50:141-146. Oxidant solutions of mostly free chlorine can be electrochemically produced on-site from brine (NaCl) solution and used to disinfect water at the household or community level. In this study electrochemical oxidant (ECO) from brine and free chlorine were evaluated under laboratory conditions for inactivation of test microbes. Purified suspensions of Escherichia coli, the rugose strain of Vibrio cholerae, Clostridium perfringens spores, MS2 coliphage and Cryptosporidium parvum oocysts were treated with 2 mg/L or 5 mg/L solutions of ECO or free chlorine at 5ºC and 25ºC and pH 6, 8, and 10 (pH 7 and 25ºC only for C. parvum oocysts) for contact times <60 min. Under nearly all conditions, inactivation kinetics were more rapid for E. coli, V. cholerae, C. perfringens spores and MS2 coliphage with ECO than with free chlorine. ECO reduced E. coli, V. cholerae and MS2 by >4 log10 within 30 min and C. perfringens spores by >2 log10 within 10 min at pH 8 and 25ºC. Contrary to previous results, however, C. parvum oocysts were not inactivated by ECO, and the reasons for this difference are uncertain. The on-site electrolytic generation of oxidants from brine provided a convenient and inexpensive disinfectant containing free chlorine that was effective against many enteric microbes, for the treatment of household and community drinking-water supplies worldwide. However, the effectiveness of such oxidants for inactivating C. parvum oocysts was variable and sometimes ineffective. [TOP OF PAGE]

  663. Molecular detection and genotyping of male-specific coliphages by reverse transcription-PCR and reverse line blot hybridization. Vinje,J., Oudejans,S.J.G., Stewart,J.R., Sobsey,M.D., Long,S.C. (2004). Appl. Environ. Microbiol. 70:5996-6004. In recent years, there has been increased interest in the use of male-specific or F+ coliphages as indicators of microbial inputs to source waters. Sero- or genotyping of these coliphages can also be used for microbial source tracking (MST). Among the male-specific coliphages, the F+ RNA (FRNA) viruses are well studied, while little is known about the F+ DNA (FDNA) viruses. We have developed a reverse line blot hybridization (RLB) assay which allows for the simultaneous detection and genotyping of both FRNA as well as FDNA coliphages. These assays included a novel generic duplex reverse transcription-PCR (RT-PCR) assay for FRNA viruses as well as a generic PCR for FDNA viruses. The RT-PCR assays were validated by using 190 field and prototype strains. Subsequent DNA sequencing and phylogenetic analyses of RT-PCR products revealed the classification of six different FRNA clusters, including the well-established subgroups I through IV, and three different FDNA clusters, including one (CH) not previously described. Within the leviviruses, a potentially new subgroup (called JS) including strains having more than 40% nucleotide sequence diversity with the known levivirus subgroups (MS2 and GA) was identified. We designed subgroup-specific oligonucleotides that were able to genotype all nine (six FRNA, three FDNA) different clusters. Application of the method to a panel of 351 enriched phage samples from animal feces and wastewater, including known prototype strains (MS2, GA, Q beta, M11, FI, and SP for FRNA and M13, f1, and fd for FDNA), resulted in successful genotyping of 348 (99%) of the samples. In summary, we developed a novel method for standardized genotyping of F+ coliphages as a useful tool for large-scale MST studies. [TOP OF PAGE]

  664. Development and evaluation of a reflective solar disinfection pouch for treatment of drinking water. Walker,D.C., Len,S.V., Sheehan,B. (2004). Appl. Environ. Microbiol. 70:2545-2550. A second-generation solar disinfection (SODIS) system (pouch) was constructed from food-grade, commercially available packaging materials selected to fully transmit and amplify the antimicrobial properties of sunlight. Depending upon the season, water source, and challenge organism, culturable bacteria were reduced between 3.5 and 5.5 log cycles. The system was also capable of reducing the background presumptive coliform population in nonsterile river water below the level of detection. Similar experiments conducted with a model virus, the F-specific RNA bacteriophage MS2, indicated that the pouch was slightly less efficient, reducing viable plaques by 3.5 log units in comparison to a 5.0 log reduction of enterotoxigenic Escherichia coli O18:H11 within the same time period. These results suggest that water of poor microbiological quality can be improved by using a freely available resource (sunlight) and a specifically designed plastic pouch constructed of food-grade packaging materials. [TOP OF PAGE]

  665. DNA bacteriophage as controls for clinical viral testing. Walkerpeach,C.R., Pasloske,B.L. (2004). Clinical chemistry 50:1970-1971. In the mid- to late-1980s, a revolution in molecular diagnostics began with the introduction of innovative methods for the detection of nucleic acids. In retrospect, the appearance of these technologies roughly coincided with the debuts of new pathogenic viruses, such as human immunodeficiency virus (HIV) and hepatitis C virus (HCV). Techniques such as PCR, transcription-mediated amplification (TMA), and branched DNA (bDNA) were applied to the detection and quantification of these viruses. Eventually, these tests were integrated into routine clinical laboratories for diagnosing and monitoring the treatment of patients infected with these viruses. Quantification data ("viral load") indicated whether the current drug cocktail was having the desired effect on the virus (i.e., lowering the viral load). If not, then another drug course could be prescribed. Today, the main advantages of these assays are great sensitivity (measuring as few as 50 copies/mL of plasma), ease of use for quantification, and early detection of viral nucleic acid in the peripheral blood before an antibody response develops, an application that has proven to be especially important in screening of human blood products. ¶ These technologies were focused not only on the newly emerging RNA viruses but also on the better-known hepatitis B virus (HBV), cytomegalovirus (CMV), herpes simplex virus (HSV), varicella zoster virus (VZV), human papillomavirus (HPV), and Epstein-Barr virus (EBV). In developing DNA-based tests for these viruses, laboratories incorporated positive controls from one of three potential sources: plasmid DNA, positive patient specimens, or commercially available viral preparations. None of these formats is ideal. Plasmid DNA cannot be used until after the specimen analyte has been extracted. Patient specimens have become more difficult to use in the US after the introduction of new "HIPAA" regulations for protection of patient privacy. Moreover, viral nucleic acids in patient specimens are degraded during multiple freeze-thaw cycles. Lastly, the... [TOP OF PAGE]

  666. Genetic diversity and population dynamics of cyanophage communities in the Chesapeake Bay. Wang,K., Chen,F. (2004). Aquat. Microb. Ecol. 34:105-116. In order to understand the genetic diversity and population dynamics of cyanophages in estuarine waters, the viral capsid assembly (g20) gene was used as a gene marker to monitor genetic variations of natural cyanomyovirus communities in the Chesapeake Bay, USA. Unique and diverse g20 sequences were found. Only 1 of 15 g20 genotypes was closely related to the known cyanomyovirus isolates. Most of the g20 genotypes in the bay were not related to the g20 clonal sequences recovered from open-ocean waters. Terminal-restriction fragment length polymorphism (T-RFLP) based on the g20 gene was developed to investigate spatial and temporal distribution of cyanomyovirus communities in the bay. The T-RFLP profiles of the g20 gene demonstrated that the cyanomyovirus population structures in the bay were more dynamic seasonally than spatially. Seasonal variation in the cyanophage community appeared to correspond to changes in host-cell density, which in turn was mainly affected by water temperature. This study represents the first effort to monitor both cyanophage titer and genetic diversity over time and space. The results of our study suggest that cyanophages could play a significant role in regulating Synechococcus biomass and population structure in the Chesapeake Bay. [TOP OF PAGE]

  667. Complete sequence and evolutionary genomic analysis of the Pseudomonas aeruginosa transposable bacteriophage D3112. Wang,P.W., Chu,L., Guttman,D.S. (2004). J. Bacteriol. 186:400-410. Bacteriophage D3112 represents one of two distinct groups of transposable phage found in the clinically relevant, opportunistic pathogen Pseudomonas aeruginosa. To further our understanding of transposable phage in P. aeruginosa, we have sequenced the complete genome of D3112. The genome is 37,611 bp, with an overall G+C content of 65%. We have identified 53 potential open reading frames, including three genes (the c repressor gene and early genes A and B) that have been previously characterized and sequenced. The organization of the putative coding regions corresponds to published genetic and transcriptional maps and is very similar to that of enterobacteriophage Mu. In contrast, the International Committee on Taxonomy of Viruses has classified D3112 as a l-like phage on the basis of its morphology. Similarity-based analyses identified 27 open reading frames with significant matches to proteins in the NCBI databases. Forty-eight percent of these were similar to Mu-like phage and prophage sequences, including proteins responsible for transposition, transcriptional regulation, virion morphogenesis, and capsid formation. The tail proteins were highly similar to prophage sequences in Escherichia coli and phage Phi12 from Staphylococcus aureus, while proteins at the right end were highly similar to proteins in Xylella fastidiosa. We performed phylogenetic analyses to understand the evolutionary relationships of D3112 with respect to Mu-like versus lambda-like bacteriophages. Different results were obtained from similarity-based versus phylogenetic analyses in some instances. Overall, our findings reveal a highly mosaic structure and suggest that extensive horizontal exchange of genetic material played an important role in the evolution of D3112. [TOP OF PAGE]

  668. Bacteriophage and phenotypic variation in Pseudomonas aeruginosa biofilm development. Webb,J.S., Lau,M., Kjelleberg,S. (2004). J. Bacteriol. 186:8066-8073. A current question in biofilm research is whether biofilm-specific genetic processes can lead to differentiation in physiology and function among biofilm cells. In Pseudomonas aeruginosa, phenotypic variants which exhibit a small-colony phenotype on agar media and a markedly accelerated pattern of biofilm development compared to that of the parental strain are often isolated from biofilms. We grew P. aeruginosa biofilms in glass flow cell reactors and observed that the emergence of small-colony variants (SCVs) in the effluent runoff from the biofilms correlated with the emergence of plaque-forming Pf1-like filamentous phage (designated Pf4) from the biofilm. Because several recent studies have shown that bacteriophage genes are among the most highly upregulated groups of genes during biofilm development, we investigated whether Pf4 plays a role in SCV formation during P. aeruginosa biofilm development. We carried out immunoelectron microscopy using anti-Pf4 antibodies and observed that SCV cells, but not parental-type cells, exhibited high densities of Pf4 filaments on the cell surface and that these filaments were often tightly interwoven into complex latticeworks surrounding the cells. Moreover, infection of P. aeruginosa planktonic cultures with Pf4 caused the emergence of SCVs within the culture. These SCVs exhibited enhanced attachment, accelerated biofilm development, and large regions of dead and lysed cells inside microcolonies in a manner identical to that of SCVs obtained from biofilms. We concluded that Pf4 can mediate phenotypic variation in P. aeruginosa biofilms. We also performed partial sequencing and analysis of the Pf4 replicative form and identified a number of open reading frames not previously recognized in the genome of P. aeruginosa, including a putative postsegregational killing operon. [TOP OF PAGE]

  669. Are viruses driving microbial diversification and diversity? Weinbauer,M.G., Rassoulzadegan,F. (2004). Environ. Microbiol. 6:1-11. Viruses can influence the genetic diversity of prokaryotes in various ways. They can affect the community composition of prokaryotes by 'killing the winner' and keeping in check competitive dominants. This may sustain species richness and the amount of information encoded in genomes. Viruses can also transfer (viral and host) genes between species. Such mechanisms have probably influenced the speciation of prokaryotes. Whole-genome sequencing has clearly revealed the importance of (virus-mediated) gene transfer. However, its significance for the ecological performance of aquatic microbial communities is only poorly studied, although the few available reports indicate a large potential. Here, we present data supporting the hypothesis that viral genes and viral activity generate genetic variability of prokaryotes and are a driving force for ecological functioning and evolutionary change. [TOP OF PAGE]

  670. Ecology of prokaryotic viruses. Weinbauer,M.G. (2004). FEMS Microbiol. Rev. 28:127-181. The finding that total viral abundance is higher than total prokaryotic abundance and that a significant fraction of the prokaryotic community is infected with phages in aquatic systems has stimulated research on the ecology of prokaryotic viruses and their role in ecosystems. This review treats the ecology of prokaryotic viruses ("phages") in marine, freshwater and soil systems from a "virus point of view". The abundance of viruses varies strongly in different environments and is related to bacterial abundance or activity suggesting that the majority of the viruses found in the environment are typically phages. Data on phage diversity are sparse but indicate that phages are extremely diverse in natural systems. Lytic phages are predators of prokaryotes, whereas lysogenic and chronic infections represent a parasitic interaction. Some forms of lysogeny might be described best as mutualism. The little existing ecological data on phage populations indicate a large variety of environmental niches and survival strategies. The host cell is the main resource for phages and the resource quality, i.e., the metabolic state of the host cell, is a critical factor in all steps of the phage life cycle. Virus-induced mortality of prokaryotes varies strongly on a temporal and spatial scale and shows that phages can be important predators of bacterioplankton. This mortality and the release of cell lysis products into the environment can strongly influence microbial food web processes and biogeochemical cycles. Phages can also affect host diversity, e.g., by "killing the winner" and keeping in check competitively dominant species or populations. Moreover, they mediate gene transfer between prokaryotes, but this remains largely unknown in the environment. Genomics or proteomics are providing us now with powerful tools in phage ecology, but final testing will have to be performed in the environment. [TOP OF PAGE]

  671. Models of phage growth and their applicability to phage therapy. Weld,R.J., Butts,C., Heinemann,J.A. (2004). J. Theor. Biol. 227:1-11. Phage therapy is complicated by the self-replicating nature of phage. It is difficult to extrapolate from in vitro phage growth data to in vivo expectations, difficult to interpret in vivo data and difficult to generalize from one in vivo situation to another. Various generic models of phage growth have been used as the theoretical basis for understanding the kinetics of phage therapy. Here, we have experimentally tested the efficacy of such simple models to predict, qualitatively and quantitatively, the growth of phage and the phage proliferation threshold in vitro. Naturally occurring, antibiotic-resistant bacteria were used to measure the growth of phage in vivo. In homogenous, in vitro environments, the models were predictive of T4 phage growth on Escherichia coli RR1. However, the models were not able to predict growth of T4 phage or K1-5 phage in the more complex environment of the rat's digestive tract. To explore fully the kinetics of phage therapy, more complex models need to be devised. We suggest that it may be necessary to consider and model the interactions between phage growth parameters and bacterial growth parameters. [TOP OF PAGE]

  672. Impact of virioplankton on archaeal and bacterial community richness as assessed in seawater batch cultures. Winter,C., Smit,A., Herndl,G.J., Weinbauer,M.G. (2004). Appl. Environ. Microbiol. 70:804-813. During cruises in the tropical Atlantic Ocean (January to February 2000) and the southern North Sea (December 2000), experiments were conducted to monitor the impact of virioplankton on archaeal and bacterial community richness. Prokaryotic cells equivalent to 10 to 100% of the in situ abundance were inoculated into virus-free seawater, and viruses equivalent to 35 to 360% of the in situ abundance were added. Batch cultures with microwave-inactivated viruses and without viruses served as controls. The apparent richness of archaeal and bacterial communities was determined by terminal restriction fragment length polymorphism (T-RFLP) analysis of PCR-amplified 16S rRNA gene fragments. Although the estimated richness of the prokaryotic communities generally was greatly reduced within the first 24 h of incubation due to confinement, the effects of virus amendment were detected at the level of individual operational taxonomic units (OTUs) in the T-RFLP patterns of both groups, Archaea and Bacteria. One group of OTUs was detected in the control samples but was absent from the virus-treated samples. This negative response of OTUs to virus amendment probably was caused by viral lysis. Additionally, we found OTUs not responding to the amendments, and several OTUs exhibited variable responses to the addition of inactive or active viruses. Therefore, we conclude that individual members of pelagic archaeal and bacterial communities can be differently affected by the presence of virioplankton. [TOP OF PAGE]

  673. Conserved translational frameshift in dsDNA bacteriophage tail assembly genes. Xu,J., Hendrix,R.W., Duda,R.L. (2004). Molecular cell 16:11-21. A programmed translational frameshift similar to frameshifts in retroviral gag-pol genes and bacterial insertion elements was found to be strongly conserved in tail assembly genes of dsDNA phages and to be independent of sequence similarities. In bacteriophage lambda, this frameshift controls production of two proteins with overlapping sequences, gpG and gpGT, that are required for tail assembly. We developed bioinformatic approaches to identify analogous -1 frameshifting sites and experimentally confirmed our predictions for five additional phages. Clear evidence was also found for an unusual but analogous -2 frameshift in phage Mu. Frameshifting sites could be identified for most phages with contractile or noncontractile tails whose length is controlled by a tape measure protein. Phages from a broad spectrum of hosts spanning Eubacteria and Archaea appear to conserve this frameshift as a fundamental component of their tail assembly mechanisms, supporting the idea that their tail genes share a common, distant ancestry. [TOP OF PAGE]

  674. Genome function--a virus-world view. Yin,J. (2004). Advances in experimental medicine and biology 547:31-46. By studying viruses one may begin to understand how static genomes can define dynamic processes of development. This talk will describe some of the approaches we are taking, using computer simulations and laboratory experiments, to account for the many molecular-level processes and interactions that occur when a common bacterium, E. coli, is infected by one of its viruses, phage T7. We accounted for processes of phage genome entry, transcription, translation, and DNA replication, including protein-DNA and protein-protein regulatory interactions, and we predicted the dynamics of phage progeny formation. The simulations have enabled us to identify limiting host-cell resources in phage growth, discover novel anti-viral strategies, and suggest frameworks for mining data from global mRNA and protein studies. [TOP OF PAGE]

  675. Luciferase reporter phage enables rapid susceptibility screening. Anonymous (2003). Medical Devices & Surgical Technology Week April 20, 81. A novel reporter phage assay enables quick susceptibility testing of Mycobacterium tuberculosis isolates. ¶ "Rapid diagnosis of drug-resistant M. tuberculosis (Mtb) is desirable worldwide," researchers in the United States noted. ¶ S. Bardarov and colleagues at the State University of New York Downstate Medical Center identified "a new luciferase reporter phage (LRP), phAE142 for this purpose," and compared it with "the automated MGIT [Mycobacteria Growth Indicator Tube] 960 for time-to-detection of Mtb in clinical specimens" and "species confirmation and antibiotic susceptibility testing (AST) of Mtb." ¶ "Twenty sputum samples were inoculated for testing by LRP, or by MGIT 960. After 'positives' were identified by either method, the LRP was used for confirmation of Mtb complex (TBC) and for AST," they said. ¶ "The LRP method proved comparably efficient to MGIT 960 at detecting Mtb," study results showed. "Using an antibiotic uniquely inhibiting TBC with LRP provided species assignment, concurrently with AST, in a median of three days, with a sensitivity of 97%." ¶ "Overall agreement in susceptibility results was 96%," Bardarov and coauthors concluded. "Reliable susceptibility results and identification of TBC can be completed in a median of 12 days (range 8 to 16 days) with LRP applied to sputum samples." ¶ Bardarov and coauthors published their study in Diagnostic Microbiology and Infectious Disease (Detection and drug-susceptibility testing of M. tuberculosis from sputum samples using luciferase reporter phage: comparison with the Mycobacteria Growth Indicator Tube (MGIT) system. Diagn Microbiol Infect Dis, 2003;45(1):53-61). [TOP OF PAGE]

  676. Glow in the dark assay illuminates TB diagnosis. Anonymous (2003). AIDS Weekly May 10, 6-7? With help from the firefly, investigators supported by the U.S. National Institute of Allergy and Infectious Diseases (NIAID) and the World Health Organization (WHO) have developed an experimental technique that holds promise for speeding the diagnosis of tuberculosis and rapidly determining which drugs can be used to kill the TB strain a patient is carrying. ¶ The technique may also offer researchers an efficient method to screen large numbers of potential anti-TB compounds, a crucial concern as scientist work to develop effective new drugs against strains of the TB bacterium resistant to the current drugs. ¶ The new approach uses luciferase, an enzyme that is part of the "glow-in-the-dark" system in the tail of fireflies, to produce light in living TB bacterium. ¶ "With the recent rise in drug-resistant tuberculosis, it is especially critical that physicians confirm a patient's TB diagnosis at the earliest possible moment and determine which drugs are effective," says Anthony S. Fauci, M.D., NIAID director. "This new laboratory technique for rapidly identifying TB and the drug susceptibility of TB isolates holds particular promise for use in clinical settings." ¶ As reported in the May 7, 1993, issue of the journal Science and at the second annual meeting of the NIAID International Centers for Disease Research in April 1993, the study investigators, led by William R. Jacobs, M.D., of the Howard Hughes Medical Institute at the Albert Einstein College of Medicine, used genetic engineering to insert the firefly luciferase gene into the genetic material of a virus specific to the TB organism. ¶ When this genetically altered virus infects the TB bacterium, the viral DNA with the luciferase gene is integrated into the bacterium's gene. The TB organism now can produce luciferase. Luciferase reacts with another substance, luciferin, to change chemical energy in cells into light. ¶ Current TB diagnostic tests rely on ceil cultures and can take weeks because the TB bacterium, Mycobacterium tuberculosis, grows slowly, multiplying only once every 24 hours. By comparison, other bacteria such as those that cause strep throat multiply every 20 minutes, rapidly forming colonies that are detected easily. ¶ While waiting for the results of TB cultures, physicians are forced to make treatment decisions with limited information. Patients may receive drugs that are not effective, while possibly transmitting the disease to others. ¶ With the new procedure, known as the luciferase phage assay, the genetically engineered virus and luciferin may be added to specimens suspected of containing TB bacteria. The luciferin diffuses easily into the cells and if TB bacteria are present, they are infected and make luciferase. Light is produced and can be measured by a light-sensitive instrument known as a luminometer. ¶ To determine the drug susceptibility of the TB strain a patient is carrying, various antiTB drugs are added to TB cultures along with the altered virus and luciferin. If light is seen, the investigators know that the antibiotic is ineffective, because the living bacterium continues to produce luciferase and, consequently, light. However, if the TB bacteria are treated with an effective antibiotic, they will be killed and no light will be emitted. ¶ "This is a quick and sensitive method for detecting drug-resistant TB strains, and it could potentially be automated to process large numbers of samples in a short time," says Dr. Jacobs. "Refinement of the luciferase phage diagnostic system for use in the clinical setting is currently under way. It is anticipated that this assay also will be used in the near future to efficiently screen compounds for anti-TB activity.". [TOP OF PAGE]

  677. Phage-enabled amperometric assay valuable for pathogen analysis. Anonymous (2003). TB & Outbreaks Week April 6, 55. A phage-enabled amperometric assay could be valuable for pathogen analysis. ¶ In a recent study from Israel, researchers described "a novel electrochemical method for the rapid identification and quantification of pathogenic and polluting bacteria." ¶ "The design incorporates a bacteriophage, a virus that recognizes, infects, and lyses only one bacterial species among mixed populations, thereby releasing intracellular enzymes that can be monitored by the amperometic measurement of enzymatic activity," explained T. Neufeld and colleagues at Tel Aviv University. ¶ "As a model system, we used virulent phage typing and cell-marker enzyme activity (beta-D-galactosidase), a combination that is specific for the bacterial strain Escherichia coli (K-12, MG1655)," Neufeld and coworkers wrote. They found that "filtration and preincubation before infecting the bacteria with the phage enabled amperometric detection at a wide range of concentrations, reaching as low as 1 colony-forming unit/100 mL within 6-8 hours." ¶ "In principle, this electrochemical method can be applied to any type of bacterium-phage combination by measuring the enzymatic marker released by the lytic cycle of a specific phage," the researchers concluded. ¶ Neufeld and coauthors published their study in Analytical Chemistry (Combined phage typing and amperometric detection of released enzymatic activity for the specific identification and quantification of bacteria. Anal Chem, 2003;75(3):580-585). [TOP OF PAGE]

  678. Phage therapy could remove E. coli O157:H7 from livestock. Anonymous (2003). TB & Outbreaks Week June 10, 15. A bacteria-killing virus found in the feces of some sheep could help remove the dangerous food-borne bacteria Escherichia coli O157:H7 from livestock. ¶ Researchers from Evergreen State College in Olympia, Washington, discussed their findings at the 103rd General Meeting of the American Society for Microbiology on May 19, 2003. ¶ "Here we report a promising new natural way of reducing pathogen concentrations in livestock. This takes advantage of bacteriophages - bacteria-killing viruses, harmless to humans and other animals, which have been used extensively as antibiotics in Eastern Europe and the former Soviet Union for over 50 years," says Michael Dyen, one of the study researchers. ¶ Dyen and his colleagues reported on a new bacteriophage (CEV1) that they isolated from the feces of sheep naturally resistant to gut colonization by E. coli O157:H7. Preliminary trials of CEV1 in the lab have shown that it can be produced easily and can efficiently infect and kill the bacteria under proper conditions. In model systems reflecting the cow/sheep gut, CEV1 completely eliminated the bacteria in 11 days. ¶ "CEV1 and other carefully-selected phages against E. coli O157:H7 could be used to develop an effective management strategy to eradicate this pathogen from livestock," says Dyen. ¶ Outbreaks of E. coli O157:H7 have been linked to the consumption of hamburger meat, alfalfa sprouts, unpasteurized fruit juice, and even drinking water; more than 75% of the cases can be directly traced to contamination from carrier ruminants. The most recent data suggest that about 28% of the cattle presented for slaughter in the U.S. harbor O157:H7, and similar numbers have been reported in Canada and Europe. Infected livestock show no signs of illness and the levels are generally low, making contaminated animals hard to identify. Current prevention methodologies have centered on reducing meat contamination in the slaughterhouse and testing all products for human consumption as they leave. ¶ "At present, there are few therapeutic treatments for victims of this potentially deadly infectious agent except supportive therapy to manage the complications of cellular damage," says Dyen. "Our work focuses on removing O157:H7 from the food chain.". [TOP OF PAGE]

  679. Genetically engineered phage delivers antimicrobial agents to bacteria. Anonymous (2003). Vaccine Weekly May 28, 5. A genetically engineered phage to deliver antimicrobial agents to bacteria provides an alternative therapy for treatment of bacterial infections. ¶ According to recent research from the United States, "The emergence and increasing prevalence of multidrug-resistant bacterial pathogens emphasizes the need for new and innovative antimicrobial strategies. Lytic phages, which kill their host following amplification and release of progeny phage into the environment, may offer an alternative strategy for combating bacterial infections. In this study, however, we describe the use of a nonlytic phage to specifically target and deliver DNA encoding bactericidal proteins to bacteria." ¶ "To test the concept of using phage as a lethal-agent delivery vehicle, we used the M13 phagemid system and the addiction toxins Gef and ChpBK," said Caroline Westwater and colleagues at the Medical University of South Carolina. "Phage delivery of lethal-agent phagemids reduced target bacterial numbers by several orders of magnitude in vitro and in a bacteremic mouse model of infection." ¶ The researchers concluded, "Given the powerful genetic engineering tools available and the present knowledge in phage biology, this technology may have potential use in antimicrobial therapies and DNA vaccine development." ¶ Westwater and her coauthors published their study in Antimicrobial Agents and Chemotherapy (Use of genetically engineered phage to deliver antimicrobial agents to bacteria: an alternative therapy for treatment of bacterial infections. [TOP OF PAGE]

  680. EFRH-phage immunization reduces beta-amyloid plaques in transgenic mouse brain. Anonymous (2003). Vaccine Weekly April 4, 4. EFRH-phage immunization reduced the number beta-amyloid plaques in the brain of transgenic mice in a study by Tel Aviv University molecular microbiologists. ¶ "Antibodies to the epitope EFRH, representing residues 3-6 within the beta-amyloid (Abeta) sequence, were previously shown to affect the solubility and disaggregation of Abeta fibrils in vitro," wrote D. Frenkel and colleagues. ¶ Their recent work centered on a new immunization strategy: they used the "EFRH peptide displayed on the surface of the filamentous phage" as antigen. ¶ Frenkel and coworkers reported that "the EFRH phage evoked effective autoimmune antibodies in amyloid precursor protein [V717I] (APP[V717I]) transgenic mice that recapitulate the amyloid plaques and vascular pathology of Alzheimer's disease (AD). ¶ "The immunization provoked a considerable reduction in the number of Abeta amyloid plaques in the brain of the transgenic mice and may serve as the basis for anti-Abeta vaccine." ¶ Frenkel and coauthors published their study in Vaccine (Reduction of beta-amyloid plaques in brain of transgenic mouse model of Alzheimer's disease by EFRH-phage immunization. Vaccine, 2003;21(11-12):1060-1065). [TOP OF PAGE]

  681. Genomics: Phage genes make bad bug worse. Anonymous (2003). Applied Genetics News 23, 0. Group A "Streptococcus" (GAS) bacteria have acquired virulence factors from bacteriophage, according to Stephen Beres and James Musser of Rocky Mountain National Laboratories (903 South 4th St., Hamilton, MT 59840; Tel: 406/363-9255, Fax: 406/363-9371). Their work, in collaboration with other colleagues, is reported in an article recently posted online in the Proceedings of National Science Foundation Early Edition. ¶ GAS bacteria are common microbes that cause a variety of nasty diseases, including strep throat, wound infections, toxic shock, "flesh-eating" disease, scarlet fever, rheumatic fever, and kidney ailments. Musser and Beres seek to understand why some GAS strains cause severe infections while others lead to milder illnesses. By comparing the complete genomes of bacterial strains isolated from people with different GAS infections, the researchers hope to identify specific genes linked with individual diseases. ¶ In their most recent study, the Rocky Mountain researchers determined the complete genetic sequence of an M3 GAS strain isolated from a person with toxic shock syndrome. M3 strains are known for causing extremely invasive infections leading to an unusually high degree of severe illness and death. The M3 strain genome contains more than 1.9 million base pairs. Approx. 1.7 million of those bases are shared with other, less deadly GAS strains, leaving approx. 10% of the genome unique to M3. When the researchers looked closely at the unique regions, they found genetic markers indicating that bacteriophages had brought in many of the additional M3 genes. ¶ "What we have discovered is that bacterial viruses have imported crucial new toxin genes to create new virulence strains," says Musser. ¶ The M3 genome that the team sequenced has phage genes that encode proteins likely to contribute to pathogenesis, such as streptococcal pyrogenic exotoxins A (SpeA) and SpeK, streptococcal superantigen (SSA), and a previously uncharacterized phospholipase A2, designated Sla. ¶ Humans infected with the M3 strain had anti-SpeK, -SSA, and -Sla antibodies, indicating that these GAS proteins are made in vivo. SpeK and SSA proved to be both pyrogenic and toxic for rabbits. ¶ Serotype M3 strains with the phage-encoded speK and sla genes increased dramatically in frequency late in the 20th century, commensurate with the rise in invasive disease caused by M3 organisms. Taken together, the results show that phage-mediated recombination has played a critical role in the emergence of a new, unusually virulent clone of serotype M3 GAS. [TOP OF PAGE]

  682. Experimental examination of bacteriophage latent-period evolution as a response to bacterial availability. Abedon,S.T., Hyman,P., Thomas,C. (2003). Appl. Environ. Microbiol. 69:7499-7506. For obligately lytic bacteriophage (phage) a trade-off exists between fecundity (burst size) and latent period (a component of generation time). This trade-off occurs because release of phage progeny from infected bacteria coincides with destruction of the machinery necessary to produce more phage progeny. Here we employ phage mutants to explore issues of phage latent-period evolution as a function of the density of phage-susceptible bacteria. Theory suggests that higher bacterial densities should select for shorter phage latent periods. Consistently, we have found that higher host densities (>_10(7) bacteria/ml) can enrich stocks of phage RB69 for variants that display shorter latent periods than the wild type. One such variant, dubbed sta5, displays a latent period that is ~70 to 80% of that of the wild type--which is nearly as short as the RB69 eclipse period--and which has a corresponding burst size that is ~30% of that of the wild type. We show that at higher host densities (>_10(7) bacteria/ml) the sta5 phage can outcompete the RB69 wild type, though only under conditions of direct (same-culture) competition. We interpret this advantage as corresponding to slightly faster sta5 population growth, resulting in multifold increases in mutant frequency during same-culture growth. The sta5 advantage is lost, however, given indirect (different-culture) competition between the wild type and mutant or given same-culture competition but at lower densities of phage-susceptible bacteria (<_10(6) bacteria/ml). From these observations we suggest that phage displaying very short latent periods may be viewed as specialists for propagation when bacteria within cultures are highly prevalent and transmission between cultures is easily accomplished. [TOP OF PAGE]

  683. Bacteriophage observations and evolution. Ackermann,H.-W. (2003). Res. Microbiol. 154:245-251. Bacteriophages are classified into one order and 13 families. Over 5100 phages have been examined in the electron microscope since 1959. At least 4950 phages (96%) are tailed. They constitute the order Caudovirales and three families. Siphoviridae or phages with long, noncontractile tails predominate (61% of tailed phages). Polyhedral, filamentous, and pleomorphic phages comprise less than 4% of bacterial viruses. Bacteriophages occur in over 140 bacterial or archaeal genera. Their distribution reflects their origin and bacterial phylogeny. Bacteriophages are polyphyletic, arose repeatedly in different hosts, and constitute 11 lines of descent. Tailed phages appear as monophyletic and as the oldest known virus group. [TOP OF PAGE]

  684. Genotoxicity of water extracts from the River Yamuna at Mathura, India. Aleem,A., Malik,A. (2003). Environmental Toxicology 18:69-77. Water samples were collected from the River Yamuna at Mathura, India, and concentrated by using XAD resins (Amberlite XAD-4 and XAD-8) and liquid-liquid extraction procedures. The genotoxicities of the extracted water samples were evaluated by the Ames Salmonella/mammalian microsome test, DNA repair of defective mutants, and bacteriophage lambda systems. The results of the Salmonella test demonstrated that the XAD-concentrated water samples had maximum mutagenicity with the TA98 strain, both with and without metabolic activation. The XAD-concentrated water samples collected in the summer showed maximum mutagenic responses compared with those collected in other seasons, whereas the liquid-liquid extracted samples exhibited maximum mutagenic activity during the postmonsoon season. The damage brought about during DNA repair of defective mutants in the presence of XAD-concentrated water samples was found to be remarkably high compared with the liquid-liquid extracted water samples at a dose level of 20 microL/mL of culture. All the mutants invariably exhibited significant decline in their colony-forming units compared with their isogenic wild-type counterparts. Survival was decreased by 86.7% and 65.1% in the polA(-) strain after 6 h of treatment with XAD-concentrated and liquid-liquid extracted water samples, respectively. A significant decrease in the survival of bacteriophage lambda was also observed when treated with test samples. The damage was more pronounced in lexA mutants when the phage was treated with XAD-concentrated samples. The recA, lexA, and polA mutants of E. coli K-12 were found to be sensitive to the test samples, suggesting damage to the DNA of exposed cells as well as to the role of recA(+), lexA(+), and polA(+) genes in coping with the hazardous effect of the pollutants. The results demonstrated substantial genotoxicity and mutagenicity in the water samples tested. [TOP OF PAGE]

  685. Immunity profiles of wild-type and recombinant Shiga-Like toxin-encoding bacteriophages and characterization of novel double lysogens. Allison,H.E., Sergeant,M.J., James,C.E., Saunders,J.R., Smith,D.L., Sharp,R.J., Marks,T.S., McCarthy,A.J. (2003). Infect. Immun. 71:3409-3418. Pathogenicity of Shiga-like toxin (stx)-producing Escherichia coli (STEC), notably serotype 0157, the causative agent of hemorrhagic colitis, hemolytic-uremic syndrome, and thrombotic thrombocytopenic purpura, is based partly on the presence of genes (stx1 and/or stx2) that are known to be carried on temperate lambdoid bacteriophages. Stx phages were isolated from different STEC strains and found to have genome sizes in the range of 48 to 62 kb and to carry either stx(1) or stx(2) genes. Restriction fragment length polymorphism patterns and sodium dodecyl sulfate-polyacrylamide gel electrophoresis protein profiles were relatively uninformative, but the phages could be differentiated according to their immunity profiles. Furthermore, these were sufficiently sensitive to enable the identification and differentiation of two different phages, both carrying the genes for Stx2 and originating from the same STEC host strain. The immunity profiles of the different Stx phages did not conform to the model established for bacteriophage lambda, in that the pattern of individual Stx phage infection of various lysogens was neither expected nor predicted. Unexpected differences were also observed among Stx phages in their relative lytic productivity within a single host. Two antibiotic resistance markers were used to tag a recombinant phage in which the stx genes were inactivated, enabling the first reported observation of the simultaneous infection of a single host with two genetically identical Stx phages. The data demonstrate that, although Stx phages are members of the lambdoid family, their replication and infection control strategies are not necessarily identical to the archetypical bacteriophage l, and this could be responsible for the widespread occurrence of stx genes across a diverse range of E. coli serotypes. [TOP OF PAGE]

  686. Survival of F-specific RNA coliphage, feline calicivirus, and Escherichia coli in water: a comparative study. Allwood,P.B., Malik,Y.S., Hedberg,C.W., Goyal,S.M. (2003). Appl. Environ. Microbiol. 69:5707-5710. The relationship between the survival of enteric viral pathogens and their indicators (coliform bacteria and coliphages) is not well understood. We compared the survival rates of feline calicivirus (FCV), Escherichia coli, and a male-specific RNA coliphage MS2 at 4, 25, and 37ºC for up to 28 days in dechlorinated water. The survival rates of E. coli and FCV, a surrogate of noroviruses (NV), had a high degree of correlation at 4 and 25ºC, while MS2 phage survived significantly longer (P < 0.05) at these two temperatures. At 37ºC, the survival rates for all three organisms were highly correlated. Decimal reduction values indicating the number of days needed for 90% reduction in titer (D values) decreased for all three organisms as storage temperatures increased. FCV had the shortest D value among all three organisms at all temperatures investigated. These findings indicate that F-specific RNA phages may be useful indicators of NV in the environment. [TOP OF PAGE]

  687. Dynamic of isolation of the Phytopathogenic bacteria's phages from A LEAF and a root of sugar beet. Andriychuk,O.M., Semchuk,L.I., Romashov,S.A., Ignatenko,T.A., Yatskovska,L.I., Boyko,A.L. (2003). Bulletin of the University of Kiev, series Biology Research on studying the dynamics of phage's isolation to indicatory cultures Pseudomonas, Xanthomonas, Erwinia, which formed groups on certain taxonomicaly attributes is carried out: 1 - some strains which represents the pathovars of the certain species (8 strain); 2 - strains which represent species of the certain genus (13 strain); 3 - certain genus of phytopathogenic bacteria (Pseudomonas, Xanthomonas, Erwinia). Such bacteria can represents the taxonomic hierarchy and represents the model of microflora field agrocenoses. Research of phage's isolation from plants and roots of sugar beet has shown the presence of various phage's isolates by a character of the lytic abilities to the used indicatory bacteria. The direct isolation of phages to 13 indicators strains and a ultra-violet induction of lysogenic microflorae were carried out. Researches carried out in dynamics during 14 months continuously. During the warm period for the analysis selected leaves of sugar beet, roots selected during all year. The reveals wide spectrum of phages was characteristic both for free, and for ultra-violet induction phages. [TOP OF PAGE]

  688. Therapy uses viruses as natural antibiotics. Anonymous (2003). The Seattle Times January 21(January 21). FORT WAYNE, Ind. - Stepping barefoot on a nail in April changed the path of Fred Bledsoe's life. ¶ The puncture wound seemed innocuous, but because he's diabetic and wounds are hard to heal, Bledsoe cleaned it carefully. ¶ The Fort Wayne man never imagined that the antibiotic-resistant bacteria that infected his foot would land him in a local hospital for 10 weeks of unsuccessful treatment, then send him halfway around the world in search of a cure. ¶ The treatment that worked, called bacteriophage, is available only in Russia and parts of Eastern Europe and the former Soviet Union. Tbilisi, Georgia, is the world's center for development and use of these naturally occurring viruses that destroy specific bacteria. ¶ It is where Bledsoe, 46, found his miracle cure. ¶ He and his family are spearheading efforts to raise awareness in the United States about phage treatment and help with research to get Food and Drug Administration approval for its use here. ¶ In September, after 2 1/2 months of intravenous antibiotics in a U.S. hospital, doctors told Bledsoe only amputation would stop the spread of staphylococcus. The bacteria was creating oozing wounds on his toes, foot and leg. Dead tissue slowly crept upward. ¶ "They actually had the amputation scheduled," he said. Then he called his sister, Saharra Bledsoe, who was out of town. ¶ "I told him, 'Don't do anything until I get home.' I heard my mother's voice say, 'You didn't come this far to fail.' I knew God had another plan," Saharra Bledsoe said. ¶ Washington connection ¶ When she returned, she happened to see a CBS "48 Hours" show called "Silent Killers." Canadian musician Alfred Gertler told of his yearlong battle with an antibiotic-resistant foot infection that was cleared using phage treatment given in Tbilisi. ¶ From the show, Saharra Bledsoe learned of Betty Kutter, a professor at The Evergreen State College in Olympia, who has done extensive phage research. The professor had connections to Eliava Institute in Georgia, a world-renowned center for developing and manufacturing therapeutic phages. ¶ Saharra Bledsoe contacted Kutter and began making arrangements to take her brother to Tbilisi. ¶ But Fred Bledsoe was skeptical when he heard where he was going and the method of treatment. His brother, Dr. Larry Bledsoe, a Fort Wayne internal medicine specialist, was doubtful, too. ¶ "On the other hand, it was intriguing, the idea of viruses fighting bacteria. So, I went and researched it and found it had been used in the past in this country. There were very few side effects. I felt it was safe," said Larry Bledsoe. ¶ The Bledsoe family chipped in and Fred Bledsoe and his sister were able to buy the $1,200-a-piece round-trip tickets to Tbilisi. ¶ "We were treated like celebrities," Saharra Bledsoe said. They were the first blacks ever to be treated at the Republic of Georgia Regional Hospital, which works closely with the Eliava Institute. ¶ And, "as far as I know, they are the first Americans to be treated there," Kutter said. ¶ Cultures of the bacteria in Fred Bledsoe's foot were taken. A phage solution, containing viruses that work against the three bacteria found in his foot, was injected into the infected areas twice a day for two weeks. Then a phage powder was used for several days. In less than three weeks, tests showed the bacteria were gone. The wounds healed. ¶ Not a new treatment ¶ Scientists have known of the existence of bacteriophages since the early 1900s. ¶ The viruses have the ability to attach to the surface of a specific bacterial cell. After a specific kind of phage finds its bacterial cell "match," the viral DNA is injected into the host cell. In minutes, the virus multiplies until it takes over and kills the cell. ¶ Phage therapy can be used to kill specific pathogens without disturbing beneficial bacteria. They pose no risk to anything other than their specific bacterial host, said Zemphira Alavidze, a phage researcher at the Eliava Institute. ¶ Phages were used in the 1930s in the United States, before penicillin was discovered. ¶ In fact, the American Medical Association did a review of bacteriophage therapy but dismissed its effectiveness at that time because, without the technological ability to see viruses, there was no proof they were living organisms. Besides, pharmaceutical companies were finding more effective antibiotics. ¶ "In Georgia, phages are the meat and potatoes of treatment," said Kutter, who has a Ph.D. in biophysics from the University of Rochester, N.Y. Kutter first traveled to Tbilisi in 1990 to examine bacteriophages of a specific E. coli bacterium. It was then that she learned bacteriophages were widely used there as antibiotics. ¶ "I'm a serious, hard-core scientist. I was very skeptical. It took a while of seeing things happen, talking to people, before I started taking it seriously," she said. ¶ A rush to bring phage to U.S. ¶ Drug-resistant bacteria, such as methicillin-resistant staph aureus (MRSA) has been a growing concern in medical circles in the United States. Staph is one of the three bacteria found in Fred Bledsoe's foot. ¶ There has been a gradual rise in MRSA since 1980, said Dr. William Jarvis of the Centers for Disease Control and Prevention in Atlanta. Some blame overuse or misuse of antibiotics. ¶ The "big gun" used against antibiotic-resistant bacteria has been vancomycin, but cases of vancomycin-resistant staph are cropping up too, according to the CDC. ¶ People should understand that phage treatment will not replace antibiotics, said Dr. Terry Brown, president of Intralytix, a Baltimore-based company researching agricultural and other uses for phage. The company is looking at how phages can keep meat-processing equipment and plants free of potentially deadly bacteria such as listeria. ¶ The company also holds the international license to make, market and distribute PhageBioDerm, a phage patch used to treat burns and other skin wounds. Although PhageBioDerm is used in Georgia, it is not yet available in the United States. ¶ Kutter said for U.S. citizens to access phage, "it will have to be manufactured here. It will not work to import it. (Georgian) standards would not meet FDA approval." ¶ According to the October 2002 issue of Science magazine, there are about two dozen companies worldwide in a frenzy to make phage treatment available in Western markets. ¶ Exponential Biotherapies Inc. of Port Washington, N.Y., has completed FDA State I, or safety, trials of a phage effective against a bacterial strain called vancomycin-resistant enterococci. ¶ The company hopes to launch clinical trials of the phage later this year. [TOP OF PAGE]

  689. Elevated abundance of bacteriophage infecting bacteria in soil. Ashelford,K.E., Day,M.J., Fry,J.C. (2003). Appl. Environ. Microbiol. 69:285-289. Here we report the first direct counts of soil bacteriophage and show that substantial populations of these viruses exist in soil (grand mean = 1.5 x 107 g-1), at least 350-fold more than the highest numbers estimated from traditional viable plaque counts. Adding pure cultures of a Serratia phage to soil showed that the direct counting methods with electron microscopy developed here underestimated the added phage populations by at least eightfold. So, assuming natural phages were similarly underestimated, virus numbers in soil averaged 1.5 x 108 g-1, which is equivalent to 4% of the total population of bacteria. This high abundance was to some extent confirmed by hybridizing colonies grown on Serratia and Pseudomonas selective media with cocktails of phage infecting these bacteria. This showed that 8.9 and 3.9%, respectively, hybridized with colonies from the two media and confirmed the presence of phage DNA sequences in the cultivable fraction of the natural population. Thus, soil phage, like their aquatic counterparts, are likely to be important in controlling bacterial populations and mediating gene transfer in soil. [TOP OF PAGE]

  690. Application of host-specific bacteriophages to the surface of chicken skin leads to a reduction in recovery of Campylobacter jejuni. Atterbury,R.J., Connerton,P.L., Dodd,C.E.R., Rees,C.E.D., Connerton,I.F. (2003). Appl. Environ. Microbiol. 69:6302-6306. Retail poultry products are widely purported as the major infection vehicle for human campylobacteriosis. Numerous intervention strategies have sought to reduce Campylobacter contamination on broiler carcasses in the abattoir. This study reports the efficacy of bacteriophage in reducing the number of recoverable Campylobacter jejuni cells on artificially contaminated chicken skin. [TOP OF PAGE]

  691. Isolation and characterization of Campylobacter bacteriophages from retail poultry. Atterbury,R.J., Connerton,P.L., Dodd,C.E.R., Rees,C.E.D., Connerton,I.F. (2003). Appl. Environ. Microbiol. 69:4511-4518. The ability of phages to survive processing is an important aspect of their potential use in the biocontrol of Campylobacter in poultry production. To this end, we have developed a procedure to recover Campylobacter bacteriophages from chilled and frozen retail poultry and have validated the sensitivity of the method by using a characterized Campylobacter phage (i.e., NCTC 12674). By using this method, we have shown that Campylobacter phages can survive on retail chicken under commercial storage conditions. Retail chicken portions purchased in the United Kingdom were screened for the presence of endogenous Campylobacter phages. Thirty-four Campylobacter bacteriophages were isolated from 300 chilled retail chicken portions, but none could be recovered from 150 frozen chicken portions. The phage isolates were characterized according to their lytic profiles, morphology, and genome size. The free-range products were significantly more likely to harbor phages (P < 0.001 by single-factor analysis of variance) than were standard or economy products. This study demonstrates that Campylobacter bacteriophages, along with their hosts, can survive commercial poultry processing procedures and that the phages exhibited a wide range of recovery rates from chicken skin stored at 4°C. [TOP OF PAGE]

  692. Association of a bacteriophage with virulence in Vibrio harveyi. Austin,B., Pride,A.C., Rhodie,G.A. (2003). Journal of Fish Diseases 26:55-58. The virulence of Vibrio harveyi, which is a serious pathogen of penaeids (Karunasagar, Pai, Malathi & Karunasagar 1994; Pizzuto & Hirst 1995; Alvarez, Austin, Alvarez & Reyes 1998) and finfish (Kraxberger-Beatty, McGarey, Grier & Lim 1990; Ishimaru & Muroga 1997), has been associated with possession of double haemolysin genes (Zhang, Meaden & Austin 2001). The study seeks to investigate a possible relationship between virulence and the previously described bacteriophage of V. harveyi (Oakey & Owens 2000). The bacteriophage, which has been determined to have an icosahedral head and rigid tail and to contain double stranded linear DNA, has been presumptively assigned to the genus Myovirus (Oakey & Owens 2000). [TOP OF PAGE]

  693. Evolution of phage with chemically ambiguous proteomes. Bacher,J.M., Bull,J.J., Ellington,A.D. (2003). BMC Evol. Biol. 3:24 BACKGROUND: The widespread introduction of amino acid substitutions into organismal proteomes has occurred during natural evolution, but has been difficult to achieve by directed evolution. The adaptation of the translation apparatus represents one barrier, but the multiple mutations that may be required throughout a proteome in order to accommodate an alternative amino acid or analogue is an even more daunting problem. The evolution of a small bacteriophage proteome to accommodate an unnatural amino acid analogue can provide insights into the number and type of substitutions that individual proteins will require to retain functionality. RESULTS: The bacteriophage Qb initially grows poorly in the presence of the amino acid analogue 6-fluorotryptophan. After 25 serial passages, the fitness of the phage on the analogue was substantially increased; there was no loss of fitness when the evolved phage were passaged in the presence of tryptophan. Seven mutations were fixed throughout the phage in two independent lines of descent. None of the mutations changed a tryptophan residue. CONCLUSIONS: A relatively small number of mutations allowed an unnatural amino acid to be functionally incorporated into a highly interdependent set of proteins. These results support the 'ambiguous intermediate' hypothesis for the emergence of divergent genetic codes, in which the adoption of a new genetic code is preceded by the evolution of proteins that can simultaneously accommodate more than one amino acid at a given codon. It may now be possible to direct the evolution of organisms with novel genetic codes using methods that promote ambiguous intermediates. [TOP OF PAGE]

  694. Bacteriophage isolation from human saliva. Bachrach,G., Leizerovici-Zigmond,M., Zlotkin,A., Naor,R., Steinberg,D. (2003). Lett. Appl. Microbiol. 36:50-53. AIMS: To detect bacteriophages for Gram-positive oral pathogens in human saliva. METHODS AND RESULTS: Saliva samples from 31 donors were screened for the presence of bacteriophages for Streptococcus sobrinus, Streptococcus mutans, Streptococcus salivarius, Actinomyces viscosus and Enterococcus faecalis. Bacteriophages for Enterococcus faecalis were found in seven samples. Enterococcus faecalis phages were still present in saliva re-collected from one donor one month, and one year after initial saliva collection. CONCLUSIONS: The presence and stability of the Enterococcus faecalis bacteriophages in human saliva suggests a possible role of these bacteriophages in the oral ecosystem. SIGNIFICANCE AND IMPACT OF THE STUDY: Phage therapy as a way to control oral bacteria might be considered. [TOP OF PAGE]

  695. Improved efficacy of newly formulated bacteriophages for management of bacterial spot on tomato. Balogh,B., Jones,J.B., Momol,M.T., Olson,S.M., Obradovic,A., Jackson,L.E. (2003). Plant Dis. 87:949-954. Bacteriophages are currently used as an alternative method for controlling bacterial spot disease on tomato incited by Xanthomonas campestris pv. vesicatoria. However, the efficacy of phage is greatly reduced due to its short residual activity on plant foliage. Three formulations that significantly increased phage longevity on the plant surface were tested in field and greenhouse trials: (i) PCF, 0.5% pregelatinized corn flour (PCF) + 0.5% sucrose; (ii) Casecrete, 0.5% Casecrete NH-400 + 0.5% sucrose + 0.25% PCF; and (iii) skim milk, 0.75% powdered skim milk + 0.5% sucrose. In greenhouse experiments, the nonformulated, PCF-, Casecrete-, and skim milk– formulated phage mixtures reduced disease severity on plants compared with the control by 1, 30, 51, and 62%, respectively. In three consecutive field trials, nonformulated phage caused 15, 20, and 9% reduction in disease on treated plants compared with untreated control plants, whereas plants treated with PCF- and Casecrete-formulated phage had 27, 32, and 12% and 30, 43, and 24% disease reduction, respectively. Plants receiving copper–mancozeb treatments were included in two field trials and had a 20% decrease in disease in the first trial and a 13% increase in the second one. Skim milk–formulated phage was tested only once and caused an 18% disease reduction. PCF-formulated phage was more effective when applied in the evening than in the morning, reducing disease on plants by 27 and 13%, respectively. The Casecrete formulated phage populations were over 1,000-fold higher than the nonformulated phage populations 36 h after phage application. [TOP OF PAGE]

  696. Do viruses form lineages across different domains of life? Bamford,D.H. (2003). Res. Microbiol. 154:231-236. The scarce characterisation of the viral world has hampered our efforts to appreciate the magnitude and diversity of the viral domain. It appears that almost every species can be infected by a number of viruses. As our knowledge of viruses increases, it appears that this myriad of viruses may be organised into a reasonably low number of viral lineages including members infecting hosts belonging to different domains of life. Viruses belonging to a lineage share a common innate "self" that refers to structural and assembly principles of the virion. This hypothesis has a few consequences. All viruses are old, maybe preceding cellular life, and virus origins are polyphyletic, as opposed to the idea of a monophyletic origin of cellular life. [TOP OF PAGE]

  697. Prophage induction and expression of prophage-encoded virulence factors in group A Streptococcus serotype M3 strain MGAS315. Banks,D.J., Lei,B., Musser,J.M. (2003). Infect. Immun. 71:7079-7086. The genome of the highly virulent group A Streptococcus (GAS) serotype M3 strain MGAS315 has six prophages that encode six proven or putative virulence factors. We examined prophage induction and expression of prophage-encoded virulence factors by this strain under in vitro conditions inferred to approximate in vivo conditions. Coculture of strain MGAS315 with Detroit 562 (D562) human epithelial pharyngeal cells induced the prophage encoding streptococcal pyrogenic exotoxin K (SpeK) and extracellular phospholipase A(2) (Sla) and the prophage encoding streptodornase (Sdn). Increased gene copy numbers after induction correlated with increased speK, sla, and sdn transcript levels. Although speK and sla are located contiguously in prophage Phi315.4, these genes were transcribed independently. Whereas production of immunoreactive SpeK was either absent or minimal during coculture of GAS with D562 cells, production of immunoreactive Sla increased substantially. In contrast, despite a lack of induction of the prophage encoding speA during coculture of GAS with D562 cells, the speA transcript level and production of immunoreactive streptococcal pyrogenic exotoxin A (SpeA) increased. Exposure of strain MGAS315 to hydrogen peroxide, an oxidative stressor, induced the prophage encoding mitogenic factor 4 (MF4), and there was a concomitant increase in the mf4 transcript. All prophages of strain MGAS315 that encode virulence factors were induced during culture with mitomycin C, a DNA-damaging agent. However, the virulence factor gene transcript levels and production of the encoded proteins decreased after mitomycin C treatment. Taken together, the results indicate that a complex relationship exists among environmental culture conditions, prophage induction, and production of prophage-encoded virulence factors. [TOP OF PAGE]

  698. Detection and drug-susceptibility testing of M. tuberculosis from sputum samples using luciferase reporter phage: comparison with the Mycobacteria Growth Indicator Tube (MGIT) system. Bardarov,S., Jr., Dou,H., Eisenach,K., Banaiee,N., Ya,S.u., Chan,J., Jacobs,W.R., Jr., Riska,P.F. (2003). Diagnostic Microbiology and Infectious Disease 45:53-61. Rapid diagnosis of drug-resistant M.tuberculosis (Mtb) is desirable worldwide. We (i) describe a new luciferase reporter phage (LRP), phAE142 for this purpose; (ii) compare it to the automated MGIT 960 for time-to-detection of Mtb in clinical specimens; and (iii) evaluate its use for species confirmation and antibiotic susceptibility testing(AST) of Mtb. Twenty sputum samples were inoculated for testing by LRP, or by MGIT 960. After "positives" were identified by either method, the LRP was used for confirmation of Mtb complex (TBC) and for AST. The LRP method proved comparably efficient to MGIT 960 at detecting Mtb. Using an antibiotic uniquely inhibiting TBC with LRP provided species assignment, concurrently with AST, in a median of 3 days, with a sensitivity of 97%. Overall agreement in susceptibility results was 96%. Reliable susceptibility results and identification of TBC can be completed in a median of 12 days (range 8 to 16d) with LRP applied to sputum samples. [TOP OF PAGE]

  699. Identification methods for Legionella from environmental samples. Bartie,C., Venter,S.N., Nel,L.H. (2003). Water Res. 37:1362-1370. Laboratories responsible for Legionella diagnostics around the world use a number of different culturing methods of non-equivalent sensitivities and specificities, to detect Legionella species in environmental samples. Specific countries usually standardize and use one approved method. For example, laboratories in Australia use the Australian Standard (AS) method and those in Europe, the International Standard method (ISO). However, no standard culturing methods have been established in South Africa to date. As a result, there is uncertainty about the true prevalence and most common species of Legionella present in the South African environment. In an attempt to provide guidelines for the development of a standard method specific for South Africa, the ISO, AS and a most probable number method were evaluated and compared. In addition, the effect of sample re-incubation with autochthonous amoebae on culture outcome was studied. Samples were collected from four environments, representing industrial water, mine water and biofilm. The samples were concentrated by membrane filtration and divided into three portions and cultured without pretreatment, after acid treatment and after heat treatment, on four culture media namely alphaBCYE, BMPA, MWY and GVPC agar. A selective approach, incorporating heat treatment, but not acid treatment, combined with culture on alphaBCYE and GVPC or MWY, was most appropriate for legionellae detection in the samples evaluated. Legionellae were cultured from 82% of the environmental samples we evaluated. In 54% of the samples tested, legionellae were present in numbers equal to or exceeding 10(2) colony-forming units per milliliter (cfu/ml). Legionella pneumophila serogroups (SGs) 1-14 were the most prevalent species and were present as single, or a combination of two or more SGs in a number of samples tested. Re-incubation of sample concentrates with autochthonous amoebae improved the culturability of legionellae in 50% of cultures on alphaBCYE and 25% on GVPC. [TOP OF PAGE]

  700. Virioplankton and microbial communities in aquatic systems: a seasonal study in two lakes of differing trophy. Bettarel,Y., Sime-Ngando,T., Amblard,C., Carrias,J.F., Portelli,C. (2003). Freshw. Biol. 48:810-822. 1. The seasonal and vertical distribution of the abundance of virus-like particles (VLPs), together with the abundances of other microbial organisms (bacteria, unpigmented and pigmented nanoflagellates and ciliates), were determined in an oligomesotrophic lake (Pavin, France) and in a eutrophic lake (Aydat, France) between March and December 2000. ¶ 2. The abundance of the viral plankton and those of other microbial taxa were significantly higher in the more productive system. The same was for the virus-to-bacteria quotient (VBQ), which averaged seven in Lake Pavin and nine in Lake Aydat. ¶ 3. The abundance of viruses increased during the period of thermal stratification in both lakes, with the highest values being recorded at the end of summer/early autumn in the epi- and the metalimnion. The seasonal pattern of abundance of viruses in both lakes in the surface layer was similar, indicating that the dynamics of viruses may be controlled by environmental factors such as light conditions. ¶ 4. There was no correlation between the abundance of viruses and protists. We found correlations between viruses and heterotrophic bacteria in the whole water column in Lake Pavin, but only in the dark bottom waters in Lake Aydat. ¶ 5. Overall, the empirical findings in this study lead us to speculate that the weaker correlation between bacteria and viruses in Lake Aydat than in Lake Pavin, as well as the higher VBQ in the former, is a consequence of the increasing relative abundance of non-bacteriophage VLPs along the trophic gradient of aquatic systems. [TOP OF PAGE]

  701. Viral lysis, flagellate grazing potential and bacterial production in Lake Pavin. Bettarel,Y., Amblard,C., Sime-Ngando,T., Carrias,J.F., Sargos,D., Garabetian,F., Lavandier,P. (2003). Microb. Ecol. 45:119-127. Abundances of different compartments of the microbial loop (i.e., viruses, heterotrophic bacteria, nonpigmented nanoflagellates, and pigmented nanoflagellates), bacterial heterotrophic production (BHP), viral lysis, and potential flagellate grazing impacts on the bacterial assemblages were estimated during a short-term study (24 h) conducted in June 1998 in the epilimnion (5 m) and metalimnion (10 m) of a moderate-altitude oligomesotrophic lake (Lake Pavin, France). Viral and bacterial abundances were higher in the metalimnion than in the epilimnion, whereas pigmented and nonpigmented nanoflagellates were more numerous in the epilimnion. The control of the BHP due to viral lysis (determined by examination of viral-containing bacteria using a transmission electron microscope) was significantly higher in the meta- (range = 6.0-33.7%, mean = 15.6%) than in the epilimnion (3.5-10.3%, 6.4%). The same was for the losses of BHP from the potential predation by nanoflagellates which ranged from 0.5 to 115.4% (mean = 38.7%) in the epilimnion, and from 0.7 to 97.5% (mean = 66.7%) in the metalimnion. Finally, estimated viral mediated mortality rates from the percentage of visibly infected cells and potential nanoflagellate grazing rates based on assumed clearance rates suggest that flagellates consumed a larger proportion of bacterial production than was lost to viral lysis. [TOP OF PAGE]

  702. AFV1, a novel virus infecting hyperthermophilic archaea of the genus Acidianus. Bettstetter,M., Peng,X., Garrett,R.A., Prangishvili,D. (2003). Virology 315:68-79. We describe a novel virus, AFV1, of the hyperthermophilic archaeal genus Acidianus. Filamentous virions are covered with a lipid envelope and contain at least five different proteins with molecular masses in the range of 23–130 kDa and a 20.8-kb-long linear double-stranded DNA. The virus has been assigned to the family Lipothrixviridae on the basis of morphotypic characteristics. Host range is confined to several strains of Acidianus and the virus persists in its hosts in a stable carrier state. The latent period of virus infection is about 4 h. Viral DNA was sequenced and sequence similarities were found to the lipothrixvirus SIFV, the rudiviruses SIRV1 and SIRV2, as well as to conjugative plasmids and chromosomes of the genus Sulfolobus. Exceptionally for the linear genomes of archaeal viruses, many short direct repeats, with the sequence TTGTT or close variants thereof, are closely clustered over 300 bp at each end of the genome. They are reminiscent of the telomeric ends of linear eukaryal chromosomes. [TOP OF PAGE]

  703. Incidence of enteric viruses in groundwater from household wells in Wisconsin. Borchardt,M.A., Bertz,P.D., Spencer,S.K., Battigelli,D.A. (2003). Appl. Environ. Microbiol. 69:1172-1180. Recent studies on the contamination of groundwater with human enteric viruses have focused on public water systems, whereas little is known about the occurrence of viruses in private household wells. The objective of the present study was to estimate the incidence of viruses in Wisconsin household wells located near septage land application sites or in rural subdivisions served by septic systems. Fifty wells in seven hydrogeologic districts were sampled four times over a year, once each season. Reverse transcriptase PCR (RT-PCR), followed by Southern hybridization, was used to detect enteroviruses, rotavirus, hepatitis A virus (HAV), and Norwalk-like viruses (NLVs). In addition, cell culture was used to detect culturable enteroviruses. Companion water samples were collected for total coliforms, Escherichia coli, fecal enterococci, F-specific RNA coliphages, nitrate, and chloride analyses. Among the 50 wells, four (8%) were positive for viruses by RT-PCR. Three wells were positive for HAV, and the fourth well was positive for both rotavirus and NLV in one sample and an enterovirus in another sample. Contamination was transient, since none of the wells was virus positive for two sequential samples. Culturable enteroviruses were not detected in any of the wells. Water quality indicators were not statistically associated with virus occurrence, although some concordance was noted for chloride. The present study is the first in the United States to systematically monitor private household wells for virus contamination and, combined with data for public wells, provides further insight on the extent of groundwater contamination with human enteric viruses. [TOP OF PAGE]

  704. Isolation and characterization of marine psychrophilic phage-host systems from Arctic sea ice. Borriss,M., Helmke,E., Hanschke,R., Schweder,T. (2003). Extremophiles 7:377-384. Phage-host systems from extreme cold environments have rarely been surveyed. This study is concerned with the isolation and characterization of three different phage-host systems from Arctic sea ice and melt pond samples collected north-west of Svalbard (Arctic). On the basis of 16S rDNA sequences, the three bacterial phage hosts exhibited the greatest similarity to the species Shewanella frigidimarina (96.0%), Flavobacterium hibernum (94.0%), and Colwellia psychrerythraea (98.4%), respectively. The host bacteria are psychrophilic with good growth at 0°C, resulting in a rapid formation of visible colonies at this temperature. The phages showed an even more pronounced adaptation to cold temperatures than the bacteria, with growth maxima below 14°C and good plaque formation at 0°C. Transmission electron microscopy (TEM) examinations revealed that the bacteriophages belonged to the tailed, double-stranded DNA phage families Siphoviridae and Myoviridae. All three phages were host-specific. [TOP OF PAGE]

  705. Prophage contribution to bacterial population dynamics. Bossi,L., Fuentes,J.A., Mora,G., Figuero-Bossi,N. (2003). J. Bacteriol. 185:6467-6471. Cocultures of Salmonella strains carrying or lacking specific prophages undergo swift composition changes as a result of phage-mediated killing of sensitive bacteria and lysogenic conversion of survivors. Thus, spontaneous prophage induction in a few lysogenic cells enhances the competitive fitness of the lysogen population as a whole, setting a selection regime that forces maintenance and spread of viral DNA. This is likely to account for the profusion of prophage sequences in bacterial genomes and may contribute to the evolutionary success of certain phylogenetic lineages. [TOP OF PAGE]

  706. Detection of phages of phytopathogenic bacterias Pseudomonas, Xanthomonas, Erwinia and Bacillus in agrocenosises. Boyko,A.L., Semchuk,L.I., Romashev,S.A., Andriychuk,O.M., Yatskovska,L.I. (2003). Agroecological magazine (3), 24-26. Studied the diffusion of phytopathogenic bacteria' bacteriophages of in [sic] Kiev area. The samplings conducted on plantations of sugar-beet. Among discharged bacteriophages the main specific weight was made by phages [of] Xanthomonas beticola. In samples [of] seeds of sugar-beet the phages are not detected. Their influencing on number of a pathogenic microflora in biocenoses is discussed. [TOP OF PAGE]

  707. Detection of the phytopathogenic bacteria' phages in Antarctica. Boyko,A.L., Semchuk,L.I., Voytsitsky,V.M., Andriychuk,O.M., Romashev,S.A., Ignatenko,T.O., Yatskovska,L.I., Vaschenko,V.M., Delimat,A. (2003). Agroeclogical magazine (4), 12-15. During expedition to the Ukrainian Antarctic station " Academician Vernadsky " in 2003 testes of a thawed snow and a ground from islands of the Argentina archipelago have been taken. The analysis on presence of phytopathogenic bacteria phages Xanthomonas, Burkholderia, Erwinia and Pseudomonas. has shown lytic activity practically in all tests. For the most of them the titres of phages was 1-10 PFU/ml, and 6 gave high spontaneous production of the investigated phages - 10(6) PFU/ml. Processing of the tests by UV-radiation with the purpose of an lysogenic microflorae induction caused the inactivation of phages up to individual negative colonies. Discussion of the received results from the point of view of the investigated region's features is offered. [TOP OF PAGE]

  708. Metagenomic analyses of an uncultured viral community from human feces. Breitbart,M., Hewson,I., Felts,B., Mahaffy,J.M., Nulton,J., Salamon,P., Rohwer,F. (2003). J. Bacteriol. 185:6220-6223. Here we present the first metagenomic analyses of an uncultured viral community from human feces, using partial shotgun sequencing. Most of the sequences were unrelated to anything previously reported. The recognizable viruses were mostly siphophages, and the community contained an estimated 1,200 viral genotypes. [TOP OF PAGE]

  709. Microbial tracers in groundwater research. Bricelj,M. (2003). RMZ-mater. Geoenviron. 50:67-70. [TOP OF PAGE]

  710. Population mixing accelerates coevolution. Brockhurst,M.A., Morgan,A.D., Rainey,P.B., Buckling,A. (2003). Ecol. Lett. 6:975-979. Theory predicts that mixing in spatially structured populations of hosts and parasites can increase the rate of antagonistic coevolution. We experimentally tested this prediction by allowing populations of bacteria (Pseudomonas fluorescens) and parasitic bacteriophage to coevolve in mixed and unmixed microcosms. Coevolution proceeded at approximately twice the rate in mixed populations compared with unmixed populations and caused the evolution of more resistant hosts and more infective parasites. [TOP OF PAGE]

  711. In vivo lysogenic conversion of Tox(-) Streptococcus pyogenes to Tox(+) with Lysogenic Streptococci or free phage. Broudy,T.B., Fischetti,V.A. (2003). Infect. Immun. 71:3782-3786. Temperate bacteriophage can transfer toxin-encoding genes between bacteria, often resulting in acquired pathogenicity. However, little is known regarding the effects of the eukaryotic host on the phage-pathogen interaction. Using Streptococcus pyogenes as a model, we demonstrate, both in vitro and in vivo, that the eukaryote mediates the efficient induction of toxin-encoding temperate phage and the resultant conversion of Tox(-) flora to Tox(+). Furthermore, we show that both phage induction and subsequent conversion need not happen in the same mammalian host, as host-to-host phage transmission can result in toxigenic conversion within the secondary host. Ultimately, our findings demonstrate that the eukaryotic host serves as an essential component in the phage-mediated evolution of virulence within the microbial population. [TOP OF PAGE]

  712. Assessing UV reactor performance for treatment of finished water. Bukhari,Z., LeChevallier,M. (2003). Water Sci. Technol. 47:179-184. Recently, use of low levels of medium- and low-pressure ultraviolet light for successful inactivation of Cryptosporidium parvum oocysts has generated tremendous excitement in the water industry. Accurate delivery of the target dose, lamp performance, sensor stability and impact of water characteristics are some factors that could impact disinfection efficacy, in turn influencing decisions on application of this technology. To this end, American Water Systems, the largest investor owned water utility in the US, has responded to some of these challenges by ascertaining the long-term feasibility of applying UV for treatment of finished water. A 4 x 1 UV reactor with a 12 inch (0.3 m) diameter was installed after granular activated carbon filtration and was operated with a finished water flow rate of 600 gpm (2,700 L/min). Over a 12-month period, various chemical (THM, HAA, UV254, DOC, TOC, metals, nitrate, nitrites) and physical measurements (lamp voltage, current, sensor measurements) were monitored to define their impact (if any) on the operation of the reactor. MS2 bacteriophage challenge studies were conducted with various lamp configurations and lamp age. These inactivation data demonstrated high levels of correlation with controlled bench scale inactivation data. For C. parvum oocysts, bench scale studies were performed with a modified in vitro infectivity assay using HCT-8 cells, an enhanced infectivity protocol and with either immunofluorescence or quantitative PCR based detection. While both assays indicated increasing infections levels of HCT-8 cells with increasing oocyst inocula, UV treatment of oocysts produced markedly different infectivity responses. Based on the data generated in this study, one in vitro infectivity assay was selected to demonstrate > 3 logs inactivation with low UV doses (5 mJ/cm(20-10 mJ/cm2). [TOP OF PAGE]

  713. Experimental evolution yields hundreds of mutations in a functional viral genome. Bull,J.J., Badgett,M.R., Rokyta,D., Molineux,I.J. (2003). J. Mol. Evol. 57:241-248. Two lines of the bacteriophage T7 were grown to fix mutations indiscriminately, using a combination of population bottlenecks and mutagenesis. Complete genome sequences revealed 404 and 299 base substitutions in the two lines, the largest number characterized in functional microbial genomes so far. Missense substitutions outnumbered silent substitutions. Silent substitutions occurred at similar rates between essential and nonessential genes, but missense substitutions occurred at a higher rate in nonessential genes than in essential genes, as expected if they were less deleterious in the nonessential genes. Viral fitness declined during this protocol, and subsequent passaging of each mutated line in large population sizes restored some of the lost fitness. Substitution levels during these recoveries were less than 6% of those during the bottleneck phase, and only two changes during recovery were reversions of the original mutations. Exchanges of genomic fragments between the two recovered lines revealed that fitness effects of some substitutions were not additive-that interactions were accumulating which could lead to incompatibility between the diverged genomes. Based on these results, unprecedented high rates of nucleotide and functional divergence in viral genomes should be attainable experimentally by using repeated population bottlenecks at a high mutation rate interspersed with recovery. [TOP OF PAGE]

  714. The future of bacteriophage biology. Campbell,A. (2003). Nature Rev. Genet. 4:471-477. After an illustrious history as one of the primary tools that established the foundations of molecular biology, bacteriophage research is now undergoing a renaissance in which the primary focus is on the phages themselves rather than the molecular mechanisms that they explain. Studies of the evolution of phages and their role in natural ecosystems are flourishing. Practical questions, such as how to use phages to combat human diseases that are caused by bacteria, how to eradicate phage pests in the food industry and what role they have in the causation of human diseases, are receiving increased attention. Phages are also useful in the deeper exploration of basic molecular and biophysical questions. [TOP OF PAGE]

  715. Phage as agents of lateral gene transfer. Canchaya,C., Fournous,G., Chibani-Chennoufi,S., Dillmann,M.L., Brüssow,H. (2003). Curr. Opin. Mirobiol. 6:417-424. When establishing lysogeny, temperate phages integrate their genome as a prophage into the bacterial chromosome. Prophages thus constitute in many bacteria a substantial part of laterally acquired DNA. Some prophages contribute lysogenic conversion genes that are of selective advantage to the bacterial host. Occasionally, phages are also involved in the lateral transfer of other mobile DNA elements or bacterial DNA. Recent advances in the field of genomics have revealed a major impact by phages on bacterial chromosome evolution. [TOP OF PAGE]

  716. Prophages and bacterial genomics: what have we learned so far? Casjens,S. (2003). Mol. Microbiol. 49:277-300. Bacterial genome nucleotide sequences are being completed at a rapid and increasing rate. Integrated virus genomes (prophages) are common in such genomes. Fifty-one of the 82 such genomes published to date carry prophages, and these contain 230 recognizable putative prophages. Prophages can constitute as much as 10-20% of a bacterium's genome and are major contributors to differences between individuals within species. Many of these prophages appear to be defective and are in a state of mutational decay. Prophages, including defective ones, can contribute important biological properties to their bacterial hosts. Therefore, if we are to comprehend bacterial genomes fully, it is essential that we are able to recognize accurately and understand their prophages from nucleotide sequence analysis. Analysis of the evolution of prophages can shed light on the evolution of both bacteriophages and their hosts. Comparison of the Rac prophages in the sequenced genomes of three Escherichia coli strains and the Pnm prophages in two Neisseria meningitidis strains suggests that some prophages can lie in residence for very long times, perhaps millions of years, and that recombination events have occurred between related prophages that reside at different locations in a bacterium's genome. In addition, many genes in defective prophages remain functional, so a significant portion of the temperate bacteriophage gene pool resides in prophages. [TOP OF PAGE]

  717. Microbiological aspects of an urban river used for unrestricted irrigation in the semi-arid region of north-east Brazil. Ceballos,B.S.O., Soares,N.E., Moraes,M.R., Catao,R.M.R., Konig,A. (2003). Water Sci. Technol. 47:51-57. This study compared the behaviour of pathogenic bacteria (Salmonella and Listeria), faecal indicators (faecal coliforms FC and faecal streptococci FS), somatic coliphages and F-specific bacteriophages in an urban river contaminated with domestic sewage and surface run-off from agricultural and cattle grazing lands. The influence of physical and chemical parameters was also investigated as well as Salmonella and Listeria serotype diversity and drug resistance patterns. Faecal contamination was high (FC = 5 x 106 - 4 x 103 CFU/100 mL; FS = 4 x 105 - 2 x 102 CFU/100 mL) but decreased along the river by up to 99.5% following 47% reduction of BOD5 and 91% increase of DO, both associated with the self purification process. Somatic coliphages (6.9 x 105 - 1 x 103 PFU/100 mL) and F-specific bacteriophages (5.8 x 104 - 65 PFU/100 mL) behaved similarly with reductions of 99.85%. Salmonella and Listeria were isolated at all sampling points with highest frequencies (91-100%) at those with sewage discharge and rural water run-off. The lowest value (35%) occurred at the end of the river where it was (a) wider and shallower, (b) it ran slower and was warmer (29-33ºC), (c) the pH was alkaline (8.2-9.9), (d) electrical conductivity (2,200-5,800 microS/cm) and DO (6-13 mg/L) were highest. Pathogen decline did not follow exactly FC and FS reduction patterns, while physical and chemical parameters apparently did not interfere with Salmonella and Listeria survival to the same extent as they did with FC and FS. Somatic coliphages and F-specific bacteriophages did not show more resistance than bacterial indicators. Catchment area contribution seemed to be more significant for pathogens than for indicators and rainy periods increased pathogenic isolation frequency. Five Salmonella serotypes and five serogroups were identified. S. hadar and serogroup E were predominant (50%); both are increasing in Brazil apparently from animal sources. Nearly 25% of Salmonella strains were resistant to at least one of twelve antimicrobials tested. Resistance to tetracycline was common (17%) followed by cefalotine (3%). Five Listeria serogroups were isolated and L. grayi (43%) and L. monocytogenes (9%) were present at all points. Listeria drug resistance rates were 100% for oxaciline followed by clindamicine (97%), tetracycline (34%) and vancomycin (32%). Both pathogenic bacterial strains presented resistance to the same drugs observed in humans and warm blood animals but the high number of sensitive strains and the low numbers of strains resistant to more than one drug was not expected because of the heavy anthropogenic impact in this basin. [TOP OF PAGE]

  718. Effect of soil properties on saturated and unsaturated virus transport through columns. Chu,Y., Jin,Y., Baumann,T., Yates,M.V. (2003). J. Environ. Qual. 32:2017-2025. Viruses from contaminant sources can be transported through porous media to drinking water wells. The objective of this study was to investigate inactivation and sorption of viruses during saturated and unsaturated transport in different soils. Bacteriophages fX174 and MS-2, and Br- tracer in a phosphate-buffered saline solution were introduced into saturated and unsaturated soil columns as a step function under constant flow rate and hydraulic conditions. Results showed that significantly greater virus removal occurred in the unsaturated columns than in the saturated columns in the two soils containing high metal oxides content. However, the increase in virus retention under unsaturated conditions was not significant in two other soils having high phosphorus and calcium contents and high pH, and in another soil with high organic matter content. The results imply that the extent of water content effect on inactivation and sorption of viruses can range from significant to minimal depending on the properties of the transport medium. We found that the presence of in situ metal oxides was a significant factor responsible for virus sorption and inactivation. Therefore, soils with high metal oxides content may have the potential to be used as hydrological barriers in preventing microbial contamination in the subsurface environments. We also found that the water content effect on virus removal and inactivation strongly depended on solid properties of the testing medium. [TOP OF PAGE]

  719. Viruses and marine pollution. Danovaro,R., Armeni,M., Corinaldesi,C., Mei,M.L. (2003). Marine Pollution Bulletin 46:301-304. This short review summarises the present knowledge on pollutant impacts on marine viruses, virus-host systems and their potential ecological implications. Excess nutrients from sewage and river effluents are a primary cause of marine eutrophication and mucilage formation, often related to the development of large viral assemblages. At the same time, hydrocarbons, polychlorinated biphenyl and pesticides alter ecosystem functioning and can determinate changes in the virus-host interactions, thus increasing the potential of viral infection. All these pollutants might have synergistic effects on the virus-host system and are able to induce prophage, thus increasing the impact of viruses on marine ecosystems. [TOP OF PAGE]

  720. Sunscreen products increase virus production through prophage induction in marine bacterioplankton. Danovaro,R., Corinaldesi,C. (2003). Microb. Ecol. 45:109-118. Classical pollutants (e.g., hydrocarbon, pesticides) have been recently recognized to induce lytic cycle in lysogenic bacteria, but information on micro-pollutants is almost completely lacking. We investigated the effects of cosmetic sun products (sunscreen and solar oil) on viral abundance and bacterial activity. We found that both sunscreen and solar oil acted as pollutants, inducing viral development and controlling bacterial abundance and production, thus leading to an increase of the virus to bacterium ratio. Short-term experiments revealed that sunscreen supplementation induced the lytic cycle in a large fraction of total bacterial abundance (13-24% of bacteria, at low and high concentrations, respectively), whereas solar oil had a lower impact (6-9%). A synchronized development of the phage-host system was observed only after sunscreen addition. The addition of sunscreen, even at low concentrations, had a significant impact on all enzymatic activities (aminopeptidase, glucosidase, and phosphatase), which increased significantly. However, when enzymatic activities were normalized per cell, a selective enhancement was observed for certain enzymes (e.g., aminopeptidase) and inhibition for others (e.g., glucosidase). These results indicate that sunscreen products can modify C, N, and P biogeochemical cycling in seawater and increase virus abundance through prophage induction in marine bacterioplankton. [TOP OF PAGE]

  721. The source of laterally transferred genes in bacterial genomes. Daubin,V., Lerat,E., Perriere,G. (2003). Genome Biol. 4:R57 BACKGROUND: Laterally transferred genes have often been identified on the basis of compositional features that distinguish them from ancestral genes in the genome. These genes are usually A+T-rich, arguing either that there is a bias towards acquiring genes from donor organisms having low G+C contents or that genes acquired from organisms of similar genomic base compositions go undetected in these analyses. RESULTS: By examining the genome contents of closely related, fully sequenced bacteria, we uncovered genes confined to a single genome and examined the sequence features of these acquired genes. The analysis shows that few transfer events are overlooked by compositional analyses. Most observed lateral gene transfers do not correspond to free exchange of regular genes among bacterial genomes, but more probably represent the constituents of phages or other selfish elements. CONCLUSIONS: Although bacteria tend to acquire large amounts of DNA, the origin of these genes remains obscure. We have shown that contrary to what is often supposed, their composition cannot be explained by a previous genomic context. In contrast, these genes fit the description of recently described genes in lambdoid phages, named 'morons'. Therefore, results from genome content and compositional approaches to detect lateral transfers should not be cited as evidence for genetic exchange between distantly related bacteria. [TOP OF PAGE]

  722. Occurrence of coliphages in urban stormwater and their fate in stormwater management systems. Davies,C.M., Yousefi,Z., Bavor,H.J. (2003). Lett. Appl. Microbiol. 37:299-303. AIMS: To investigate the occurrence of coliphages in, and their removal from, urban stormwater. METHODS AND RESULTS: Inflow and outflow concentrations of somatic and f-specific RNA coliphages to two stormwater treatment systems were determined on 21 occasions over a period of 5 months. Somatic coliphages were detected in 19 (90%) of the constructed wetland inlet samples, 13 (62%) of the pond inlet samples, and less frequently at the outlets of the two systems. F-specific RNA coliphages were detected at the inlets but only occasionally at the pond outlet. Somatic coliphages were found to attach preferentially to particles <5 microm in size and persisted in the sediments of the two systems. CONCLUSIONS: Treatment systems providing conditions that are conducive to the settlement of fine particles may effectively remove sediment-bound coliphages and, therefore, possibly enteric viruses from stormwater. SIGNIFICANCE AND IMPACT OF THE STUDY: The results will aid the design of systems for effective removal of viral contaminants from urban stormwater. [TOP OF PAGE]

  723. Filamentous phages linked to virulence of Vibrio cholerae. Davis,B.M., Waldor,M.K. (2003). Curr. Opin. Mirobiol. 6:35-42. The pathogenicity of Vibrio cholerae depends upon its production of two key virulence factors: the toxin co-regulated pilus (TCP), a colonization factor, and cholera toxin, an exotoxin. Genes encoding both virulence factors were introduced into V. cholerae by horizontal gene transfer. The toxin genes are contained within the genome of CTXf, an integrated filamentous phage identified in 1996. In the past few years, it has been shown that CTXf relies on novel processes for phage DNA integration, replication and secretion. In addition, expression of CTXf genes--including the toxin genes--and transmission of CTXf were recently found to be promoted by the antirepressor RstC, which is encoded within RS1, a newly described satellite phage of CTXf. The genetic island that encodes TCP has also been described as a filamentous phage; however, these sequences are unlike the genome of any previously characterized filamentous phage. [TOP OF PAGE]

  724. Identification of new phages to type Staphylococcus aureus strains and comparison with a genotypic method. de Gialluly,C., Loulergue,J., Bruant,G., Mereghetti,L., Massuard,S., van der Mee,N., Audurier,A., Quentin,R. (2003). J. Hosp. Infect. 55:61-67. The aim of this study was to optimize the epidemiological monitoring of strains of Staphylococcus aureus, a major cause of hospital-acquired infections. From September to December 1998 47 S. aureus strains isolated from swabs taken from orthopaedic and trauma patients were in studied. Thirty-five isolates were sensitive to methicillin (MSSA) and 12 were methicillin-resistant (MRSA). Ten of the 47 isolates could not be phage-typed using the international set of typing phages: five of these isolates were MSSA and five were MRSA. These MRSA isolates, which were also not typeable by the phages currently recommended for phage-typing MRSA, were lysed by locally isolated experimental phages 584 and 1814. Phage 1814 lysed the gentamicin-resistant MRSA and phage 584 acted on the gentamicin-sensitive MRSA. Both new phages were inactive against the methicillin-sensitive isolates. Cloning of certain isolates was confirmed by macrorestriction genomic profiles obtained by pulsed-field gel electrophoresis analysis (PFGE). The results showed good discriminatory ability of antibiotic-resistance pattern phenotyping and phage-typing when the phages used were adapted to epidemic-associated MRSA strains. [TOP OF PAGE]

  725. A detection method for Legionella spp in (cooling) water: fluorescent in situ hybridisation (FISH) on whole bacteria. Declerck,P., Verelst,L., Duvivier,L., Van Damme,A., Ollevier,F. (2003). Water Sci. Technol. 47:143-146. Although traditional culture methods are appropriate for detection of Legionella species, such culture takes several days. Rapid detection (< 24 h) of individual Legionella is possible using fluorescent in situ hybridisation (FISH) on whole bacteria. Water samples were filtered and the concentrated bacteria were immediately detected (without culture) with a fluorescence microscope following appropriate labelling. The detection level was very high and quantification was possible. For the detection of all Legionella spp. the probe LEG705 was used, complementary to a 16S rRNA sequence conserved in all Legionella spp. For specific detection of L. pneumophila the probe LEGPNE1 was used. This probe is designed against a variable domain of the 16S rRNA sequence from L. pneumophila. CY3 and FLUOS labels were tested and CY3 showed clearly detectable bacteria with minimum background staining. This FISH technique is very sensitive, fast, reliable and individual bacteria are easily detected. [TOP OF PAGE]

  726. PCR as a test for the presence or absence of Legionella in (cooling) water. Declerck,P., Verelst,L., Duvivier,L., Van Damme,A., Ollevier,F. (2003). Water Sci. Technol. 47:103-107. Legionella pneumophila, a Gram-negative bacterium, is the causative agent of legionellosis. Traditionally, culture methods are normally used to detect Legionella species in different types of water (e.g. surface or tap water, circulating systems, air conditioners and their cooling devices). In this study the PCR conditions to detect Legionella were optimised based on the EnviroAmp Legionella kit (Perkin-Elmer) which is no longer commercially available. The PCR is very sensitive and specific in indicating the presence or absence (no quantification with classical PCR) of Legionella spp in general and more specifically L. pneumophila. To identify L. pneumophila. DNA sequences from the mip (macrophage infectivity potentiator) gene were amplified. The mip gene is conserved and specific for L. pneumophila although mip-like genes are also present in other Legionella spp. The PCR techniques were able to detect small amounts of Legionella in tap water samples. Cooling water, however, often contained PCR-inhibiting substances that could result in false negative PCR results for Legionella. [TOP OF PAGE]

  727. The diversity and evolution of the T4-type bacteriophages. Desplats,C., Krisch,H.M. (2003). Res. Microbiol. 154:259-267. Recent studies suggest that viruses are the most numerous entities in the biosphere; bacteriophages, the viruses that infect Eubacteria and Archaea, constitute a substantial fraction of this population. In spite of their ubiquity, the vast majority of phages in the environment have never been studied and nothing is known about them. For the last 10 years our research has focused on an extremely widespread group of phages, the T4-type. It has now become evident that phage T4 has a myriad of relatives in nature that differ significantly in their host range. The genomes of all these phages have homology to the T4 genes that determine virion morphology. Although phylogenetically related, these T4-type phages can be subdivided into four groups that are increasingly distant from T4: the T-evens, the pseudo T-evens, the schizo T-evens and the exo T-evens. Genomic comparisons between the various T4-type phages and T4 indicate that these genomes share homology not only for virion structural components but also for most of the essential genes involved in the T4 life cycle. This suggests that horizontal transmission of the genetic information may have played a less general role in the evolution of these phages than has been supposed. Nevertheless, we have identified several regions of the T4-type genome, such as the segment containing the tail fiber genes that exhibit evidence of extensive modular shuffling during evolution. The T4-type genomes appear to be a mosaic containing a large and fixed group of essential genes as well as highly variable set of non-essential genes. These non-essential genes are probably important for the adaptation of these phages to their particular life-style. Furthermore, swapping autonomous domains within the essential proteins may slightly modify their function(s) and contribute to the adaptive ability of the T4-type phage family. Regulatory sequences also display considerable evolutionary plasticity and this too may facilitate the adaptation of phage gene expression to new environments and stresses. [TOP OF PAGE]

  728. Early lysis of Lactobacillus helveticus CNRZ 303 in Swiss cheese is not prophage-related. Deutsch,S.M., Neveu,A., Guezenec,S., Ritzenthaler,P., Lortal,S. (2003). Int. J. Food Microbiol. 81:147-157. Lactobacillus helveticus is mainly used as starter in Swiss-type cheeses. Often, lysogenic strains are eliminated because of the risk of early lysis and acidification failure due to phage expression. On the other hand, L. helveticus lysis was shown to positively influence cheese proteolysis during ripening. In order to better assess the relationship between lysis and lysogeny, a prophage-cured derivative of L. helveticus CNRZ 303 was isolated (LH 303-G11) and relysogenised (LH 303-G11R), as demonstrated by hybridisation using the whole phage DNA as probe. The growth, lysis in buffered solutions and lytic activities in zymogram using either Micrococcus luteus or L. helveticus as substrate were identical between the mother strain and its cured derivatives. Only morphological differences were observed by scanning electron microscopy: the cells of the cured derivative were shorter in length. The mother strain and its cured and relysogenised derivatives were assayed in triplicate in experimental Swiss cheeses (scale 1:100). No differences were noted during the cheese making: the three strains exhibited identical kinetics of acidification, leading to similar cheeses at day 1 in terms of gross composition and pH. Phages were detected only in the cheeses made with the mother strain and the relysogenised derivative. The lysis of L. helveticus, estimated by viability decrease and release of the intracellular marker D-lactate deshydrogenase, started early before brining and continued during the cold room ripening. No obvious differences of lysis extent were observed. These results demonstrated for the first time that, in the case of LH 303, the extensive lysis observed in cheese is mainly due to autolysin activity and not to prophage induction. [TOP OF PAGE]

  729. On the insertion of foreign DNA into mammalian genomes: mechanism and consequences. Doerfler,W., Grtraud,O., Rainer,S., Katja,F., Hilde,H., Petra,W., Jörg,S. (2003). Gene 157:241-245. We have studied the integration of adenovirus type 12 (Ad12) DNA in transformed and hamster tumor cells over many years. Upon infection of hamster cells with Ad12, viral DNA has been found in association with hamster chromosomes, possibly in part integrated into the host genome. Ad12 DNA integration is not sequence specific. Transcriptionally active sites of the host genome show a preponderance for foreign DNA insertion. We are pursuing the mechanism of Ad12 DNA integrative recombination in a cell-free system prepared from hamster cell nuclear extracts. In a number of Ad12-transformed hamster cell lines or in cell lines carrying foreign DNA, we have located the inserted Ad12 DNA copies on hamster chromosomes by fluorescent in situ hybridization (FISH). Among the consequences of Ad12 DNA integration, we have studied the de novo methylation of the integrated foreign (Ad12) DNA and increases in DNA methylation in several cellular genes and DNA segments in Ad12-transformed and hamster tumor cells. Several lines of evidence argue for the notion that parameters in addition to nucleotide sequence, in particular site of integration and/or the chromatin configuration of the integrated DNA, are important in generating de novo methylation patterns. The de novo methylation of integrated foreign DNA can be interpreted as an old cellular defense mechanism against the activity of foreign genes in an established genome. Pursuing this concept, we have asked for the most likely portal of entry of foreign DNA, supposedly the gastroinstetinal tract in most animals. This hypothesis has been tested by feeding mice linearized or circular, double-stranded bacteriophage M13mp18 DNA. A small amount of this DNA transiently survives the digestive regime of the animals' GI tract, although in a heavily fragmented form. A minute proportion of the fed M13mp18 DNA can be retrieved from the bloodstream of mice between 2 and 8 h after feeding, mainly associated with the leukocyte population. [TOP OF PAGE]

  730. Levels of male-specific RNA bacteriophage and Escherichia coli in molluscan bivalve shellfish from commercial harvesting areas. Dore,W.J., Mackie,M., Lees,D.N. (2003). Lett. Appl. Microbiol. 36:92-96. AIMS: Current measures for controlling the public health risks associated with bivalve molluscan shellfish consumption rely on the use of Escherichia coli to indicate the sanitary quality of shellfish harvesting areas. However, it has been demonstrated that E. coli is an inadequate indicator of the viral risk associated with shellfish. An alternative indicator organism, male-specific RNA (FRNA) bacteriophage has been proposed for this role. This study compared the distribution of E. coli and FRNA bacteriophage in shellfish harvesting areas. METHODS AND RESULTS: A total of 608 shellfish samples from 49 shellfish harvesting areas were analysed for E. coli and FRNA bacteriophage using standard published methods. The geometric mean concentration of FRNA bacteriophage in all samples was over three times greater than that of E. coli (1800 and 538 counts/100 g for FRNA bacteriophage and E. coli, respectively). In contrast to E. coli, FRNA bacteriophage concentrations were strongly influenced by season with a geometric mean count of 4503 PFU/100 g in the winter (October-March) compared with 910 PFU/100 g in the summer (April-September). CONCLUSIONS: FRNA bacteriophage were present in shellfish at higher concentrations than E. coli. Elevated levels of FRNA bacteriophage observed in the winter concur with the known increased viral risk associated with shellfish harvested at that time of year in the UK. Levels of FRNA bacteriophage found in many shellfish from category B harvesting areas would not be eliminated by conventional treatment processes. SIGNIFICANCE AND IMPACT OF THE STUDY: Data from this study will inform future proposals to introduce FRNA bacteriophage as an indicator of the viral risk associated with shellfish. [TOP OF PAGE]

  731. Comparative electrochemical inactivation of bacteria and bacteriophage. Drees,K.P., Abbaszadegan,M., Maier,R.M. (2003). Water Res. 37:2291-2300. Electric fields and currents have been shown to be capable of disinfecting drinking water and reducing the numbers of bacteria and yeast in food. However, little research has been conducted regarding the effectiveness of electric fields and currents in the inactivation of viruses. The objective of this study was to compare the ability of bacteria and bacteriophage to survive exposure to direct electric current in an electrochemical cell, where they would be subject to irreversible membrane permeabilization processes, direct oxidation of cellular/viral constituents by electric current, and disinfection by electrochemically generated oxidants. Suspensions of the bacteria Escherichia coli and Pseudomonas aeruginosa and bacteriophage MS2 and PRD1 at both high (approximately 1 x 106CFU or PFU/mL) and low (approximately 1 x 103CFU or PFU/mL) population densities were exposed to currents ranging from 25 to 350mA in 5 s pulses. Post-exposure plaque counts of the bacteriophage were proportionally higher than bacterial culturable counts at corresponding current exposures. E. coli and MS2 were then exposed to 5mA for 20 min at both high and low population densities. The inactivation rate of E. coli was 2.1-4.3 times greater than that of MS2. Both bacteria and bacteriophage were more resistant to exposure to direct current at higher population densities. Also, amelioration of inactivation within the electrochemical cell by the reducing agent glutathione indicates the major mechanism of inactivation in the electrochemical cell is disinfection by electrochemically generated oxidants. The implications of these results are that technologies relying upon direct current to reduce the numbers of microbes in food and water may not be sufficient to reduce the numbers of potentially pathogenic viruses and ensure the safety of the treated food or water. [TOP OF PAGE]

  732. Usefulness of different groups of bacteriophages as model micro-organisms for evaluating chlorination. Duran,A.E., Muniesa,M., Moce-Llivina,L., Campos,C., Jofre,J., Lucena,F. (2003). J. Appl. Microbiol. 95:29-37. AIMS: To assess the usefulness of bacterial and viral indicators in chlorination processes and to collect quantitative information necessary for risk assessment analysis in water disinfection processes based on chlorination. METHODS AND RESULTS: Naturally occurring bacterial indicators, bacteriophages and enteroviruses were determined to evaluate the effect of chlorination in groundwater and secondary sewage effluents. Additionally, the effect of chlorinating on selected bacteriophages, enteroviruses and Escherichia coli was also tested in spiked samples of bottled water and sewage effluents. Results indicate that chlorination inactivates more efficiently bacteria than phages and enteroviruses. Among the human viruses, phages infecting Bacteroides fragilis and selected somatic coliphages belonging to the Siphoviridae family were the most persistent to chlorination. CONCLUSIONS: The three groups of bacteriophages studied were all more resistant to chlorination than bacteria and some of the phages were more resistant than enteroviruses. Results presented here indicate that it is very risky to generalize from information obtained with inactivation experiments done with single isolates of any phage or virus. If possible, inactivation studies should be done with naturally occurring populations. Phages offer a good opportunity for studying naturally occurring populations. Thus, the bacteriophages offer a range of resistance to chlorination that may represent most of the viruses that can be found in water. SIGNIFICANCE AND IMPACT OF THE STUDY: Data reported in this study support the inclusion of bacteriophages as additional indicators of the efficiency of water chlorination processes and water quality. [TOP OF PAGE]

  733. Haloarchaeal viruses: how diverse are they? Dyall-Smith,M., Tang,S.-L., Bath,C. (2003). Res. Microbiol. 154:309-313. Hypersaline lakes are highly productive microbial environments that provide many advantages for microbial ecologists, including stable communities of relatively low diversity (mainly haloarchaea). An important component of these communities is comprised of their noncellular parasites, i.e., their viruses. Few viruses of halobacteria (haloviruses) have been isolated and studied even though a wide selection of host species have been formally described (and easily cultured) for ten years. Hypersaline waters have been shown to contain very high concentrations of virus-like particles (at least 10(7) particles/ml), particularly fusiform particles, but laboratory isolations of new haloviruses have been very slow and the detailed study of selected examples even slower. Here we provide an outline of the reported haloviruses, including fusiform and unpublished isolates from this laboratory, and we discuss their diversity and the future directions for this research. [TOP OF PAGE]

  734. Real-time PCR provides improved detection and titer determination of bacteriophage. Edelman,D.C., Barletta,J. (2003). BioTechniques 35:368-375. The plaque assay is the traditional method for the quantification of bacteriophage, particularly for l cloning vectors. Unfortunately, this technique is fraught with procedural difficulties, and the quality of the data obtained from this gold standard assay may be inaccurate due to the subjective interpretation of the results. The application of quantitative real-time PCR (QPCR) technology can address these issues and be a more accurate platform to evaluate phage growth conditions and quantify viral titers in phage preparations. QPCR, with an improved primer set specific for l phage and coupled with fluorescent dye detection of PCR products, was used to detect and quantify phages in lysates with no prior DNA purification. Phages were detected below one plaque-forming unit, and at least 89 viral copies were detected from a purified DNA sample. When unknown concentrations of various phage preparations were assessed using QPCR, they were attained more efficiently, with greater sensitivity and precision, and the method produced more accurate quantitative data spanning a wider linear range than those obtained by the plaque assay (six logs vs. one log, respectively). Finally, QPCR for the detection of phage has multiple applications, including conventional cloning and in alternative fields of study such as environmental sciences. [TOP OF PAGE]

  735. Male-specific coliphages as an additional fecal contamination indicator for screening fresh carrots. Endley,S., Lu,L., Vega,E., Hume,M.E., Pillai,S.D. (2003). J. Food Prot. 66:88-93. The objective of this study was to evaluate the efficacy of male-specific (F+) coliphages as a fecal-contamination indicator for fresh carrots. The prevalence of specific pathogens and indicator organisms on the surface of carrots obtained from a farm, truck, and processing shed was studied. Twenty-five carrot samples collected from each of these locations were washed, and aliquots of the wash were analyzed for the presence of F+ coliphages, Escherichia coli, Salmonella, and Shigella. Additionally, the Salmonella isolates were genotyped using pulsed-field gel electrophoresis (PFGE). Our studies detected the presence of F+ coliphages, E. coli, and Salmonella on carrots. All samples, however, tested negative for Shigella. Although none of the carrot samples from the field were positive for E. coli, one sample was positive for Salmonella, and another was positive for F+ coliphages. From the truck, two carrot samples (8%) were positive for Salmonella, four (16%) were positive for F+ coliphages, and four (16%) were positive for E. coli. None of the carrot samples from the processing shed were positive for Salmonella. However, 2 carrot samples (8%) were positive for E. coli, and 14 carrot samples (56%) were positive for F+ coliphages. The PFGE results suggest that there were three distinct Salmonella genotypes among the carrot samples from the truck and that the Salmonella isolates identified on carrot samples from the field and truck locations were different. Microbiological screening of fresh produce such as carrots (which can be exposed to fecal contaminants in soils and water) should ensure the detection of both viral and bacterial contaminants. Overall, in this study, F+ coliphages were detected in 25% of the carrot samples, compared to E. coli (8%), Salmonella (4%), and Shigella (0%). The results suggest F+ coliphages can serve as a conservative indicator of fecally associated viruses on carrots. This suggests that in addition to E. coli screening, F+ coliphages should be included when produce such as carrots that are vulnerable to fecal contaminants are screened. Since the detection of specific enteric viral pathogens is expensive, screening for viral indicators of fecal contamination using F+ coliphages can be an economical approach to providing an additional level of assurance about the microbiological quality of fresh carrots. [TOP OF PAGE]

  736. Phage conversion of exfoliative toxin A in Staphylococcus aureus isolated from cows with mastitis. Endo,Y., Yamada,T., Matsunaga,K., Hayakawa,Y., Kaidoh,T., Takeuchi,S. (2003). Vet Microbiol 96:81-90. An exfoliative toxin produced by Staphylococcus aureus is the causative agent of staphylococcal scalded-skin syndrome (SSSS) in young children. Recently, we reported that only few isolates of S. aureus from bovine mastitis contained the eta gene encoding exfoliative toxin A (ETA) and produced ETA in vitro. In this study, we isolated temperate phages from two ETA-positive bovine isolates of S. aureus by treatment with mitomycin C. Polymerase chain reaction (PCR) assay of the phage genomes suggested that the temperate phages carried the structural gene for ETA. Moreover, the nucleotide sequence analysis of the PCR products revealed that the eta gene was located very close to an amidase gene on the phage genomes. The nucleotide sequence for the amidase gene of the bovine phage (bovine phi ETA) differed at nine positions from that of the amidase gene of phi ETA from a human isolate reported by Yamaguchi et al. [Mol. Microbiol. 38 (2000) 694], suggesting that eta-converting phages are heterogeneous. Bovine phi ETA had a head with a hexagonal outline and a non-contractile and flexible tail. Bovine phi ETA was able to lysogenize ETA-negative bovine isolates of S. aureus, and the lysogenized S. aureus isolates had the ability to produce ETA. These results suggest the possibility of horizontal transmission of the eta gene by temperate bacteriophages among bovine isolates of S. aureus. [TOP OF PAGE]

  737. Direct estimates of the contribution of viral lysis and microzooplankton grazing to the decline of a Micromonas spp. population. Evans,C., Archer,S.D., Jacquet,S., Wilson,W.H. (2003). Aquat. Microb. Ecol. 30:207-219. During a mesocosm study in Raunefjorden, Norway, a Micromonas spp. population, initially showing exponential net growth, dramatically declined after Day 4 of the experiment. Using a modification of the dilution approach originally developed to quantify grazing by microzooplankton on phytoplankton, it was possible to partition the mortality of Micromonas spp. between grazing and viral lysis on Days 5, 6 and 7 during the population decline. Parallel dilution experiments were carried out in which 0.2 µm- and 10 kDa-filtered water was used as the diluents. In this way, gradients of grazing pressure (0.2 µm series) and grazing pressure + viral concentration (10 kDa series) were produced. Model 1 linear regression of the fraction of whole water versus the apparent growth rate of chlorophyll a and Micromonas spp. provided an estimate of mortality in the 0.2 µm and 10 kDa dilution series. On Days 5, 6 and 7, the slopes of the linear regressions of 0.2 µm and 10 kDa dilution series were significantly different at p = 0.083, 0.001 and 0.093 respectively. From the differences in slope between the series, estimates of viral mortality amounted to a turnover rate of the Micromonas spp. standing stocks of 10, 25 and 9% d(-1). This compares to a turnover rate by the microzooplankton of 48, 26 and 23% d(-1). On all 3 d the combined viral lysis and grazing mortality exceeded estimates of the potential production of Micromonas spp., in part accounting for the population decline. This study demonstrates that the dilution approach can be adapted to directly determine virus-induced mortality rates of specific phytoplankton. However, further work is required to determine how the specificity of viral infection and variety of viral infection cycles affect the results of this modified dilution approach when applied to other phytoplankton taxa and communities. [TOP OF PAGE]

  738. Host range of chlamydiaphages fCPAR39 and Chp3. Everson,J.S., Garner,S.A., Lambden,P.R., Fane,B.A., Clarke,I.N. (2003). J. Bacteriol. 185:6490-6492. The host range of fCPAR39 is limited to four Chlamydophila species: C. abortus, C. caviae, C. pecorum, and C. pneumoniae. Chp3 (a newly discovered bacteriophage isolated from C. pecorum) shares three of these hosts (C. abortus, C. caviae, and C. pecorum) but can additionally infect Chlamydophila felis. The ability to support replication was directly correlated with the binding properties of the respective bacteriophages with their host species. Binding studies also show that fCPAR39 and Chp3 use different host receptors to infect the same host cells: cell binding is sensitive to proteinase K treatment, confirming that the chlamydiaphage receptors are proteinaceous in nature. [TOP OF PAGE]

  739. CTXphi-independent production of the RS1 satellite phage by Vibrio cholerae. Faruque,S.M., Kamruzzaman,M., Sack,D.A., Mekalanos,J.J., Nair,G.B. (2003). Proc. Natl. Acad. Sci. USA 100:1280-1285. The cholera toxin genes of Vibrio cholerae are encoded by the filamentous phage, CTXphi. Chromosomal CTXphi prophage DNA is often found flanked by copies of a related genetic element designated RS1, and RS1 DNA can be packaged into filamentous phage particles (designated RS1phi) by using the CTXphi morphogenesis genes. RS1phi is a satellite phage that further controls expression and dissemination of CTXphi. Here we describe a CTXphi-independent mechanism for production of RS1phi. A nontoxigenic environmental V. cholerae strain (55V71) was identified that supports production of RS1phi. However, newly infected CTX-negative strains did not produce RS1phi, indicating that additional 55V71 genes were involved in production of RS1phi. Analysis of nucleic acids from phage preparations of 55V71 revealed a 7.5-kb single-stranded DNA, whose corresponding replicative form was found in plasmid preparations. This DNA likely corresponds to the genome of a new filamentous phage, which we have designated KSF-1phi. The replicative form DNA of KSF-1phi was cloned into pUC18, and the resulting construct pKSF-1.1 supported the production of RS1phi particles by CTX-negative V. cholerae strains. RS1phi particles produced in this way infect recipient V. cholerae strains by a mechanism that is independent of the CTXphi receptor, the toxin-coregulated pilus. Thus, KSF-1phi is capable of facilitating the transfer of the RS1 element to strains that do not express toxin coregulated pilus. Given that RS1phi can enhance coproduction of CTXphi particles, KSF-1phi-mediated dissemination of RS1 may indirectly promote the spread of toxin genes among V. cholerae strains. This study also shows that filamentous phages can package diverse DNA elements and thus may play a role in horizontal transfer of more genes than previously appreciated. [TOP OF PAGE]

  740. Pathogenicity islands and phages in Vibrio cholerae evolution. Faruque,S.M., Mekalanos,J.J. (2003). Trends Microbiol. 11:505-510. The identification of accessory genetic elements (plasmids, phages and chromosomal 'pathogenicity islands') encoding virulence-associated genes has facilitated our efforts to understand the origination of pathogenic microorganisms. Toxigenic Vibrio cholerae, the etiologic agent of cholera, represents a paradigm for this process in that this organism evolved from environmental nonpathogenic V. cholerae by acquisition of virulence genes. The major virulence genes in V. cholerae, which are clustered in several chromosomal regions, appear to have been recently acquired from phages or through undefined horizontal gene transfer events. Evidence is accumulating that the interactions of phages with each other can also influence the emergence of pathogenic clones of V. cholerae. Therefore, to track the evolution of pathogens from their nonpathogenic progenitors, it is also crucial to identify and characterize secondary genetic elements that mediate lateral transfer of virulence genes in trans. Understanding the evolutionary events that lead to the emergence of pathogenic clones might provide new approaches to the control of cholera and other infectious diseases. [TOP OF PAGE]

  741. Application of a novel immunomagnetic separation-bacteriophage assay for the detection of Salmonella enteritidis and Escherichia coli O157:H7 in food. Favrin,S.J., Jassim,S.A., Griffiths,M.W. (2003). Int. J. Food Microbiol. 85:63-71. Salmonella infection is the second most prevalent cause of foodborne illness in most developing countries. Meat, poultry, and dairy products are frequently implicated in outbreaks. The objective of this study was to apply a novel immunomagnetic separation (IMS)-bacteriophage assay to the detection of Salmonella enteritidis in artificially inoculated skimmed milk powder, chicken rinses, and ground beef. In all food types tested, the IMS-bacteriophage assay was able to detect an average of 3 CFU of S. enteritidis in 25 g or ml of food sample. Total assay time including pre-enrichment is about 20 h. The results indicate that the IMS-bacteriophage assay is a rapid and sensitive means of detecting S. enteritidis in these foods. The assay was successfully adapted to the detection of Escherichia coli O157:H7 and was able to detect E. coli in ground beef at the lowest inoculation level tested, 2 CFU/g. The assay was also adapted to the simultaneous detection of S. enteritidis and E. coli. The results indicate that the IMS-bacteriophage assay shows promise for the simultaneous detection of these pathogens, but further development work would be necessary to improve sensitivity and produce reliable results at low inoculation levels. [TOP OF PAGE]

  742. Effects of pH and temperature on the survival of coliphages MS2 and Qb. Feng,Y.Y., Ong,S.L., Hu,J.Y., Tan,X.L., Ng,W.J. (2003). J. Indust. Microbiol. Biotechnol. 30:549-552. The RNA F-specific coliphages, MS2 and Qb, have been used as virus indicators in water and wastewater studies. It is therefore useful to have a good understanding concerning the effects of environmental factors on their survival in order to choose an appropriate candidate for assessing microbial safety in relation to water quality management. The effects of pH and temperature on the survival of these two coliphages were investigated. MS2 survived better in acidic conditions than in an alkaline environment. In contrast, Qb had a better survival rate in alkaline conditions than in an acidic environment. The inactivation rates of both coliphages were lowest within the pH range 6-8 and the temperature range 5-35°C. The inactivation rates of both coliphages increased when the pH was decreased to below 6 or increased to above 8. The inactivation rates of both coliphages increased with increasing temperature. Qb behaved peculiarly in extreme pH buffers, i.e. it was inactivated very rapidly initially when subjected to an extreme pH environment, although the inactivation rate subsequently decreased. In general, MS2 was a better indicator than Qb. However, within the pH range 6-9 and at temperatures not above 25°C, either MS2 or Qb could be used as a viral indicator. [TOP OF PAGE]

  743. The role played by viruses in the evolution of their hosts: a view based on informational protein phylogenies. Filée,J., Forterre,P., Laurent,J. (2003). Res. Microbiol. 154:237-243. Viruses are often considered as fragments of cellular RNA or DNA that escaped a long time ago from cellular chromosomes and that evolved later on by capturing additional genes from the genomes of their hosts. However, this view has now been challenged by the discovery of surprising homology between viruses with very distantly related hosts, and by phylogenetic analyses suggesting that genes might also have flown from viruses to cells. We present here phylogenetic analyses of four proteins involved in DNA replication and synthesis of DNA precursors (DNA polymerases delta, ribonucleotide reductases, thymidylate synthases and replicative helicases) and we discuss the reciprocal roles of cells and viruses during the evolutionary history of these enzymes. These analyses revealed numerous lateral gene transfer events between cells and viruses, in both directions. We suggest that lateral gene transfers from viruses to cells and nonorthologous gene replacements of cellular genes by viral ones are an important source of "genetic novelties" in the evolution of cellular lineages. Thus, viruses have definitively to be considered as major players in the evolution of cellular genomes. [TOP OF PAGE]

  744. Evaluation of potential indicators of viral contamination in shellfish and their applicability to diverse geographical areas. Formiga-Cruz,M., Allard,A.K., Conden-Hansson,A.C., Henshilwood,K., Hernroth,B.E., Jofre,J., Lees,D.N., Lucena,F., Papapetropoulou,M., Rangdale,R.E., Tsibouxi,A., Vantarakis,A., Girones,R. (2003). Appl. Environ. Microbiol. 69:1556-1563. The distribution of the concentration of potential indicators of fecal viral pollution in shellfish was analyzed under diverse conditions over 18 months in diverse geographical areas. These microorganisms have been evaluated in relation to contamination by human viral pathogens detected in parallel in the analyzed shellfish samples. Thus, significant shellfish-growing areas from diverse countries in the north and south of Europe (Greece, Spain, Sweden, and the United Kingdom) were defined and studied by analyzing different physicochemical parameters in the water and the levels of Escherichia coli, F-specific RNA bacteriophages, and phages infecting Bacteroides fragilis strain RYC2056 in the shellfish produced, before and after depuration treatments. A total of 475 shellfish samples were studied, and the results were statistically analyzed. According to statistical analysis, the presence of human viruses seems to be related to the presence of all potential indicators in the heavily contaminated areas, where E. coli would probably be suitable as a fecal indicator. The F-RNA phages, which are present in higher numbers in Northern Europe, seem to be significantly related to the presence of viral contamination in shellfish, with a very weak predictive value for hepatitis A virus, human adenovirus, and enterovirus and a stronger one for Norwalk-like virus. However, it is important to note that shellfish produced in A or clean B areas can sporadically contain human viruses even in the absence of E. coli or F-RNA phages. The data presented here will be useful in defining microbiological parameters for improving the sanitary control of shellfish consumed raw or barely cooked. [TOP OF PAGE]

  745. The great virus comeback—from an evolutionary perspective. Forterre,P. (2003). Res. Microbiol. 154:223-225. [first paragraph] Viruses have played a critical role in the development of the field we now know as molecular biology [15]. The study of bacteriophages and animal viruses became fashionable when molecular biologists realized that it was too difficult to directly attack their favorite biological problem in cellular organisms. However, the interest in viruses as model systems has declined with the ensuing years, as powerful experimental tools (often provided by viruses themselves) made it much easier to work directly on "serious" things (the cellular ones). Since we are cellular organisms, this egocentric trend is understandable. The viruses truly belong to another living world, one whose only "raison d'être" seems to be to eat us. Most research on viruses now focuses on this conflict for resources between viruses and cells. For many, the only viruses worth studying are "pathogens" that can harm or kill us, in order to understand their vicious tricks and to effectively protect ourselves from them. For a long time, only a small handful of biologists had the scientific curiosity to be interested in viruses for their own sake. This was reinforced by the prejudice, shared by many, that viruses are not really alive, since they are not autonomous. Problems such as the origin of viruses were never considered as serious topics of scientific research. The extent of viral ubiquity and diversity was largely unknown and ignored. The studies on the mechanisms of viral evolution were mainly focused on a small number of "emerging pathogens". Most evolutionists ignored viruses because of the "Woesian revolution" that resulted in the first large-scale experimental studies on the evolution of microorganisms. Indeed, since viruses contain no ribosomal RNA, they had no obvious place in the "universal" tree of life. The development of genomic techniques also had a role in casting viruses aside from evolutionary models. Although viral genomes were the first to be completely sequenced, the nearly exclusive focus of subsequent analysis on cellular genomes shifted the attention of evolutionists toward the cellular world. [TOP OF PAGE]

  746. The physical environment affects cyanophage communities in British Columbia inlets. Frederickson,C.M., Short,S.M., Suttle,C.A. (2003). Microb. Ecol. 46:348-357. Little is known about the natural distribution of viruses that infect the photosynthetically important group of marine prokaryotes, the cyanobacteria. The current investigation reveals that the structure of cyanophage communities is dependent on water column structure. PCR was used to amplify a fragment of the cyanomyovirus gene (g) 20, which codes for the portal vertex protein. Denaturing gradient gel electrophoresis (DGGE) of PCR amplified g20 gene fragments was used to examine variations in cyanophage community structure in three inlets in British Columbia, Canada. Qualitative examination of denaturing gradient gels revealed cyanophage community patterns that reflected the physical structure of the water column as indicated by temperature and salinity. Based on mobility of PCR fragments in the DGGE gels, some cyanophages appeared to be widespread, while others were observed only at specific depths. Cyanophage communities within Salmon Inlet were more related to one another than to communities from either Malaspina Inlet or Pendrell Sound. As well, surface communities in Malaspina Inlet and Pendrell Sound were different when compared to communities at depth. In the same two locations, distinct differences in community composition were observed in communities that coincided with depths of high chlorophyll fluorescence. The observed community shifts over small distances (only a few meters in depth or inlets separated by less than 100 km) support the idea that cyanophage communities separated by small spatial scales develop independently of each other as a result isolation by water column stratification or land mass separation, which may ultimately lead to changes in the distribution or composition of the host community. [TOP OF PAGE]

  747. Reduction of ß-amyloid plaques in brain of transgenic mouse model of Alzheimer's disease by EFRH-phage immunization. Frenkel,D., Dewachter,I., Van Leuven,F., Solomon,B. (2003). Vaccine 21:1060-1065. Antibodies to the epitope EFRH, representing residues 3–6 within the ß-amyloid (Aß) sequence, were previously shown to affect the solubility and disaggregation of Aß fibrils in vitro. Here, we describe a novel method of immunization, using as antigen the EFRH peptide displayed on the surface of the filamentous phage. The EFRH phage evoked effective auto-immune antibodies in amyloid precursor protein [V717I] (APP[V717I]) transgenic mice that recapitulate the amyloid plaques and vascular pathology of Alzheimer's disease (AD). The immunization provoked a considerable reduction in the number of Aß amyloid plaques in the brain of the transgenic mice and may serve as the basis for anti-Aß vaccine. [TOP OF PAGE]

  748. Viral influence on aquatic bacterial communities. Fuhrman,J.A., Schwalbach,M. (2003). Biol. Bull. 204:192-195. Bacterial viruses, or bacteriophages, have numerous roles in marine systems. Although they are now considered important agents of mortality of bacteria, a second possible role of regulating bacterial community composition is less well known. The effect on community composition derives from the presumed species-specificity and density-dependence of infection. Although models have described the "kill the winner" hypothesis of such control, there are few observational or experimental demonstrations of this effect in complex natural communities. We report here on some experiments that demonstrate that viruses can influence community composition in natural marine communities. Although the effect is subtle over the time frame suitable for field experiments (days), the cumulative effect over months or years would be substantial. Other virus roles, such as in genetic exchange or microbial evolution, have the potential to be extremely important, but we know very little about them. [TOP OF PAGE]

  749. Improvement of phage defence in Lactococcus lactis by introduction of the plasmid encoded restriction and modification system LlaAI. Gabs,S., Josephsen,J. (2003). Lett. Appl. Microbiol. 36:332-336. AIMS: To study the ability of the plasmid-encoded restriction and modification (R/M) system LlaAI to function as a bacteriophage resistance mechanism in Lactococcus lactis during milk fermentations. METHODS AND RESULTS: Plasmid pAIcat4, carrying the R/M system LlaAI and a chloramphenicol resistance cassette, was introduced into the plasmid-free strain L. lactis MG1614 and the industrial strain L. lactis 964. By measuring changes in conductivity the influence of different phage on the growth was determined. CONCLUSIONS: The plasmid-encoded R/M system LlaAI significantly improves the bacteriophage resistance of L. lactis during milk fermentations. SIGNIFICANCE AND IMPACT OF THE STUDY: It is essential to determine the potential of a phage defence mechanism in L. lactis starter culture strains during growth in milk before steps are taken to improve starter cultures. This study shows that LlaAI is useful for improvement of starter cultures. [TOP OF PAGE]

  750. Nonpathogenic Escherichia coli can contribute to the production of Shiga toxin. Gamage,S., Strasser,J.E., Chalk,C.L., Weiss,A.A. (2003). Infect. Immun. 71:3107-3115. The food-borne pathogen, Escherichia coli O157:H7, has been associated with gastrointestinal disease and the life-threatening sequela hemolytic uremic syndrome. The genes for the virulence factor, Shiga toxin 2 (Stx2), in E. coli O157:H7 are encoded on a temperate bacteriophage under the regulation of the late gene promoter. Induction of the phage lytic cycle is required for toxin synthesis and release. We investigated the hypothesis that nonpathogenic E. coli could amplify Stx2 production if infected with the toxin-encoding phage. Toxin-encoding phage were incubated with E. coli that were either susceptible or resistant to the phage. The addition of phage to phage-susceptible bacteria resulted in up to 40-fold more toxin than a pure culture of lysogens, whereas the addition of phage to phage-resistant bacteria resulted in significantly reduced levels of toxin. Intestinal E. coli isolates incubated with Shiga toxin-encoding phage produced variable amounts of toxin. Of 37 isolates, 3 produced significantly more toxin than was present in the inoculum, and 1 fecal isolate appeared to inactivate the toxin. Toxin production in the intestine was assessed in a murine model. Fecal toxin recovery was significantly reduced when phage-resistant E. coli was present. These results suggest that the susceptibility of the intestinal flora to the Shiga toxin phage could exert either a protective or an antagonistic influence on the severity of disease by pathogens with phage-encoded Shiga toxin. Toxin production by intestinal flora may represent a novel strategy of pathogenesis. [TOP OF PAGE]

  751. Bacteriophages as viral indicators for radiation processing of water: a chemical approach. Gehringer,P., Eschweiler,H., Leth,H., Pribil,W., Pfleger,S., Cabaj,A., Haider,T., Sommer,R. (2003). Applied Radiation and Isotopes 58:651-656. Inactivation of the bacteriophages PHI X 174 (somatic coliphage), MS2 (F-specific coliphage) and B40-8 (phage infecting Bacteroides fragilis) suspended in tap water was studied applying gamma and electron beam irradiation as well. PHI X 174 phage was found to be a suitable viral indicator for water disinfection by means of ionizing radiation. The nutrient broths introduced simultaneously with the bacteriophages into the water when it is spiked with the phages for the experiments did not significantly change the scavenging capacity of the water matrix. No dose rate effect was observed with MS2 and B40-8 phages but PHI X 174 phage showed a clear dose rate effect. It was found that in water MS2 phage is significantly more sensitive to ionizing radiation than Escherichia coli. [TOP OF PAGE]

  752. Removal of Encephalitozoon intestinalis, calicivirus, and coliphages by conventional drinking water treatment. Gerba,C.P., Riley,K.R., Nwachuku,N., Ryu,H., Abbaszadegan,M. (2003). J. Environ. Sci. Health Part A Tox. /Haz. Subst. Environ. Engin. 38:1259-1268. The removal of the Microsporidia, Encephalitozoon intestinalis, feline calicivirus and coliphages MS-2, PRD-1, and Fr were evaluated during conventional drinking water treatment in a pilot plant. The treatment consisted of coagulation, sedimentation, and mixed media filtration. Fr coliphage was removed the most (3.21 log), followed by feline calicivirus (3.05 log), E. coli (2.67 log), E. intestinalis (2.47 log), MS-2 (2.51 log). and PRD-1 (1.85 log). With the exception of PRD-1 the greatest removal of the viruses occurred during the flocculation step of the water treatment process. [TOP OF PAGE]

  753. Observation of virus-like particles in high temperature enrichment cultures from deep-sea hydrothermal vents. Geslin,C., Le Romancer,M., Gaillard,M., Erauso,G., Prieur,D. (2003). Res. Microbiol. 154:303-307. A systematic search was carried out on samples collected in various geographically distant hydrothermal sites located on the East Pacific Rise (EPR 9ºN and 13ºN) and Mid-Atlantic Ridge (MAR 36ºN and 37ºN) to investigate the diversity of virus-like particles (VLPs) from deep-sea vents. Eighty-nine positive enrichment cultures were obtained from one hundred and one crude samples at 85ºC. VLPs were detected by electron microscopy in fifteen different enrichments. Among the different morphotypes observed, the lemon-shaped type prevailed but rods and novel pleomorphic morphologies were also observed. Several observations strongly suggested that host strains of the novel VLPs belong to the hyperthermophilic euryarchaeal order Thermococcales. [TOP OF PAGE]

  754. Bacteriophage ecology and plants. Gill,J.J., Abedon,S.T. (2003). APSnet Feature http://www.apsnet.org/online/feature/phages/ Plant biology cannot be fully appreciated absent microbial flora, and plant-associated bacteria are incompletely understood without an awareness of phage -- the viruses of prokaryotes. Phage have been found in association with "buds, leaves, root nodules (leguminous plants), roots, rotting fruit, seeds, stems and straw; crown gall tumors… healthy or diseased alfalfa, barley, beans, broccoli, Brussels sprouts, buckwheat, clover, cotton, cucumber, lucerne, mulberry, oats peas, peach trees, radish, rutabaga, ryegrass, rye, timothy, tobacco, tomatoes, [and] wheat" (4). In this overview we consider the myriad ways that phage can impact ecologically on plant-associated bacteria. [TOP OF PAGE]

  755. Bacteriophages of Erwinia amylovora. Gill,J.J., Svircev,A.M., Smith,R., Castle,A.J. (2003). Appl. Environ. Microbiol. 69:2133-2138. Fifty bacteriophage isolates of Erwinia amylovora, the causal agent of fire blight, were collected from sites in and around the Niagara region of southern Ontario and the Royal Botanical Gardens, Hamilton, Ontario. Forty-two phages survived the isolation, purification, and storage processes. The majority of the phages in the collection were isolated from the soil surrounding trees exhibiting fire blight symptoms. Only five phages were isolated from infected aerial tissue in pear and apple orchards. To avoid any single-host selection bias, six bacterial host strains were used in the initial isolation and enrichment processes. Molecular characterization of the phages with a combination of PCR and restriction endonuclease digestions showed that six distinct phage types, described as groups 1 to 6, were recovered. Ten phage isolates were related to the previously characterized E. amylovora PEa1, with some divergence of molecular markers between phages isolated from different sites. A study of the host ranges of the phages revealed that certain types were unable to efficiently lyse some E. amylovora strains and that some isolates were able to lyse the epiphytic bacterium Pantoea agglomerans. Representatives from the six molecular groups were studied by electron microscopy to determine their morphology. The phages exhibited distinct morphologies when examined by an electron microscope. Group 1 and 2 phages were tailed and contractile, and phages belonging to groups 3 to 6 had short tails or openings with thin appendages. Based on morphotypes, the bacteriophages of E. amylovora were placed in the order Caudovirales, in the families Myoviridae and Podoviridae. [TOP OF PAGE]

  756. Reduction of experimental Salmonella and Campylobacter contamination of chicken skin by application of lytic bacteriophages. Goode,D., Allen,V.M., Barrow,P.A. (2003). Appl. Environ. Microbiol. 69:5032-5036. Lytic bacteriophages, applied to chicken skin that had been experimentally contaminated with Salmonella enterica serovar Enteritidis or Campylobacter jejuni at a multiplicity of infection (MOI) of 1, increased in titer and reduced the pathogen numbers by less than 1 log10 unit. Phages applied at a MOI of 100 to 1,000 rapidly reduced the recoverable bacterial numbers by up to 2 log10 units over 48 h. When the level of Salmonella contamination was low (< log10 2 per unit area of skin) and the MOI was 10^5, no organisms were recovered. By increasing the number of phage particles applied (i.e., MOI of 10^7), it was also possible to eliminate other Salmonella strains that showed high levels of resistance because of restriction but to which the phages were able to attach. [TOP OF PAGE]

  757. Bacteriophage biocontrol and bioprocessing: application of phage therapy to industry. Goodridge,L., Abedon,S.T. (2003). SIM News 53:254-262. Here we take a slightly different tack from the mostly clinical considerations of phage therapy, emphasizing instead the role of phages as a means of selectively reducing bacterial loads in nonclinical settings. Since the phrase phage therapy carries a connotation of medical doctors administering phages as living drugs to suffering patients, we instead employ the alliterations bacteriophage biocontrol and bacteriophage bioprocessing to describe, as we review here, the more generalized application of phages as everything from biocontrol agents on the farm to the bioprocessing of certain foods. We also provide a primer on phage-based methods of bacterial diagnosis. [TOP OF PAGE]

  758. Morphological, host range, and genetic characterization of two coliphages. Goodridge,L., Gallaccio,A., Griffiths,M.W. (2003). Appl. Environ. Microbiol. 69:5364-5371. Two coliphages, AR1 and LG1, were characterized based on their morphological, host range, and genetic properties. Transmission electron microscopy showed that both phages belonged to the Myoviridae; phage particles of LG1 were smaller than those of AR1 and had an isometric head 68 nm in diameter and a complex contractile tail 111 nm in length. Transmission electron micrographs of AR1 showed phage particles consisting of an elongated isometric head of 103 by 74 nm and a complex contractile tail 116 nm in length. Both phages were extensively tested on many strains of Escherichia coli and other enterobacteria. The results showed that both phages could infect many serotypes of E. coli. Among the enterobacteria, Proteus mirabilis, Shigella dysenteriae, and two Salmonella strains were lysed by the phages. The genetic material of AR1 and LG1 was characterized. Phage LG1 had a genome size of 49.5 kb compared to 150 kb for AR1. Restriction endonuclease analysis showed that several restriction enzymes could degrade DNA from both phages. The morphological, genome size, and restriction endonuclease similarities between AR1 and phage T4 were striking. Southern hybridizations showed that AR1 and T4 are genetically related. The wide host ranges of phages AR1 and LG1 suggest that they may be useful as biocontrol, therapeutic, or diagnostic agents to control and detect the prevalence of E. coli in animals and food. [TOP OF PAGE]

  759. New insights into the possible role of bacteriophages in host defense and disease. Gorski,A., Dabrowska,K., Switala-Jele,K., Nowaczyk,M., Weber-Dabrowska,B., Boratynski,J., Wietrzyk,J., Opolski,A. (2003). Medical Immunology 2:2 Background: While the ability of bacteriophages to kill bacteria is well known and has been used in some centers to combat antibiotics - resistant infections, our knowledge about phage interactions with mammalian cells is very limited and phages have been believed to have no intrinsic tropism for those cells. ¶ Presentation of the hypothesis: At least some phages (e.g., T4 coliphage) express Lys-Arg-Gly (KGD) sequence which binds b3 integrins (primarily aIIbb3). Therefore, phages could bind b3+ cells (platelets, monocytes, some lymphocytes and some neoplastic cells) and downregulate activities of those cells by inhibiting integrin functions. ¶ Testing the hypothesis: Binding of KGD+ phages to b3 integrin+ cells may be detected using standard techniques involving phage - mediated bacterial lysis and plaque formation. Furthermore, the binding may be visualized by electron microscopy and fluorescence using labelled phages. Binding specificity can be confirmed with the aid of specific blocking peptides and monoclonal antibodies. In vivo effects of phage - cell interactions may be assessed by examining the possible biological effects of b3 blockade (e.g., anti-metastatic activity). ¶ Implication of the hypothesis: If, indeed, phages can modify functions of b3+ cells (platelets, monocytes, lymphocytes, cancer cells) they could be important biological response modifiers regulating migration and activities of those cells. Such novel understanding of their role could open novel perspectives in their potential use in treatment of cardiovascular and autoimmune disease, graft rejection and cancer. [TOP OF PAGE]

  760. New insights into the possible role of bacteriophages in transplantation. Gorski,A., Nowaczyk,M., Weber-Dabrowska,B., Kniotek,M., Boratynski,J., Ahmed,A., Dabrowska,K., Wierzbicki,P., Switala-Jelen,K., Opolski,A. (2003). Transplant. Proc. 35:2372-2373. Due to the increasing prevalence of drug-resistant bacterial infections in the "post-antibiotic era," bacteriophages (bacterial viruses, BP) may be useful to administer to transplant recipients without exposing them to an increased risk of rejection, which occurs consequent to some viral infections. Herein we present evidence that at least some coliphages (T4) do not pose such risk. Interestingly, they may produce immunosuppressive effects extending transplant survival. Our data suggest that BP may be used in clinical transplantation to treat drug-resistant bacterial infections and perhaps as an adjunct to immunosuppressive therapy. [TOP OF PAGE]

  761. Evaluation of microbial source tracking methods using mixed fecal sources in aqueous test samples. Griffith,J.F., Weisberg,S.B., McGee,C.D. (2003). Water Hlth. 1:141-151. Microbiological source tracking (MST) methods are increasingly being used to identify fecal contamination sources in surface waters, but these methods have been subjected to limited comparative testing. In this study, 22 researchers employing 12 different methods were provided sets of identically prepared blind water samples. Each sample contained one to three of five possible fecal sources (human, dog, cattle, seagull or sewage). Researchers were also provided with portions of the fecal material used to inoculate the blind water samples for use as library material. No MST method that was tested predicted the source material in the blind samples perfectly. Host-specific PCR performed best at differentiating between human and non-human sources, but primers are not yet available for differentiating between all of the non-human sources. Virus and F+ coliphage methods reliably identified sewage, but were unable to identify fecal contamination from individual humans. Library-based isolate methods correctly identified the dominant source in most samples, but also had frequent false positives in which fecal sources not in the samples were incorrectly identified as being present. Among the library-based methods, genotypic methods generally performed better than phenotypic methods. [TOP OF PAGE]

  762. Study on interaction between T4 phage and Escherichia coli B by microcalorimetric method. Guosheng,L., Yi,L., Xiangdong,C., Peng,L., Ping,S., Songsheng,Q. (2003). J. Virol. Meth. 112:137-143. The process that T4 phages multiply in host cells of Escherichia coli B was determined using LKB-2277 Bioactivity Monitor by means of stopped-flow method, and the growth was measured turbidometrically at the same time at 37ºC. By analyzing thermo-curves, quantitative parameters could be obtained to characterize the interactions of host cells and phages. The parameters such as k(a), P(max), G etc. change regularly with the decrease of multiplicity of infection (MOI) value. Infection-lysis equations were fitted and the lytic rate constant k(L) was obtained. The results show that the metabolic activity of infected cells is more intensive than that of normal cells. The phenomenon of lysis inhibition (LIN) was first detected with the microcalorimetric method, and the mechanism is discussed. [TOP OF PAGE]

  763. Genetically modified filamentous phage as bactericidal agents: a pilot study. Hagens,S., Bläsi,U. (2003). Lett. Appl. Microbiol. 37:318-323. AIMS: To evaluate the ability of a filamentous phage encoding lethal proteins to kill bacteria without host-cell lysis. METHODS AND RESULTS: Bacterial survival was determined after infection of a growing Escherichia coli culture with phage M13 encoding either the restriction endonuclease BglII gene or modified phage lambda S holin genes. The genetically engineered phage exerted a high killing efficiency while leaving the cells structurally intact. When compared with a lytic phage, the release of endotoxin was minimized after infection with the genetically modified phages. CONCLUSIONS: Genetically engineered phage can be used for efficient killing, concomitantly minimizing endotoxin release. SIGNIFICANCE AND IMPACT OF THE STUDY: This feasibility study provides a possible strategy for the use of genetically engineered phage as bactericidal agents by optimizing the advantages and minimizing potential risks such as release of pyrogenic cell wall components. [TOP OF PAGE]

  764. The complete sequence of marine bacteriophage VpV262 infecting Vibrio parahaemolyticus indicates that an ancestral component of a T7 viral supergroup is widespread in the marine environment. Hardies,S.C., Comeau,A.M., Serwer,P., Suttle,C.A. (2003). Virology 310:359-371. The 46,012-bp sequence of the marine bacteriophage VpV262 infecting the bacterium Vibrio parahaemolyticus is reported. The VpV262 sequence reveals that it is a distant relative of marine Roseophage SIO1, and an even more distant relative of coliphage T7. VpV262 and SIO1 appear to represent a widespread marine phage group that lacks an RNA polymerase gene and is ancestral to the T7-like phages. We propose that this group together with the T7-like phages be designated as the T 7 supergroup. The ancestral head structure gene module for the T7 supergroup was reconstructed by using sensitive biased Psi-blast searches supplemented by statistical support derived from gene order. In the early and replicative segments, these phages have participated in extensive interchange with the viral gene pool. VpV262 carries a different replicative module than SIO1 and the T7-like phages. [TOP OF PAGE]

  765. Identification of an inducible bacteriophage in a virulent strain of Streptococcus suis serotype 2. Harel,J., Martinez,G., Nassar,A., Dezfulian,H., Labrie,S.J., Brousseau,R., Moineau,S., Gottschalk,M. (2003). Infect. Immun. 71:6104-6108. Streptococcus suis infection is considered to be a major problem in the swine industry worldwide. Most virulent Canadian isolates of S. suis serotype 2 do not produce the known virulence markers for this pathogen. PCR-based subtraction hybridization was adapted to isolate unique DNA sequences which were specific to virulent strains of S. suis isolated in Canada. Analysis of some subtracted DNA clones revealed significant homology with bacteriophages of gram-positive bacteria. An inducible phage (named Ss1) was observed in S. suis following the incubation of the virulent strain 89-999 with mitomycin C. Phage Ss1 has a long noncontractile tail and a small isometric nucleocapsid and is a member of the Siphoviridae family. Ss1 phage DNA appears to be present in most Canadian S. suis strains tested in this study, which were isolated from diseased pigs or had proven virulence in mouse or pig models. To our knowledge, this is the first report of the isolation of a phage in S. suis. [TOP OF PAGE]

  766. Can an arbitrary sequence evolve towards acquiring a biological function? Hayashi,Y., Sakata,H., Makino,Y., Urabe,I., Yomo,T. (2003). J. Mol. Evol. 56:162-168. To explore the possibility that an arbitrary sequence can evolve towards acquiring functional role when fused with other pre-existing protein modules, we replaced the D2 domain of the fd-tet phage genome with the soluble random polypeptide RP3-42. The replacement yielded an fd-RP defective phage that is six-order magnitude lower infectivity than the wild-type fd-tet phage. The evolvability of RP3-42 was investigated through iterative mutation and selection. Each generation consists of a maximum of ten arbitrarily chosen clones, whereby the clone with highest infectivity was selected to be the parent clone of the generation that followed. The experimental evolution attested that, from an initial single random sequence, there will be selectable variation in a property of interest and that the property in question was able to improve over several generations. fd-7, the clone with highest infectivity at the end of the experimental evolution, showed a 240-fold increase in infectivity as compared to its origin, fd-RP. Analysis by phage ELISA using anti-M13 antibody and anti-T7 antibody revealed that about 37-fold increase in the infectivity of fd-7 was attributed to the changes in the molecular property of the single polypeptide that replaced the D2 domain of the g3p protein. This study therefore exemplifies the process of a random polypeptide generating a functional role in rejuvenating the infectivity of a defective bacteriophage when fused to some preexisting protein modules, indicating that an arbitrary sequence can evolve toward acquiring a functional role. Overall, this study could herald the conception of new perspective regarding primordial polypeptides in the field of molecular evolution. [TOP OF PAGE]

  767. Validation of phage T7 biological dosimeter by quantitative polymerase chain reaction using short and long segments of phage T7 DNA. Hegedus,M., Modos,K., Ronto,G., Fekete,A. (2003). Photochem Photobiol 78:213-219. Phage T7 can be used as a biological dosimeter; its reading, the biologically effective dose (BED), is proportional to the inactivation rate |ln (n/n0)|. For the measurement of DNA damage in phage T7 dosimeter, a quantitative polymerase chain reaction (QPCR) methodology has been developed using 555 and 3826 bp fragments of phage T7 DNA. Both optimized reactions are so robust that an equally good amplification was obtained when intact phage T7 was used in the reaction mixture. In the biologically relevant dose range a good correlation was obtained between the BED of the phage T7 dosimeter and the amount of ultraviolet (UV) photoproducts determined by QPCR with both fragments under the effect of five various UV sources. A significant decrease in the yield of photoproducts was detected by QPCR in isolated T7 DNA and in heated phage compared with intraphage DNA with all irradiation sources. Because the yield of photoproducts was the same in B, C and A conformational states of T7 DNA, a possible explanation for modulation of photoproduct frequency in intraphage T7 DNA is that the presence of bound phage proteins induces an alteration in DNA structure that can result in increased induction of photoproducts. [TOP OF PAGE]

  768. Fate of physical, chemical, and microbial contaminants in domestic wastewater following treatment by small constructed wetlands. Hench,K.R., Bissonnette,G.K., Sexstone,A.J., Coleman,J.G., Garbutt,K., Skousen,J.G. (2003). Water Res. 37:921-927. In order to evaluate the efficacy of constructed wetlands for treatment of domestic wastewater for small communities located in rural areas, small-scale wetland mesocosms (400 L each) containing two treatment designs (a mixture of Typha, Scirpus, and Juncus species; control without vegetation) were planted into two depths (45 or 60 cm) with pea gravel. Each mesocosm received 19 L/day of primary-treated domestic sewage. Mesocosms were monitored (inflow and outflow samples) on a monthly basis over a 2-year period for pH, total suspended solids (TSS), 5-day biochemical oxygen demand (BOD(5)), total Kjeldahl nitrogen (TKN), dissolved oxygen (DO), and conductivity. Microbiological analyses included enumeration of fecal coliforms, enterococci, Salmonella, Shigella, Yersinia, and coliphage. Significant differences between influent and effluent water quality for the vegetated wetlands (p<0.05) were observed in TSS, BOD(5), and TKN. Increased DO and reduction in fecal coliform, enterococcus, Salmonella, Shigella, Yersinia, and coliphage populations also were observed in vegetated wetlands. Greatest microbial reductions were observed in the planted mesocosms compared to those lacking vegetation. Despite marked reduction of several contaminants, wetland-treated effluents did not consistently meet final discharge limits for receiving bodies of water. Removal efficiencies for bacteria and several chemical parameters were more apparent during the initial year compared to the second year of operation, suggesting concern for long-term efficiency and stability of such wetlands. [TOP OF PAGE]

  769. Bacteriophages with tails: chasing their origins and evolution. Hendrix,R.W., Hatfull,G.F., Smith,M.C.M. (2003). Res. Microbiol. 154:253-257. Comparative genomic analysis of the tailed bacteriophages shows that they are genetically mosaic with respect to each other, implying that horizontal exchange of sequences is an important component of their evolution. Horizontal exchange occurs intensively among closely related phages but also at reduced frequency across the entire population of tailed phages. It results in exchange of homologous functions, exchange of analogous but non-homologous functions as with the prophage integrases, and introduction of novel functions into the genome as with the morons. Extrapolation of these processes back in evolutionary time leads to a speculative model for the origins and early evolution of phages. [TOP OF PAGE]

  770. Bacteriophage genomics. Hendrix,R.W. (2003). Curr. Opin. Microbiol. 6:506-511. Comparative genomic studies of bacteriophages, especially the tailed phages, together with environmental studies, give a dramatic new picture of the size, genetic structure and dynamics of this population. Sequence comparisons reveal some of the detailed mechanisms by which these viruses evolve and influence the evolution of their bacterial and archaeal hosts. We see rampant horizontal exchange of sequences among genomes, mediated by both homologous and nonhomologous recombination. High frequency exchange among phages occupying similar ecological niches leads to a high degree of mosaic diversity in local populations. Horizontal exchange also takes place at lower frequency across the entire span of phage sequence space. [TOP OF PAGE]

  771. Bacterial diversity in shallow oligotrophic marine benthos and overlying waters: Effects of virus infection, containment, and nutrient enrichment. Hewson,I., Vargo,G.A., Fuhrman,J.A. (2003). Microb. Ecol. 46:322-336. Little is known of the factors shaping sediment bacterial communities, despite their high abundance and reports of high diversity. Two factors hypothesized to shape bacterial communities in the water column are nutrient (resource) availability and virus infection. The role these factors play in benthic bacterial diversity was assessed in oligotrophic carbonate-based sediments of Florida Bay (USA). Sediment-water mesocosm enclosures were made from 1-m diameter clear polycarbonate cylinders which were pushed into sediments to 201 cm sediment depth enclosing similar to80 L of water. Mesocosms were amended each day for 14 d with 10 muM NH4+ and 1 muM PO43-. In a second experiment, viruses from a benthic flocculent layer were concentrated and added back to flocculent layer samples which were collected near the mesocosm enclosures. Photosynthesis by microalgae in virus-amended incubations was monitored by pulse-amplitude modulated (PAM) fluorescence. In both experiments, bacterial diversity was estimated using automated rRNA intergenic spacer analysis (ARISA), a high-resolution fingerprinting approach. Initial sediment bacterial operational taxonomic unit (OTU) richness (236 +/- 3) was higher than in the water column (148 +/- 9), where an OTU was detectable when its amplified DNA represented >0.09% of the total amplified DNA. Effects on bacterial diversity and operational taxonomic unit (OTU) richness in nutrient-amended mesocosms may have been masked by the effects of containment, which stimulated OTU richness in the water column, but depressed OTU richness and diversity in sediments. Nutrient addition significantly elevated virus abundance and the ratio of viruses to bacteria (p < 0.05 for both) in the sediments, concomitant with elevated bacterial diversity. However, water column bacterial diversity (in unamended controls) was not affected by nutrient amendments, which may be due to rapid nutrient uptake by sediment organisms or adsorption of P to carbonate sediments. Addition of live viruses to benthic flocculent layer samples increased bacterial OTU diversity and richness compared with heat-killed controls; however, cluster analyses showed that the community structure in the virus-amended mesocosms varied greatly between replicates. Despite the strong effects upon eubacterial communities, photosynthesis of co-occurring protists and cyanobacteria was not significantly altered by the presence of virus concentrates. This study supports the hypothesis that nutrient availability plays a key role in shaping sediment bacterial communities, and also that viruses may regulate the abundance of the dominant competitors and allow less dominant organisms to maintain or increase their abundance in a community due to decreased competition for resources. [TOP OF PAGE]

  772. Viriobenthos production and virioplankton sorptive scavenging. Hewson,I., Fuhrman,J.A. (2003). Microb. Ecol. 46:337-347. Virus production in oxic surface sediments and virioplankton sorption to suspended particles was estimated across three stations in the Southern California region (33.4°N, 118.6°W). Viriobenthos production was estimated using a sterile sediment and filtered porewater dilution technique that targeted production from both attached bacteria and bacteria living free in the porewater, and attached bacteria alone. Potential virus production rates by bacteria free in the porewater ranged from 1.7 to 4.6 x 10(8) VLP cm(-3) h(-1), while attached bacteria had slower potential production rates of between 0.4 and 1.1 x 10(8) VLP cm(-3) h(-1), suggesting turnover rates of viruses in sediments (1-5 h) which are significantly higher than those of virioplankton (similar to24-48 h). Virioplankton adsorbed to small (<150 mum) suspended sediments at stations with high ambient suspended solid concentrations. Virioplankton scavenging rates combined with published sedimentation rates demonstrate that this mechanism of virus arrival could only account for 0.01% of daily benthic virus production. Calculated mortality rates of benthic bacteria (4-14% h(-1)) suggest viruses may play an important role in sediment carbon cycling. [TOP OF PAGE]

  773. Evaluation of biotracers to monitor effluent retention time in constructed wetlands. Hodgson,C.J., Perkins,J., Labadz,J.C. (2003). Lett. Appl. Microbiol. 36:362-371. AIMS: With concern surrounding the environmental impact of chemical tracers on the aquatic environment, this paper presents the initial evaluation of biotracers used to determine the effluent retention time, an important performance indicator, in a Free Water Surface Constructed Wetland. METHODS AND RESULTS: Production of the biotracers, coliphage MS2, and the bacteriophage of Enterobacter cloacae and antibiotic resistant endospores of Bacillus globigii is described in detail. Their subsequent use in three separate tracer experiments - January, March and June (2000) - revealed the variability of retention time with respect to effluent flow. The biotracer MS2 showed the constructed wetland had a retention time of 8-9 h at a mean discharge of 0.9 l s-1, increasing to 10-12 h at a mean discharge 0.3 l s-1. A similar retention of 9-10 h at a mean discharge of 0.3 l s-1 was calculated for the Ent. cloacae phage. In contrast, use of endospores revealed considerably longer retention times at these mean discharge rates; 12-24 h and 36-48 h, respectively. CONCLUSION: Biotracers could provide a useful and environmentally friendly technique to monitor effluent retention in constructed wetlands. At this stage the phage tracers appear particularly promising due to ease of isolation and recovery. SIGNIFICANCE AND IMPACT OF THE STUDY: Initial results are encouraging and have highlighted the potential of biotracers as alternatives to chemical tracers, even in microbially-rich waters. [TOP OF PAGE]

  774. Evaluation of Bacillus subtilis and coliphage MS2 as indicators of advanced water treatment efficiency. Huertas,A., Barbeau,B., Desjardins,C., Galarza,A., Figueroa,M.A., Toranzos,G.A. (2003). Water Sci. Technol. 47:255-259. The assessment of water treatment facilities for their efficiency using alternate indicators is of paramount importance. Current methods for assessing efficiency are limited by the specific characteristics of the microorganisms, such as their different sensitivities to disinfectants. A pilot study was carried out to compare different treatment scenarios for the future upgrade of the Sergio Cuevas Water Treatment plant (the largest in the Caribbean) in San Juan, Puerto Rico. The treatment units under investigation included a coagulation-flocculation-sedimentation unit, dual-media filters, micro-filtration units, intermediate ozone injection and contact columns as well as a biological filtration unit. The plant was challenged at different stages of treatment with Bacillus subtilis spores and MS2 coliphages in an attempt to test them as possible alternate indicators of treatment plant performance. These organisms were chosen because of their resistance to disinfection and desiccation, their low analysis costs and ease of detection. The removal of spores and coliphages by each treatment unit tested was calculated by seeding a known concentration (5-7 log10) of spores and coliphages and following the removal or disinfection rates. The seeded indicators were detected using traditional culture techniques. Ballasted clarification was shown to be highly efficient at removing 99.1% (approximately 3 log10) of the spores and 85.1% (approximately 0.86 log10) of MS2. Ozone treatment inactivated 80.37% (approximately 1.4 log10) spores and 99.95% (approximately 3.07 log10) coliphages. The coliphage inactivation rate obtained confirmed data obtained by previous studies indicating that MS2 was less resistant to ozonation than B subtilis spores. The membrane technology had the best efficiency in terms of physical removal of spores achieving over 99.9% (> 3 log10) removal. Coliphage removal mechanisms remain to be determined and will be a future focus of the study. Preliminary results indicate that aerobic spores and coliphages may be useful as indicators to determine the efficiency of different drinking water treatment technologies. [TOP OF PAGE]

  775. Bacteriophage treatment of a severe Escherichia coli respiratory infection in broiler chickens. Huff,W.E., Huff,G.R., Rath,N.C., Balog,J.M., Donoghue,A.M. (2003). Avian Diseases 47:1399-1405. A bacteriophage to a serotype 02, nonmotile Escherichia coli was isolated from municipal waste treatment facilities and poultry processing plants. A study was conducted to determine the efficacy of multiple vs. single intramuscular (i.m.) injections of bacteriophage to treat a severe E. coli respiratory infection. The birds were challenged at 7 days of age by injection of 6 x 10(4) colony-forming units (cfu) of E. coli into the thoracic air sac followed by an i.m. injection into the thigh with either heat-killed or active bacteriophage. There were 16 treatments with three replicate pens of 10 birds. There were four control treatments, which included untreated birds, birds injected with either heat-killed or active bacteriophage, and birds challenged only with E. coli. In the remaining treatments, birds were injected with heat-killed or active bacteriophage either once immediately after E. coli challenge or immediately after challenge and at 8 and 9 days of age, once at 8 days of age or at 8, 9, and 10 days of age, and once at 9 days of age or at 9, 10, and 11 days of age. Mortality was significantly decreased from 57% to 13% in the birds given a single i.m. injection of bacteriophage immediately after E. coli challenge, and there was complete recovery in birds treated immediately after challenge and at 8 and 9 days of age, which was a significant improvement from the single injection treatment. There was a significant reduction in mortality from 57% to 10% in the birds treated with bacteriophage once at 8 days of age and those birds treated at 8, 9, and 10 days of age, with no difference between single or multiple treatments. The mortality in the single or multiple phage treated birds that started at 9 days of age was reduced from 57% to 28% and 27%, respectively, but was not statistically different from the control. These data suggest that bacteriophage can be an effective treatment when administered early in this experimental E. coli respiratory disease and that early multiple treatments are better than a single treatment. The efficacy of bacteriophage treatment diminishes as it is delayed, with no difference between single or multiple treatments. Bacteriophage may provide an effective alternative to antibiotics, but like antibiotic therapy, the effectiveness of phage to rescue animals decreases the longer treatment is delayed in the disease process. [TOP OF PAGE]

  776. Evaluation of aerosol spray and intramuscular injection of bacteriophage to treat an Escherichia coli respiratory infection. Huff,W.E., Huff,G.R., Rath,N.C., Balog,J.M., Donoghue,A.M. (2003). Poult. Sci. 82:1108-1112. Two studies were conducted to determine the efficacy of either aerosol or i.m. injection of bacteriophage to treat an Escherichia coli respiratory infection in broiler chickens. An additional two studies were conducted to enumerate the bacteriophage in the blood of birds at 1, 2, 3, 4, 5, 6, 24, and 48 h after being sprayed or injected i.m. with bacteriophage. Five birds were bled at each period. In study 1, there were 10 treatments with three replicate pens of 10 birds. The treatments consisted of an untreated control, heat-killed bacteriophage spray, active bacteriophage spray, E. coli challenge at 7 d of age, and E. coli challenge followed by spraying the birds with heat-killed bacteriophage or active bacteriophage at 2, 24, or 48 h after challenge. In study 2 there were 11 treatments with three replicate pens of 10 birds per pen. The treatments were untreated controls, birds injected i.m. in the thigh with heat-killed or active bacteriophage, E. coli challenge at 7 d of age, PBS challenge, E. coli challenge followed by injection of heat-killed or active bacteriophage immediately after challenge or at 24 or 48 h after challenge. In both studies the E. coli challenge consisted of injecting 10(4) cfu into the thoracic air sac. Treatment of this severe E. coli infection with the bacteriophage aerosol spray significantly reduced mortality from 50 to 20% when given immediately after the challenge but had little treatment efficacy when administered 24 or 48 h after challenge. The i.m. injection of bacteriophage significantly reduced mortality from 53 to 17%, 46 to 10%, and 44 to 20% when given immediately, 24, or 48 h after challenge, respectively. Only a few birds sprayed with bacteriophage had detectable bacteriophage in their blood with an average of 96 pfu/mL 1 h after bacteriophage administration, and no bacteriophage was detected 24 and 48 h after bacteriophage administration. All birds injected i.m. with bacteriophage had detectable levels of bacteriophage in their blood at levels of 10(4) pfu/mL of blood up to 6 h after bacteriophage administration, and four of the five birds had detectable bacteriophage in their blood at an average level of 70 pfu/mL of blood 24 h after bacteriophage administration. The relative inefficiency of the spray treatment to the i.m. injection treatment may be due to the inability to get bacteriophage into the blood at high concentrations when the birds are sprayed versus the consistent high titers achieved with the i.m. injection of bacteriophage. These data provide support to the concept that bacteriophage may be an effective alternative to antibiotics in animal production when they are administered in a way that delivers high titers of the bacteriophage to the critical site of the bacterial infection. [TOP OF PAGE]

  777. [Bacteriophage therapy]. Huovinen,P. (2003). Duodecim; laaketieteellinen aikakauskirja 119:581-583. [TOP OF PAGE]

  778. Bacteriophage assay. Hyman,L.J., Toth,I.K. (2003). 674537(6,555,331). There is provided an assay suitable for the typing of bacterial strains. In the assay a predetermined amount of phage is combined with a bacterial isolate of unknown strain, the mixture being located in a suitable container. The mixture of phage and bacteria is conveniently held in a liquid or semi-liquid medium facilitating interaction of the two species. The extent of bacterial growth in the presence of the phage is measured by conventional means, preferably by means of an OD reading. Desirably the phage is retained in the selected container, which is conveniently a micro-titer plate, through use of a fixant such as 5% gelatin. [TOP OF PAGE]

  779. Effects of lysogeny of Shiga toxin 2-encoding bacteriophages on pulsed-field gel electrophoresis fragment pattern of Escherichia coli K-12. Iguchi,A., Osawa,R., KAWANO,J., Shimizu,A., Terajima,J., Watanabe,H. (2003). Curr. Microbiol. 46:224-227. Escherichia coli K-12 lysogens of three different Shiga toxin 2 (Stx2)-encoding bacteriophages were examined for variability in their pulsed-field gel electrophoresis (PFGE) fragment patterns. The PFGE fragment patterns could be classified into three types (i.e., PFGE types B, C, and D). For the PFGE type D, a 255-kbp fragment present in the original K-12 strain was apparently shifted by the size of Stx 2-encoding phage genomic DNA (ca. 65 kbp) to the position at 320 kbp. In contrast, the types B and C showed the above fragment shift plus further 6- and 10-fragment differences, respectively, from the original K-12 strain. The evidence suggests that even a single genetic event like lysogeny can cause marked genotypic modification of the host strain. [TOP OF PAGE]

  780. Efficient release of overproduced gene products from Escherichia coli BL21(DE3) by lytic infection with newly isolated bacteriophages. Iida,Y., Matsudo,Y., Saito,R., Nakasato,M., Nonomura,T., Kakutani,K., Tosa,Y., Mayama,S., Toyoda,H. (2003). Biosci. Biotech. Biochem. 67:198-202. Overproduced proteins from Escherichia coli BL21(DE3) were efficiently released with virulent bacteriophages. Leviviridae-like bacteriophages were isolated from soil and used to lyse BL21(DE3) cells transformed with beta-glucosidase, chitinase, or chitosanase genes. This method caused lysis of bacterial cells similar to that by conventional sonication and enabled us to effectively recover and purify the enzymes. [TOP OF PAGE]

  781. Phage therapy: a reappraisal of bacteriophages as antibiotics. Inal,J.M. (2003). Arch. Immunol. Ther. Exp. 51:237-244. The concept of phage therapy to treat bacterial infections was born with the discovery of the bacteriophage almost a century ago. After a chequered history, its current renaissance is fueled by the dangerous appearance of antibiotic-resistant bacteria on a global scale. As a mark of this renewed interest, the unanswered problems of phage therapy are now being addressed, especially for human use. Phage therapy in the agricultural, food-processing and fishery industries is already being successfully applied, and this review, whilst being aware of the potential drawbacks, emphasizes the need for further carefully controlled empirical data on its efficacy and safety in treating human and animal disease, especially in view of its numerous advantages over antibiotics. Finally the potential of phage therapy against bioterrorism and the emergence of second generation phage antibacterials based on phage-derived single-protein lysis systems are addressed. [TOP OF PAGE]

  782. A fluoroquinolone induces a novel mitogen-encoding bacteriophage in Streptococcus canis. Ingrey,K.T., Ren,J., Prescott,J.F. (2003). Infect. Immun. 71:3028-3033. This study investigated whether the recently recognized emergence of canine streptococcal toxic shock syndrome (STSS) and necrotizing fasciitis (NF) might be partly attributed to the use of fluoroquinolones to treat Streptococcus canis infections in dogs. Both mitomycin and the fluoroquinolone enrofloxacin caused bacteriophage-induced lysis of S. canis strain 34, an isolate from a case of canine STSS and NF. Fluoroquinolone-evoked, bacteriophage-induced lysis occurred over a range of concentrations similar to those that would occur after treatment of dogs with these agents. To search for a possible bacteriophage-encoded streptococcal superantigen gene(s), a library of the 36.5 (±1.1)-kb bacteriophage, designated phisc1, was made by ligating 3- to 7-kb Tsp5091-digested phisc1 fragments into an EcoRI-digested lambdaZapII vector. Recombinants were screened for mitogenic activity by using canine peripheral blood lymphocytes. Of 800 recombinants screened, 11 recombinants with mitogenic effects were identified, and their inserts were sequenced. The highest homology of 11.6 kb of sequenced phisc1 DNA was to the completely sequenced Streptococcus pneumoniae bacteriophage MM1. Seven of the 11 phisc1 sequenced inserts contained a 552-bp open reading frame, scm, with 27% amino acid similarity to pokeweed (Phytolacca americana) mitogen. PCR showed this gene to be present in 22 of 23 S. canis isolates tested. Quantitative reverse transcription-PCR showed that bacteriophage induction was associated with a 58-fold enhancement of expression of this gene relative to that in a noninduced culture of a similar age. The presence of this gene on a fluoroquinolone-induced bacteriophage may explain the association observed between fluoroquinolone use in dogs and the development of canine STTS and NF. [TOP OF PAGE]

  783. Maintenance of species identity and controlling speciation of bacteria: a new function for restriction/modification systems? Jeltsch,A. (2003). Gene 317:13-16. Bacteria frequently exchange DNA among each other by horizontal gene transfer. However, maintenance of species identity and in particular speciation requires a certain barrier against an unregulated uptake of foreign DNA. Here it is suggested that formation of such a barrier is one important biological function of restriction/modification systems, in addition to the classical function of protection of bacteria against bacteriophage infection. This model explains the extreme variability and wide distribution of restriction/modification systems among prokaryotes, the prevalence of RM-systems in pathogenic bacteria and the existence of several RM-systems in single bacterial strains. [TOP OF PAGE]

  784. The vertical distribution and diversity of marine bacteriophage at a station off Southern California. Jiang,S., Fu,W., Chu,W., Fuhrman,J.A. (2003). Microb. Ecol. 45:399-410. Sixty-two bacteriophages were isolated on eight indigenous bacteria from a Pacific Ocean station spanning 887-m vertical depth, on two occasions between 1999 and 2000. On the basis of 16S rRNA sequences, six hosts were tentatively identified to be in the genus Vibrio and the other two were closely related to Altermonas macleodii (W9a) and Pseudoalteromonas spp. (W13a). Restriction fragment length polymorphism (RFLP) analysis of phage genomes using AccI and HapI showed that 16 phages infecting host C4a (Vibrio) displayed 14 unique RFLP patterns. However, identical phages infecting host C4b, C6a, and C6b (all Vibrio) were obtained from both the surface layer and the hypoxic zone at 850 m. Most phage isolates from the second year had a different RFLP pattern but shared genetic similarity to the phages infecting the same host from the previous year based on a hybridization study using phage genome probes. Cluster analysis of RFLP patterns and hybridization results also indicated that phages infecting the same or genetically related hosts, in general, shared higher degrees of homology in spite of the diverse RFLP patterns. Pulsed field gel electrophoresis (PFGE) analysis of native viral genomes indicated a range in genome size from less than 40 to 200 kb, and the dominant band shifted up by about 5-10 kb in the deep samples compared to the shallow ones. Hybridization of phage genome probes with total viral community DNA from various depths suggests these isolates, or at least some of their genes, represent a detectable portion of the natural viral community and were distributed throughout the water column. Thus, the results of this study demonstrated that the genetic diversity of bacteriophage in the ocean is far greater than that of their bacterial hosts. However, host range may have contributed to the evolution of the diverse phage population in the marine environment. [TOP OF PAGE]

  785. Alternatives to antibiotics: bacteriocins, antimicrobial peptides and bacteriophages. Joerger,R.D. (2003). Poult. Sci. 82:647640 Bacteriocins, antimicrobial peptides, and bacteriophage have attracted attention as potential substitutes for, or as additions to, currently used antimicrobial compounds. This publication will review research on the potential application of these alternative antimicrobial agents to poultry production and processing. Bacteriocins are proteinaceous compounds of bacterial origin that are lethal to bacteria other than the producing strain. It is assumed that some of the bacteria in the intestinal tract produce bacteriocins as a means to achieve a competitive advantage, and bacteriocin-producing bacteria might be a desirable part of competitive exclusion preparations. Purified or partially purified bacteriocins could be used as preservatives or for the reduction or elimination of certain pathogens. Currently only nisin, produced by certain strains of Lactococcus lactis subsp. lactis, has regulatory approval for use in certain foods, and its use for poultry products has been studied extensively. Exploration of the application of antimicrobial peptides from sources other than bacteria to poultry has not yet commenced to a significant extent. Evidence for the ability of chickens to produce such antimicrobial peptides has been provided, and it is likely that these peptides play an important role in the defense against various pathogens. Bacteriophages have received renewed attention as possible agents against infecting bacteria. Evidence from several trials indicates that phage therapy can be effective under certain circumstances. Numerous obstacles for the use of phage as antimicrobials for poultry or poultry products remain. Chiefly among them are the narrow host range of many phages, the issue of phage resistance, and the possibility of phage-mediated transfer of genetic material to bacterial hosts. Regulatory issues and the high cost of producing such alternative antimicrobial agents are also factors that might prevent application of these agents in the near future. [TOP OF PAGE]

  786. Microbiological indicators of water quality in the Xochimilco canals, Mexico City. Juarez-Figueroa,L.A., Silva-Sanchez,J., Uribe-Salas,F.J., Cifuentes-Garcia,E. (2003). Salud publica de Mexico 45:389-395. OBJECTIVE: To quantify microbiology indicators of fecal contamination in the effluents of two waste water treatment plants and in samples collected in several canals in Xochimilco. MATERIAL AND METHODS: A cross sectional study was performed. Ten sites, 5 from plant effluents and 5 from canals, were selected for sampling during November and December 2001. Fecal coliforms and enterococci were quantified by membrane filtration, male specific (F+) and somatic coliphages by double agar layer technique, and Cryptosporidium oocysts and Giardia cysts by concentration with Envirocheck filter followed by immunofluorescence microscopy quantification. The average of organisms counts from effluents and canal water were compared with t Student test. RESULTS: Treated water discharge in canals showed a low count of Fecal Coliforms (average 40.4/100 ml), enterococci (average 58.8/100 ml) and Cryptosporidium oocysts (average 13.2/100 l), while coliphages and Giardia cyst rendered higher counts (average 1467.5/100 ml and 1199.8/100 l, respectively) suggesting the water treatment methods could fail to remove these agents. A significant lower count of Giardia cysts (average 45/100 l) and no Cryptosporidium oocysts were found in irrigation canals, which suggests a natural clearance of these pathogens. Strains of Escherichia coli isolated in one of the canals contaminated with sewage had antimicrobial multi-resistance that was transferred by conjugation suggesting that resistance is encoded in a plasmid potentially transferable to other pathogenic bacteria. CONCLUSIONS: Cost effective and culturally acceptable waste treatment methods will require careful planning and consultation if they are to be adopted and mantained by local populations. [TOP OF PAGE]

  787. ??? Kamiunten,H., Wakimoto,S. (2003). Ann. Phytopath. Soc. Japan 46:315-321. [TOP OF PAGE]

  788. [Bacteriophage therapy: Stalin's forgotten medicine]. Kaulen,H. (2003). Deutsche medizinische Wochenschrift 128:307 [TOP OF PAGE]

  789. Lack of correlation between O-serotype, bacteriophage susceptibility and genomovar status in the Burkholderia cepacia complex. Kenna,D.T., Barcus,V.A., Langley,R.J., Vandamme,P., Govan,J.R.W. (2003). FEMS Immunol. Med. Microbiol. 35:87-92. The Burkholderia cepacia complex comprises at least nine phylogenetically related genomic species (genomovars) which cause lifethreatening infection in immunocompromised humans, particularly individuals with cystic fibrosis or chronic granulomatous disease. Prior to recognition that 'B. cepacia' comprise multiple species, in vitro studies revealed that the lipopolysaccharide (LPS) of these Gramnegative bacteria is strongly endotoxic. In this study, we used 117 B. cepacia complex isolates to determine if there is a correlation between O-antigen serotype and genomovar status. Isolates were also tested for their ability to act as bacterial hosts for the LPS-binding bacteriophages NS1 and NS2. The absence of genomovar II (Burkholderia multivorans) in 'historical B. cepacia' isolates was notable. Neither O-serotype nor phage susceptibility correlated with genomovar status. We conclude that variability in LPS may contribute to the success of these highly adaptable bacteria as human pathogens. [TOP OF PAGE]

  790. Evidence for horizontal transfer of the EcoT38I restriction-modification gene to chromosomal DNA by the P2 phage and diversity of defective P2 prophages in Escherichia coli TH38 strains. Kita,K., Kawakami,H., Tanaka,H. (2003). J. Bacteriol. 185:2296-2305. A DNA fragment carrying the genes coding for a novel EcoT38I restriction endonuclease (R.EcoT38I) and EcoT38I methyltransferase (M.EcoT38I), which recognize G(A/G)GC(C/T)C, was cloned from the chromosomal DNA of Escherichia coli TH38. The endonuclease and methyltransferase genes were in a head-to-head orientation and were separated by a 330-nucleotide intergenic region. A third gene, the C.EcoT38I gene, was found in the intergenic region, partially overlapping the R.EcoT38I gene. The gene product, C.EcoT38I, acted as both a positive regulator of R.EcoT38I gene expression and a negative regulator of M.EcoT38I gene expression. M.EcoT38I purified from recombinant E. coli cells was shown to be a monomeric protein and to methylate the inner cytosines in the recognition sequence. R.EcoT38I was purified from E. coli HB101 expressing M.EcoT38I and formed a homodimer. The EcoT38I restriction (R)-modification (M) system (R-M system) was found to be inserted between the A and Q genes of defective bacteriophage P2, which was lysogenized in the chromosome at locI, one of the P2 phage attachment sites observed in both E. coli K-12 MG1655 and TH38 chromosomal DNAs. Ten strains of E. coli TH38 were examined for the presence of the EcoT38I R-M gene on the P2 prophage. Conventional PCR analysis and assaying of R activity demonstrated that all strains carried a single copy of the EcoT38I R-M gene and expressed R activity but that diversity of excision in the ogr, D, H, I, and J genes in the defective P2 prophage had arisen. [TOP OF PAGE]

  791. The view from Les Treilles on the origins, evolution and diversity of viruses. Krisch,H.M. (2003). Res. Microbiol. 154:227-229. [first two paragraphs] The traces of the Les Treilles meeting on Origins, Diversity and Evolution of Viruses go back to early 1998. At that time I raised the idea of a meeting on this topic with two of my phage genomics colleagues, Roger Hendrix of Pittsburgh and Harald Brüssow of Lausanne. Although there was enthusiasm for such an endeavour, our efforts in France, Switzerland and the United States to raise the necessary funds were not successful. The idea languished until 1999 when Patrick Forterre of Paris came to Toulouse to give a seminar. During dinner together that evening we decided, based on Patrick's recent successful experience with them, to apply for funding from the Fondation des Treilles in southern France. Less than a year later the meeting became a reality and it was a wonderful experience for all concerned. ¶ The chapters that follow capture the flavour of the results reported during the formal sessions. But for me, and many other participants, the most important aspect of the Les Treilles experience was the ad-libbed discussions among the participants on themes that were too "difficult" to be the subject of formal talks. At one point during the meeting Michael Balter (from the journal Science) accused me of being among the rare participants at Les Treilles not liking to speculate. Not true, but unlike some others, I am somewhat shy about doing it in public. Regardless of personal timidity, the ambiance at the Les Treilles meeting made us all think about hard questions that are too easy to dismiss by simply saying: "We will never know". My mentality rapidly became: "Who cares if we never really know, can we at least come up with a plausible scenario?" I now think we can, and that is a significant step forward. What follows is my scenario of the origins, evolution and diversity of viruses. It is my view from Les Treilles: a synthesis of what I heard, said, thought and maybe even dreamed during those too few days and nights near Tourtour. Needless to say it took some additional time and reflection to come up with even this only semi-complete story line. [TOP OF PAGE]

  792. Myoviridae bacteriophages of Pseudomonas aeruginosa: a long and complex evolutionary pathway. Krylov,V., Pleteneva,E., Bourkaltseva,M., Shaburova,O., Volckaert,G., Sykilinda,N., Kurochkina,L., Mesyanzhinov,V. (2003). Res. Microbiol. 154:269-275. Recently we have accomplished the entire DNA sequence of bacteriophage fKZ, a giant virus infecting Pseudomonas aeruginosa. The 280 334-bp of fKZ genome is a linear, circularly permutated and terminally redundant, AT-rich dsDNA molecule that contains no sites for NotI, PstI, SacI, SmaI, XhoI and XmaIII endonucleases. Limited homology to other bacteriophages on the DNA and protein levels indicated that fKZ represents a distinct branch of the Myoviridae family. In this work, we analyzed a group of six P. aeruginosa phages (Lin68, Lin21, PTB80, NN, EL, and RU), which are morphologically similar to fKZ, have similar genome size and low G + C content. All phages have a broad host range among P. aeruginosa strains, and they are resistant to the inhibitory action of many P. aeruginosa plasmids. The analysis of the genomic DNA by restriction enzymes and DNA-DNA hybridization shows that phages are representative of three fKZ-like species: fKZ-type (öKZ, Lin21, NN and PTB80), EL-type (EL and RU) and Lin68 which has a shorter tail than other phages. Except for related phages EL and RU, all fKZ-like phages have identical N-terminal amino acid sequences of the major capsid protein. Random genome sequencing shows that the EL and RU phages have no homology to the fKZ-like phages on DNA level. We propose that the fKZ, Lin21, NN, PTB80 and Lin68 phages can be included in a new fKZ genus, and that the EL and RU phages belong to a separate genus within the Myoviridae family. Based on the resistance to many restriction enzymes and the transduction ability, there are indications that over the long pathway of evolution, the fKZ-like phages probably inherited the capacity to infect different bacterial species. [TOP OF PAGE]

  793. The role of horizontal gene transfer by bacteriophages in the origin of pathogenic bacteria. Krylov,V. (2003). Rus. J. Genet. 39:483-504. The review considers the involvement of bacteriophages in transferring genes, which determine bacterial pathogenicity, and the increasing role of comparative genomics and genetics of bacteria and bacteriophages in detecting new cases of horizontal gene transfer. Examples of phage participation in this process proved to a different extent are described. Emphasis is placed on the original work carried out in Russia and focused on bacteriophages (temperate transposable phages and giant virulent fKZ-like phages) of conditional pathogen Pseudomonas aeruginosa. Consideration is given to the possible lines of further research of the role of bacteriophages in the infection process and, in particular, the role of virulent phages, whose products are sim-ilar to those of pathogenic bacteria, in modification of clinical signs of infectious diseases and in evolution. An attempt is made to predict the possible direction of pathogen evolution associated with development of new treatment strategies and generation of new specific niches. [TOP OF PAGE]

  794. [Role of horizontal gene transfer by bacteriophages in the origin of pathogenic bacteria]. Krylov,V.N. (2003). Genetika 39:595-620. The review considers the involvement of bacteriophages in transferring genes, which determine bacterial pathogenicity, and the increasing role of comparative genomics and genetics of bacteria and bacteriophages in detecting new cases of horizontal gene transfer. Examples of phage participation in this process proved to a different extent are described. Emphasis is placed on the original work carried out in Russia and focused on bacteriophages (temperate transposable phages and giant virulent phi KZ-like phages) of conditional pathogen Pseudomonas aeruginosa. Consideration is given to the possible lines of further research of the role of bacteriophages in the infection process and, in particular, the role of virulent phages, whose products are similar to those of pathogenic bacteria, in modification of clinical signs of infectious diseases and in evolution. An attempt is made to predict the possible direction of pathogen evolution associated with development of new treatment strategies and generation of new specific niches. [TOP OF PAGE]

  795. Lysogeny and bacteriophage host range within the Burkholderia cepacia complex. Langley,R., Kenna,D.T., Vandamme,P., Ure,R., Govan,J.R.W. (2003). J. Med. Microbiol. 52:483-490. The Burkholderia cepacia complex comprises a group of nine closely related species that have emerged as life-threatening pulmonary pathogens in immunocompromised patients, particularly individuals with cystic fibrosis or chronic granulomatous disease. Attempts to explain the genomic plasticity, adaptability and virulence of the complex have paid little attention to bacteriophages, particularly the potential contribution of lysogenic conversion and transduction. In this study, lysogeny was observed in 10 of 20 representative strains of the B. cepacia complex. Three temperate phages and five lytic phages isolated from soils, river sediments or the plant rhizosphere were chosen for further study. Six phages exhibited T-even morphology and two were lambda-like. The host range of individual phages, when tested against 66 strains of the B. cepacia complex and a representative panel of other pseudomonads, was not species-specific within the B. cepacia complex and, in some phages, included Burkholderia gladioli and Pseudomonas aeruginosa. These new data indicate a potential role for phages of the B. cepacia complex in the evolution of these soil bacteria as pathogens of plants, humans and animals, and as novel therapeutic agents. [TOP OF PAGE]

  796. The genome of bacteriophage fKMV, a T7-like virus infecting Pseudomonas aeruginosa. Lavigne,R., Burkal'tseva,M.V., Robben,J., Sykilinda,N.N., Kurochkina,L.P., Grymonprez,B., Jonckx,B., Krylov,V.N., Mesyanzhinov,V.V., Volckaert,G. (2003). Virology 312:49-59. The complete DNA sequence of a new lytic T7-like bacteriophage fKMV is presented. It is the first genome sequence of a member of the Podoviridae that infects Pseudomonas aeruginosa. The linear G + C-rich (62.3%) double-stranded DNA genome of 42,519 bp has direct terminal repeats of 414 bp and contains 48 open reading frames that are all transcribed from the same strand. Despite absence of homology at the DNA level, 11 of the 48 fKMV-encoded putative proteins show sequence similarity to known T7-type phage proteins. Eighteen open reading frame products have been assigned, including an RNA polymerase, proteins involved in DNA replication, as well as structural, phage maturation, and lysis proteins. Surprisingly, the major capsid protein completely lacks sequence homology to any known protein. Also, the strong virulence toward many clinical P. aeruginosa isolates and a short replication time make fKMV attractive for phage therapy or a potential source for antimicrobial proteins. [TOP OF PAGE]

  797. [Bacteriophages for treatment and prophylaxis of infectious diseases]. Lazareva,E.B. (2003). Antibiot. Khimoter. 48:36-40. [TOP OF PAGE]

  798. Environmental factors influencing the microbiological contamination of commercially harvested shellfish. Lee,R.J., Morgan,O.C. (2003). Water Sci. Technol. 47:65-70. Filter-feeding bivalve molluscs (such as oysters, clams, mussels and cockles) can concentrate contaminants from the water column. The extent of faecal contamination in shellfish is usually estimated by determining the concentration of faecal coliforms and/or Escherichia coli. Three sample points in each of three geographically separate commercial shellfisheries were selected for analysis for the effect of season, spring/neap and high/low tidal cycles, rainfall and wind direction on the results of routine E. coli monitoring. General linear modelling was used for the analyses. The principle factors affecting the contamination of shellfisheries were season, high/low tidal cycle and rainfall. The effects varied between harvesting areas and between individual sampling points within harvesting areas. Undertaking such analyses for all harvesting areas would contribute to the management of monitoring programmes and assist in the evaluation of potentially contaminating sources, such as sewage discharges. The type of analyses undertaken on E. coli monitoring data would also be pertinent for the analysis of putative viral indicators, such as F+ coliphage, and could be extended to data on bacterial and viral pathogens. [TOP OF PAGE]

  799. Biocontrol of Listeria monocytogenes on fresh-cut produce by treatment with lytic bacteriophages and a bacteriocin. Leverentz,B., Conway,W.S., Camp,M.J., Janisiewicz,W.J., Abuladze,T., Yang,M., Saftner,R., Sulakvelidze,A. (2003). Appl. Environ. Microbiol. 69:4519-4526. The fresh-cut produce industry has been the fastest-growing portion of the food retail market during the past 10 years, providing consumers with convenient and nutritious food. However, fresh-cut fruits and vegetables raise food safety concerns, because exposed tissue may be colonized more easily by pathogenic bacteria than intact produce. This is due to the higher availability of nutrients on cut surfaces and the greater potential for contamination because of the increased amount of handling. We found that applied Listeria monocytogenes populations survived and increased only slightly on fresh-cut Red Delicious apples stored at 10°C but increased significantly on fresh-cut honeydew melons stored at 10°C over 7 days. In addition, we examined the effect of lytic, L. monocytogenes-specific phages via two phage application methods, spraying and pipetting, on L. monocytogenes populations in artificially contaminated fresh-cut melons and apples. The phage mixture reduced L. monocytogenes populations by 2.0 to 4.6 log units over the control on honeydew melons. On apples, the reduction was below 0.4 log units. In combination with nisin (a bacteriocin), the phage mixture reduced L. monocytogenes populations by up to 5.7 log units on honeydew melon slices and by up to 2.3 log units on apple slices compared to the control. Nisin alone reduced L. monocytogenes populations by up to 3.2 log units on honeydew melon slices and by up to 2.0 log units on apple slices compared to the control. The phage titer was stable on melon slices, but declined rapidly on apple slices. The spray application of the phage and phage plus nisin reduced the bacterial numbers at least as much as the pipette application. The effectiveness of the phage treatment also depended on the initial concentration of L. monocytogenes. [TOP OF PAGE]

  800. Comparative genomic analyses of the vibrio pathogenicity island and cholera toxin prophage regions in nonepidemic serogroup strains of Vibrio cholerae. Li,M., Kotetishvili,M., Chen,Y., Sozhamannan,S. (2003). Appl. Environ. Microbiol. 69:1728-1738. Two major virulence factors are associated with epidemic strains (O1 and O139 serogroups) of Vibrio cholerae: cholera toxin encoded by the ctxAB genes and toxin-coregulated pilus encoded by the tcpA gene. The ctx genes reside in the genome of a filamentous phage (CTXphi), and the tcpA gene resides in a vibrio pathogenicity island (VPI) which has also been proposed to be a filamentous phage designated VPIphi. In order to determine the prevalence of horizontal transfer of VPI and CTXphi among nonepidemic (non-O1 and non-O139 serogroups) V. cholerae, 300 strains of both clinical and environmental origin were screened for the presence of tcpA and ctxAB. In this paper, we present the comparative genetic analyses of 11 nonepidemic serogroup strains which carry the VPI cluster. Seven of the 11 VPI(+) strains have also acquired the CTXphi. Multilocus sequence typing and restriction fragment length polymorphism analyses of the VPI and CTXphi prophage regions revealed that the non-O1 and non-O139 strains were genetically diverse and clustered in lineages distinct from that of the epidemic strains. The left end of the VPI in the non-O1 and non-O139 strains exhibited extensive DNA rearrangements. In addition, several CTXphi prophage types characterized by novel repressor (rstR) and ctxAB genes and VPIs with novel tcpA genes were found in these strains. These data suggest that the potentially pathogenic, nonepidemic, non-O1 and non-O139 strains identified in our study most likely evolved by sequential horizontal acquisition of the VPI and CTXphi independently rather than by exchange of O-antigen biosynthesis regions in an existing epidemic strain. [TOP OF PAGE]

  801. Population dynamics and gene transfer in genetically modified bacteria in a model microcosm. Lilley,A.K., Bailey,M.J., Barr,M., Kilshaw,K., Timms-Wilson,T.M., Day,M.J., Norris,S.J., Jones,T.H., Godfray,H.C.J. (2003). Molecular Ecology 12:3097-3107. The horizontal transfer and effects on host fitness of a neutral gene cassette inserted into three different genomic loci of a plant-colonizing pseudomonad was assessed in a model ecosystem. The KX reporter cassette (kanamycin resistance, aph, and catechol 2, 3, dioxygenase, xylE) was introduced on the disarmed transposon mini-Tn5 into: (I) the chromosome of a spontaneous rifampicin resistant mutant Pseudomonas fluorescens SBW25R; (II) the chromosome of SBW25R in the presence of a naturally occurring lysogenic-phage (phage Phi101); and (III) a naturally occurring plasmid pQBR11 (330 kbp, tra+, Hgr) introduced into SBW25R. These bacteria were applied to Stellaria media (chickweed) plants as seed dressings [c. 5 x 104 colony-forming units (cfu)/seed] and the seedlings planted in 16 microcosm chambers containing model plant and animal communities. Gene transfer to pseudomonads in the phyllosphere and rhizosphere was found only in the plasmid treatment (III). Bacteria in the phage treatment (II) initially declined in density and free phage was detected, but populations partly recovered as the plants matured. Surprisingly, bacteria in the chromosome insertion treatment (I) consistently achieved higher population densities than the unmanipulated control and other treatments. Plasmids were acquired from indigenous bacterial populations in the control and chromosome insertion treatments. Plasmid acquisition, plasmid transfer from inocula and selection for plasmid carrying inocula coincided with plant maturation. [TOP OF PAGE]

  802. A role for bacteriophage T4 rI gene function in the control of phage development during pseudolysogeny and in slowly growing host cells. Los,M., Wegrzyn,G., Neubauer,P. (2003). Res. Microbiol. 154:547-552. Although most studies on bacteriophages have been performed under laboratory conditions that are optimal for host cell growth, in nature, bacteria and bacteriophages coexist in different habitats. Here, by using different growth rates in carbon-limited chemostats, we investigated the development of phage T4 in its host Escherichia coli. Our results strongly suggest that T4 can form pseudolysogens not only when bacterial growth is completely inhibited, but also in growing host cells. The rI gene, previously known to be indispensable for lysis inhibition, seems to play an important role in optimization of phage development in slowly growing cells as well as during establishment and maintenance of pseudolysogeny. [TOP OF PAGE]

  803. Isolation and characterization of a Lactobacillus plantarum bacteriophage, phiJL-1, from a cucumber fermentation. Lu,Z., Breidt,F.J., Fleming,H.P., Altermann,E., Klaenhammer,T.R. (2003). Int. J. Food Microbiol. 84:225-235. A virulent Lactobacillus plantarum bacteriophage, PhiJL-1, was isolated from a commercial cucumber fermentation. The phage was specific for two related strains of L. plantarum, BI7 and its mutant (deficient in malolactate fermenting ability) MU45, which have been evaluated as starter cultures for controlled cucumber fermentation and as biocontrol microorganisms for minimally processed vegetable products. The phage genome of PhiJL-1 was sequenced to reveal a linear, double-stranded DNA (36.7 kbp). Sodium dodecyl sulfate-polyacryamide gel electrophoresis (SDS-PAGE) profiles indicated that PhiJL-1 contains six structural proteins (28, 34, 45, 50, 61, and 76 kDa). Electron microscopy revealed that the phage has an isometric head (59 nm in diameter), a long non-contractile tail (182 nm in length and 11 nm in width), and a complex base plate. The phage belongs to the Bradley group B1 or Siphoviridae family. One-step growth kinetics of the phage showed that the latent period was 35 min, the rise period was 40 min, and the average burst size was 22 phage particles/infected cell. Phage particles (90%) adsorbed to the host cells 20 min after infection. Calcium supplementation (up to 30 mM CaCl(2)) in MRS media did not affect the first cycle of phage adsorption, but promoted rapid phage propagation and cell lysis in the infection cycle subsequent to adsorption. The D values of PhiJL-1 at pH 6.5 were estimated to be 2.7 min at 70 ºC and 0.2 min at 80 ºC by a thermal inactivation experiment. Knowledge of the properties of L. plantarum bacteriophage PhiJL-1 may be important for the development of controlled vegetable fermentations. [TOP OF PAGE]

  804. Bacteriophage ecology in commercial sauerkraut fermentations. Lu,Z., Breidt,F., Plengvidhya,V., Fleming,H.P. (2003). Appl. Environ. Microbiol. 69:3192-3202. Knowledge of bacteriophage ecology in vegetable fermentations is essential for developing phage control strategies for consistent and high quality of fermented vegetable products. The ecology of phages infecting lactic acid bacteria (LAB) in commercial sauerkraut fermentations was investigated. Brine samples were taken from four commercial sauerkraut fermentation tanks over a 60- or 100-day period in 2000 and 2001. A total of 171 phage isolates, including at least 26 distinct phages, were obtained. In addition, 28 distinct host strains were isolated and identified as LAB by restriction analysis of the intergenic transcribed spacer region and 16S rRNA sequence analysis. These host strains included Leuconostoc, Weissella, and Lactobacillus species. It was found that there were two phage-host systems in the fermentations corresponding to the population shift from heterofermentative to homofermentative LAB between 3 and 7 days after the start of the fermentations. The data suggested that phages may play an important role in the microbial ecology and succession of LAB species in vegetable fermentations. Eight phage isolates, which were independently obtained two or more times, were further characterized. They belonged to the family Myoviridae or Siphoviridae and showed distinct host ranges and DNA fingerprints. Two of the phage isolates were found to be capable of infecting two Lactobacillus species. The results from this study demonstrated for the first time the complex phage ecology present in commercial sauerkraut fermentations, providing new insights into the bioprocess of vegetable fermentations. [TOP OF PAGE]

  805. Reduction of poliovirus 1, bacteriophages, Salmonella montevideo, and Escherichia coli O157:H7 on strawberries by physical and disinfectant washes. Lukasik,J., Bradley,M.L., Scott,T.M., Dea,M., Koo,A., Hsu,W.Y., Bartz,J.A., Farrah,S.R. (2003). J. Food Prot. 66:188-193. The efficacy levels of different physical and chemical washing treatments in the reduction of viral and bacterial pathogens from inoculated strawberries were evaluated. Escherichia coli O157:H7, Salmonella Montevideo, poliovirus 1, and the bacteriophages PRD1, phiX174, and MS2 were used as model and surrogate organisms. Chemicals readily available to producers and/or consumers were evaluated as antimicrobial additives for the production of washes. The gentle agitation of contaminated strawberries in water for 2 min led to reductions in microbial populations ranging from 41 to 79% and from 62 to 90% at water temperatures of 22 and 43 degrees C, respectively. Significant reductions (> 98%) in numbers of bacteria and viruses were obtained with sodium hypochlorite (50 to 300 ppm of free chlorine), Oxine or Carnebon (200 ppm of product generating "stabilized chlorine dioxide"), Tsunami (100 ppm of peroxyacetic acid), and Alcide (100 or 200 ppm of acidified sodium chlorite) washes. Overall, 200 ppm of acidified sodium chlorite produced the greatest reductions of microorganisms. Hydrogen peroxide (0.5%) was slightly less effective than free chlorine in a strawberry wash and caused slight fruit discoloration. Cetylpyridinium chloride (0.1%) was effective in the reduction of bacterial species, while trisodium phosphate (1%) was effective against viruses. The consumer-oriented produce wash Fit was very effective (> 99%) in reducing the numbers of bacteria but not in reducing the numbers of viruses. Another wash, Healthy Harvest, was significantly less effective than Fit in reducing bacterial pathogens but more effective for viruses. The performance of automatic dishwashing detergent was similar to that of Healthy Harvest and significantly better than that of liquid dishwashing detergent. Solutions containing table salt (2% NaCl) or vinegar (10%) reduced the numbers of bacteria by about 90%, whereas only the vinegar wash reduced the numbers of viruses significantly (ca. 95%). [TOP OF PAGE]

  806. Use of real-time quantitative PCR for the analysis of phiLC3 prophage stability in lactococci. Lunde,M., Blatny,J.M., Lillehaug,D., Aastveit,A.H., Nes,I.F. (2003). Appl. Environ. Microbiol. 69:41-48. Bacteriophages are a common and constant threat to proper milk fermentation. It has become evident that lysogeny is widespread in lactic acid bacteria, and in this work the temperate lactococcal bacteriophage phi LC3 was used as a model to study prophage stability in lactococci. The stability was analyzed in six phi LC3 lysogenic Lactococcus lactis subsp. cremoris host strains when they were growing at 15 and 30 degrees C. In order to perform these analyses, a real-time PCR assay was developed. The stability of the phi LC3 prophage was found to vary with the growth phase of its host L. lactis IMN-C1814, in which the induction rate increased during the exponential growth phase and reached a maximum level when the strain was entering the stationary phase. The maximum spontaneous induction frequency of the phi LC3 prophage varied between 0.32 and 9.1% (28-fold) in the six lysogenic strains. No correlation was observed between growth rates of the host cells and the spontaneous prophage induction frequencies. Furthermore, the level of extrachromosomal phage DNA after induction of the prophage varied between the strains (1.9 to 390%), and the estimated burst sizes varied up to eightfold. These results show that the host cells have a significant impact on the lytic and lysogenic life styles of temperate bacteriophages. The present study shows the power of the real-time PCR technique in the analysis of temperate phage biology and will be useful in work to reveal the impact of temperate phages and lysogenic bacteria in various ecological fields. [TOP OF PAGE]

  807. Characterisation of technologically proficient wild Lactococcus lactis strains resistant to phage infection. Madera,C., Garcia,P., Janzen,T., Rodriguez,A., Suarez,J.E. (2003). Int. J. Food Microbiol. 86:213-222. The aim of this work was to establish whether Lactococcus lactis strains isolated from spontaneous dairy fermentations exhibited useful milk-processing capabilities and resistance to bacteriophage infection in order to be used as components in starter formulations. The 33 out of 100 isolates of L. lactis, originated from farmhouse cheeses, were found to be resistant to a collection of 34 phages belonging to the c2 and 936 groups. Six of the isolates were discarded as potential starters because they were lysogenic and other five because they produced tyramine. Plasmid and chromosomal profiles of the 22 remaining isolates allowed their classification into 16 different strains. All of these were good lactic acid producers from lactose, moderately proteolytic and, in eight cases, diacetyl production from citrate was observed. The mechanism(s) leading to the phenotype of phage resistance was identified for all the strains used in this study. Inhibition of adsorption was the most frequent one, although genetic determinants for some abortive infection systems were also detected (abiB, abiG and abiI). Frequently, more than one mechanism was present in the same strain. One of the strains, L. lactis IPLA542, was selected as a model starter for pilot fermentations. It clotted milk normally both in the absence and in the presence of phage at concentrations that completely abolished the process when promoted by a phage-susceptible strain. [TOP OF PAGE]

  808. Detection of Escherichia coli using immunomagnetic separation and bacteriophage amplification coupled with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Madonna,A.J., Van Cuyk,S., Voorhees,K.J. (2003). Rapid Commun. Mass Spectrom. 17:257-263. The application of whole cell analysis by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has emerged as a valuable tool for rapidly identifying/detecting bacteria. This technique requires minimal sample preparation and is simple to perform, but is generally limited to purified samples of bacteria at concentrations greater than 1.0 x 10(6) cells/mL. In this paper, we describe a bacterial detection method that integrates immunomagnetic separation with bacteriophage amplification prior to MALDI-MS analysis. The developed method consists of three main stages: (1) isolation of a target bacterium by immunomagnetic separation; (2) infection of the immuno-captured bacterium with a lytic bacteriophage; and (3) assay of infected medium for bacteriophage progeny using MALDI-MS to produce a molecular weight signal for the virus capsid protein. With this technique, the presence of Escherichia coli in broth was determined in less then 2 h total analysis time at a concentration of approximately 5.0 x 10(4) cells/mL. [TOP OF PAGE]

  809. Diagnostic and therapeutic applications of lytic phages. Mandeville,R., Griffiths,M., Goodridge,L., McIntyre,L., Ilenchuk,T.T. (2003). Analytical Letters 36:3241-3259. The ability of lytic phages to rapidly kill and lyse infected bacteria, the specificity of phages for particular bacteria, and the ability of phages to increase in number during the infection process make phages excellent potential diagnostic and therapeutic agents for fighting bacterial disease. However, temperate phages are of little use in phage diagnostics and therapy. [TOP OF PAGE]

  810. Bacterial photosynthesis genes in a virus. Mann,N.H., Cook,A., Millard,A., Bailey,S., Clokie,M. (2003). Nature 424:741 A bacteriophage may protect itself and its host against a deadly effect of bright sunlight. [TOP OF PAGE]

  811. Phages of the marine cyanobacterial picophytoplankton. Mann,N.H. (2003). FEMS Microbiol. Rev. 27:17-34. Cyanobacteria of the genera Synechococcus and Prochlorococcus dominate the prokaryotic component of the picophytoplankton in the oceans. It is still less than 10 years since the discovery of phages that infect marine Synechococcus and the beginning of the characterisation of these phages and assessment of their ecological impact. Estimations of the contribution of phages to Synechococcus mortality are highly variable, but there is clear evidence that phages exert a significant selection pressure on Synechococcus community structure. In turn, there are strong selection pressures on the phage community, in terms of both abundance and composition. This review focuses on the factors affecting the diversity of cyanophages in the marine environment, cyanophage interactions with their hosts, and the selective pressures in the marine environment that affect cyanophage evolutionary biology. [TOP OF PAGE]

  812. Genetic diversity and temporal variation in the cyanophage community infecting marine Synechococcus species in Rhode Island's coastal waters. Marston,M.F., Sallee,J.L. (2003). Appl. Environ. Microbiol. 69:4639-4647. The cyanophage community in Rhode Island's coastal waters is genetically diverse and dynamic. Cyanophage abundance ranged from over 10(4) phage ml(-1) in the summer months to less then 10(2) phage ml(-1) during the winter months. Thirty-six distinct cyanomyovirus g20 genotypes were identified over a 3-year sampling period; however, only one to nine g20 genotypes were detected at any one sampling date. Phylogenetic analyses of g20 sequences revealed that the Rhode Island cyanomyoviral isolates fall into three main clades and are closely related to other known viral isolates of Synechococcus spp. Extinction dilution enrichment followed by host range tests and PCR restriction fragment length polymorphism analysis was used to detect changes in the relative abundance of cyanophage types in June, July, and August 2002. Temporal changes in both the overall composition of the cyanophage community and the relative abundance of specific cyanophage g20 genotypes were observed. In some seawater samples, the g20 gene from over 50% of isolated cyanophages could not be amplified by using the PCR primer pairs specific for cyanomyoviruses, which suggested that cyanophages in other viral families (e.g., Podoviridae or Siphoviridae) may be important components of the Rhode Island cyanophage community. [TOP OF PAGE]

  813. Bacteriophage therapy: an alternative to conventional antibiotics. Mathur,M.D., Vidhani,S., Mehndiratta,P.L. (2003). The Journal of the Association of Physicians of India 51:593-596. Bacteriophage therapy is an important alternative to antibiotics in the current era of multidrug resistant pathogens. We reviewed the studies that dealt with the therapeutic use of phages from 1966-1996 and few latest ongoing phage therapy projects via internet. Phages were used topically, orally or systemically in Polish and Soviet studies. The success rate found in these studies was 80-95% with few gastrointeslinal or allergic side effects. British studies also demonstrated significant efficacy of phages against Escherichia coli, Acinetobacter spp., Pseudomonas spp and Staphylococcus aureus. US studies dealt with improving the bioavailability of phage. Problems faced in these studies have also been discussed. In conclusion, phage therapy may prove as an important alternative to antibiotics for treating multidrug resistant pathogens. [TOP OF PAGE]

  814. Experimental protection of mice against lethal Staphylococcus aureus infection by novel bacteriophage fMR11. Matsuzaki,S., Yasuda,M., Nishikawa,H., Kuroda,M., Ujihara,T., Shuin,T., Shen,Y., Jin,Z., Fujimoto,S., Nasimuzzaman,M.D., Wakiguchi,H., Sugihara,S., Sugiura,T., Koda,S., Muraoka,A., Imai,S. (2003). J. Infect. Dis. 187:613-624. The protective effects of bacteriophages were assessed against experimental Staphylococcus aureus infection in mice. Of the S. aureus phages isolated in the study, phi MR11 was representatively used for all testing, because its host range was the most broad and it carries no genes for known toxins or antibiotic resistance. Intraperitoneal injections (8 x 108 cells) of S. aureus, including methicillin-resistant bacteria, caused bacteremia and eventual death in mice. In contrast, subsequent intraperitoneal administration of purified phi MR11 (MOI > or = 0.1) suppressed S. aureus-induced lethality. This lifesaving effect coincided with the rapid appearance of phi MR11 in the circulation, which remained at substantial levels until the bacteria were eradicated. Inoculation with high-dose phi MR11 alone produced no adverse effects attributable to the phage. These results uphold the efficacy of phage therapy against pernicious S. aureus infections in humans and suggest that phi MR11 may be a potential prototype for gene-modified, advanced therapeutic S. aureus phages. [TOP OF PAGE]

  815. [Coliphages as indicators of fecal contamination in sea water]. Meloni,P., Isola,D., Loi,N., Schintu,M., Contu,A. (2003). Annali di igiene : medicina preventiva e di comunita 15:111-116. Assessment of water quality has traditionally relied on faecal indicator organisms, which however do not necessarily correlate well with the presence of pathogenic organisms. Coliphages are regarded as possible alternative indicators. Although they can be detected in water by rapid, simple and reliable procedures, any agreement about a standard method has not yet been reached. Moreover guidelines for the levels of bacteriophages have not yet been set as for coliform bacteria, making difficult to evaluate results. In this work both bacteriophages anti E. coli and traditional indicators of fecal contamination were detected on 274 seawater samples taken from 23 sampling stations located along the coast of southern Sardinia (Italy). The results confirm the usefulness of coliphages as indicators of fecal contamination and suggest a level which could be considered a guideline value for their presence in seawater. [TOP OF PAGE]

  816. [Development of cyanobacterial phages at the Institute of Microbiology and Virology of the National Academy of Sciences of Ukraine (History and perspectives)]. Mendzhul,M.I., Lysenko,T.G., Syrchin,S.A. (2003). Mikrobiol. Zh. 65:133-140. The paper deals with the basic trends of fundamental investigations of the Department of Algae Viruses in the field of cyanophagia-ecology, biological and physico-chemical properties of cyanophages as well as interrelation with the host cells. Such problems as a possibility to use the system cyanophage-cyanobacteria as the experimental model for development of the unified functional model of productive infection, efficient methods of prophylaxis and therapy of virus infections as well as the solution of various biotechnological problems are discussed. [TOP OF PAGE]

  817. The prospect for bacteriophage therapy in Western Medicine. Merril,C.R., Scholl,D., Adhya,S.L. (2003). Nat. Rev. 2:489-497. Bacteriophage (phage) have been used for clinical applications since their initial discovery at the beginning of the twentieth century. However, they have never been subjted to the scrutiny -- in terms of the determination of efficacy and pharmaco-kinites of therapeutic agents -- that is required in countries that enforce certification of marketed pharmaceuticals. There are a number of historical reasons for this deficiency, including the overshadowing discovery of antibiotics. Neverhteless, present efforts to develop phage into reliable antibacterial agents have been substantially enhanced by knowledge gained concerning the genetics and physiology of phage in molecular detail during the past 50 years. Such efforts will be of importance given the emergence of antibiotic-resistant bacteria. [TOP OF PAGE]

  818. Antibacterial therapy with bacteriophage genotypically modified to delay inactivation by the host defense system. Merril,C.R., Carlton,R.M., Adhya,S.L. (2003). Exponential Biotherapies, Inc. and The United States of America as represented by the Department of Health. 464412(5,660,812). New York, NY; Washington, DC. The present invention is directed to bacteriophage therapy, using methods that enable the bacteriophage to delay inactivation by any and all parts of the host defense system (HDS) against foreign objects that would tend to reduce the numbers of bacteriophage and/or the efficiency of those phage at killing the host bacteria in an infection. Disclosed is a method of producing bacteriophage modified for anti-HDS purposes, one method being selection by serial passaging, and the other method being genetic engineering of a bacteriophage, so that the modified bacteriophage will remain active in the body for longer periods of time than the wild-type phage. [TOP OF PAGE]

  819. Comparative reduction of Norwalk virus, poliovirus type 1, F+ RNA coliphage MS2 and Escherichia coli in miniature soil columns. Meschke,J.S., Sobsey,M.D. (2003). Water Sci. Technol. 47:85-90. Norwalk-like viruses (NLVs) are important agents of waterborne illness and have been linked to several groundwater-related outbreaks. The presence of human enteric viruses, in particular the presence of NLVs, is difficult to detect in the environment. Consequently, surrogate organisms are typically used as indicators of viruses from faecal contamination. Whether traditional bacterial indicators are reliable indicators for viral pathogens remains uncertain. Few studies have directly compared mobility and reduction of bacterial indicators (e.g. coliforms, Escherichia coli) and other surrogate indicators (coliphages) with pathogenic human viruses in soil systems. In this study the mobility and comparative reduction of the prototype NLV, Norwalk Virus (NV), was compared to poliovirus 1 (PV1), a bacterial indicator (E coli, EC) and a viral indicator (coliphage MS2) through miniature soil columns. Replicate, 10 cm deep, miniature columns were prepared using three soils representing a range of soil textures (sand, organic muck, and clay). Columns were initially conditioned, then incubated at 10-14 degrees C, dosed twice weekly for 8 weeks with one column pore volume of virus-seeded groundwater per dose, followed by 8 weeks of dosing with one column pore volume per dose of unseeded, simulated rainwater. Columns were allowed to drain after each dosing until an effluent volume equivalent to an applied dose was collected. Column effluents and doses were assayed for all viruses and EC. Rapid mobility with minimal reduction was observed for all organisms in the sand. Similar reductions were observed in organic muck for most organisms but NV showed a greater reduction. No organisms were shown to pass through the clay columns. Elution of viruses, in particular PV1, from the columns was gradual. After cessation of microbe dosing, E. coli was less detectable than viruses in column effluents and, therefore, unreliable as a virus indicator. [TOP OF PAGE]

  820. Distribution of viruses and bacteria in relation to diagenetic activity in an estuarine sediment. Middelboe,M., Glud,R.N., Finster,K. (2003). Limnol. Oceanogr. 48:1447-1456. The distribution of viruses and bacteria was investigated in relation to bacterial sulfate reduction and total respiration (production of dissolved inorganic carbon, [DIC]) in a coastal sediment. Viral and bacterial abundance ranged from about 0.5 X 10(8) to 8 X 10(8) viruses cm(-3) and 0.1 X 10(8) to 4 X 10(8) bacteria cm(-3) in the upper 16 cm of the sediment and showed large and systematic changes within scales of a few centimeters. In general, viral abundance was highest in the sediment surface (0-1 em); however subsurface peaks at 3-5 cm depth associated with increased diagenetic activity were also observed. The virus-bacterium ratio ranged from 1.4 to 7.8 and increased significantly with depth in the upper 6 cm (P < 0.001). Viral abundance showed significant positive correlation with both bacterial abundance and activity (P much less than 0.001), suggesting that the distribution and abundance of viruses were closely coupled to the activity of the bacterial community and that viruses are produced by bacteria within the sediment. The significant coupling between viral abundance and sulfate reduction rate and DIC production is the first indication of viral production associated with diagenetic active bacteria in marine sediments. This coupling between viral abundance and bacterial activity and the distinct pattern of vertical distribution show that viruses are a dynamic component of the benthic community. The morphological analysis indicated that interstitial viral communities were dominated by long (>1 mum) filamentous forms with a helical symmetry. Several types of these filamentous forms were observed, as well as a variety of tailed forms with icosahedral symmetry. Filamentous forms are rarely found in the water column, which suggests that they are adapted to the benthic environment and specific to the interstitial hosts. [TOP OF PAGE]

  821. Virus-induced transfer of organic carbon between marine bacteria in a model community. Middelboe,M., Riemann,L., Steward,G.L., Hansen,V., Nybroe,O. (2003). Aquat. Microb. Ecol. 33:1-10. Viral lysis results in the transformation of living cells into dissolved and colloidal organic matter referred to as lysate. When viruses are included in food web models it is generally assumed that lysates are readily metabolized by bacteria in the community. We hypothesized that the production of lysate by viruses could also influence microbial community composition by mediating the diversification of carbon sources. To test this hypothesis, we established simple model communities containing various combinations of 2 marine bacteria (Cellulophaga sp. and Photobacterium sp.) and 2 viruses (one specific to each bacterial type) grown in a seawater-based medium with lactose as the sole carbon source. This medium supported vigorous growth of Cellulophaga sp. but not of Photobacterium sp. In control experiments, where Photobacterium sp. was cultured with either Cellulophaga sp. or Cellulophaga-specific virus, the biomass of Photobacteriurn sp. increased by 50% or less. In contrast, the Photobacterium sp. biomass significantly increased by 8-fold (p < 0.001, n = 3) in co-cultures with the Cellulophaga sp. virus-host pair. These data indicate that the substrate supporting growth by Photobacteriurn sp. was primarily Cellulophaga lysate and not material introduced with the host and virus inocula nor material secreted by Cellulophaga during normal growth. Estimates of the trophic transfer suggested that 28% of the Cellulophaga sp. lysate was converted into new bacterial biomass, which indicated that at least 62% of the lysate was metabolized by Photo bacterium sp. Our results from this simple marine model community illustrate that the activity of a virus-host system can effect the transfer of organic material from one bacterial type to another whose growth would otherwise be limited by a lack of suitable substrates. [TOP OF PAGE]

  822. Role for bacteriophages? Viruses that fight bacteria. Miller,F. (2003). WATT Poultry USA April(April), 8. Faced with the potential loss of traditional antibiotics, scientists at the University of Arkansas Division of Agriculture and the USDA Agriculture Research Service are updating century-old technology to fight illness-causing bacteria in poultry by infecting them with viruses known as bacteriophages. [TOP OF PAGE]

  823. Bacteriophage and the evolution of epidemic cholera. Miller,J.F. (2003). Infect. Immun. 71:2981-2982. [first paragraph] In January 1991, the seventh pandemic of cholera reached the west coast of Peru. It spread rapidly throughout South and Central America, and within a period of 11 months, over 300,000 cases of diarrhea were reported to the Pan American Health Organization (11). The Vibrio cholerae strain responsible was an O1 serogroup El Tor biotype, a close relative of the isolate that initiated the same pandemic 30 years earlier on the Indonesian island of Sulawesi (4). As South America was contending with an explosive outbreak of secretory diarrhea, a new epidemic began in 1992 in Madras, India, and Southern Bangladesh. The cause was a non-O1 V. cholerae strain representing an entirely new serogroup, later designated O139 (5). This came as a major surprise; it had previously been assumed that only O1 serogroup strains had the capacity to cause epidemic or endemic disease. In retrospect this assumption was naïve, considering the capacity of bacterial pathogens to undergo horizontal gene exchange. O139 strains continue to cause outbreaks with expanding geographic distribution, potentially heralding the eighth pandemic of cholera. [TOP OF PAGE]

  824. Coevolution of bacteriophage PP01 and Escherichia coli O157:H7 in continuous culture. Mizoguchi,K., Morita,M., Fischer,C.R., Yoichi,M., Tanji,Y., Unno,H. (2003). Appl. Environ. Microbiol. 69:170-176. The interaction between Escherichia coli O157:H7 and its specific bacteriophage PP01 was investigated in chemostat continuous culture. Following the addition of bacteriophage PP01, E. coli O157:H7 cell lysis was observed by over 4 orders of magnitude at a dilution rate of 0.876 h-1 and by 3 orders of magnitude at a lower dilution rate (0.327 h-1). However, the appearance of a series of phage-resistant E. coli isolates, which showed a low efficiency of plating against bacteriophage PP01, led to an increase in the cell concentration in the culture. The colony shape, outer membrane protein expression, and lipopolysaccharide production of each escape mutant were compared. Cessation of major outer membrane protein OmpC production and alteration of lipopolysaccharide composition enabled E. coli O157:H7 to escape PP01 infection. One of the escape mutants of E. coli O157:H7 which formed a mucoid colony (Mu) on Luria-Bertani agar appeared 56 h postincubation at a dilution rate of 0.867 h-1 and persisted until the end of the experiment (approximately 200 h). Mu mutant cells could coexist with bacteriophage PP01 in batch culture. Concentrations of the Mu cells and bacteriophage PP01 increased together. The appearance of mutant phage, which showed a different host range among the O157:H7 escape mutants than wild-type PP01, was also detected in the chemostat culture. Thus, coevolution of phage and E. coli O157:H7 proceeded as a mutual arms race in chemostat continuous culture. [TOP OF PAGE]

  825. Bacteriophage ST64B, a genetic mosaic of genes from diverse sources isolated from Salmonella enterica serovar Typhimurium DT 64. Mmolawa,P.T., Schmieger,H., Heuzenroeder,M.W. (2003). J. Bacteriol. 185:6481-6485. The complete sequence of the double-stranded DNA (dsDNA) genome of the Salmonella enterica serovar Typhimurium ST64B bacteriophage was determined. The 40,149-bp genomic sequence of ST64B has an overall G+C content of 51.3% and is distinct from that of P22. The genome architecture is similar to that of the lambdoid phages, particularly that of coliphage lambda. Most of the putative tail genes showed sequence similarity to tail genes of Mu, a nonlambdoid phage. In addition, it is likely that these tail genes are not expressed due to insertions of fragments of genes related to virulence within some of the open reading frames. This, together with the inability of ST64B to produce plaques on a wide range of isolates, suggests that ST64B is a defective phage. In contrast to the tail genes, most of the head genes showed similarity to those of the lambdoid phages HK97 and HK022, but these head genes also have significant sequence similarities to those of several other dsDNA phages infecting diverse bacterial hosts, including Escherichia, Pseudomonas, Agrobacterium, Caulobacter, Mesorhizobium, and Streptomyces: This suggests that ST64B is a genetic mosaic that has acquired significant portions of its genome from sources outside the genus Salmonella. [TOP OF PAGE]

  826. Survival of bacterial indicator species and bacteriophages after thermal treatment of sludge and sewage. Moce-Llivina,L., Muniesa,M., Pimenta-Vale,H., Lucena,F., Jofre,J. (2003). Appl. Environ. Microbiol. 69:1452-1456. The inactivation of naturally occurring bacterial indicators and bacteriophages by thermal treatment of a dewatered sludge and raw sewage was studied. The sludge was heated at 80 degrees C, and the sewage was heated at 60 degrees C. In both cases phages were significantly more resistant to thermal inactivation than bacterial indicators, with the exception of spores of sulfite-reducing clostridia. Somatic coliphages and phages infecting Bacteroides fragilis were significantly more resistant than F-specific RNA phages. Similar trends were observed in sludge and sewage. The effects of thermal treatment on various phages belonging to the three groups mentioned above and on various enteroviruses added to sewage were also studied. The results revealed that the variability in the resistance of phages agreed with the data obtained with the naturally occurring populations and that the phages that were studied were more resistant to heat treatment than the enteroviruses that were studied. The phages survived significantly better than Salmonella choleraesuis, and the extents of inactivation indicated that naturally occurring bacteriophages can be used to monitor the inactivation of Escherichia coli and Salmonella. [TOP OF PAGE]

  827. Isolated DNA encoding enzyme for phage resistance. Moineau,S., Walker,S., Vedamuthu,E.R., Vandenbergh,P.A. (2003). Quest International Flavors & Food Ingredients Company. 820980(5,925,388). Bridgewater, NJ. An isolated DNA of a Lactococcus lactis showing a SEQ ID NO:1 encoding a restriction and twp modification enzymes (R/M SEQ ID NO: 2, 3 and 4). The isolated DNA is used to transform sensitive dairy cultures, such as Lactococcus lactis and Streptococcus thermophilus, to provide phage resistance. Escherichia coli can be used to produce endonucleases. [TOP OF PAGE]

  828. Use of fluorescently labeled phage in the detection and identification of bacterial species. Mosier-Boss,P.A., Lieberman,S.H., Andrews,J.M., Rohwer,F.L., Wegley,L.E., Breitbart,M. (2003). Applied spectroscopy 57:1138-1144. Phages are viruses whose hosts are bacterial cells. They identify their hosts by specific receptor molecules on the outside of the host cell. Once the phages find their specific receptors, they bind to the bacterial cell and inject their nucleic acid inside the cell. The binding between phage and host can be so specific that only certain strains of a single species can be infected. In this communication, the specificity of phage P22 for Salmonella typhimurium LT2 is exploited to allow the detection of Salmonella in the presence of other bacterial species. In particular, the dsDNA of P22 is bound to SYBR gold, a highly sensitive, fluorescent nucleic acid stain. When multiple phages infect the same cell, the fluorescence emissions of the phage DNA inside the cell allow it to be imaged using an epifluorescence microscope. The advantages of using phages as the bacterial recognition element in a sensor over antibodies are discussed. [TOP OF PAGE]

  829. Bacterial host strains that support replication of somatic coliphages. Muniesa,M., Moce-Llivina,L., Katayama,H., Jofre,J. (2003). Antonie van Leeuwenhoek J. Microbiol. 83:305-315. Somatic coliphages detected by Escherichia coli strain WG5 have been proposed as potential indicators of water quality. Their potential replication in the water environment is considered a drawback for their use as indicators. However, the contribution of replication outside the gut to the total numbers has never been quantified. It has not been determined either the fraction of bacterial strains that might support replication of phages detected by strain WG5 in the water environment. We examined the sensitivity of 291 host strains to 25 phages by streaking slants of the presumptive host strain onto an agar layer that contains bacteriophages, which gives a total of 7275 combinations (sensitivity tests). Only a 3.02% of the tests showed sensitivity. Additionally, six environmental strains were used as hosts to count phages in sewage and seawater. Phages isolated on these strains were used to infect strain WG5. The environmental strains detected 1 log10 fewer phages than strain WG5 in sewage and seawater. The fraction of phages that were detected by the six strains and that also infected strain WG5 ranged from < 0.07% to < 2.0% of the total amount of bacteriophages detected by strain WG5 in the same samples. Our results confirm that less than 3% of naturally occurring hosts support replication of phages infecting E. coli. We conclude that the contribution of replication to the number of somatic coliphages detected in the aquatic environment is negligible. [TOP OF PAGE]

  830. Shiga toxin 2-converting bacteriophages associated with clonal variability in Escherichia coli O157:H7 strains of human origin isolated from a single outbreak. Muniesa,M., de Simon,M., Prats,G., Ferrer,D., Panella,H., Jofre,J. (2003). Infect. Immun. 71:4554-4562. Shiga toxin 2 (Stx2)-converting bacteriophages induced from 49 strains of Escherichia coli O157:H7 isolated during a recent outbreak of enterocolitis in Spain were examined in an attempt to identify the variability due to the stx(2)-converting phages. The bacterial isolates were divided into low-, medium-, and high-phage-production groups on the basis of the number of phages released after mitomycin C induction. Low- and medium-phage-production isolates harbored two kinds of phages but released only one of them, whereas high-phage-production isolates harbored only one of the two phages. One of the phages, fSC370, which was detected only in the isolates with two phages, showed similarities with phage 933W. The second phage, fLC159, differed from fSC370 in morphology and DNA structure. When both phages were present in the same bacterial chromosome, as occurred in most of the isolates, only SC370 was detected in the supernatants of the induced cultures. If fLC159 was released, its presence was masked by fSC370. When fSC370 was absent, large amounts of fLC159 were released, suggesting that there was some regulation of phage expression between the two phages. To our knowledge, this is the first description of clonal variability due to phage loss. The higher level of phage production was reflected in the larger amounts of Stx2 toxin produced by the cultures. Some relationship between phage production and the severity of symptoms was observed, and consequently these observations suggest that the virulence of the isolates studied could be related to the variability of the induced stx(2)-converting phages. [TOP OF PAGE]

  831. Experimental bacteriophage-mediated virulence in strains of Vibrio harveyi. Munro,J., Oakey,J., Bromage,E., Owens,L. (2003). Dis. Aquat. Org. 54:187-194. Vibriosis is a major disease problem in prawn aquaculture. Until now there has been no clear explanation why some strains of Vibrio are pathogenic, while others are not. This study demonstrated that the presence of the bacteriophage V. harveyi myovirus like (VHML) may confer virulence to V. harveyi Strain 642. This was demonstrated by infecting naive avirulent V. harveyi Strains 12, 20, 45 and 645 with the bacteriophage and converting them into virulent strains. The previously naive strains of Vibrio infected with Bacteriophage VHML from V. harveyi Strain 642 demonstrated up-regulation of haemolysin, up-regulation of protein excretion, additional proteins which were recognised as toxic proteins from Strain 642 by monoclonal antibodies specific to the exotoxin sub-units, and a significant increase in mortality of larval Penaeus monodon. It was concluded that Bacteriophage VHML conferred virulence to V. harveyi Strains 12, 20, 45 and 645 and that Bacteriophage VHML either fully or partly confers virulence in V. harveyi Strain 642. [TOP OF PAGE]

  832. Inhibitory action of telithromycin against Shiga toxin and endotoxin. Nakagawa,S., Kojio,S., Taneike,I., Iwakura,N., Tamura,Y., Kushiya,K., Gondaira,F., Yamamoto,T. (2003). Biochem. Biophys. Res. Com. 310:1194-1199. Shiga toxin (Stx)-producing Escherichia coli (STEC) is associated with hemolytic uremic syndrome (HUS). High inflammatory cytokine [interleukin (IL)-6 and IL-8] levels and low anti-inflammatory cytokine (IL-10) levels are indicators of a high risk for developing HUS in STEC-infected children. In this study, we investigated inhibitory action of telithromycin, a ketolide, against STEC and against Stx and lipopolysaccharide (LPS). Telithromycin inhibited in vitro STEC growth without inducing Stx phage, in marked contrast to norfloxacin. Stx markedly induced inflammatory (but not anti-inflammatory) cytokine production in human peripheral blood monocytes, while LPS induced both inflammatory and anti-inflammatory cytokine production. Telithromycin selectively inhibited the IL-6 and IL-8 production from Stx-stimulated (but not LPS-stimulated) monocytes. The drug did not significantly inhibit IL-10 production. Our data suggest that Stx plays a crucial role in the stimulation of inflammatory cytokines and such inflammatory response is inhibited by telithromycin, an anti-bacterial agent. [TOP OF PAGE]

  833. Method for detecting bacteria with bacteriaphage [sic]. Nakayama,H. (2003). Matsushita Electric Industrial Co., Ltd. 510072(6,555,312). Osaka, JP. A method for detecting a bacterium for measurement, including the steps of: allowing a bacteriophage to bind to the bacterium, the bacteriophage being capable of specifically binding to the bacterium and growing in the bacterium, whereby a gene within the bacteriophage which expresses a light-emission protein is introduced into the bacterium so that a protein is produced within the bacterium as a product of the gene; and providing an external factor in a non-invasive manner from outside of the bacterium, thereby causing only the actually-present bacterium to emit light in a specific manner. [TOP OF PAGE]

  834. Genomic sequence of C1, the first streptococcal phage. Nelson,D., Schuch,R., Zhu,S., Tscherne,D.M., Fischetti,V.A. (2003). J. Bacteriol. 185:3325-3332. C(1), a lytic bacteriophage infecting group C streptococci, is one of the earliest-isolated phages, and the method of bacterial classification known as phage typing was defined by using this bacteriophage. We present for the first time a detailed analysis of this phage by use of electron microscopy, protein profiling, and complete nucleotide sequencing. This virus belongs to the Podoviridae family of phages, all of which are characterized by short, noncontractile tails. The C(1) genome consists of a linear double-stranded DNA molecule of 16,687 nucleotides with 143-bp inverted terminal repeats. We have assigned functions to 9 of 20 putative open reading frames based on experimental substantiation or bioinformatic analysis. Their products include DNA polymerase, holin, lysin, major capsid, head-tail connector, neck appendage, and major tail proteins. Additionally, we found one intron belonging to the HNH endonuclease family interrupting the apparent lysin gene, suggesting a potential splicing event yielding a functional lytic enzyme. Examination of the C(1) DNA polymerase suggests that this phage utilizes a protein-primed mechanism of replication, which is prominent in the phi29-like members of Podoviridae. Consistent with this evidence, we experimentally determined that terminal proteins are covalently attached to both 5' termini, despite the fact that no homology to known terminal proteins could be elucidated in any of our open reading frames. Likewise, comparative genomics revealed no close evolutionary matches, suggesting that the C(1) bacteriophage is a unique member of the Podoviridae. [TOP OF PAGE]

  835. Concentrations and inactivation of Ascaris eggs and pathogen indicator organisms in wastewater stabilization pond sludge. Nelson,K.L. (2003). Water Sci. Technol. 48:89-95. During treatment in wastewater stabilization ponds (WSPs) many pathogens, in particular helminth eggs, are concentrated in the sludge layer. Because periodic removal of the sludge is often required, information is needed on the concentrations and inactivation of pathogens in the sludge layer to evaluate the public health risk they pose upon removal of the sludge. In this paper, previous reports on the sludge concentrations of various pathogen indicator organisms and helminth eggs are reviewed and results from our own recent experiments are reported. The advantages and disadvantages of several methods for studying inactivation in the sludge layer are discussed, as well as implications for the management of WSP sludge. In our recent experiments, which were conducted at three WSPs in central Mexico, sludge cores, dialysis chambers, and batch experiments were used to measure the inactivation rates of fecal coliform bacteria, fecal enterococci, F+ coliphage, somatic coliphage, and Ascaris eggs. The first-order inactivation rate constants were found to be approximately 0.1, 0.1, 0.01, 0.001, and 0.001 d(-1), respectively. The concentrations of all the organisms were found to vary both vertically and horizontally in the sludge layer; therefore, to determine the maximum and average concentration of organisms in the sludge layer of a WSP, complete sludge cores must be collected from representative locations throughout the pond. [TOP OF PAGE]

  836. [Bacteriophage therapy and colleague Martin Arrowsmith]. Nevasaari,K. (2003). Duodecim; laaketieteellinen aikakauskirja 119:1367 [TOP OF PAGE]

  837. Biology of the temperate Streptococcus thermophilus bacteriophage TP-J34 and physical characterization of the phage genome. Neve,H., Freudenberg,W., Diestel-Feddersen,F., Ehlert,R., Heller,K.J. (2003). Virology 315:184-194. The temperate Streptococcus thermophilus bacteriophage TP-J34 was identified in the lysogenic host strain J34. The majority of phage particles produced upon induction was defective and noninfectious, consisting of DNA-filled heads lacking tails. A physical map (45.6 kb) was established. Analysis of minor restriction bands of the DNA isolated from phage particles as well as the analysis of the protein pattern indicated that phage TP-J34 is a pac-type phage. This was confirmed by immunoelectron microscopy using antisera raised against virulent cos- and pac-type S. thermophilus phages. The lysogenic host J34 but not its noninducible derivate J34-12 contained phage DNA in the nonintegrated state and exhibited autolysis at elevated temperatures. Prophage-carrying strains grew homogeneously while 16 of 20 prophage-cured derivatives aggregated and sedimented rapidly. When phage TP-J34 was propagated lytically on a prophage-cured host strain, a 2.7-kb site-specific deletion occurred in the phage genome. This deletion was also identified in the prophage DNAs of relysogenized strains. [TOP OF PAGE]

  838. [Evaluation of relations between plasmids and phage host range among clinical isolates of Enterobacter cloacae]. Nieradko,J., Kurlenda,J. (2003). Med Dosw Mikrobiol 55:343-349. The aim of this study was evaluation the plasmid influence on phage host range of clinical strains of Enterobacter cloacae. We found that strains included in restrictive pattern A, displayed reduced host range. Such reduced sensitivity make these strains excellent candidates for search restrictive-modification systems. High discriminative efficacy of isolated phages (specific for strains Enterobacter cloacae) make them useful tool for phage typing in epidemiological investigations. [TOP OF PAGE]

  839. Use of viral pathogens and indicators to differentiate between human and non-human fecal contamination in a microbial source tracking comparison study. Noble,R.T., Allen,S.M., Blackwood,A.D., Chu,W., Jiang,S.C., Lovelace,G.L., Sobsey,M.D., Stewart,J.R., Wait,D.A. (2003). Water Hlth. 1:195-207. Assays for the detection and typing of adenoviruses, enteroviruses and F+ specific coliphages were performed on samples created as part of a national microbial source tracking methods comparison study. The samples were created blind to the researchers, and were inoculated with a variety of types of fecal contamination source (human, sewage, dog, seagull and cow) and mixtures of sources. Viral tracer and pathogen assays demonstrated a general ability to discriminate human from non-human fecal contamination. For example, samples inoculated with sewage were correctly identified as containing human fecal contamination because they contained human adenovirus or human enterovirus. In samples containing fecal material from individual humans, human pathogen analysis yielded negative results probably because the stool samples were taken from healthy individuals. False positive rates for the virus-based methods (0-8%) were among the lowest observed during the methods comparison study. It is suggested that virus-based source tracking methods are useful for identification of sewage contamination, and that these methods may also be useful as an indication of the public health risk associated with viral pathogens. Overall, virus-based source tracking methods are an important approach to include in the microbial source tracking 'toolbox'. [TOP OF PAGE]

  840. Microbial water quality improvement by small scale on-site subsurface wetland treatment. Nokes,R.L., Gerba,C.P., Karpiscak,M.M. (2003). J. Environ. Sci. Health Part A Tox. /Haz. Subst. Environ. Engin. 38:1849-1855. It has been demonstrated that large constructed wetlands used for domestic wastewater treatment are useful in the reduction of enteric microorganisms. This study evaluated the ability of three small-scale, on-site subsurface wetlands with different vegetation densities to remove total coliforms, fecal coliforms, coliphage, Giardia and Cryptosporidium. These wetlands were found to be equally efficient in the removal of enteric bacteria and coliphage as larger constructed wetlands. Giardia and Cryptosporidium were usually undetectable after passage of the wastewater through the subsurface wetlands. Coliphage removal increased with increasing vegetation density. [TOP OF PAGE]

  841. Scope of potential bacterial agents of diarrhoea and microbial assessment of quality of river water sources in rural Venda communities in South Africa. Obi,C.L., Potgieter,N., Bessong,P.O., Matsaung,G. (2003). Water Sci. Technol. 47:59-64. The microbial quality of several, usually untreated, surface domestic water sources, used by rural communities in the Venda Region of South Africa, was assessed to gauge their fitness for human consumption and to highlight the possible impact of waterborne diseases. The water sources studied were six points on the Levubu River and the rivers Mutale, Ngwedi, Tshinane, Makonde, Mutshindudi and Mudaswali. Total and faecal coliform, heterotrophic bacteria, enterococci and coliphage counts were used as indicators/surrogates to estimate the degree of bacterial and viral contamination respectively by standard methods. The presence of potential bacterial agents of diarrhoea such as Salmonella, Shigella, Campylobacter, Plesiomonas, Aeromonas and Vibrio was also determined. Results showed that the ranges of counts with regard to all the water sources investigated were 2.9 x 10(2) - 6.3 x 10(4) CFU/100 mL for faecal coliforms, 6.0 x 10(2) - 3.7 x 10(4) CFU/100 mL for total coliforms, 1.8 x 10(2) - 1.3 x 10(6) CFU/mL for heterotrophic plate count, 1.0 x 10(1) - 3.7 x 10(4) CFU/100 mL for enterococci and 0-13 PFU/100 mL for coliphages. These values are far higher than the acceptable maximum limits prescribed for South Africa by the Dept of Water & Forestry and the Water Research Commission - 0 CFU/100 mL, 5 CFU/100 mL, 1.0 x 10(2) CFU/mL, 0 CFU/100 mL and 1 PFU/100 mL for faecal coliforms, total coliforms, heterotrophic bacteria, enterococci and coliphages respectively. Salmonella, Shigella, Vibrio cholerae, Campylobacter, Aeromonas and Plesiomonas were isolated from several of the water sources investigated. The use of these water sources for drinking and domestic purposes poses a serious threat to the health and well being of the users and calls for urgent government intervention. [TOP OF PAGE]

  842. Genome organization and molecular analysis of the temperate bacteriophage MM1 of Streptococcus pneumoniae. Obregon,V., Garcia,J.L., Garcia,E., Lopez,R., Garcia,P. (2003). J. Bacteriol. 185:2362-2368. The genome of MM1 (40,248 bp), a temperate bacteriophage from the Spain(23F)-1 multiresistant epidemic clone of Streptococcus pneumoniae, is organized in 53 open reading frames (ORFs) and in at least five functional clusters. Bioinformatic and N-terminal amino acid sequence analyses enabled the assignment of possible functions to 26 ORFs. Analyses comparing the MM1 genome with those of other bacteriophages revealed similarities, mainly with genomes of phages infecting gram-positive bacteria, which suggest recent exchange of genes between species colonizing the same habitat. [TOP OF PAGE]

  843. [How do bacteria acquire the resistance to antibiotics]. Ohno,A. (2003). Nippon Rinsho - Japanese Journal of Clinical Medicine 61 Suppl 3:158-163. [TOP OF PAGE]

  844. Suppression of leaf feeding and oviposition of phytophagous ladybird beetles (Coleoptera: Coccinellidae) by chitinase gene-transformed phylloplane bacteria and their specific bacteriophages entrapped in alginate gel beads. Otsu,Y., Mori,H., Komuta,K., Shimizu,H., Nogawa,S., Matsuda,Y., Nonomura,T., Sakuratani,Y., Tosa,Y., Mayama,S., Toyoda,H. (2003). J. Econ. Entom. 96:555-563. The chitinase gene-transformed strain KPM-007E/chi of Enterobacter cloacae was vitally entrapped in sodium alginate gel beads with its specific virulent bacteriophage EcP-01 to provide a new method for microbially digesting chitinous peritrophic membranes of phytophagous ladybird beetles Epilachna vigintioctopunctata. First, chitinase SH1 from a gram-positive bacterium Kurthia zopfii was overproduced by Escherichia coli cells and purified by affinity column chromatography. The purified enzyme effectively digested peritrophic membranes dissected from the ladybird beetles to expose epithelial tissues beneath the peritrophic membrane, and the beetles that had ingested chitinase after submergence in chitinase solution had considerably reduced their feeding on tomato leaves. KPM-007E/chi, entrapped in the alginate beads, released the chitinase. More chitinase was released when KPM-007E/chi was present with their specific virulent bacteriophage EcP-01 in the beads because of lysis of bacterial cells infected with the bacteriophages. This chitinase release from the microbial beads (containing KPM-007E/chi and EcP-01) was sufficient to digest the peritrophic membrane as well as to suppress feeding of bead-sprayed tomato leaves by the ladybird beetles. A daily supply of tomato leaves treated with the microbial beads considerably suppressed leaf feeding and oviposition by the ladybird beetles, suggesting a possible application of chitinase-secreting bacteria for suppressing herbivorous insect pests. [TOP OF PAGE]

  845. Faecal contamination of greywater and associated microbial risks. Ottoson,J., Stenstrom,T.A. (2003). Water Res. 37:645-655. The faecal contamination of greywater in a local treatment system at Vibyasen, north of Stockholm, Sweden was quantified using faecal indicator bacteria and chemical biomarkers. Bacterial indicator densities overestimated the faecal load by 100-1000-fold when compared to chemical biomarkers. Based on measured levels of coprostanol, the faecal load was estimated to be 0.04 g person(-1) day(-1). Prevalence of pathogens in the population and the faecal load were used to form the basis of a screening-level quantitative microbial risk assessment (QMRA) that was undertaken for rotavirus, Salmonella typhimurium, Campylobacter jejuni, Giardia lamblia and Cryptosporidium parvum. The different exposure scenarios simulated--direct contact, irrigation of sport fields and groundwater recharge--gave unacceptably high rotavirus risks (0.04 < Pinf < 0.60) despite a low faecal load. The poor reduction of somatic coliphages, which were used as a virus model, in the treatment was one main reason and additional treatment of the greywater is suggested. Somatic coliphages can under extreme circumstances replicate in the wastewater treatment system and thereby underestimate the virus reduction. An alternative QMRA method based on faecal enterococci densities estimated similar risks as for rotavirus. Growth conditions for Salmonella in greywater sediments were also investigated and risk modelling based on replication in the system increased the probability of infection from Salmonella 1000-fold, but it was still lower than the risk of a rotavirus infection. [TOP OF PAGE]

  846. Growth and reduction of microorganisms in sediments collected from a greywater treatment system. Ottosson,J., Stenstrom,T.A. (2003). Lett. Appl. Microbiol. 36:168-172. AIMS: To study the effects of competitive microbiota, temperature and nutrient availability on Salmonella, Enterococcus, Campylobacter[,] spores of sulphite reducing anaerobes and bacteriophages MS2 and phiX174 in sediments from a greywater treatment system. METHODS AND RESULTS: Standard culture methods were used. Bacteria died off rapidly under normal conditions (20 degrees C, competitive microbiota) but remained stable or grew in the other conditions studied. When the sediments became nutrient depleted after 2 weeks, a log-linear die-off was observed for Salmonella, which was higher at 20 degrees C than at 4 degrees C. Bacteriophage decay was shown to be log-linear from day 0, with T90 values ranging from 9 (phiX174, 20 degrees C) to 55 days (phiX174, 4 degrees C). The MS2 phage had a significantly higher decay rate in tyndallized sediments (T90 = 17 days) than in original sediments (T90 = 47 days) (P < 0.001), with temperature not shown to affect the decay rate. Spores of sulphite-reducing anaerobes were not significantly reduced during the study period (35 days). Campylobacter died-off rapidly or entered a viable but non-culturable state and subsequently results were not provided. CONCLUSIONS: Competition was the most important factor to suppress pathogenic bacterial growth in an eutrophic environment. When nutrient depleted conditions prevailed, temperature was more important and log-linear decay of microorganisms could be observed. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings suggest that the normally occurring microbiota will suppress pathogenic bacterial growth in nutrient rich sediments. With lower nutrient status, temperature is the more important factor in reducing pathogens. [TOP OF PAGE]

  847. Response of bacterial and viral communities to nutrient manipulations in seawater mesocosms. Øvreås,L., Bourne,D., Sandaa,R.-A., Casamayer,E.O., Bnlloch,S., Goddard,V., Smerdon,G., Heldal,M., Thingstad,T.F. (2003). Aquat. Microb. Ecol. 31:109-121. Changes in natural bacterial and viral assemblages were studied in seawater mesocosms manipulated with inorganic (nitrate + phosphate) and inorganic + organic (glucose) nutrient additions. As inferred from the gel band patterns obtained by DGGE, only moderate changes within the bacterial community took place when mineral nutrients were added alone. Supplementing the mineral nutrients with glucose in excess of what the bacteria could consume led, however, to major changes in band patterns. Based on fluorescence in situ hybridisation (FISH), the major bacterial response was identified as an increase in the population of gamma-Proteobacteria with a smaller response in alpha-Proteobacteria. Sequencing of bands from the DGGE gels indicated that glucose + mineral nutrients led to a Vibrio-dominated bacterial community. A specific FISH probe was designed from a band sequence affiliated to Vibrio splendidus, and linked a large-celled bacterial morphotype to the DGGE-gel bands dominating in glucose-amended mesocosms. A similar difference in the response of the viral populations among treatments was demonstrated using pulsed field gel electrophoresis (PFGE). The number of bands on DGGE gels and PFGE gels were similar (mean ratio 0.98). We suggest an interpretation of these results where coexistence of nutrient-competing bacterial hosts is controlled by viral lysis. We also suggest that the success of large bacteria in glucose-replete treatments was not based on superior glucose-utilisation abilities, but rather on an advantage in competition for limiting mineral nutrients derived from the combination of a large cell surface with a low cellular content of the limiting element, possible for cells with large C-rich inclusion bodies. [TOP OF PAGE]

  848. Yersiniophages. Special reference to phi YeO3-12. Pajunen,M.I., Molineux,I.J., Skurnik,M. (2003). Advances in experimental medicine and biology 529:233-240. [TOP OF PAGE]

  849. Phage evolution: new worlds of genomic diversity. Papke,R.T., Doolittle,W.F. (2003). Curr. Biol. 13:R606-R607 A recent comparative survey of genomes of phages infecting mycobacteria reveals a vast combinatorial network of gene rearrangements and may provide general models for pattern and process in genome evolution. [TOP OF PAGE]

  850. Use of a phage-based assay for phenotypic detection of mycobacteria directly from sputum. Park,D.J., Drobniewski,F.A., Meyer,A., Wilson,S.M. (2003). J. Clin. Microbiol. 41:680-688. The phage amplified biologically assay is a new method for rapid and low-cost phenotypic determination of the drug sensitivities of Mycobacterium tuberculosis isolates and the detection of viable organisms in patient specimens. Infection of slowly growing mycobacteria with phage (phage D29) was followed by chemical virucide destruction of extracellular phage. Infected mycobacteria were mixed in culture with rapidly growing sensor cells, which the phage can also infect; i.e., lytic amplification of phage occurs. The aims of the present study were to optimize the speed and sensitivity of the assay and reduce its cost for developing countries by using an M. tuberculosis-spiked sputum model with (i). identification of inhibitory components of sputum and optimization of decontamination methods; (ii). simplification of the washing and development steps; (iii). reduction of the use of high-cost components, e.g., oleate-albumin-dextrose-catalase (OADC) supplement; and (iv). optimization of virucide treatment. The following results were obtained. (i). An inhibitory factor in sputum which could be removed by treatment of the sample with sodium dodecyl sulfate or NaOH decontamination was identified. (ii). A microcentrifuge-based approach with thixotropic silica as a bedding and resuspension agent was developed as an alternative to conventional centrifugation medium exchange. The yield was increased 228-fold, with increased speed and reduced cost. (iii). At present, after extracellular inactivation of phage, the ferrous ammonium sulfate (FAS) virucide is sequestered by dilution with an expensive supplement, OADC. Sodium citrate with calcium chloride was found to be a cost-effective after treatment with the FAS protectant and offered greater protection than OADC. Kinetic-lysis experiments indicated that an infection time of 1 to 3 h prior to FAS addition was optimal. (iv). Amplification of the signal (which corresponded to the burst size) was shown by allowing lysis prior to plating in a spiked medium model (up to 20-fold) and a spiked sputum model (up to 10-fold). A liquid culture detection method capable of detecting approximately 60 viable M. tuberculosis organisms in 1 ml of sputum was developed. Taken together, these improvements support the routine application of the assay to sputum specimens. [TOP OF PAGE]

  851. Bacteriophage control of Pseudomonas plecoglossicida infection in ayu Plecoglossus altivelis. Park,S.C., Nakai,T. (2003). Dis. Aquat. Org. 53:33-39. Two previously isolated phages were used to examine the therapeutic effects against Pseudomonas plecoglossicida infection in ayu Plecoglossus altivelis. Phage PPp-W4 (Podoviridae) inhibited the in vitro growth of P. plecoglossicida more effectively than Phage PPpW-3 (Myoviridae), and a mixture (PPpW-3/W-4) of the 2 phages exhibited the highest inhibitory activity. In phage therapy experiments, ayu were fed P. plecoglossicida-impregnated feed (10(7) CFU fish(-1)) and then fed phage-impregnated feed (10(7) PFU fish(-1)). Mortalities of fish receiving PPpW-3, PPpW-4, PPpW-3/W-4, and a control fish receiving no phages were 53.3, 40.0, 20.0 and 93.3%, respectively. Phage (PPpW-3/W-4)-receiving fish also showed high protection against water-borne infection with P. plecoglossicida. In a field trial, when phage (PPpW-3/W-4)-impregnated feed was administered to ayu in a pond where the disease occurred naturally, daily mortality of fish decreased at a constant level (5% d(-1)) to one-third after a 2 wk period. The causal relationship of phages in this phenomenon was verified by the long-lasting appearance of administered phages in the kidneys of the fish, and a disappearance of P. plecoglossicida from apparently healthy fish. Neither phage-resistant organisms nor phage-neutralizing antibodies were detected in diseased fish or apparently healthy fish, respectively. These results indicate the potential for phage control of the disease. [TOP OF PAGE]

  852. Pharmacokinetic principles of bacteriophage therapy. Payne,R.J.H., Jansen,V.A.A. (2003). Clinical Pharmacokinetics 42:315-325. Use of bacteriophage to control bacterial infections, including antibiotic-resistant infections, shows increasing therapeutic promise. Effective bacteriophage therapy requires awareness of various novel kinetic phenomena not known in conventional drug treatments. Kinetic theory predicts that timing of treatment could be critical, with the strange possibility that inoculations given too early could be less effective or fail completely. Another paradoxical result is that adjuvant use of an antibiotic can sometimes diminish the efficacy of phage therapy. For a simple kinetic model, mathematical formulae predict the values of critical density thresholds and critical time points, given as functions of independently measurable biological parameters. Understanding such formulae is important for interpreting data and guiding experimental design. Tailoring pharmacokinetic models for specific systems needs to become standard practice in future studies. [TOP OF PAGE]

  853. Origins of highly mosaic mycobacteriophage genomes. Pedulla,M.L., Ford,M.E., Houtz,J.M., Karthikeyan,T., Wadsworth,C., Lewis,J.A., Jacobs-Sera,D., Falbo,J., Gross,J., Pannunzio,N.R., Brucker,W., Kumar,V., Kandasamy,J., Keenan,L., Bardarov,S., Jr., Kriakov,J., Lawrence,J.G., Jacobs,W.R., Jr., Hendrix,R.W., Hatfull,G.F. (2003). Cell 113:171-182. Bacteriophages are the most abundant organisms in the biosphere and play major roles in the ecological balance of microbial life. The genomic sequences of ten newly isolated mycobacteriophages suggest that the bacteriophage population as a whole is amazingly diverse and may represent the largest unexplored reservoir of sequence information in the biosphere. Genomic comparison of these mycobacteriophages contributes to our understanding of the mechanisms of viral evolution and provides compelling evidence for the role of illegitimate recombination in horizontal genetic exchange. The promiscuity of these recombination events results in the inclusion of many unexpected genes including those implicated in mycobacterial latency, the cellular and immune responses to mycobacterial infections, and autoimmune diseases such as human lupus. While the role of phages as vehicles of toxin genes is well established, these observations suggest a much broader involvement of phages in bacterial virulence and the host response to bacterial infections. [TOP OF PAGE]

  854. Development of a novel method of lytic phage delivery by use of a bacteriophage P22 site-specific recombination system. Platt,R., Reynolds,D.L., Phillips,G.J. (2003). FEMS Microbiol. Lett. 223:259-265. Bacteriophage therapy represents a potential alternative to the use of antibiotics to control proliferation of pathogenic bacteria. As an alternative to the strategy where a limited number of doses of large numbers of lytic bacteriophages are administered, a novel method delivery system was developed so that phages are continually released into the culture. Specifically, a non-pathogenic Escherichia coli strain was constructed that was lysogenic for a lytic mutant of bacteriophage lambda. This lysogen was shown to be effective at decreasing the number of lambda-sensitive E. coli in vitro. Construction of this E. coli strain was accomplished by development of a plasmid-based system utilizing the site-specific recombination machinery of bacteriophage P22 to integrate DNA constructs into the host chromosome. This recombination system is useful for strain construction and other genetic manipulations in both E. coli and Salmonella enterica serovars. [TOP OF PAGE]

  855. Microbial contamination of two urban sandstone aquifers in the UK. Powell,K.L., Taylor,R.G., Cronin,A.A., Barrett,M.H., Pedley,S., Sellwood,J., Trowsdale,S.A., Lerner,D.N. (2003). Water Res. 37:339-352. Development of urban groundwater has historically been constrained by concerns about its quality. Rising urban water tables and overabstraction from rural aquifers in the UK have led to a renewed interest in urban groundwater, particularly the possibility of finding water of acceptable quality at depth. This study assessed the microbial quality of groundwater collected from depth-specific intervals over a 15-month period within the Permo-Triassic Sherwood Sandstone aquifers underlying the cities of Nottingham and Birmingham. Sewage-derived bacteria (thermotolerant coliforms, faecal streptococci and sulphite-reducing clostridia) and viruses (enteroviruses, Norwalk-like viruses, coliphage) were regularly detected to depths of 60 m in the unconfined sandstone and to a depth of 91 m in the confined sandstone. Microbial concentrations varied temporally and spatially but increased frequency of contamination with depth coincided with geological heterogeneities such as fissures and mudstone bands. Significantly, detection of Norwalk-like viruses and Coxsackievirus B4 in groundwater corresponded with seasonal variations in virus discharge to the sewer system. The observation of low levels of sewage-derived microbial contaminants at depth in the Triassic Sandstone aquifer is explained by the movement of infinitesimal proportions of bulk (macroscopic) groundwater flow along preferential pathways (e.g., fissures, bedding planes). The existence of very high microbial populations at source (raw sewage) and their extremely low detection limits at the receptor (multilevel piezometer) enable these statistically extreme (microscopic) flows to be traced. Rapid penetration of microbial contaminants into sandstone aquifers, not previously reported, highlights the vulnerability of sandstone aquifers to microbial contamination. [TOP OF PAGE]

  856. Evolutionary insights from studies on viruses from hot habitats. Prangishvili,D. (2003). Res. Microbiol. 154:289-294. The morphological diversity of viruses which parasitize hyperthermophilic archaea thriving at temperatures >=80 °C appears to exceed that of viruses of prokaryotes living at lower temperatures. Based on assumptions of the existence of viruses in the prebiotic phase of evolution and hot origins of cellular life, we suggest that this remarkable diversity could have its source in ancestral diversity of viral morphotypes in hot environments. Attempts are made to trace evolutionary relationships of viruses of hyperthermophilic archaea with other viruses. [TOP OF PAGE]

  857. Virus removal during simulated soil-aquifer treatment. Quanrud,D.M., Carroll,S.M., Gerba,C.P., Arnold,R.G. (2003). Water Res. 37:753-762. Removals of indigenous coliphage and seeded poliovirus type 1 during simulated soil-aquifer treatment were evaluated during transport of secondary effluent under unsaturated flow conditions in 1-m soil columns. Independent variables included soil type (river sand or sandy loam) and infiltration rate. Removal of coliphage was in all cases less than removal of poliovirus type 1 (strain LSc-2ab), supporting contentions that indigenous coliphage can act as a conservative indicator of groundwater contamination by viral pathogens of human origin. Coliphage retention was significantly more efficient (p<0.001) in the finer-grained sandy loam (93%) than in sand (76%). Increasing reactor detention time from 5 to 20 h increased coliphage attenuation from 70% to 99% in a 1-m sand column. There was a significant linear correlation (p=0.012) between log-transformed (fractional) coliphage concentration [log(C/C(0))] and reactor detention time. Re-mobilization of attached coliphage occurred during simulated rainfall using low-ionic-strength water. Inhibition of aerobic respiration resulted in significantly less efficient coliphage attenuation (p=0.033), suggesting the involvement of aerobic microorganisms in the survival/retention of this virus. [TOP OF PAGE]

  858. Homogeneity of the morphological groups of bacteriophages infecting Bacteroides fragilis strain HSP40 and strain RYC2056. Queralt,N., Jofre,J., Araujo,R., Muniesa,M. (2003). Curr. Microbiol. 46:163-168. Bacteriophages infecting Bacteroides fragilis strains RYC2056 and HSP40 have been proposed as indicators of water quality. To accomplish this function, homogeneity of the group of phages detected by these strains is necessary to ensure that the final results are not due to the different kinetics of inactivation of the phages. To evaluate homogeneity, we observed by electron microscopy bacteriophages isolated from sewage with two Bacteroides fragilis strains (HSP40 and RYC2056). A predominant group of phages was observed, Siphoviridae with slightly curved tails. Detection of other minority groups, which could be present in the sample, was done with neutralization experiments by using antiserum against the majority group and with host mutants resistant to infection with the predominant phage. Although two other minority groups were observed, results showed that bacteriophages infecting B. fragilis strain HSP40 and strain RYC2056 form a homogeneous group, Siphoviridae with slightly curved tails being the most predominant in sewage. [TOP OF PAGE]

  859. Inactivation of Lactobacillus delbrueckii bacteriophages by heat and biocides. Quiberoni,A., Guglielmotti,D.M., Reinheimer,J.A. (2003). Int. J. Food Microbiol. 84:51-62. The effect of several biocides and thermal treatments on the viability of four Lactobacillus delbrueckii phages was investigated. Time to achieve 99% inactivation of phages at 63 and 72 degrees C in three suspension media (Tris Magnesium Gelatin (TMG) buffer, Man Rogosa Sharpe (MRS) broth and reconstituted nonfat dry skim milk (RSM)) was calculated. Thermal resistance depended on the phage considered, but a marked heat-resistance was exhibited by one phage (Ib(3)) since its high titre suspensions were completely inactivated only after 45 min at 72 degrees C or 15 min at 90 degrees C. A clear protective effect of the milk was revealed when the three suspension media were compared. As regards to the effects of biocides on phages, only peracetic acid was found to be effective for inactivating high titre suspensions. Ethanol, even at a concentration of 100%, was not suitable to assure no surviving phage particles and isopropanol turned out to be less effective than ethanol. Sodium hypochlorite at 200-400 ppm inactivated the phages completely, except phage Ib(3), which was only destroyed after treatments with 1200 ppm. The diversity observed in the heat and biocide resistance of L. delbrueckii phages is useful to establish a basis for adopting the most effective thermal and chemical treatments for inactivating them in dairy plants and laboratory environments. [TOP OF PAGE]

  860. The VirB4 family of proposed traffic nucleoside triphosphatases: common motifs in plasmid RP4 TrbE are essential for conjugation and phage adsorption. Rabel,C., Grahn,A.M., Lurz,R., Lanka,E. (2003). J. Bacteriol. 185:1045-1058. Proteins of the VirB4 family are encoded by conjugative plasmids and by type IV secretion systems, which specify macromolecule export machineries related to conjugation systems. The central feature of VirB4 proteins is a nucleotide binding site. In this study, we asked whether members of the VirB4 protein family have similarities in their primary structures and whether these proteins hydrolyze nucleotides. A multiple-sequence alignment of 19 members of the VirB4 protein family revealed striking overall similarities. We defined four common motifs and one conserved domain. One member of this protein family, TrbE of plasmid RP4, was genetically characterized by site-directed mutagenesis. Most mutations in trbE resulted in complete loss of its activities, which eliminated pilus production, propagation of plasmid-specific phages, and DNA transfer ability in Escherichia coli. Biochemical studies of a soluble derivative of RP4 TrbE and of the full-length homologous protein R388 TrwK revealed that the purified forms of these members of the VirB4 protein family do not hydrolyze ATP or GTP and behave as monomers in solution. [TOP OF PAGE]

  861. Bacterial debris—an ecological mechanism for coexistence of bacteria and their viruses. Rabinovitch,A., Aviram,I., Zaritsky,A. (2003). J. Theor. Biol. 224:377-383. A model of bacteria and phage survival is developed based on the idea of shielding by bacterial debris in the system. This model is mathematically formulated by a set of four nonlinear difference equations for susceptible bacteria, contaminated bacteria, bacterial debris and phages. Simulation results show the possibility of survival, and domains of existence of stable and unstable solutions. [TOP OF PAGE]

  862. Removal of microbes from municipal wastewater effluent by rapid sand filtration and subsequent UV irradiation. Rajala,R.L., Pulkkanen,M., Pessi,M., Heinonen-Tanski,H. (2003). Water Sci. Technol. 47:157-162. The elimination of wastewater microbes is often necessary when effluent receiving waters are reused for different purposes e.g. for irrigation or as a raw water source of drinking water. In the present study, rapid sand filtration (SF) combined with the use of polyaluminium chloride coagulation was used as a pre-treatment to improve the quality of wastewater effluent before further treatment with UV irradiation. Pilot-scale experiments were run in four treatment plants in Finland. Treatment performance was followed by measuring physical and microbial parameters. Rapid sand filtration reduced suspended solids, turbidity and colour of effluents by about 90%, 70-80% and 20-50% respectively. It also improved the UV transmittance of water by up to 20%. Microbes and phosphorus were reduced by 90-99% and to 0.05 mg/L respectively. UV irradiation further reduced the number of microbes up to 99.9%. The efficiency of UV doses in pilot UV reactors was confirmed with collimated-beam device determinations and with added FRNA phages. More than 99.9% reduction of MS2 was achieved with the dose of 140mWs/cm2 in pilot UV reactors. Rapid sand filtration and the subsequent UV irradiation reduced the number of all the tested microbes to a low level, often below the detection limit. Suspended solids and the water turbidity were reduced to 1-2 mg/L and approximately 1 NTU respectively. [TOP OF PAGE]

  863. Virus cleans up food poisoning bug. Randerson,J. (2003). NewScientist. com April 23:A virus that kills the food-poisoning bacterium E. coli O157:H7 has been discovered in sheep. The virus could help eliminate the bacterium in farm animals, greatly reducing the chance of human infections. ¶ O157:H7, a toxic strain of the normally harmless gut bug E. coli, is a major cause of food poisoning. Three-quarters of cases can be traced directly to livestock, which harbour the bug without becoming ill. Meat can be contaminated when the animals are slaughtered, and manure can also be a source of infections. ¶ So Andrew Brabban at Evergreen State College in Washington state and his team wanted to test different antibiotics to find those which would eliminate the bugs from farm animals. First, they had to infect sheep with E. coli. But they hit an unexpected problem: the bacteria just kept disappearing from the animals. The team re-infected the sheep three times, and every time the bacteria mysteriously vanished. ¶ It turned out that the sheep harbour a bacteria-killing virus, or bacteriophage, that infects certain E. coli strains. When the team tested the phage against the food-poisoning bug in the lab, they found it kills 16 out of 18 toxic strains. "That includes all the big ones you've ever heard about," says Brabban, such as the strain responsible for an outbreak at Jack in the Box fast-food outlets in the US in 1993, which left four people dead. But the phage, christened CEV1, only kills eight out of 73 harmless E. coli strains. ¶ Exponential infection ¶ Brabban now hopes to use the phage to wipe out O157:H7 in herds and flocks. In a small trial in sheep, the phage reduced numbers of the toxic bacterium by 99 per cent in just two days, he told a meeting of the Society for General Microbiology in Edinburgh earlier in April. ¶ And using bacteriophages has all sorts of advantages. Phages are far more discriminating than antibiotics, so the natural microbial flora of animals' guts should not be affected. Also, while antibiotics are expensive and must be given to every animal, infecting just one animal with the CEV1 phage is likely to be enough to pass the phage to a whole herd or flock - and the numbers of the phage will rise exponentially as long as there are host bacteria left to infect. ¶ What is more, the phage seems to persist in animals, suggesting it continues to replicate in a harmless E. coli strain after all the O157:H7 bacteria have been destroyed. Finally, while bacteria can develop resistance as they do to antibiotics, the phage can out-evolve them. ¶ Brabban thinks that giving the phage to animals is more practical than using it to treat people. For instance, killing E. coli 0157:H7 releases large quantities of its toxin, which can make a patient's condition worse. And animal treatment would not have to meet the strict safety standards for human therapies, one reason why phage still are not used in the West. However, the team will need to show that the phage will not have an adverse effect on human gut flora if they are passed to people via food. [TOP OF PAGE]

  864. Quantitative detection of specific nucleic acid sequences using lambdoid bacteriophages linked by oligonucleotides to solid support. Ray,B.L., Lin,E.C.C. (2003). Hybridon, Inc. 368870(5,679,510). Cambridge, MA. The present invention provided compositions, methods and kits for detection and quantitation of pathogenic organisms. The composition of the invention is an oligonucleotide probe comprising a bacteriophage covalently linked to one site on an oligonucleotide probe complementary to a conserved region of a pathogenic organism. At a second site, the oligonucleotide probe is linked to a matrix. The oligonucleotide probe contains a region complementary to one strand of a restriciton endonuclease recognition site or an oligoribonucleotide moiety. The number of pathogenic organisms present in a biological fluid sample may be quantitated in accordance with the method of the invention by combining the composition of the invention with the sample, allowing hybridization to occur. Hybridization generates a DNA-RNA hybrid, and by adding the appropriate nucleolytic enzyme capable of cleaving DNA-RNA hybrids; bacteriophage will be released for measurement. The kit of the invention provides components which allow the method of the invention to be performed. [TOP OF PAGE]

  865. Comparison of shiga toxin production by hemolytic-uremic syndrome-associated and bovine-associated shiga toxin-producing Escherichia coli isolates. Ritchie,J.M., Wagner,P.L., Acheson,D.W.K., Waldor,M.K. (2003). Appl. Environ. Microbiol. 69:1059-1066. There is considerable diversity among Shiga toxin (Stx)-producing Escherichia coli (STEC) bacteria, and only a subset of these organisms are thought to be human pathogens. The characteristics that distinguish STEC bacteria that give rise to human disease are not well understood. Stxs, the principal virulence determinants of STEC, are thought to account for hemolytic-uremic syndrome (HUS), a severe clinical consequence of STEC infection. Stxs are typically bacteriophage encoded, and their production has been shown to be enhanced by prophage-inducing agents such as mitomycin C in a limited number of clinical STEC isolates. Low iron concentrations also enhance Stx production by some clinical isolates; however, little is known regarding whether and to what extent these stimuli regulate Stx production by STEC associated with cattle, the principal environmental reservoir of STEC. In this study, we investigated whether toxin production differed between HUS- and bovine-associated STEC strains. Basal production of Stx by HUS-associated STEC exceeded that of bovine-associated STEC. In addition, following mitomycin C treatment, Stx2 production by HUS-associated STEC was significantly greater than that by bovine-associated STEC. Unexpectedly, mitomycin C treatment had a minimal effect on Stx1 production by both HUS- and bovine-associated STEC. However, Stx1 production was induced by growth in low-iron medium, and induction was more marked for HUS-associated STEC than for bovine-associated STEC. These observations reveal that disease-associated and bovine-associated STEC bacteria differ in their basal and inducible Stx production characteristics. [TOP OF PAGE]

  866. Global phage diversity. Rohwer,F. (2003). Cell 113:141 Ten new mycobacteriophage genomes presented by Pedulla et al. (2003) show that most phage diversity remains uncharacterized. Extrapolation suggests that less than 0.0002% of the global phage metagenome has been sampled. The new genomes also contain a number of potential virulence factors that may be important in pathogenesis. [TOP OF PAGE]

  867. Los bacteriófagos como herramienta para combatir infecciones en acuicultura [abstract is in English, manuscript is in Spanish]. Ronda,C., Vázquez,M., López,R. (2003). Revista AquaTIC 18:3-10. Bacteriophages (phages), the most abundant entities in nature, have been proposed as therapeutic agents since they were isolated in the early years of the last century. The current antibiotic resistance of most pathogenic microorganisms together with the technical achievements in the study of phages has led to reconsider the work carried out for scientists of the former Soviet Union and to propose the use of bacterial viruses as a real therapeutical alternative. In this minireview we analyze the most relevant contribution on phage therapy in Aquaculture as well as the new possibility that offer the use of phage and phage products, like the lytic enzymes, named enzybiotics, as an alternative tool in therapy. [TOP OF PAGE]

  868. Virioplankton community structure along a salinity gradient in a solar saltern. Sandaa,R.-A., Skjoldal,E., Bratbak,G. (2003). Extremophiles 7:347-351. The virioplankton community structure along a salinity gradient from near seawater (40‰) to saturated sodium chloride brine (37‰) in a solar saltern was investigated by pulsed-field gel electrophoresis. Viral populations with genome sizes varying from 10 kb to 533 kb were detected. The viral community structure changed along the salinity gradient. Cluster analysis of the viral genome-banding pattern resulted in two main clusters. The virioplankton diversity within the samples with salinity from 40‰ to 15‰ was on the same cluster of a cladogram. The other group consisted of virioplankton from samples with salinity above 220‰. The virioplankton diversity in the different samples was calculated using the Shannon index. The diversity index demonstrated an increase in diversity in the samples along the gradient from 40‰ to 15‰ salinity, followed by a decrease in the diversity index along the rest of the salinity gradient. These results demonstrate how viral diversity changes from habitats that are considered one of the most common (seawater) to habitats that are extreme in salt concentrations (saturated sodium brine). The diversity index was highest in the environments that lie in between the most extreme and the most common. [TOP OF PAGE]

  869. Genome analysis of a novel Shiga toxin 1 (Stx1)-converting phage which Is closely related to Stx2-converting phages but not to other Stx1-converting phages. Sato,T., Shimizu,T., Watarai,M., Kobayashi,M., Kano,S., Hamabata,T., Takeda,Y., Yamasaki,S. (2003). J. Bacteriol. 185:3966-3971. Two Stx-converting phages, designated Stx1f and Stx2f-II, were isolated from an Escherichia coli O157:H7 strain, Morioka V526, and their entire nucleotide sequences were determined. The genomes of both phages were similar except for the stx gene-flanking regions. Comparing these phages to other known Stx-converting phages, we concluded that Stx1f is a novel Stx1-converting phage closely related to Stx2-converting phages so far reported. [TOP OF PAGE]

  870. Bioluminescent biosensor device. Sayler,G.S., Ripp,S.A., Applegate,B. (2003). University of Tennessee and Purdue Research Foundation. 910360(6,544,729). Knoxville, TN; West Lafayette, IN. Disclosed are methods and devices for detection of bacteria based on recognition and infection of one or more selected strains of bacteria with bacteriophage genetically modified to cause production of an inducer molecule in the bacterium following phage infection. The inducer molecule is released from the infected bacterium and is detected by genetically modified bacterial bioreporter cells designed to emit bioluminescence upon stimulation by the inducer. Autoamplification of the bioluminescent signal permits detection of low levels of bacteria without sample enrichment. Also disclosed are methods of detection for select bacteria, and kits for detection of select bacteria based on the described technology. [TOP OF PAGE]

  871. Bacteriophages and Clostridium spores as indicator organisms for removal of pathogens by passage through saturated dune sand. Schijven,J.F., de Bruin,H.A.M., Hassanizadeh,S.M., Roda Husman,A.M. (2003). Water Res. 37:2186-2194. In a field study on the efficiency of dune recharge for drinking water production, bacteriophage MS2 was shown to be removed 8 log(10) by passage through the dune sand. The question of whether pathogenic viruses would be removed as much as MS2 was studied by comparing complete breakthrough curves of MS2 with those of the human viruses Coxsackievirus B4 (CB4) and Poliovirus 1 (PV1) in laboratory columns. The columns were designed to closely simulate the field conditions: same sand, water, porewater velocity and temperature. Employing a two-site kinetic model to simulate breakthrough curves, attachment/detachment to two types of kinetic sites as well as inactivation of free and attached viruses were evaluated. It was found that attachment to only one of the sites is of significance for determining overall removal. At field scale, removal of the less negatively charged PV1 was extrapolated to be about 30 times greater than that of MS2, but removal of CB4 would be only as much as that of MS2. Also, removal of spores of Clostridium perfringens D10, a potential surrogate for Cryptosporidium oocysts, was studied. The attachment rate coefficient of the spores was 7.5 times greater than that of MS2. However, this does not imply that the removal of the spores is 7.5 times greater than that of MS2. Due to negligible inactivation in combination with detachment of previously attached spores, the actual removal rate of the spores depends on the duration of contamination and eventually all spores will break through. Provided no irreversible attachment or physical straining occurs, this may also be the case for other persistent microorganisms, like oocysts of Cryptosporidium. [TOP OF PAGE]

  872. Virus succession observed during an Emiliania huxleyi bloom. Schroeder,D., Oke,J., Malin,G., Wilson,W.H. (2003). Appl. Environ. Microbiol. 69:2484-2490. Denaturing gradient gel electrophoresis was used as a molecular tool to determine the diversity and to monitor population dynamics of viruses that infect the globally important coccolithophorid Emiliania huxleyi. We exploited variations in the major capsid protein gene from E. huxleyi-specific viruses to monitor their genetic diversity during an E. huxleyi bloom in a mesocosm experiment off western Norway. We reveal that, despite the presence of several virus genotypes at the start of an E. huxleyi bloom, only a few virus genotypes eventually go on to kill the bloom. [TOP OF PAGE]

  873. Genome sequence of two closely related Vibrio parahaemolyticus phages, VP16T and VP16C. Seguritan,V., Feng,I.-W., Rohwer,F., Swift,M., Segall,A.M. (2003). J. Bacteriol. 185:6434-6447. Two bacteriophages of an environmental isolate of Vibrio parahaemolyticus were isolated and sequenced. The VP16T and VP16C phages were separated from a mixed lysate based on plaque morphology and exhibit 73 to 88% sequence identity over about 80% of their genomes. Only about 25% of their predicted open reading frames are similar to genes with known functions in the GenBank database. Both phages have cos sites and open reading frames encoding proteins closely related to coliphage lambda's terminase protein (the large subunit). Like in coliphage lambda and other siphophages, a large operon in each phage appears to encode proteins involved in DNA packaging and capsid assembly and presumably in host lysis; we refer to this as the structural operon. In addition, both phages have open reading frames closely related to genes encoding DNA polymerase and helicase proteins. Both phages also encode several putative transcription regulators, an apparent polypeptide deformylase, and a protein related to a virulence-associated protein, VapE, of Dichelobacter nodosus. Despite the similarity of the proteins and genome organization, each of the phages also encodes a few proteins not encoded by the other. We did not identify genes closely related to genes encoding integrase proteins belonging to either the tyrosine or serine recombinase family, and we have no evidence so far that these phages can lysogenize the V. parahaemolyticus strain 16 host. Surprisingly for active lytic viruses, the two phages have a codon usage that is very different than that of the host, suggesting the possibility that they may be relative newcomers to growth in V. parahaemolyticus. The DNA sequences should allow us to characterize the lifestyles of VP16T and VP16C and the interactions between these phages and their host at the molecular level, as well as their relationships to other marine and nonmarine phages. [TOP OF PAGE]

  874. Wastewater reuse in on-site wastewater treatment: bacteria and virus movement in unsaturated flow through sand filter. Selas,B., Lakel,A., Andres,Y., Le Cloirec,P. (2003). Water Sci. Technol. 47:59-64. In on-site wastewater treatment plants, effluents are pre-treated by septic tank and treated by soil infiltration or sand filtration systems, with unsaturated flow conditions. These systems remove efficiently carbon, nitrogen and suspended solids. But for microbial pollution, the treatment efficiency depends on the hydrodynamic behaviour and filtering media characteristics. Contamination of superficial water and groundwater due to pathogenic viruses and pathogenic bacteria is responsible for many diseases. The objective of this study is to approach the mechanisms and operating conditions to control bacteria and virus release in the environment. Experiments were carried out on reactors of different length packed with sand. Hydraulic load of 90 cm x d(-1) with a pulse periodic flow was used. The influence of chemical composition of the solution on the treatment efficiency has also been studied. For the first time, the residence time distribution (RTD) has been studied using a conservative tracer (KI), to determine the main hydrodynamic parameters. For the second time, the RTD with bacterial and viral tracers (E. coli, bacteriophage MS2) was applied, with the aim to define microbial behaviour in filtering media, including adsorption and filtration phenomena. This work allowed us to determine retardation factors according to the hydraulic loads and chemical composition. [TOP OF PAGE]

  875. Ecological aspects of circulation of the phytopathogevic bacteria' phages in biocenoses. Semchuk,L.I., Andriychuk,O.M., Romashev,S.A., Ignatenko,T.O., Yatskovska,L.I. (2003). Ecological Bulletin Special release:498-501. [TOP OF PAGE]

  876. Detection of the phytopathogenic bacteria phages in the gills of the Black Sea fishes. Semchuk,L.I., Stepanova,O.A., Boyko,A.L., Romashev,S.A., Andriychuk,O.M., Ignatenko,T.O., Yatskovska,L.I. (2003). Bulletin of the University of Kiev. , series Biology A biological activity of phages, isolated from the gills of Black Sea fishes, to phytopathogenic bacteria was studied. It was shown the capacity of the testing cultures to support the phages reproduction during the definite time. [TOP OF PAGE]

  877. Escherichia coli O157:H7 Shiga toxin-encoding bacteriophages: integrations, excisions, truncations, and evolutionary implications. Shaikh,N., Tarr,P.I. (2003). J. Bacteriol. 185:3596-3605. As it descended from Escherichia coli O55:H7, Shiga toxin (Stx)-producing E. coli (STEC) O157:H7 is believed to have acquired, in sequence, a bacteriophage encoding Stx2 and another encoding Stx1. Between these events, sorbitol-fermenting E. coli O157:H(-) presumably diverged from this clade. We employed PCR and sequence analyses to investigate sites of bacteriophage integration into the chromosome, using evolutionarily informative STEC to trace the sequence of acquisition of elements encoding Stx. Contrary to expectations from the two currently sequenced strains, truncated bacteriophages occupy yehV in almost all E. coli O157:H7 strains that lack stx(1) (stx(1)-negative strains). Two truncated variants were determined to contain either GTT or TGACTGTT sequence, in lieu of 20,214 or 18,895 bp, respectively, of the bacteriophage central region. A single-nucleotide polymorphism in the latter variant suggests that recombination in that element extended beyond the inserted octamer. An stx(2) bacteriophage usually occupies wrbA in stx(1)(+)/stx(2)(+) E. coli O157:H7, but wrbA is unexpectedly unoccupied in most stx(1)-negative/stx(2)(+) E. coli O157:H7 strains, the presumed progenitors of stx(1)(+)/stx(2)(+) E. coli O157:H7. Trimethoprim-sulfamethoxazole promotes the excision of all, and ciprofloxacin and fosfomycin significantly promote the excision of a subset of complete and truncated stx bacteriophages from the E. coli O157:H7 strains tested; bile salts usually attenuate excision. These data demonstrate the unexpected diversity of the chromosomal architecture of E. coli O157:H7 (with novel truncated bacteriophages and multiple stx(2) bacteriophage insertion sites), suggest that stx(1) acquisition might be a multistep process, and compel the consideration of multiple exogenous factors, including antibiotics and bile, when chromosome stability is examined. [TOP OF PAGE]

  878. Temporal dynamics of natural communities of marine algal viruses and eukaryotes. Short,S.M., Suttle,C.A. (2003). Aquat. Microb. Ecol. 32:107-119. The composition of algal virus communities in relation to temperature, salinity, chlorophyll a (chl a) concentration and eukaryotic community composition was monitored at a single location for 14 mo. Changes in algal virus and eukaryote communities were determined using polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE) to generate genetic fingerprints. Sequence analysis of bands extracted from denaturing gradient gels revealed the presence of at least 5 distinct viruses as well as temporally dynamic and diverse communities of eukaryotes that included taxa from the viridiplantae, fungi and metazoa. Comparison of algal virus fingerprints with environmental conditions revealed that, at certain times, changes in algal virus community composition were coincident with changes in tide height, salinity or chl a concentration. However, algal virus community changes were not often coupled to eukaryote community changes. The lack of coincidence between changes in virus and eukaryote communities can be explained by the presence of organisms that were not hosts of the detected viruses. It is likely that the uncoupling of 18S and AVS fingerprints was due to succession among non-host eukaryotes. Although algal virus fingerprint patterns were stable throughout most of the study, stable eukaryote fingerprint patterns were observed only during the winter months. Furthermore, specific taxa of algal viruses persisted in fluctuating physical and biological environments. We concluded that the constant production of, and mortality from, some taxa of algal viruses provide further evidence that algal viruses affect phytoplankton community structure and dynamics. [TOP OF PAGE]

  879. The imprint of wild viruses on freshwater microbial ecology. Sime-Ngando,T., Bettarel,Y., Chartogne,C., Sean,K. (2003). Recent Research Development in Microbiology 7:481-497. Viruses are the most abundant and, probably, the most diverse biological items in aquatic systems. There is still little information on these biological entities in freshwaters, although some of the fundamental works on viruses are known from these ecosystems. This review focuses on the structure (diversity, abundance) and the functional role (bacterivory, impact on bacterial diversity) of viruses, in the context of freshwater environments. Freshwater viruses typically exceed 10 million per millilitre in mesotrophic waters, the majority being represented by Siphoviridae with small capsid (<60 nm) and genome (<100 kb) size, generally regarded as bacteriophages. Their relative contribution to bacterial mortality is variable but generally is lower than the impact of protists. There is evidence that phages exert a significant pressure on the community structure and the diversity of their hosts. In turn, they are sensitive to environmental factors, both in terms of integrity and infectivity. Future research directions are identified, among which the microbial ecology of cyanophages and other phytophages is critical to freshwater microbial ecology. [TOP OF PAGE]

  880. Survival of indicator organisms during enrichment on tetrachloroethene. Skramstad,J.D., Hurst,C.J., Novak,P.J. (2003). Water Environ Res 75:368-376. A laboratory study was performed as the basis for a full-scale bioaugmentation project at a site contaminated with chlorinated ethenes. The objectives of this study were to 1) develop a protocol to enrich for a tetrachloroethene (PCE)-dechlorinating culture from waste activated sludge and anaerobic digester biosolids and 2) monitor the survival of fecal coliform bacteria and bacteriophage, which model enteric viruses, during the enrichment process. A culture was enriched in 8 days with the ability to degrade 6-microM PCE to cis-dichloroethene. Using the enrichment protocol in two identical experiments, significant inactivation of fecal coliform bacteria (2 log) and somatic coliphage (0.33 log) was observed in one of the experiments; no inactivation occurred in the second experiment. The number of F-specific coliphage decreased in both experiments (0.87 and 1.26 log inactivation). Despite the decrease in some of the coliform and bacteriophage numbers, the quantity of organisms and phage particles present after enrichment was still high (approximately 7.5 x 10(5) most probable number/L, 6.9 x 10(6) plaque-forming units (PFU)/L, and 3.3 x 10(5) PFU/L, for fecal coliform bacteria, somatic coliphage, and F-specific coliphage, respectively). This may be cause for concern, depending on the current and future groundwater use at or near a site undergoing bioaugmentation with cultures derived from waste activated sludge and anaerobic digester biosolids. [TOP OF PAGE]

  881. Development of a microplate assay for the detection of single plaque-forming units of bacteriophage FX174 in crude lysates. Slattery,S.D., Valentine,C.R. (2003). Environmental and Molecular Mutagenesis 41:121-125. Mice containing the FX174 am3 transgene can be used for measuring in vivo mutation; however, the single burst analysis method used for distinguishing in vivo mutations from mutations generated during sample processing is labor-intensive. A liquid microplate assay was developed that detects a single mutant plaque-forming unit (PFU) of FX174 bacterial virus in the presence of excess nonmutant virus. The assay is based on inhibiting reduction of the tetrazolium dye, MTS, by bacterial cells selective for mutant virus. The assay is performed with crude lysates of infected bacteria and is as accurate as scoring viral plaques on a bacterial lawn. This microplate assay may have application in increasing throughput of the single burst analysis of FX174 in transgenic mouse mutation assays. [TOP OF PAGE]

  882. Viruses of hyperthermophilic Archaea. Snyder,J.C., Stedman,K., Rice,G., Wiedenheft,B., Spuhler,J., Young,M.J. (2003). Res. Microbiol. 154:474-482. The viruses of Archaea are likely to be useful tools for studying host evolution, host biochemical pathways, and as tools for the biotechnology industry. Many of the viruses isolated from Archaea show distinct morphologies and genes. The euryarchaeal viruses show morphologies similar to the head-and-tail phage isolated from Bacteria; however, sequence analysis of viral genomes from Crenarchaea shows little or no similarity to previously isolated viruses. Because viruses adapt to host organism characteristics, viruses may lead to important discoveries in archaeal biochemistry, genetics, and evolution. [TOP OF PAGE]

  883. Identification of catabolite repression as a physiological regulator of biofilm formation by Bacillus subtilis by use of DNA microarrays. Stanley,N.R., Britton,R.A., Grossman,A.D., Lazazzera,B.A. (2003). J. Bacteriol. 185:1951-1957. Biofilms are structured communities of cells that are encased in a self-produced polymeric matrix and are adherent to a surface. Many biofilms have a significant impact in medical and industrial settings. The model gram-positive bacterium Bacillus subtilis has recently been shown to form biofilms. To gain insight into the genes involved in biofilm formation by this bacterium, we used DNA microarrays representing >99% of the annotated B. subtilis open reading frames to follow the temporal changes in gene expression that occurred as cells transitioned from a planktonic to a biofilm state. We identified 519 genes that were differentially expressed at one or more time points as cells transitioned to a biofilm. Approximately 6% of the genes of B. subtilis were differentially expressed at a time when 98% of the cells in the population were in a biofilm. These genes were involved in motility, phage-related functions, and metabolism. By comparing the genes differentially expressed during biofilm formation with those identified in other genomewide transcriptional-profiling studies, we were able to identify several transcription factors whose activities appeared to be altered during the transition from a planktonic state to a biofilm. Two of these transcription factors were Spo0A and sigma-H, which had previously been shown to affect biofilm formation by B. subtilis. A third signal that appeared to be affecting gene expression during biofilm formation was glucose depletion. Through quantitative biofilm assays and confocal scanning laser microscopy, we observed that glucose inhibited biofilm formation through the catabolite control protein CcpA. [TOP OF PAGE]

  884. Relationships between fuselloviruses infecting the extremely thermophilic archaeon Sulfolobus: SSV1 and SSV2. Stedman,K.M., She,Q., Phan,H., Arnold,H.P., Hoz,I., Garrett,R.A., Zillig,W. (2003). Res. Microbiol. 154:295-302. The fusellovirus SSV2 from an Icelandic Sulfolobus strain was isolated, characterized and its complete genomic sequence determined. SSV2 is very similar in morphology, replication, genome size and number of open reading frames (ORFs) to the type virus of the family, SSV1 from Japan, except in its high level of uninduced virus production. The nucleotide sequences are, however, only 55% identical to each other, much less than related bacteriophage, related animal viruses and the rudiviruses of Sulfolobus, SIRV1 and SIRV2. Nevertheless the genome architecture is very similar between the two viruses, indicating that despite this genomic dissimilarity the virus genomes are mostly homologous. Unlike SSV1, the sequence of SSV2 indicates integration into a glycyl tRNA gene and is completely missing a DNA packaging gene. There is a unique, perfectly tandemly directly repeated sequence of 62 nucleotides in SSV2 that has no similarity to known sequences or structures. By comparison to the SSV2 genome, an integrated partial fusellovirus genome was found in the Sulfolobus solfataricus P2 genome further confirming the dynamism of the Sulfolobus genome. Clustering of cysteine codon containing ORFs both in SSV1 and SSV2 indicates that these Fuselloviridae arose from a genome fusion event. [TOP OF PAGE]

  885. Enteric virions and microbial biofilms--a secondary source of public health concern? Storey,M.V., Ashbolt,N.J. (2003). Water Sci. Technol. 48:97-104. Through their many sorption sites, microbial biofilms can accumulate both organic and inorganic particulate and colloidal material from bulk water environments. An application of such first principles to the ecology of "biocolloidal" enteric virions would suggest that they too may be concentrated by biofilms in a similar way. Though previous studies have isolated human gastrointestinal (enteric) virions from microbial biofilms, the exact human health significance of this has been neither fully investigated nor completely understood. Through an assessment of the location, accumulation and persistence of model enteric virion (fX174, MS2 and B40-8 bacteriophages as well as 20 nm fluorescent latex microspheres) within biofilms, the aim of the current study was to investigate whether the interaction of enteric virions with distribution pipe biofilms could provide a secondary source of public health concern to consumers. Model enteric virions were found to be incorporated into biofilms at concentrations representing 1% of those present in the adjacent bulk water environment. A sub-population (0.01%) of these persisted throughout an experimental period of 30 days, inferring their potential to accumulate over time. Furthermore, model enteric virions were partitioned into bacterial microcolonies, environments where biofilm bacteria can persist and re-grow, even in the presence of "acceptable" levels of disinfection. A risk model for enteric virion accumulation and release from distribution pipe biofilms suggested that associated risks may exceed USEPA benchmark values. These findings could have wide-reaching implications in water treatment and distribution strategies, and necessitate a re-appraisal of current water guideline values. [TOP OF PAGE]

  886. Cyanophages infecting the oceanic cyanobacterium Prochlorococcus. Sullivan,M.B., Waterbury,J.B., Chisholm,S.W. (2003). Nature 424:1047-1051. Prochlorococcus is the numerically dominant phototroph in the tropical and subtropical oceans, accounting for half of the photosynthetic biomass in some areas. Here we report the isolation of cyanophages that infect Prochlorococcus, and show that although some are host-strain-specific, others cross-infect with closely related marine Synechococcus as well as between high-light- and low-light-adapted Prochlorococcus isolates, suggesting a mechanism for horizontal gene transfer. High-light-adapted Prochlorococcus hosts yielded Podoviridae exclusively, which were extremely host-specific, whereas low-light-adapted Prochlorococcus and all strains of Synechococcus yielded primarily Myoviridae, which has a broad host range. Finally, both Prochlorococcus and Synechococcus strain-specific cyanophage titres were low (< 10(3) ml(-1)) in stratified oligotrophic waters even where total cyanobacterial abundances were high (> 10(5) cells x ml(-1)). These low titres in areas of high total host cell abundance seem to be a feature of open ocean ecosystems. We hypothesize that gradients in cyanobacterial population diversity, growth rates, and/or the incidence of lysogeny underlie these trends. [TOP OF PAGE]

  887. Characterization of a Vibrio cholerae phage isolated from the coastal water of Peru. Talledo,M., Rivera,I.N.G., Lipp,E.K., Neale,A., Karaolis,D., Huq,A., Colwell,R.R. (2003). Environ. Microbiol. 5:350-354. A Vibrio cholerae bacteriophage, family Myoviridae, was isolated from seawater collected from the coastal water of Lima, Peru. Genome size was estimated to be 29 kbp. The temperate phage was specific to V. cholerae and infected 12/13 V. cholerae O1 strains and half of the four non-O1/non-O139 strains tested in this study. Vibrio cholerae O139 strains were resistant to infection and highest infection rates were obtained in low nutrient media amended with NaCl or prepared using seawater as diluent. [TOP OF PAGE]

  888. [Transduction of plasmid antibiotic resistance determinants with pseudo-T-even bacteriophages]. Taniashin,V.I., Zimin,A.A., Shliapnikov,M.G., Boronin,A.M. (2003). Genetika 39:914-926. Transduction of antibiotic resistance determinants of the plasmid pBR322 with pseudoT-even bacteriophages RB42, RB43, and RB49 was studied. It is established that antibiotic resistance determinants of plasmid pBR322 from Escherichia coli recA(+)- and recA(-)-donor strains do not differ significantly in respect to the efficiency of transduction. Amber mutants RB43-21, RB43-33, and a double amber mutant RB43am21am33 were obtained. These mutants facilitated transduction experiments in some cases. Transduction of antibiotic resistance markers of the vector plasmid pBR325 and recombinant plasmid pVT123, containing a DNA fragment with hoc segE uvsW genes of phage T4, was studied. The frequency of appearance of transductants resistant to pseudoT-even bacteriophages used in transduction was determined, and the sensitivity of resistant transductants to 32 RB bacteriophages and also to phages lambda, T2, T4, T5, T6, T7, and BF23 was estimated. The efficiency of plating pseudoT-even bacteriophages RB42 and RB43 on strain E. coli 802 himA hip carrying mutations in genes that encode subunits of the Integration Host Factor (IHF) was shown to be higher than on isogenic strain E. coli 802. The growth of pseudoT-even bacteriophages limited in vivo by modification-restriction systems of chromosomal (EcoKI, EcoBI), phage (EcoP1I), and plasmid (EcoRI, EcoR124I, and EcoR124II) localization was analyzed. It was shown that these phages were only slightly restricted by the type I modification-restriction systems EcoBI, EcoR124I, and EcoR124II. Phage RB42 was restricted by systems EcoKI, EcoP1I, and EcoRI; phage RB43, by systems EcoKI and EcoRI; and phage RB49, by the EcoRI modification-restriction system. [TOP OF PAGE]

  889. Seasonal change and fate of coliphages infected to Escherichia coli O157:H7 in a wastewater treatment plant. Tanji,Y., Mizoguchi,K., Yoichi,M., Morita,M., Kijima,N., Kator,H., Unno,H. (2003). Water Res. 37:1136-1142. Seasonal change of virulent phage infected to two E. coli O157:H7 strains (O:157-phage) in the influent of a domestic wastewater treatment plant in the central part of Japan and fate of O:157-phage in the plant were monitored almost monthly from March 2001 to February 2002. Coliphage infected to nonpathogenic E. coli O157:H7 ATCC43888 (43888-phage) was detected for 1 year. On the other hand, phage infected to pathogenic E. coli O157:H7 EDL933 (EDL-phage) was detected intermittently. Concentration of EDL-phage was almost one-tenth of that of 43888-phage. The progressive decrease in phage concentration with the treatment steps was observed. No phage was detected in the supernatant from the secondary settling tank and effluent. PCR amplification of the Stx 2 gene that encodes Shiga toxin (Stx) was observed when O:157-phage concentration in the influent was high x103 PFU/ml order. Concentration and percentage of suspended O:157-phage decreased with the progress of the wastewater treatment. 933W phage, which encodes Stx 2 gene, was more fragile and sensitive to chlorination than T4 phage. However, addition of 0.02 mg/l chlorine, in conformance with the required concentration of the plant, did not affect the viability of T4 and 933 W phages. On the other hand, 1mg/l chlorine inactivated the 933 W phage significantly. [TOP OF PAGE]

  890. Set a microbe to kill a microbe: drug resistance renews interest in phage therapy. Thacker,P.D. (2003). J. Am. Med. Assoc. 290:3183-3185. Phage therapy predated antibiotics by decades, but was largely supplanted when these drugs became available. Now, however, the emerging threat posed by antibiotic-resistant pathogens is spurring a sesurgence of interest in phage, as a potential therapy to cure or prevent infetions and as a tool to kill food-borne pathogens. [TOP OF PAGE]

  891. Stable coexistence in marine algal host-virus systems. Thyrhaug,R., Larsen,A., Thingstad,T.F., Bratbak,G. (2003). Mar. Ecol. Prog. Ser. 254:27-35. All microalgal host-virus systems isolated to date are lytic: the viruses lyse the infected hosts within hours after infection. Moreover, current models of phytoplankton host-virus interactions predict rapid extinction of both host and virus. Nevertheless, marine phytoplankton and their respective viruses do coexist in marine ecosystems. To investigate this apparent paradox we performed a series of experiments which show that phytoplankton populations always recover after virus-induced lysis and that endemic viral infections may promote survival of the host population. We hypothesize that phenotypic plasticity of algal susceptibility to viral infection makes such coexistence of host and virus possible. [TOP OF PAGE]

  892. Survival of indicators of bacterial and viral contamination in wastewater subjected to low temperatures and freezing: application to cold climate waste stabilisation ponds. Torrella,F., Lopez,J.P., Banks,C.J. (2003). Water Sci. Technol. 48:105-112. The survival of bacterial and viral pollution indicators and Salmonella in urban wastewaters under freezing conditions (-14ºC for up to 60 days) is reported. Presumptive, total and faecal coliforms (PC, TC, FC), salmonellae and coliphages were tested. The dynamics of somatic coliphage (E. coli C) and F-pili specific coliphage inactivation were compared at 4ºC and 25ºC over various run times. On freezing of the wastewater, it was found that PC, TC and FC showed a first rapid phase (days) of inactivation followed by a slower second phase (up to 4 weeks) and then stabilisation at between 1-10% of the initial population size, depending on the wastewater sample used. Salmonella spp. were detectable in 0.1 ml of raw wastewater and were still detected up to 2 days after freezing but none were detected in 100 ml samples after 4, 42 and 60 days, although microbiologically similar but antigenically different forms were found. Viral indicators of pollution showed a slow but constant decrease in viability during the first month but then stabilised at between 10-20% survivors (10% in somatic E. coli C phages, 15.8% in somatic Salmonella phages and 17.9% in F-pili specific coliphages). Using electron microscopy, no difference in susceptibility to freezing could be detected with respect to morphological phage types, which were either small icosahedral particles or complex tailed phages. The study of viral indicators at 4ºC versus 25ºC showed a higher survival of the various coliphages over time at 4ºC. F-pili specific leviviridae were particularly susceptible to the antiviral factors at 25ºC and no viable units per ml were detected after one month at that temperature, whereas somatic coliphages were detected in higher numbers after this period, especially at 4ºC. [TOP OF PAGE]

  893. Transduction of porcine enteropathogenic Escherichia coli with a derivative of a shiga toxin 2-encoding bacteriophage in a porcine ligated ileal loop system. Tóth,I., Schmidt,H., Dow,M., Malik,A., Oswald,E., Nagy,B. (2003). Appl. Environ. Microbiol. 69:7242-7247. In this study, we have investigated the ability of detoxified Shiga toxin (Stx)-converting bacteriophages F3538 (Dstx(2)::cat) (H. Schmidt et al., Appl. Environ. Microbiol. 65:3855-3861, 1999) and H-19B::Tn10d-bla (D. W. Acheson et al., Infect. Immun. 66:4496-4498, 1998) to lysogenize enteropathogenic Escherichia coli (EPEC) strains in vivo. We were able to transduce the porcine EPEC strain 1390 (O45) with F3538 (Dstx(2)::cat) in porcine ligated ileal loops but not the human EPEC prototype strain E2348/69 (O127). Neither strain 1390 nor strain E2348/69 was lysogenized under these in vivo conditions when E. coli K-12 containing H-19B::Tn10d-bla was used as the stx1 phage donor. The repeated success in the in vivo transduction of an Stx2-encoding phage to a porcine EPEC strain in pig loops was in contrast to failures in the in vitro trials with these and other EPEC strains. These results indicate that in vivo conditions are more effective for transduction of Stx2-encoding phages than in vitro conditions. [TOP OF PAGE]

  894. A virus booster for game theory. Turner,P.E. (2003). ASM News 69:289-295. Experiments with a selfish genotype of RNA bacteriophage f6-a parasite of a parasite-provide evidence for prisoner's dilemma. [TOP OF PAGE]

  895. Searching for the advantages of virus sex. Turner,P.E. (2003). Origins of Life and Evolution of the Biosphere 33:95-108. Sex (genetic exchange) is a nearly universal phenomenon in biological populations. But this is surprising given the costs associated with sex. For example, sex tends to break apart co-adapted genes, and sex causes a female to inefficiently contribute only half the genes to her offspring. Why then did sex evolve? One famous model poses that sex evolved to combat Muller's ratchet, the mutational load that accrues when harmful mutations drift to high frequencies in populations of small size. In contrast, the Fisher-Muller Hypothesis predicts that sex evolved to promote genetic variation that speeds adaptation in novel environments. Sexual mechanisms occur in viruses, which feature high rates of deleterious mutation and frequent exposure to novel or changing environments. Thus, confirmation of one or both hypotheses would shed light on the selective advantages of virus sex. Experimental evolution has been used to test these classic models in the RNA bacteriophage f6, a virus that experiences sex via reassortment of its chromosomal segments. Empirical data suggest that sex might have originated in f6 to assist in purging deleterious mutations from the genome. However, results do not support the idea that sex evolved because it provides beneficial variation in novel environments. Rather, experiments show that too much sex can be bad for f6; promiscuity allows selfish viruses to evolve and spread their inferior genes to subsequent generations. Here I discuss various explanations for the evolution of segmentation in RNA viruses, and the added cost of sex when large numbers of viruses co-infect the same cell. [TOP OF PAGE]

  896. Escape from Prisoner's Dilemma in RNA phage phi6. Turner,P.E., Chao,L. (2003). Am. Nat. 161:497-505. We previously examined competitive interactions among viruses by allowing the RNA phage phi6 to evolve at high and low multiplicities of infection (ratio of infecting viruses to bacterial cells). Derived high-multiplicity phages were competitively advantaged relative to their ancestors during coinfection, but their fixation caused population fitness to decline. These data conform to the evolution of lowered fitness in a population of defectors, as expected from the Prisoner's Dilemma of game theory. However, the generality of this result is unknown; the evolution of viruses at other multiplicities may alter the fitness payoffs associated with conflicting strategies of cooperation and defection. Here we examine the change in matrix variables by propagating the ancestor under strictly clonal conditions, allowing cooperation the chance to evolve. In competitions involving derived cooperators and their selfish counterparts, our data reveal a new outcome where the two strategies are predicted to coexist in a mixed polymorphism. Thus, we demonstrate that the payoff matrix is not a constant in phi6. Rather, clonal selection allows viruses the opportunity to escape the Prisoner's Dilemma. We discuss mechanisms that may afford selfish genotypes an advantage during intrahost competition and the relevance in our system for alternative ecological interactions among viruses. [TOP OF PAGE]

  897. Calcium hypochlorite as a disinfecting additive for dental stone. Twomey,J.O., Abdelaziz,K.M., Combe,E.C., Anderson,D.L. (2003). The Journal of prosthetic dentistry 90:282-288. STATEMENT OF PROBLEM: Dental casts come into direct contact with impression materials and other items that are contaminated by saliva and blood from a patient's mouth, leaving the casts susceptible to cross-contamination. Topical methods of disinfecting casts are difficult to control, while immersion methods are potentially destructive. Thus, an additional method to control cross-contamination between patients and laboratory personnel is needed. PURPOSE: This study was undertaken in an attempt to develop a dental stone with disinfecting properties and adequate compressive and tensile strengths. MATERIAL AND METHODS: Calcium hypochlorite [Ca(OCl)(2)] in aqueous solution in concentrations from 0 to 1.5% was tested as a disinfecting additive to type V dental stone. The compressive and tensile strength properties of the modified stone were measured (MPa) using a universal testing machine at a consistency similar to unmodified stone. Strength data were analyzed by 1-way ANOVA and post hoc Tukey-Kramer procedure (alpha < or =.05). To measure the disinfecting ability, the effect on Bacillis subtilis bacteriophage phi29 was tested in triplicate to find the minimum concentration at which no phage was detected. Additionally, 3 impressions were disinfected with CaviCide, and 3 impressions rinsed in water served as controls. RESULTS: In general, the effect of adding the disinfectant to the stone was a decrease in strength. Exceptions were the dry compressive strength, for which there was a significant increase in strength (P=.048) at 0.5%, and the wet compressive and wet tensile strength, which showed no significant difference between the 1.5% and the control. When Ca(OCl)(2) was added at the concentration 0.5% (2765 ppm available chlorine), the gypsum had acceptable mechanical properties; dry compressive strength was 78.86 +/- 4.12 MPa, and dry tensile strength was 10.64 +/- 1.27 MPa, compared to control values of 67.85 +/- 6.28 and 13.41 +/- 1.24 MPa, respectively. At concentrations of 0.3% and higher (36 1650 ppm of available chlorine), calcium hypochlorite was able to completely inactivate phi29. CONCLUSION: It is possible to prepare a type V dental stone that contains a disinfectant, has adequate mechanical properties, and will reduce numbers of residual microorganisms. For example, stone mixed with water containing 0.5% Ca(OCl)(2) meets these criteria. [TOP OF PAGE]

  898. Comparative analyses of the complete genome sequences of Pierce's disease and citrus variegated chlorosis strains of Xylella fastidiosa. Van Sluys,M.A., et al (2003). J. Bacteriol. 185:1018-1026. Xylella fastidiosa is a xylem-dwelling, insect-transmitted, gamma-proteobacterium that causes diseases in many plants, including grapevine, citrus, periwinkle, almond, oleander, and coffee. X. fastidiosa has an unusually broad host range, has an extensive geographical distribution throughout the American continent, and induces diverse disease phenotypes. Previous molecular analyses indicated three distinct groups of X. fastidiosa isolates that were expected to be genetically divergent. Here we report the genome sequence of X. fastidiosa (Temecula strain), isolated from a naturally infected grapevine with Pierce's disease (PD) in a wine-grape-growing region of California. Comparative analyses with a previously sequenced X. fastidiosa strain responsible for citrus variegated chlorosis (CVC) revealed that 98% of the PD X. fastidiosa Temecula genes are shared with the CVC X. fastidiosa strain 9a5c genes. Furthermore, the average amino acid identity of the open reading frames in the strains is 95.7%. Genomic differences are limited to phage-associated chromosomal rearrangements and deletions that also account for the strain-specific genes present in each genome. Genomic islands, one in each genome, were identified, and their presence in other X. fastidiosa strains was analyzed. We conclude that these two organisms have identical metabolic functions and are likely to use a common set of genes in plant colonization and pathogenesis, permitting convergence of functional genomic strategies. [TOP OF PAGE]

  899. Transport and survival of bacterial and viral tracers through submerged-flow constructed wetland and sand-filter system. Vega,E., Lesikar,B., Pillai,S.D. (2003). Bioresource Technology 89:49-56. Untreated or improperly treated wastewater has often been cited as the primary contamination source of groundwater. The use of decentralized wastewater treatment systems has applicability around the world since it obviates the need for extensive infrastructure development and expenditures. The use of a submerged flow constructed wetland (CW) and a sand filter to remove bacterial and viral pathogens from wastewater streams was evaluated in this study Salmonella sp. and a bacteriophages tracer were used in conjunction with the conservative bromide tracer to understand the fate and transport of these organisms in these treatment systems. Viral breakthrough numbers in the sand filter and CW were similar with a Spearman Rank correlation of 0.8 (P<0.05). In the CW, the virus exhibited almost a 3-log reduction, while in the sand filter, the viruses exhibited a 2-log reduction. The bacterial tracers, however, did not exhibit similar reductions. Low numbers of bacteria and viruses were still detectable in the effluent streams suggesting that disinfection of the effluent is critical. The survival of the tracer bacteria and viruses was as expected dependent on the biotic and abiotic conditions existing within the wastewater. The results suggest that the microbial removal characteristics of decentralized wastewater treatment systems can vary and depend on factors such as adsorption, desorption and inactivation which in turn depend on the design specifics such as filter media characteristics and local climatic conditions. [TOP OF PAGE]

  900. The prophage sequences of Lactobacillus plantarum strain WCFS1. Ventura,M., Canchaya,C., Kleerebezem,M., de Vos,W.M., Siezen,R.J., Brüssow,H. (2003). Virology 316:245-255. The Lactobacillus plantarum commensal WCFS1 contains four prophage elements in its genome. Lp1 and Lp2 are two about 40-kb-long uninducible prophages that share closely related DNA packaging, head and tail genes defining a second lineage of pac-site Siphoviridae in L. plantarum, distinct from L. plantarum phage phig1e, but related to Bacillus phage SPP1 and Lactococcus phage TP901-1. Northern analysis revealed transcribed prophage genes exclusively near both attachment sites. Comparative genomics identified candidate lysogenic conversion genes (LCG) downstream of the lysis cassette and within the lysogeny module. Notable are genes with sequence similarities to putative LCG from Streptococcus pyogenes prophages and to a Bacillus plasmid. Both prophages harbored tRNA genes. R-Lp3 and R-Lp4 represent short prophage remnants; R-Lp3 abuts Lp2 and displays sequence links to cos-site Siphoviridae. [TOP OF PAGE]

  901. Integration and distribution of Lactobacillus johnsonii prophages. Ventura,M., Canchaya,C., Pridmore,D., Berger,B., Brüssow,H. (2003). J. Bacteriol. 185:4603-4608. In Lactobacillus johnsonii strain NCC533, two prophages were integrated into tRNA genes and one was disrupted by integration. In a survey, the prophages were restricted to strains sharing an essentially identical restriction pattern. Microarray analysis showed that the prophage DNA represents about 50% of the NCC533 strain-specific DNA. [TOP OF PAGE]

  902. pGIL01, a linear tectiviral plasmid prophage originating from Bacillus thuringiensis serovar israelensis. Verheust,C., Jensen,G., Mahillon,J. (2003). Microbiology (Reading England) 149:2083-2092. Bacillus thuringiensis serovar israelensis harbours, in addition to several circular plasmids, a small linear molecule of about 15 kb. Sequence analysis of this molecule, named pGIL01, showed the presence of at least 30 ORFs, five of which displayed similarity with proteins involved in phage systems: a B-type family DNA polymerase, a LexA-like repressor, two potential muramidases and a DNA-packaging protein (distantly related to the P9 protein of the tectiviral phage PRD1). Experimental evidence confirmed that pGIL01 indeed corresponds to the linear prophage of a temperate phage. This bacteriophage, named GIL01, produces small turbid plaques and is sensitive to organic solvents, which suggests the presence of lipid components in its capsid. Experiments using proteases and exonucleases also revealed that proteins are linked to the genomes of both pGIL01 prophage and GIL01 phage at their 5' extremities. Altogether, these features are reminiscent of those of phages found in the Tectiviridae family, and more specifically of those of PRD1, a broad-host-range phage of Gram-negative bacteria. Dot-blot hybridization, PFGE, PCR and RFLP analyses also showed the presence of pGIL01 variants in the Bacillus cereus group. [TOP OF PAGE]

  903. Selection of bacteriophage-resistant mutants of Streptococcus thermophilus. Viscardi,M., Capparelli,R., Di Matteo,R., Carminati,D., Giraffa,G., Iannelli,D. (2003). J. Microbiol. Meth. 55:109-119. Phage-resistant mutants have been isolated from Streptococcus thermophilus. Selection was carried out using anti-phage antibodies or Hoechst 33258-labelled phages. Two mutants out of eight tested displayed reduced acidifying capacity. Selection of the bacteria that extruded more rapidly the fluorochrome 5-6-carboxyfluorescein diacetate (CFDA) restored the acidifying capacity of these two mutants to the level of the parental strains. Mutants displaying phage resistance and good acidifying capacity were obtained in 4-5 days. New phages that are able to overcome the protection mechanisms of the existing bacteria arise continually in the dairy environment. The procedures described here permit to replace promptly the starter culture susceptible to newly emerged phages with a resistant one. [TOP OF PAGE]

  904. Rapid selection of phage-resistant mutants in Streptococcus thermophilus by immunoselection and cell sorting. Viscardi,M., Capparelli,R., Iannelli,D. (2003). Int. J. Food Microbiol. 89:223-231. Immunoselection and flow cytometry allowed the isolation from Streptococcus thermophilus strain Str31 of double mutants displaying resistance to the phage f31 and good acid production. Strain Str31 is very sensitive to phage f31. This phage-host system seemed therefore particularly suitable to test the validity of the selection method adopted in this study. Mutants were stable with respect to both characters. The isolation of the double mutants required 4 to 5 days. The approach does not involve genetic manipulations and can therefore be an alternative to genetic engineering when this technology cannot be applied. [TOP OF PAGE]

  905. Viral and bacterioplankton dynamics in two lakes with different humic contents. Vrede,K., Stensdotter,U., Lindstrom,E.S. (2003). Microb. Ecol. 46:406-415. Viral and bacterioplankton dynamics were investigated, together with the temporal variation of phage-infected bacterioplankton in two oligotrophic lakes, one humic and the other clearwater. Bacterial abundance was significantly higher in the humic lake, while the abundance of virus-like particles (VLP) was significantly higher in the clearwater lake. There were no differences in either the frequency of infected bacterial cells (FIC), or in burst size between the lakes. Because of the higher bacterial abundance in the humic lake, a larger number of bacteria were lyzed in this lake. FIC showed large seasonal changes, varying between 9 and 43%, which covers almost the entire range of previously published data from both lacustrine and marine environments. The temporal changes in VLP abundance and FIC were slow in both the humic and clearwater lakes. The burst size was low in both lakes (average value, nine in each case), probably because of the oligotrophic status of the lakes. The chlorophyll a concentrations were higher and positively correlated with VLP numbers in the clearwater lake, indicating that a significant proportion of the viruses in this lake may be phytoplankton viruses. [TOP OF PAGE]

  906. Risk management in biological evolution. Wagner,A. (2003). J. Theor. Biol. 225:45-57. I present a framework to study the evolution of traits that allow an organism to survive life-threatening but rare risks. Specifically, I am concerned with risks so rare that any one individual in a population may not experience the risk-causing event in its lifetime. A theory of rare risk management is virtually absent in evolutionary biology, although it is well developed in economics. This is surprising because of the great influence economics had on evolutionary biology, and because biology is full of examples for evolved risk management traits. They include the ability of bacteria to sporulate, of pathogens to survive antibiotic treatment, of temperate bacteriophages to enter a lytic life cycle, as well as traits that allow higher organisms to survive rare environmental disasters, such as sporadic wildfires and irregular flooding. I make predictions about the sustenance of risk management traits under two scenarios, one where the catastrophic events cause individual deaths, and another one where catastrophic events cause population extinction. A well-developed theory of risk management will not only predict the distribution of risk management traits, but may also serve other purposes, such as to reconstruct the spectrum of environments that an organism encountered in its evolutionary history from the record stored in its genome's memory. [TOP OF PAGE]

  907. Three Bacillus anthracis bacteriophages from topsoil. Walter,M.H., Baker,D.D. (2003). Curr. Microbiol. 47:55-58. Three Bacillus anthracis bacteriophages from Iowa topsoil are characterized as to latent period, morphology, structural proteins, DNA size, and restriction endonuclease digestion. Electron micrographs indicate that the three isolates include two members of the Myoviridae and one smaller phage belonging to the Podoviridae. Phages Nk and DB resemble Myoviridae phage SP50 in morphology, but host range studies, protein, and DNA analysis indicate that both differ from SP50. Phage MH is very similar to phage phi 29, but differs in terms of host range, structural protein, and DNA characteristics. [TOP OF PAGE]

  908. Efficacy and durability of Bacillus anthracis bacteriophages used against spores. Walter,M.H. (2003). J. Environ. Health 66:9-15, 24. Antibiotics and vaccines help fight anthrax disease, but there are no anthrax spore control methods suitable for use in environments where humans are present. The work reported in this article indicates that bacteriophages may help reduce risk from anthrax spores. Dose-response studies demonstrated that higher concentrations of mixed Bacillus anthracis bacteriophages (3.5 x 10(8) plaque-forming units per milliliter) inhibited subsequent growth of bacteria when sprayed on B. anthracis spores. Phages also were tested for durability under conditions designed to simulate environments possibly encountered during mass phage production, storage, and use against anthrax spores. They remained infectious at temperatures from -20ºC to 37ºC, under filtration, aerosolization, and treatments with perspiration and blood. Phages were sensitive to temperatures over 55ºC and to desiccation. Ultraviolet light reduced spore viability more than phage infectivity under similar conditions. The potential for personal or environmental decontamination of anthrax spores with phages is discussed. [TOP OF PAGE]

  909. Cell death in Pseudomonas aeruginosa biofilm development. Webb,J.S., Thompson,L.S., James,S., Charlton,T., Tolker-Nielsen,T., Koch,B., Givskov,M., Kjelleberg,S. (2003). J. Bacteriol. 185:4585-4592. Bacteria growing in biofilms often develop multicellular, three-dimensional structures known as microcolonies. Complex differentiation within biofilms of Pseudomonas aeruginosa occurs, leading to the creation of voids inside microcolonies and to the dispersal of cells from within these voids. However, key developmental processes regulating these events are poorly understood. A normal component of multicellular development is cell death. Here we report that a repeatable pattern of cell death and lysis occurs in biofilms of P. aeruginosa during the normal course of development. Cell death occurred with temporal and spatial organization within biofilms, inside microcolonies, when the biofilms were allowed to develop in continuous-culture flow cells. A subpopulation of viable cells was always observed in these regions. During the onset of biofilm killing and during biofilm development thereafter, a bacteriophage capable of superinfecting and lysing the P. aeruginosa parent strain was detected in the fluid effluent from the biofilm. The bacteriophage implicated in biofilm killing was closely related to the filamentous phage Pf1 and existed as a prophage within the genome of P. aeruginosa. We propose that prophage-mediated cell death is an important mechanism of differentiation inside microcolonies that facilitates dispersal of a subpopulation of surviving cells. [TOP OF PAGE]

  910. Bacteriophages as an efficient therapy for antibiotic-resistant septicemia in man. Weber-Dabrowska,B., Mulczyk,M., Gorski,A. (2003). Transplant. Proc. 35:1385-1386. [first paragraph] Acute bacterial infection-induced sepsis, with shock, metabolic acidosis, oliguria, or hypoxemia, remains a major medical challenge, especially at a time when experts believe that we may be returning to the pre-antibiotic era, arising from increasing antibiotic resistance In the USA alone there are at least 500,000 cases of sepsis annually, with mortality rates ranging from 30% to 50% (ie, 150,000 to 250,000 deaths). Assuming a U.S. population of approximately 270 million and a total world population of >6 billion, this would mean at least 11 million cases of sepsis worldwide with at least 3 to 5 million deaths annually (or probably more, as American health-care standards are generally much higher than in many other countries). Several treatments designed to reduce sepsis-associated mortality have been unsuccessful; therefore, finding an effective new therapy for sepsis is urgently needed. [TOP OF PAGE]

  911. Lysogeny and virus-induced mortality of bacterioplankton in surface, deep, and anoxic waters. Weinbauer,M., Brettar,I., Höfle,M. (2003). Limnol. Oceanogr. 48:1457-1465. Lysogeny (bacteria containing inducible prophages) and lytic viral infection (bacteria in a lytic stage of infection) were investigated at the community level in contrasting marine environments such as estuarine versus offshore waters, surface versus deep waters, and oxic versus anoxic waters in the Mediterranean and Baltic Seas. The frequency of lysogenic cells (FLC) in bacterioplankton communities ranged from not detectable to 84% as estimated by prophage induction due to mitomycin C, and highest values were typically found in deep waters (800–2,000 m). Transmission electron microscopy based estimates of virus-induced mortality of bacterioplankton (VMB) ranged from a few percent to 71%, and highest values were found in anoxic waters of the Baltic Sea. FLC and the frequency of infected cells (FIC) were related in form of a negative power function indicating that environments exist where one of the two viral life strategies prevails. Across all investigated environments, FLC was negatively related to bacterial abundance and production, whereas FIC showed a positive relationship with viral and bacterial parameters. FIC was higher and FLC was lower in moderately productive estuarine and offshore surface waters than in less productive mesopelagic and deep waters. Thus, lysogeny seems to be a survival strategy at low host abundance and activity, whereas high host abundance and activity seems to favor the lytic life cycle. The key process for the prevalence of lytic infection compared to prophage replication at high host abundance could be competition due to outnumbering. Between 11% and 88% (average, 35%) of the bacteria contained a functional (lytic or lysogenic) viral genome. [TOP OF PAGE]

  912. Comparing the effects of resource enrichment and grazing on viral production in a meso-eutrophic reservoir. Weinbauer,M.G., Christaki,U., Nedoma,J., Simek,K. (2003). Aquat. Microb. Ecol. 31:137-144. As viral production depends on bacteria, factors which influence bacterial production should also impact viral production. Likewise, viruses and heterotrophic nanoflagellates (HNF) both exploit bacterial prey, so HNF grazing could influence interactions between viruses and bacteria. To examine these relationships, we examined samples from experiments in which natural bacterial populations were subjected to relaxation of nutrient limitation and different levels of grazing pressure from HNF. We observed that stimulation of bacterial production and abundance with the relaxation of nutrient limitation resulted in a higher standing stock of viruses, higher viral production and also a higher virus-induced lysis rate of bacterioplankton. These relationships suggest that the relative effect of virus-induced mortality is higher in more productive environments. We found that viral abundance, viral production and virus-induced mortality of bacteria was highest in the treatments in which grazing rates on bacteria by HNF were highest, and lowest in the treatments where no eukaryotic predators were present. Thus, high grazing rates were associated with high virus production rates. The resource enrichment had a stronger effect on viral production and infection of bacteria than grazing. Averaged over time for single treatments, viruses lysed a significant portion (range, 18 to 66%) of the bacterial production per day. [TOP OF PAGE]

  913. Use of genetically engineered phage to deliver antimicrobial agents to bacteria: an alternative therapy for treatment of bacterial infections. Westwater,C., Kasman,L.M., Schofield,D.A., Werner,P.A., Dolan,J.W., Schmidt,M.G., Norris,J.S. (2003). Antimicrob. Agents Chemother. 47:1301-1307. The emergence and increasing prevalence of multidrug-resistant bacterial pathogens emphasizes the need for new and innovative antimicrobial strategies. Lytic phages, which kill their host following amplification and release of progeny phage into the environment, may offer an alternative strategy for combating bacterial infections. In this study, however, we describe the use of a nonlytic phage to specifically target and deliver DNA encoding bactericidal proteins to bacteria. To test the concept of using phage as a lethal-agent delivery vehicle, we used the M13 phagemid system and the addiction toxins Gef and ChpBK. Phage delivery of lethal-agent phagemids reduced target bacterial numbers by several orders of magnitude in vitro and in a bacteremic mouse model of infection. Given the powerful genetic engineering tools available and the present knowledge in phage biology, this technology may have potential use in antimicrobial therapies and DNA vaccine development. [TOP OF PAGE]

  914. Suppression of Salmonella growth by wild-type and large-plaque variants of bacteriophage Felix O1 in liquid culture and on chicken frankfurters. Whichard,J.M., Sriranganathan,N., Pierson,F.W. (2003). J. Food Prot. 66:220-225. The bacteriophage Felix O1, a member of Myoviridae, is specific for, and possesses a broad host range within, the genus Salmonella. This work explores a Felix O1 phage-based intervention for Salmonella enterica serotype Typhimurium DT104 that is potentially applicable at several stages of animal production and processing. A variant of Felix O1 was obtained that produces a larger, clearer plaque phenotype (LP) on Salmonella Typhi than wild-type Felix O1 (WT) does, not unlike r mutants of phage T4. LP exhibited slightly more extensive overall suppression of Salmonella Typhi in brain heart infusion (BHI) broth, as ascertained on the basis of culture turbidity (optical density at 600 nm). Both phage variants suppressed log phase BHI broth cultures containing 8.2 x 10(6) CFU of Salmonella Typhimurium DT104 per ml. A PFU/CFU ratio of 1.0 was effective for WT and LP, whereas increasing the PFU/CFU ratio to 5.0 did not increase suppression. Untreated Salmonella-contaminated frankfurters were compared with treated samples (PFU/CFU ratio, 1.9 x 10(4)) to test WT and LP for their ability to suppress Salmonella growth on chicken frankfurters contaminated with 300 CFU of Salmonella Typhimurium DT104. Suppression levels of 1.8 and 2.1 log units were achieved with WT and LP, respectively (P = 0.0001), but no difference was found between the performances of the two variants (P = 0.5088). [TOP OF PAGE]

  915. Sampling natural viral communities from soil for culture-independent analyses. Williamson,K.E., Wommack,K.E., Radosevich,M. (2003). Appl. Environ. Microbiol. 69:6628-6633. An essential first step in investigations of viruses in soil is the evaluation of viral recovery methods suitable for subsequent culture-independent analyses. In this study, four elution buffers (10% beef extract, 250 mM glycine buffer, 10 mM sodium pyrophosphate, and 1% potassium citrate) and three enumeration techniques (plaque assay, epifluorescence microscopy [EFM], and transmission electron microscopy [TEM]) were compared to determine the best method of extracting autochthonous bacteriophages from two Delaware agricultural soils. Beef extract and glycine buffer were the most effective in eluting viable phages inoculated into soils (up to 29% recovery); however, extraction efficiency varied significantly with phage strain. Potassium citrate eluted the highest numbers of virus-like particles from both soils based on enumerations by EFM (mean, 5.3 x 10(8) g of dry soil(-1)), but specific soil-eluant combinations posed significant problems to enumeration by EFM. Observations of virus-like particles under TEM gave confidence that the particles were, in fact, phages, but TEM enumerations yielded measurements of phage abundance (mean, 1.5 x 10(8) g of dry soil(-1)) that were about five times lower. Clearly, the measurement of phage abundance in soils varies with both the extraction and enumeration methodology; thus, it is important to assess multiple extraction and enumeration approaches prior to undertaking ecological studies of phages in a particular soil. [TOP OF PAGE]

  916. Wide geographic distribution of bacteriophages that lyse the same indigenous freshwater isolate (Sphingomonas sp. strain B18). Wolf,A., Wiese,J., Jost,G., Witzel,K.P. (2003). Appl. Environ. Microbiol. 69:2395-2398. An indigenous freshwater bacterium (Sphingomonas sp. strain B18) from Lake Plubetasee (Schleswig-Holstein, Germany) was used to isolate 44 phages from 13 very different freshwater and brackish habitats in distant geographic areas. This bacterial strain was very sensitive to a broad spectrum of phages from different aquatic environments. Phages isolated from geographically distant aquatic habitats, but also those from the same sample, were diverse with respect to morphology and restriction pattern. Some phages were widely distributed, while different types coexisted in the same sample. It was concluded that phages could be a major factor in shaping the structure of bacterial communities and maintaining a high bacterial diversity. [TOP OF PAGE]

  917. Sorption of MS2 bacteriophage to layered double hydroxides: effects of reaction time, pH, and competing anions. You,Y., Vance,G.F., Sparks,D.L., Zhuang,J., Jin,Y. (2003). J. Environ. Qual. 32:2046-2053. Batch sorption and column breakthrough studies were conducted to investigate the potential of layered double hydroxides (LDHs) to remove bacteriophage MS2 from contaminated waters. All four of the LDHs evaluated in this study had very high retention capacities for MS2. Sorption results showed that MS2 could be completely removed from 5.2 x 10(2) plaque-forming units (pfu)/mL solution by Mg-Al LDH 2 (i.e., 2:1 Mg to Al ratio LDH), with the highest sorption capacity observed in this study of 1.51 x 10(10) pfu/g. Attachment of MS2 to LDHs was a rapid process and reached quasi-equilibrium after a 1-h reaction time. Within the pH range studied (pH 4-9), Mg-Al LDH 2 showed high sorption potential for MS2 at all pH values but sorption decreased slightly with increasing solution pH. Background solution anions influenced virus sorption, with SO4(2-) and HPO4(2-) decreasing sorption significantly whereas the presence of NO3- had little effect on the attachment of MS2 to Mg-Al LDH 2. The addition of another virus (phiX174) only caused a slight decrease in the retention of MS2 by Mg-Al LDH 2, suggesting that there was insignificant competitive sorption between MS2 and phiX174 on LDH surfaces. Results from column experiments indicate that there was no MS2 breakthrough from columns packed with Mg-Al LDH 2-coated sand, suggesting complete MS2 retention at the virus concentration tested. The high mass recovery by beef extract solution revealed that the removal of viruses by the LDH was due to sorption of MS2 to LDH surfaces, rather than inactivation. [TOP OF PAGE]

  918. Genome of Xanthomonas oryzae bacteriophage Xp10: an odd T-odd phage. Yuzenkova,J., Nechaev,S., Berlin,J., Rogulja,D., Kuznedelov,K., Inman,R., Mushegian,A., Severinov,K. (2003). J. Mol. Biol. 330:735-748. Xp10 is a lytic bacteriophage of the phytopathogenic bacterium Xanthomonas oryzae. Though morphologically Xp10 belongs to the Syphoviridae family, it encodes its own single-subunit RNA polymerase characteristic of T7-like phages of the Podoviridae family. Here, we report the determination and analysis of the 44,373 bp sequence of the Xp10 genome. The genome is a linear, double-stranded DNA molecule with 3' cohesive overhangs and no terminal repeats or redundancies. Half of the Xp10 genome contains genes coding for structural proteins and host lysis functions in an arrangement typical for temperate dairy phages that are related to the Escherichia coli lambda phage. The other half of the Xp10 genome contains genes coding for factors of host gene expression shut-off, enzymes of viral genome replication and expression. The two groups of genes are transcribed divergently and separated by a regulatory region, which contains divergent promoters recognized by the host RNA polymerase. Xp10 has apparently arisen through a recombination between genomes of widely different phages. Further evidence of extensive gene flux in the evolution of Xp10 includes a high fraction (10%) of genes derived from an HNH-family endonuclease, and a DNA-dependent DNA polymerase that is closer to a homolog from Leishmania than to DNA polymerases from other phages or bacteria. [TOP OF PAGE]

  919. Virus retention and transport as influenced by different forms of soil organic matter. Zhuang,J., Jin,Y. (2003). J. Environ. Qual. 32:816-823. Organic materials are widespread in natural soil and aquatic environments. Their effect on virus transport is very important in assessing the risk for contamination of ground water by viruses. This study aimed to determine how different forms (mineral-associated and dissolved) of natural organic matter influence the retention and transport of two bacteriophages (MS-2 and fX174) in two porous media (a sand and a soil). We found that mineral-associated organic matter significantly promoted the transport of one virus (MS-2) but not the other (fX174) in a phosphate-buffered saline solution. Similarly, MS-2 was retained less in sand columns with increasing concentrations of dissolved humic acid, while little effect was observed for fX174 under the same conditions. The two viruses have different surface properties and thus exhibited different reactivity to the metal oxides present on sand particles and were affected differently by organic matter. Because the organic matter used in the study was negatively charged and hydrophilic, blocking of virus sorption sites and increasing of virus-medium electrostatic repulsion arising from modification of the sand and virus surface by organic matter are probably responsible for the facilitated transport. For dissolved humic acid, its competition for sorption sites with viruses was an additional mechanism involved. This study suggests that the effect of organic matter varied depending on the organic material properties and the type of viruses involved. As a general trend, the effect of organic matter was dominated by electrostatic rather than hydrophobic interactions. [TOP OF PAGE]

  920. [Bacteriophages in the milk industry (they are no small enemy)] Los bacteriofagos en la industria láctea (no hay enemigo pequeño) [Spanish]. Anonymous (2002). Industrias Lácteas Españolas 32-35. [TOP OF PAGE]

  921. Marschall Italian & Specialty Cheese Seminars. Anonymous (2002). [TOP OF PAGE]

  922. A new phage may help control pathogens on fresh-cut produce. Anonymous (2002). Journal of Environmental Health 64(9), 59. Those fresh-cut fruits and vegetables in the grocery store are convenient, but they also have the potential to become another channel for human pathogens. So two Agricultural Research Service (ARS) scientists are testing the concept of using phages--viruses that infect and kill only bacteria--to control foodborne pathogens on produce. Early results are promising. ¶ ARS plant pathologists Britta Leverentz and Bill Conway have been the first to test phages on fruits and vegetables. While the peel or rind of intact fruit provides a physical and chemical barrier, microbes can multiply rapidly on cut surfaces, especially if those surfaces are not too acidic. ¶ The scientists are working under a cooperative agreement with Intralytix, of Baltimore, Maryland, which is providing known phages for Salmonella strains as a model. Phages are very selective about their host bacteria. Those specific for Salmonella, for instance, would leave beneficial bacteria free to multiply on freshcut produce and crowd out potential pathogens. ¶ The researchers tested a cocktail of four anti-Salmonella phages on fresh-cut melons, which have low acidity and on apples, which have higher acidity. The phages consistently reduced Salmonella more than a 1,000-fold on melon chunks stored at 40[degrees]F and 50[degrees]F, and more than 100-fold on fruit stored at room temperature. ¶ Those results come closer to the industry's goal of the 100,000-fold reductions than occurs with chlorine and other sanitizers now in use. The food industry is looking for alternatives because bacteria are developing resistance. Also, chlorine can be irritating to users, and solutions are often too dilute to reduce bacteria more than 10- to 100-fold. ¶ On apples, the cocktail was ineffective. Researchers are looking for acid-tolerant phages or a way to buffer the inoculum for high-acid produce. An article on this research appears in the July 2001 issue of Agricultural Research. [TOP OF PAGE]

  923. Bacteria and bacteriophage detection using immobilized enzyme substrates. Adams,C.A., Krejcarek,G.E., Wicks,J.H. (2002). 3M Innovative Properties Company. 548157(6,436,661). St. Paul, MN. Methods of detecting bacteria including the use of an immobilized enzyme substrate, and the immobilized enzyme substrate. [TOP OF PAGE]

  924. Bacteriophage, a process for the isolation thereof, and a universal growth medium useful in the process thereof. Agrawal,P., Soni,V. (2002). Council of Scientic and Industrial Research. 295851(6,482,632). New Delhi, IN. The present invention provides a isolated bacteriophage useful as a tool for studying biological, biochemical, physiological and genetic properties of actinomycetes and other organisms which comprises a novel strain of Saccharomonospora having certain specified characteristics. The invention also relates to a process for the isolation of the said bacteriophage and/or DNA phage and to a novel universal growth medium which is particularly useful in the said process. Another embodiment of the process relates to a cloning vector which comprises a plasmid or bacteriophage comprising the phage DNA of the invention. [TOP OF PAGE]

  925. Treatment of post-burns bacterial infections by bacteriophages, specifically ubiquitous Pseudomonas spp. notoriously resistant to antibiotics. Ahmad,S.I. (2002). Med Hypotheses 58:327-331. Post-burn microbial infections are a major problem in recovering from the trauma of third-degree burns, and the survival of patients can depend upon the severity of the burn and the infections encountered. Within 24 hours, patients can start suffering from opportunistic bacterial attacks, which can vary from simple infection, such as those easily treatable by antibiotics, to more complicated types, which may have natural or acquired resistance to drugs. Infection by multiple drug-resistant bacteria can create additional complexity to the problem. As an alternative to treating bacterial infections by antibiotics, bacteriophages have been in use in certain parts of the world, such as at Tbilisi in Georgia and in Poland, and this approach has now been more widely recognized. Results have shown that phage therapy has an 80% success rate against Enterococcus infections and up to 90% against Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli and Klebsiella pneumoniae. Here it is proposed that bacteriophages can effectively be used for the treatment of post-burn infections, particularly the ubiquitous opportunistic pathogens, Pseudomonas spp., known to be notoriously resistant to a variety of antibiotics. This kind of treatment may be of particular importance in Third World countries where the incidence of burns and infections, due to lack of stringent safety regulations and proper hygiene respectively, may be more common and where cocktails of antibiotics may be less affordable. Phages that can possibly be employed in the treatment and their advantages compared to the use of antibiotics are also highlighted. [TOP OF PAGE]

  926. Complete genomic sequence of SfV, a serotype-converting temperate bacteriophage of Shigella flexneri. Allison,G.E., Angeles,D., Tran-Dinh,N., Verma,N.K. (2002). J. Bacteriol. 184:1974-1987. Bacteriophage SfV is a temperate serotype-converting phage of Shigella flexneri. SfV encodes the factors involved in type V O-antigen modification, and the serotype conversion and integration-excision modules of the phage have been isolated and characterized. We now report on the complete sequence of the SfV genome (37,074 bp). A total of 53 open reading frames were predicted from the nucleotide sequence, and analysis of the corresponding proteins was used to construct a functional map. The general organization of the genes in the SfV genome is similar to that of bacteriophage lambda, and numerous features of the sequence are described. The superinfection immunity system of SfV includes a lambda-like repression system and a P4-like transcription termination mechanism. Sequence analysis also suggests that SfV encodes multiple DNA methylases, and experiments confirmed that orf-41 encodes a Dam methylase. Studies conducted to determine if the phage-encoded methylase confers host DNA methylation showed that the two S. flexneri strains analyzed encode their own Dam methylase. Restriction mapping and sequence analysis revealed that the phage genome has cos sites at the termini. The tail assembly and structural genes of SfV show homology to those of phage Mu and Mu-like prophages in the genome of Escherichia coli O157:H7 and Haemophilus influenzae. Significant homology (30% of the genome in total) between sections of the early, regulatory, and structural regions of the SfV genome and the e14 and KpLE1 prophages in the E. coli K-12 genome were noted, suggesting that these three phages have common evolutionary origins. [TOP OF PAGE]

  927. Steps toward mapping the human vasculature by phage display. Arap,W., et al (2002). Nat. Med. 8:121-127. The molecular diversity of receptors in human blood vessels remains largely unexplored. We developed a selection method in which peptides that home to specific vascular beds are identified after administration of a peptide library. Here we report the first in vivo screening of a peptide library in a patient. We surveyed 47,160 motifs that localized to different organs. This large-scale screening indicates that the tissue distribution of circulating peptides is nonrandom. High-throughput analysis of the motifs revealed similarities to ligands for differentially expressed cell-surface proteins, and a candidate ligand-receptor pair was validated. These data represent a step toward the construction of a molecular map of human vasculature and may have broad implications for the development of targeted therapies. [TOP OF PAGE]

  928. Characterization of Serratia isolates from soil, ecological implications and transfer of Serratia proteamaculans subsp. quinovora Grimont et al. 1983 to Serratia quinivorans corrig., sp. nov. Ashelford,K.E., Fry,J.C., Bailey,M.J., Day,M.J. (2002). Int J Syst Evol Microbiol 52:2281-2289. Eleven strains of Serratia were isolated from different soils and the guts of invertebrates and characterized by their sensitivity to eight indigenous bacteriophages. They were also classified according to bacteriocin production and sensitivity, BiOLOG plate and API 20E strip profiles and 16S rRNA sequence information. One strain was thus identified as Serratia plymuthica, another as Serratia fonticola. The remaining strains were shown to be closely related to Serratia proteamaculans subsp. quinovora Grimont et al. 1983 after DNA-DNA cross-hybridization demonstrated relatedness greater than 70% with the type strain of this subspecies. From an ecological perspective, our results illustrated the wide variation in sensitivity that closely related Serratia strains have towards various indigenous soil phages and that these phages have broad host ranges within the genus. Furthermore, the phage and bacteriocin interactions within the Serratia strains examined were intricate and did not reflect phylogenetic relationships. These results together imply that complex interactions will occur in soil within the natural community of Serratia strains and their bacteriophages. DNA-DNA cross-hybridization and phenotypic characterization showed that S. proteamaculans subsp. quinovora strains formed a cohesive group at the species level. It is therefore concluded that these strains should be designated as Serratia quinivorans corrig., sp. nov. [TOP OF PAGE]

  929. Particle transport in a karst aquifer: natural and artificial tracer experiments with bacteria, bacteriophages and microspheres. Auckenthaler,A., Raso,G., Huggenberger,P. (2002). Water Sci. Technol. 46:131-138. Fast changes in spring water quality in karst areas are a major concern for production of drinking water and require detailed knowledge of the complex interaction between karst aquifer, transport behavior of microorganisms and water treatment. We have conducted artificial and natural particle transport experiments at a karst spring with bacteria, bacteriophages, microspheres, and pathogens. Transport of the investigated microorganisms, turbid matter and chemical pullutants as well as increase in discharge are strongly related to precipitation and the heterogeneity of the aquifer. The indicator bacteria E. coli revealed a significant correlation to verotoxin-producing E coli and Cryptosporidium spp. We conclude that artificial particle tracers can help identify 'hot spots' for microbial recharge and that system parameters in spring water such as turbidity, UV-extinction and increase in discharge can be key parameters for efficient raw water management. [TOP OF PAGE]

  930. Epigenetics as a first exit problem. Aurell,E., Sneppen,K. (2002). Phys. Rev. Lett. 88:048101 We develop a framework to discuss the stability of epigenetic states as first exit problems in dynamical systems with noise. We consider in particular the stability of the lysogenic state of the lambda prophage. The formalism defines a quantitative measure of robustness of inherited states. [TOP OF PAGE]

  931. Bacteriophage composition useful in treating food products to prevent bacterial contamination. Averback,P., Gemmell,J. (2002). Nymox Pharmaceutical Corporation. 718093(6,461,608). St. Laurent, CA. The present invention is directed to novel bacteriophage compositions useful in treating food products to prevent bacterial contamination. [TOP OF PAGE]

  932. The hyaluronan lyase of Streptococcus pyogenes bacteriophage H4489A. Baker,J.R., Dong,S., Pritchard,D.G. (2002). Biochem. J. 365:317-322. Many pathogenic streptococci produce extracellular hyaluronan lyases which are thought to aid the spread of the organism in host tissues. In addition, several phages of group A streptococci are known to synthesize a bound form of hyaluronidase. It has been suggested that the function of this hyaluronidase is to facilitate penetration of the hyaluronan capsule by phage and thus to gain access for the phage to the cell surface of the host streptococcus [Hynes, Hancock and Ferretti (1995) Infect. Immun. 63, 3015-3020]. In the present work, the hyaluronidase of Streptococcus pyogenes bacteriophage H4489A, expressed in E. coli, has been purified and characterized. The enzyme was shown to be a lyase with a distributive action pathway. Unlike most bacterial hyaluronidases that have been characterized, the phage enzyme was found to specifically cleave hyaluronan, which adds credence to the view that its function is to digest the hyaluronan capsule of the host organism. This bacteriophage lyase may provide a practical alternative to the lyase from Streptomyces hyalurolyticus as a reagent for the specific cleavage of hyaluronan. [TOP OF PAGE]

  933. Fundamental changes in light scattering associated with infection of marine bacteria by bacteriophage. Balch,W.M., Vaughn,J.M., Novotny,J.F., Drapeau,D.T., Goes,J.I., Booth,E., Lapierre,J.M., Vining,C.L., Ashe,A., Vaughn,J.M., Jr. (2002). Limnol. Oceanogr. 47:1554-1561. Bacteria and phytoplankton are key determinants of the ocean's inherent optical properties. Despite their high abundance, marine viruses have generally been thought to play a minor role in ocean optics because of their small scattering cross-sections. Nevertheless, the role of specific viral infection on the optical properties of bacteria and phytoplankton has remained unknown (i.e., as viruses disrupt micron-sized host cells to produce submicron cell debris). Here, we used laboratory and mesocosm cultures of marine bacteria for virus infection experiments in which growth conditions and host-virus specificity were controlled. We report that the chief optical impact of viruses is associated with infection and lysis of their hosts. We quantitatively describe, for the first time, two optical changes associated with infection and lysis of marine bacteria by bacteriophage: (1) rapid, strong shifts in the magnitude and shape of the optical volume scattering function and (2) rapid production of colored dissolved organic material. Qualitatively, these changes result in nearly complete clearing of turbid host bacterial suspensions. Although some optical differences would be expected between infection of bacteria in laboratory cultures versus field populations (mainly because of differences in cell size), these results are applicable to the field, especially for dense host suspensions such as in blooms. Even in nonbloom situations, as long as the host bacteria contribute a significant amount of the total particle backscattering, we expect that virus-induced backscattering changes would be detectable by use of satellite or aircraft remote-sensing techniques. [TOP OF PAGE]

  934. Strategies for improving the efficacy of bacteriophages for controlling bacterial spot of tomato. Balogh,B. (2002). University of Florida. Bacterial spot, caused by the bacterium pv. vesicatoria, is one of the major tomato diseases in Florida. The disease is routinely controlled by the application of copper-mancozeb, a mixture of chemical pesticides; however, there is no adequate control measure when the environmental conditions are conducive for disease development. A novel method for controlling this disease is the application of a mixture of bacteriophages, viruses that infect bacteria. However, these control agents are rapidly degraded by harmful environmental factors such as sunlight or desiccation, which delimits the efficacy of phage treatment. It has been hypothesized that the efficacy of phage treatment could be enhanced if the longevity of the viruses was increased. ¶ Three formulations were developed that enhanced the longevity of bacteriophages on plant foliage. These formulations were (i) PCF (0.5% pregelatinized corn flour (PCPF 400, Lauhoff Grain Co., Danville, IL ) + 0.5% sucrose), (ii) Casecrete (0.5% Casecrete NH-400, a water-soluble casein protein polymer (American Casein Company, Burlington, NJ)+ 0.5% sucrose + 0.25% PCPF 400)), and (iii) skim milk (0.75% powdered skim milk + 0.5% sucrose). The use of these formulations resulted in a 4,700, 38,500 and 100,000-fold increase in phage populations two days after application compared to the non-formulated phage populations. ¶ The PCF, Casecrete and skim milk formulations and the non-formulated phages all significantly reduced disease severity in field trials on tomato compared to the standard copper-mancozeb treatment by 22, 33, 27 and 19%, respectively. The PCF and the Casecrete formulations reduced the disease severity compared to the non-formulated phage by 11 and 21% in average in three field experiments, respectively. The skim milk formulation reduced the disease severity by 10% compared to the non-formulated phage application in one field experiment. ¶ The co-application of skim milk-formulated phages and copper-mancozeb treatment resulted in a superior disease control efficacy, which was significantly better than the control achieved by any of the treatments. The integration of phage application with Actigard treatment resulted in a significant increase in efficacy only with the PCF formulation but not with Casecrete formulation. The lowest phage titer that significantly reduced disease development in greenhouse experiment was 106 PFU/ml. [TOP OF PAGE]

  935. The fundamental contribution of phages to GAS evolution, genome diversification and strain emergence. Banks,D.J., Beres,S.B., Musser,J.M. (2002). Trends Microbiol. 10:515-521. The human bacterial pathogen group A Streptococcus (GAS) causes many different diseases including pharyngitis, tonsillitis, impetigo, scarlet fever, streptococcal toxic shock syndrome, necrotizing fasciitis and myositis, and the post-infection sequelae glomerulonephritis and rheumatic fever. The frequency and severity of GAS infections increased in the 1980s and 1990s, but the cause of this increase is unknown. Recently, genome sequencing of serotype M1, M3 and M18 strains revealed many new proven or putative virulence factors that are encoded by phages or phage-like elements. Importantly, these genetic elements account for an unexpectedly large proportion of the difference in gene content between the three strains. These new genome-sequencing studies have provided evidence that temporally and geographically distinct epidemics, and the complex array of GAS clinical presentations, might be related in part to the acquisition or evolution of phage-encoded virulence factors. We anticipate that new phage-encoded virulence factors will be identified by sequencing the genomes of additional GAS strains, including organisms non-randomly associated with particular clinical syndromes. [TOP OF PAGE]

  936. Characterization of six Leuconostoc fallax bacteriophages isolated from an industrial sauerkraut fermentation. Barrangou,R., Yoon,S.S., Breidt,F.J., Fleming,P., Klaenhammer,T.R. (2002). Appl. Environ. Microbiol. 68:5452-5458. Six bacteriophages active against Leuconostoc fallax strains were isolated from industrial sauerkraut fermentation brines. These phages were characterized as to host range, morphology, structural proteins, and genome fingerprint. They were exclusively lytic against the species L. fallax and had different host ranges among the strains of this species tested. Morphologically, three of the phages were assigned to the family Siphoviridae, and the three others were assigned to the family Myovidae: Major capsid proteins detected by electrophoresis were distinct for each of the two morphotypes. Restriction fragment length polymorphism analysis and randomly amplified polymorphic DNA fingerprinting showed that all six phages were genetically distinct. These results revealed for the first time the existence of bacteriophages that are active against L. fallax and confirmed the presence and diversity of bacteriophages in a sauerkraut fermentation. Since a variety of L. fallax strains have been shown to be present in sauerkraut fermentation, bacteriophages active against L. fallax are likely to contribute to the microbial ecology of sauerkraut fermentation and could be responsible for some of the variability observed in this type of fermentation. [TOP OF PAGE]

  937. Genome sequence of a serotype M3 strain of group A Streptococcus: phage-encoded toxins, the high-virulence phenotype, and clone emergence. Beres,S.B., Sylva,G.L., Barbian,K.D., Lei,B., Hoff,J.S., Mammarella,N.D., Liu,M.Y., Smoot,J.C., Porcella,S.F., Parkins,L.D., Campbell,D.S., Smith,T.M., McCormick,J.K., Leung,D.Y.M., Schlievert,P.M., Musser,J.M. (2002). Proc. Natl. Acad. Sci. USA 99:10078-10083. Genome sequences are available for many bacterial strains, but there has been little progress in using these data to understand the molecular basis of pathogen emergence and differences in strain virulence. Serotype M3 strains of group A Streptococcus (GAS) are a common cause of severe invasive infections with unusually high rates of morbidity and mortality. To gain insight into the molecular basis of this high-virulence phenotype, we sequenced the genome of strain MGAS315, an organism isolated from a patient with streptococcal toxic shock syndrome. The genome is composed of 1,900,521 bp, and it shares approximately 1.7 Mb of related genetic material with genomes of serotype M1 and M18 strains. Phage-like elements account for the great majority of variation in gene content relative to the sequenced M1 and M18 strains. Recombination produces chimeric phages and strains with previously uncharacterized arrays of virulence factor genes. Strain MGAS315 has phage genes that encode proteins likely to contribute to pathogenesis, such as streptococcal pyrogenic exotoxin A (SpeA) and SpeK, streptococcal superantigen (SSA), and a previously uncharacterized phospholipase A(2) (designated Sla). Infected humans had anti-SpeK, -SSA, and -Sla antibodies, indicating that these GAS proteins are made in vivo. SpeK and SSA were pyrogenic and toxic for rabbits. Serotype M3 strains with the phage-encoded speK and sla genes increased dramatically in frequency late in the 20th century, commensurate with the rise in invasive disease caused by M3 organisms. Taken together, the results show that phage-mediated recombination has played a critical role in the emergence of a new, unusually virulent clone of serotype M3 GAS. [TOP OF PAGE]

  938. The functional importance of bacteriophages in the microbial loop of an oligomesotrophic lake over a diel cycle. Bettarel,Y., Sime-Ngando,T., Amblard,C., Carrias,J.F., Sargos,D., Garabetian,F., Lavandier,P. (2002). Annales de Limnology - International Journal of Limnology 38:263-269. The abundances of the different compartments of the microbial loop (i.e., viruses, heterotrophic bacteria, heterotrophic nanoflagellates, and pigmented nanoflagellates), total (TPP) and excreted (EPP) primary production, bacterial production (BP), viral lytic activity (LA), and bacterivory by nanoflagellates (FG) were measured on lune 15 and 16, 1998, in a moderate-altitude oligomesotrophic lake (Lac Pavin, France), at 5 and 10 m depths. At both depths, losses of the bacterial community by virallysis (LA5m = 1.7 X 106 cells.l-1.h-1, LAC = 2.0 X 106 cells.l-1.h-1) were, on average, lower than those due to the grazing activity of flagellates (FG5m = 10.3 X 106 cells.l-1.h-1, FG10m= 8.4 X 106 cells.l-1.h-1). A carbon budget exercise indicated that, for the sampling period and depths, 17.8 % of C from TPP (= 38.1 % of EPP) was used by bacteria. On the other hand, 52.7 % of BP (= 2.15% of TPP) was grazed by nanoflagellates, while 11.0% of BP (= 0.45% of TPP) was lysed by viruses. [TOP OF PAGE]

  939. Bacteriophage therapy rescues mice bacteremic from a clinical isolate of vancomycin-resistant Enterococcus faecium. Biswas,B., Adhya,S., Washart,P., Paul,B., Trostel,A.N., Powell,B., Carlton,R., Merril,C.R. (2002). Infect. Immun. 70:204-210. Colonization of the gastrointestinal tract with vancomycin-resistant Enterococcus faecium (VRE) has become endemic in many hospitals and nursing homes in the United States. Such colonization predisposes the individual to VRE bacteremia and/or endocarditis, and immunocompromised patients are at particular risk for these conditions. The emergence of antibiotic-resistant bacterial strains requires the exploration of alternative antibacterial therapies, which led our group to study the ability of bacterial viruses (bacteriophages, or phages) to rescue mice with VRE bacteremia. The phage strain used in this study has lytic activity against a wide range of clinical isolates of VRE. One of these VRE strains was used to induce bacteremia in mice by intraperitoneal (i.p.) injection of 109 CFU. The resulting bacteremia was fatal within 48 h. A single i.p. injection of 3 x 108 PFU of the phage strain, administered 45 min after the bacterial challenge, was sufficient to rescue 100% of the animals. Even when treatment was delayed to the point where all animals were moribund, approximately 50% of them were rescued by a single injection of this phage preparation. The ability of this phage to rescue bacteremic mice was demonstrated to be due to the functional capabilities of the phage and not to a nonspecific immune effect. The rescue of bacteremic mice could be effected only by phage strains able to grow in vitro on the bacterial host used to infect the animals, and when such strains are heat inactivated they lose their ability to rescue the infected mice. [TOP OF PAGE]

  940. Trade-offs and coexistence in microbial microcosms. Bohannan,B.J.M., Kerr,B., Jessup,C.M., Hughes,J.B., Sandvik,G. (2002). Antonie van Leeuwenhoek J. Microbiol. 81:107-115. Trade-offs among the abilities of organisms to respond to different environmental factors are often assumed to play a major role in the coexistence of species. There has been extensive theoretical study of the role of such trade-offs in ecological communities but it has proven difficult to study such trade-offs experimentally. Microorganisms are ideal model systems with which to experimentally study the causes and consequences of ecological trade-offs. In model communities of E. coli B and T-type bacteriophage, a trade-off in E. coli between resistance to bacteriophage and competitive ability is often observed. This trade-off can allow the coexistence of different ecological types of E. coli. The magnitude of this trade-off affects, in predictable ways, the structure, dynamics and response to environmental change of these communities. Genetic factors, environmental factors, and gene-by-environment interactions determine the magnitude of this trade-off. Environmental control of the magnitude of trade-offs represents one avenue by which environmental change can alter community properties such as invasability, stability and coexistence. [TOP OF PAGE]

  941. Fate of bacterial indicators, viruses and protozoan parasites in a wastewater multi-component treatment system. Bonadonna,L., Briancesco,R., Cataldo,C., Divizia,M., Donia,D., Pana,A. (2002). New. Microbiol. 25:413-420. The extent of reduction in selected microrganisms was tested at a multi-component wastewater treatment plant that treats sewage for a potential re-use in agriculture. The aim of the investigation was to evaluate possible reciprocal correlation among the different microrganisms and to compare the removal of two encysted pathogenic protozoa with that of microbial indicators, Clostridium perfringens spores, enteroviruses and bacteriophages. Samples collected included the raw wastewater, the chlorinated effluent and the effluent after an ultraviolet light treatment. All of the raw sewage samples were positive for Cryptosporidium oocysts and Giardia cysts, as well as for the other microorganisms tested but the bacteriophage B40-8. The data obtained confirm the removal efficiency of the entire process for indicator bacteria but also show the low and variable removal efficiency for the other microbial parameters, such as Giardia and Cryptosporidium, enteroviruses and Clostridium perfringens spores. Reciprocal correlation between Cryptosporidium and Giardia (oo)cysts and the other microbial groups was not demonstrated. The results confirm the resistance of Clostridium perfringens spores, enteroviruses and protozoa to chlorination and demonstrate the relative persistence of these organisms in the effluents even during the ultraviolet light treatment. The yields also emphasise the influence of the analytical method for the determination of protozoan parasites. [TOP OF PAGE]

  942. Characterization of the two-component abortive phage infection mechanism AbiT from Lactococcus lactis. Bouchard,J.D., Dion,E., Bissonnette,F., Moineau,S. (2002). J. Bacteriol. 184:6325-6332. During the production of fermented dairy products, virulent bacteriophages infecting Lactococcus lactis can delay or stop the milk acidification process. A solution to this biological problem consists of introducing natural phage barriers into the strains used by the dairy industry. One such hurdle is called abortive infection (Abi) and causes premature cell death with no or little phage progeny. Here, we describe the isolation and characterization of a novel Abi mechanism encoded by plasmid pED1 from L. lactis. The system is composed of two constitutively cotranscribed genes encoding putative proteins of 127 and 213 amino acids, named AbiTi and AbiTii, respectively. Site-directed mutagenesis indicated that a hydrophobic region at the C-terminal extremity of AbiTi is essential to the antiphage phenotype. The AbiT system is effective against phages of the 936 and P335 species (efficiency of plaquing between 10(-5) and 10(-7)) and causes a 20-fold reduction in the efficiency to form centers of infection as well as a 10- to 12-fold reduction in the burst size. Its efficacy could be improved by raising the plasmid copy number, but changing the intrinsic ratio of AbiTi and AbiTii did not greatly affect the antiphage activity. The monitoring of the intracellular phage infection process by DNA replication, gene expression, and electron microscopy as well as the study of phage mutants by genome mapping indicated that AbiT is likely to act at a later stage of the phage lytic cycle. [TOP OF PAGE]

  943. Common themes among bacteriophage-encoded virulence factors and diversity among the bacteriophages involved. Boyd,E.F., Brüssow,H. (2002). Trends Microbiol. 10:521-529. There are common themes among bacteriophage-encoded virulence factors, which include the well-characterized bacterial toxins and proteins that alter antigenicity as well as several new classes of bacteriophage-encoded proteins such as superantigens, effectors translocated by a type III secretion system, and proteins required for intracellular survival and host cell attachment. These virulence factors are encoded by a diversity of bacteriophages, members of the viral families Siphoviridae, Podoviridae, Myoviridae and Inoviridae, with some bacteriophages having characteristics of more than one virus family. The location of virulence genes within the bacteriophage genomes is non-random and consistent with an origin via imprecise prophage excision or as either transferable cassettes or integral components of the bacteriophage genome. [TOP OF PAGE]

  944. Genomic analysis of uncultured marine viral communities. Breitbart,M., Salamon,P., Andresen,B., Mahaffy,J.M., Segall,A.M., Mead,D., Azam,F., Rohwer,F. (2002). Proc. Natl. Acad. Sci. USA 99:14250-14255. Viruses are the most common biological entities in the oceans by an order of magnitude. However, very little is known about their diversity. Here we report a genomic analysis of two uncultured marine viral communities. Over 65% of the sequences were not significantly similar to previously reported sequences, suggesting that much of the diversity is previously uncharacterized. The most common significant hits among the known sequences were to viruses. The viral hits included sequences from all of the major families of dsDNA tailed phages, as well as some algal viruses. Several independent mathematical models based on the observed number of contigs predicted that the most abundant viral genome comprised 2-3% of the total population in both communities, which was estimated to contain between 374 and 7,114 viral types. Overall, diversity of the viral communities was extremely high. The results also showed that it would be possible to sequence the entire genome of an uncultured marine viral community. [TOP OF PAGE]

  945. Microviridae, a family divided: isolation, characterization, and genome sequence of fMH2K, a bacteriophage of the obligate intracellular parasitic bacterium Bdellovibrio bacteriovorus. Brentlinger,K.L., Hafenstein,S., Novak,C.R., Fane,B.A., Borgon,R., McKenna,R., Agbandje-McKenna,M. (2002). J. Bacteriol. 184:1089-1094. A novel single-stranded DNA phage, fMH2K, of Bdellovibrio bacteriovorus was isolated, characterized, and sequenced. This phage is a member of the Microviridae, a family typified by bacteriophage fX174. Although B. bacteriovorus and Escherichia coli are both classified as proteobacteria, fMH2K is only distantly related to fX174. Instead, phiMH2K exhibits an extremely close relationship to the Microviridae of Chlamydia in both genome organization and encoded proteins. Unlike the double-stranded DNA bacteriophages, for which a wide spectrum of diversity has been observed, the single-stranded icosahedral bacteriophages appear to fall into two distinct subfamilies. These observations suggest that the mechanisms driving single-stranded DNA bacteriophage evolution are inherently different from those driving the evolution of the double-stranded bacteriophages. [TOP OF PAGE]

  946. F-specific RNA coliphages: occurrence, types, and survival in natural waters. Brion,G.M., Meschke,J.S., Sobsey,M.D. (2002). Water Res. 36:2419-2425. A small, well-defined watershed was investigated over a 2-year period to determine the prevalence of F-specific RNA coliphage (F + RNA) serotypes as indicators of animal fecal contamination. Sampling sites collected runoff from areas of urban and agricultural land use patterns. F-specific coliphages were concentrated from 2-L freshwater samples by polyethylene glycol precipitation, isolated using the double agar layer (DAL) method, confirmed as F + RNA by RNAse suppression, and serotyped. A subset of serotyped F + RNA were confirmed by genotyping. To determine relative survival, 10 confirmed F + RNA field isolates and 5 prototypic F + RNA were spiked into surface water and incubated at 25 degrees C for 36 days. F-specific coliphage isolation was strongly associated with rainfall events and was infrequent from primarily animal impacted surface waters. Field isolates were predoffiinantly Type I F + RNA (81%) and raw sewage isolates were predominantly Type III F + RNA (57%). Genotyping from either the watershed or raw sewage samples never positively identified Type IV F + RNA. Results from laboratory studies showed that F + RNA differ in their survival in water and that Type IV strains were the least persistent. Type III F + RNA were found to be reliably related to the release of uncontrolled human fecal material in the watershed, but the results of this study suggest that further study is required before utilizing for fecal source identification in natural waters. [TOP OF PAGE]

  947. The in vitro interaction of Streptococcus pyogenes with human pharyngeal cells induces a phage-encoded extracellular DNase. Broudy,T.B., Pancholi,V., Fischetti,V.A. (2002). Infect. Immun. 70:2805-2811. The role lysogenic bacteriophage play in the pathogenesis of the host bacterium is poorly understood. In a previous study, we found that streptococcal coculture with human pharyngeal cells resulted in the induction of lysogenic bacteriophage as well as the phage-associated streptococcal pyrogenic exotoxin C (SpeC). In this study, we have determined that in addition to SpeC induction, a number of other streptococcal proteins are also released by the bacteria during coculture with pharyngeal cells. Among these, we identified and characterized a novel 27-kDa secreted protein. Sequence analysis of this novel protein demonstrated it to be encoded by the same lysogenic bacteriophage which harbors speC. Protein sequence analysis revealed varied homologies with several streptococcal DNases. Further biochemical characterization of the recombinantly expressed protein verified it to be a divalent cation-dependent streptococcal phage-encoded DNase (Spd1). Although functionally distinct, SpeC and Spd1 are associated by a number of parameters, including genetic proximity and transcriptional regulation. Finally, we speculate on the induction of phage-encoded DNase (Spd1) enhancing the fitness of both bacteria and phage. [TOP OF PAGE]

  948. Performance of a novel Viresolve NFR virus filter. Brough,H., Antoniou,C., Carter,J., Jakubik,J., Xu,Y., Lutz,H. (2002). Biotechnol. Prog. 18:782-795. Mammalian cell-expressed therapeutic proteins are particularly vulnerable to contamination by endogenous retrovirus-like particles (RVLPs). The Viresolve NFR filter was designed to meet the critical requirement of manufacturing a safe and virus-free therapeutic by retaining RVLPs by a minimum of six log reduction value (LRV). The NFR designation refers to retrovirus removal in a normal flow format. To qualify the product, we tested two model viruses: the 78 nm diameter phi6 bacteriophage and the 80-110 nm diameter Xenotropic Murine Leukemia Virus (X-MuLV). Robust retention was demonstrated over a wide range of process parameters. Viresolve NFR filters also retain other model adventitious viruses including 70-85 nm diameter Reovirus 3 (Reo3), 70-90 nm diameter Adenovirus 2 (Ad2), and 53 nm diameter PR772 by >6 LRV. In addition to these model viruses, the filter retains >7 LRV of both the mycoplasma Acholeplasma laidlawii and the bacterium Brevundimonas diminuta. Protein passage is shown to be consistently high (95-100%) for a variety of therapeutic protein products, including monoclonal antibodies. Characterization of the filter in specific applications is made simple by availability of ultralow surface area (5 cm(2)) disks, which are shown to scale linearly to the manufacturing scale pleated-filters. Viresolve NFR filters provide consistent water permeability performance (34-37 LMH/psi) and show very little plugging for all feedstocks evaluated. The Viresolve NFR filter incorporates Retropore, a unique asymmetric polyethersulfone membrane, the surface of which has been modified to minimize protein binding. [TOP OF PAGE]

  949. RNA bacteriophage capsid-mediated drug delivery and epitope presentation. Brown,W.L., Mastico,R.A., Wu,M., Heal,K.G., Adams,C.J., Murray,J.B., Simpson,J.C., Lord,J.M., Taylor-Robinson,A.W., Stockley,P.G. (2002). Intervirology 45:371-380. OBJECTIVE: To use our knowledge of the three-dimensional structure and self-assembly mechanism of RNA bacteriophage capsids to develop novel virus-like particles (VLPs) for drug delivery and epitope presentation. METHODS: Site-directed mutagenesis of a recombinant MS2 coat protein expression construct has been used to generate translational fusions encompassing short epitope sequences. These chimeric proteins still self-assemble in vivo into T = 3 shells with the foreign epitope in an accessible location. Covalent conjugation has also been used to generate RNA stem-loops attached to the toxin, ricin A chain, or to nucleotide-based drugs, that are still capable of stimulating self-assembly of the capsid in vitro. These packaged drugs can then be directed to specific cells in culture by further covalent decoration of the capsids with targeting molecules. RESULTS: Chimeric VLPs are strongly immunogenic when carrying either B or T cell epitopes, the latter generating cytokine profiles consistent with memory responses. Immune responses to the underlying phage epitopes appear to be proportional to the area of the phage surface accessible. Phage shells effectively protect nucleic acid-based drugs and, for the toxin construct, make cell-specific delivery systems with LD50 values in culture sub-nanomolar. CONCLUSION: VLP technology has potential for therapeutic and prophylactic intervention in disease. [TOP OF PAGE]

  950. Killing of Mycobacterium avium and Mycobacterium tuberculosis by a mycobacteriophage delivered by a nonvirulent mycobacterium: A model for phage therapy of intracellular bacterial pathogens. Broxmeyer,L., Sosnowskai,D., Miltner,E., Chacón,O., Wagner,D., McGarvey,J., Barletta,R.G., Bermudez,L.E. (2002). J. Infect. Dis. 186:1155-1160. Mycobacterium avium causes disseminated infection in patients with acquired immune deficieny syndrome. Mycobacterium tuberculosis is a pathogen associated with the deaths of millions of people worldwide annually. Effective therapeutic regimens exist that are limited by the emergence of drug resistance and the inability of antibiotics to kill dormant organisms. The present study describes a system using Mycobacterium smegmatis, an avirulent mycobacterium, to deliver the lytic phage TM4 where both M. avium and M. tuberculosis reside within macrophages. These results showed that treatment of M. aviuminfected, as well as M. tuberculosis infected, RAW 264.7 macrophages, with M. smegmatis transiently infected with TM4, resulted in a significant time- and titer-dependent reduction in the number of viable intracellular bacilli. In addition, the M. smegmatis vacuole harboring TM4 fuses with the M. avium vacuole in macrophages. These results suggest a potentially novel concept to kill intracellular pathogenic bacteria and warrant future development. [TOP OF PAGE]

  951. Phage genomics: Small is beautiful. Brüssow,H., Hendrix,R.W. (2002). Cell 108:13-16. The Age of Genomics dawned only gradually for bacteriophages. It was 1977 when the genome of phage phiX174 was published and 1983 when the "large" genome of phage lambda hit the streets. More recently, the pace has quickened, so that we now have over 100 complete phage genomes and can expect thousands in a very few years. These sequences have been marvelously informative for the biology of the individual phages, but with the advent of high volume sequencing technology, the real excitement for phage biology is that it is now possible to analyze the sequences together and thereby address-for the first time at whole genome resolution-a set of fundamental biological questions related to populations: What is the structure of the global phage population? What are its dynamics? How do phages evolve? This is Comparative Genomics with a capital "C". [TOP OF PAGE]

  952. Antagonistic coevolution between a bacterium and a bacteriophage. Buckling,A., Rainey,P.B. (2002). Proc. R. Soc. Lond. B Biol. Sci. 269:931-936. Antagonistic coevolution between hosts and parasites is believed to play a pivotal role in host and parasite population dynamics, the evolutionary maintenance of sex and the evolution of parasite virulence. Furthermore, antagonistic coevolution is believed to be responsible for rapid differentiation of both hosts and parasites between geographically structured populations. Yet empirical evidence for host-parasite antagonistic coevolution, and its impact on between-population genetic divergence, is limited. Here we demonstrate a long-term arms race between the infectivity of a viral parasite (bacteriophage; phage) and the resistance of its bacterial host. Coevolution was largely driven by directional selection, with hosts becoming resistant to a wider range of parasite genotypes and parasites infective to a wider range of host genotypes. Coevolution followed divergent trajectories between replicate communities despite establishment with isogenic bacteria and phage, and resulted in bacteria adapted to their own, compared with other, phage populations. [TOP OF PAGE]

  953. The role of parasites in sympatric and allopatric host diversification. Buckling,A., Rainey,P.B. (2002). Nature 420:496-499. Exploiters (parasites and predators) are thought to play a significant role in diversification, and ultimately speciation, of their hosts or prey. Exploiters may drive sympatric (within-population) diversification if there are a variety of exploiter-resistance strategies or fitness costs associated with exploiter resistance. Exploiters may also drive allopatric (between-population) diversification by creating different selection pressures and increasing the rate of random divergence. We examined the effect of a virulent viral parasite (phage) on the diversification of the bacterium Pseudomonas fluorescens in spatially structured microcosms. Here we show that in the absence of phages, bacteria rapidly diversified into spatial niche specialists with similar patterns of diversity across replicate populations. In the presence of phages, sympatric diversity was greatly reduced, as a result of phage-imposed reductions in host density decreasing competition for resources. In contrast, allopatric diversity was greatly increased as a result of phage-imposed selection for resistance, which caused populations to follow divergent evolutionary trajectories. These results show that exploiters can drive diversification between populations, but may inhibit diversification within populations by opposing diversifying selection that arises from resource competition. [TOP OF PAGE]

  954. Dynamics of success and failure in phage and antibiotic therapy in experimental infections. Bull,J.J., Levin,B.R., DeRouin,T., Walker,N., Bloch,C.A. (2002). BMC Microbiol. 2:35 BACKGROUND: In 1982 Smith and Huggins showed that bacteriophages could be at least as effective as antibiotics in preventing mortality from experimental infections with a capsulated E. coli (K1) in mice. Phages that required the K1 capsule for infection were more effective than phages that did not require this capsule, but the efficacies of phages and antibiotics in preventing mortality both declined with time between infection and treatment, becoming virtually ineffective within 16 hours. RESULTS: We develop quantitative microbiological procedures that (1) explore the in vivo processes responsible for the efficacy of phage and antibiotic treatment protocols in experimental infections (the Resistance Competition Assay, or RCA), and (2) survey the therapeutic potential of phages in vitro (the Phage Replication Assay or PRA). We illustrate the application and utility of these methods in a repetition of Smith and Huggins' experiments, using the E. coli K1 mouse thigh infection model, and applying treatments of phages or streptomycin. CONCLUSIONS: 1) The Smith and Huggins phage and antibiotic therapy results are quantitatively and qualitatively robust. (2) Our RCA values reflect the microbiological efficacies of the different phages and of streptomycin in preventing mortality, and reflect the decline in their efficacy with a delay in treatment. These results show specifically that bacteria become refractory to treatment over the term of infection. (3) The K1-specific and non-specific phages had similar replication rates on bacteria grown in broth (based on the PRA), but the K1-specific phage had markedly greater replication rates in mouse serum. [TOP OF PAGE]

  955. [Phenogenetic characterization of a group of giant Phi KZ-like bacteriophages of Pseudomonas aeruginosa]. Burkal'tseva,M.V., Krylov,V.N., Pleteneva,E.A., Shaburova,O.V., Krylov,S.V., Volkart,G., Sykilinda,N.N., Kurochkina,L.P., Mesianzhinov,V.V. (2002). Genetika 38:1470-1479. A comparative study was made of a group of Pseudomonas aeruginosa virulent giant DNA bacteriophages similar to phage phi KZ in several genetic and phenotypic properties (particle size, particle morphology, genome size, appearance of negative colonies, high productivity, broad spectrum of lytic activity, ability to overcome the suppressing effect of plasmids, absence of several DNA restriction sites, capability of general transduction, pseudolysogeny). We have recently sequenced the phage phi KZ genome (288,334 bp) [J. Mol. Biol., 2002, vol. 317, pp. 1-19]. By DNA homology, the phages were assigned to three species (represented by phage phi KZ, Lin68, and EL, respectively) and two new genera (phi KZ and EL). Restriction enzyme analysis revealed the mosaic genome structure in four phages of the phi KZ species (phi KZ, Lin21, NN, and PTB80) and two phages of the EL species (EL and RU). Comparisons with respect to phage particle size, number of structural proteins, and the N-terminal sequences of the major capsid protein confirmed the phylogenetic relatedness of the phages belonging to the phi KZ genus. The origin and evolution of the phi KZ-like phages are discussed. Analysis of protein sequences encoded by the phage phi KZ genome made it possible to assume wide migration of the phi KZ-like phages (wandering phages) among various prokaryotes and possibly eukaryotes. Since the phage phi KZ genome codes for potentially toxic proteins, caution must be exercised in the employment of large bacteriophages in phage therapy. [TOP OF PAGE]

  956. Phenogenetic characterization of a group of giant fKZ-like bacteriophages of Pseudomonas aeruginosa. Burkal'tseva,M.V., Krylov,V.N., Pleteneva,E.A., Shaburova,O.V., Krylov,S.V., Volkart,G., Sykilinda,N.N., Kurochkina,L.P., Mesyanzhinov,V.V. (2002). Rus. J. Genet. 38:1242-1250. A comparative study was made of a group of Pseudomonas aeruginosa virulent giant DNA bacteriophages similar to KZ in several genetic and phenotypic properties (particle size, particle morphology, genome size, appearance of negative colonies, high productivity, broad spectrum of lytic activity, ability to overcome the suppressing effect of plasmids, absence of several DNA restriction sites, capability of general transduction, pseudolysogeny). We have recently sequenced the phage KZ genome (288 334 bp) [J. Mol. Biol., 2002, vol. 317, pp. 1-19]. By DNA homology, the phages were assigned to three species (represented by phages KZ, Lin68, and EL, respectively) and two new genera (KZ, LIN21, NN, and PTB80) and two phages of the EL species (EL and RU). Comparisons with respect to phage particle size, number of structural proteins, and the N-terminal sequences of the major capsid protein confirmed the phylogenetic relatedness of the phages belonging to the KZ genus. The origin and evolution of the KZ-like phages are discussed. Analysis of protein sequences encoded by the phage KZ genome made it possible to assume wide migration of the KZ-like phages (wandering phages) among various prokaryotes and possibly eukaryotes. Since the phage KZ genome codes for potentially toxic proteins, caution must be exercise in the empoyment of large bacteriophages in phage therapy. [TOP OF PAGE]

  957. Integrity of powdered and powder-free latex examination gloves. Calhoun,A.J., Rodrick,G.E., Brown,F.H. (2002). J. Public Health Dent. 62:170-172. OBJECTIVES: The difference in permeability between one brand of powdered and another of powder-free latex examination gloves was evaluated to determine leak rates. METHODS: Thirty-one of each type of glove were tested for each of three different conditions: usage by dental personnel (1) for 15 minutes or longer, (2) for less than 15 minutes, and (3) directly from the manufacturer's packaging (zero usage time). Each glove was evaluated in the fingers and the palm. The phiX-174 viral solution in the glove was allowed to penetrate for 15 minutes. Powder (cornstarch) was subsequently added to 20 powder-free gloves, and 15 of these were pierced with a 30-gauge needle. RESULTS: Powdered gloves showed no leakage rates. Because of this, 30-, 27-, and 25-gauge needles were used to pierce five gloves each. One glove with 27- and 25-gauge needle holes showed leakage. Leakage rates for powder-free gloves: 45.1 percent for more than 15 minutes of use, 25.8 percent for less than 15 minutes of use, and 16.1 percent for zero minutes of use. Two of the 20 pierced and one of the five unpierced powder-free gloves with added cornstarch leaked. CONCLUSION: Significant differences in leak results between powdered and powder-free gloves suggest further study is needed. [TOP OF PAGE]

  958. Genome plasticity in Lactococcus lactis. Campo,N., Dias,M.J., Daveran-Mingot,M.L., Ritzenthaler,P., Le Bourgeois,P. (2002). Antonie van Leeuwenhoek J. Microbiol. 82:123-132. Comparative genome analyses contribute significantly to our understanding of bacterial evolution and indicate that bacterial genomes are constantly evolving structures. The gene content and organisation of chromosomes of lactic acid bacteria probably result from a strong evolutionary pressure toward optimal growth of these microorganisms in milk. The genome plasticity of Lactococcus lactis was evaluated at inter- and intrasubspecies levels by different experimental approaches. Comparative genomics showed that the lactococcal genomes are not highly plastic although large rearrangements (a.o. deletions, inversions) can occur. Experimental genome shuffling using a new genetic strategy based on the Cre-loxP recombination system revealed that two domains are under strong constraints acting to maintain the original chromosome organisation: a large region around the replication origin, and a smaller one around the putative terminus of replication. Future knowledge of the rules leading to an optimal genome organisation could facilitate the definition of new strategies for industrial strain improvement. [TOP OF PAGE]

  959. Removal of bacterial and viral faecal indicator organisms in a waste stabilization pond system in Choconta, Cundinamarca (Colombia). Campos,C., Guerrero,A., Cardenas,M. (2002). Water Sci. Technol. 45:61-66. A major objective for domestic wastewater treatment using waste stabilization pond systems is the removal of pathogenic microorganisms. Traditional evaluation parameters for faecal contamination are the total and faecal coliforms. However, epidemiological studies, environmental resistance and the behaviour in the treatment systems, show that viruses are an important disease agent and even more resistant to disinfection than bacteria. Therefore, it is important to introduce viruses as a faecal indicator and to compare them with the traditional bacterial indicators. A waste stabilization pond system was evaluated in the municipality of Choconta, Cundinamarca (Colombia), for the removal of faecal indicators (such as Escherichia coli, Streptococcus faecalis, Clostridium perfringens) and viruses like F+, somatic and Bacteroides fragilis phages. The system includes two facultative ponds in series with a flow of 1555 m3/day. Samples were collected at the entrance of the system, in the two ponds and from the final effluent. Results show a decrease between 0.3 and 4.7 logarithmic units in the bacterial indicators and between 1 and 4.6 logarithmic units with viral indicators. [TOP OF PAGE]

  960. Genome analysis of an inducible prophage and prophage remnants integrated in the Streptococcus pyogenes strain SF370. Canchaya,C., Desiere,F., McShan,W.M., Ferretti,J.J., Parkhill,J., Brüssow,H. (2002). Virology 302:245-258. The mitomycin C inducible prophage SF370.1 from the highly pathogenic M1 serotype Streptococcus pyogenes isolate SF370 showed a 41-kb-long genome whose genetic organization resembled that of SF11-like pac-site Siphoviridae. Its closest relative was prophage NIH1.1 from an M3 serotype S. pyogenes strain, followed by S. pneumoniae phage MM1 and Lactobacillus phage phig1e, Listeria phage A118, and Bacillus phage SPP1 in a gradient of relatedness. Sequence similarity with the previously described prophages SF370.2 and SF370.3 from the same polylysogenic SF370 strain were mainly limited to the tail fiber genes. As in these two other prophages, SF370.1 encoded likely lysogenic conversion genes between the phage lysin and the right attachment site. The genes encoded the pyrogenic exotoxin C of S. pyogenes and a protein sharing sequence similarity with both DNases and mitogenic factors. The screening of the SF370 genome revealed further prophage-like elements. A 13-kb-long phage remnant SF370.4 encoded lysogeny and DNA replication genes. A closely related prophage remnant was identified in S. pyogenes strain Manfredo at a corresponding genome position. The two prophages differed by internal indels and gene replacements. Four phage-like integrases were detected; three were still accompanied by likely repressor genes. All prophage elements were integrated into coding sequences. The phage sequences complemented the coding sequences in all cases. The DNA repair genes mutL and mutS were separated by the prophage remnant SF370.4; prophage SF370.1 and S. pneumoniae phage MM1 integrated into homologous chromosomal locations. The prophage sequences were interpreted with a hypothesis that predicts elements of cooperation and an arms race between phage and host genomes. [TOP OF PAGE]

  961. Evaluation of the international phage typing set and some experimental phages for typing of Listeria monocytogenes from poultry in Spain. Capita,R., Alonso-Calleja,C., Mereghetti,L., Moreno,B., del Camino,G. (2002). J. Appl. Microbiol. 92:90-96. Aims: The validity of the international phage set and 13 experimental phages for subtyping Listeria monocytogenes strains isolated from poultry in Spain was investigated. Methods and Results: Ninety-six L. monocytogenes strains (52 from serogroup 1/2 and 44 from serogroup 4) were phage-typed using the international phage set, 10 experimental phages for typing serogroup 1/2 strains (seven isolated in France: 1313, 9425, 1807, 351, 881, 717 and 586-, and three from Denmark: 5775, 12682 and 6223-) and three experimental phages isolated in France for typing serogroup 4 strains (2425 A, 4286 and 197). Percentages of serogroup 1/2, serogroup 4 and total phage-typeable strains were 57.7%, 52.3% and 55.2%, respectively. Important differences in the behaviour of the phages tested were found. The typeability rate, the specificity index and the percentage of strong reactions were greater in the phages of international set than in the experimental phages. The number of phage typeable strains and the number of phage types (42) were not modified by the use of experimental phages. Conclusions: The phage set used was not effective for typing L. monocytogenes strains from poultry in Spain, because a low typeability rate was found. Significance and Impact of the Study: Our results suggest the importance of the availability of new phages specific to a geographical area in order to improve the typeability of the system. [TOP OF PAGE]

  962. On the stability properties of a stochastic model for phage-bacteria interaction in open marine environment. Carletti,M. (2002). Math. Biosci. 175:117-131. In this paper we extend the deterministic model for the epidemics induced by virulent phages on bacteria in marine environment introduced by Beretta and Kuang [Math. Biosci. 149 (1998) 57], allowing random fluctuations around the positive equilibrium. The stochastic stability properties of the model are investigated both analytically and numerically suggesting that the deterministic model is robust with respect to stochastic perturbations. [TOP OF PAGE]

  963. Memory in bacteria and phage. Casadesus,J., D'Ari,R. (2002). Bioessays 24:512-518. Whenever the state of a biological system is not determined solely by present conditions but depends on its past history, we can say that the system has memory. Bacteria and bacteriophage use a variety of memory mechanisms, some of which seem to convey adaptive value. A genetic type of heritable memory is the programmed inversion of specific DNA sequences, which causes switching between alternative patterns of gene expression. Heritable memory can also be based on epigenetic circuits, in which a system with two possible steady states is locked in one or the other state by a positive feedback loop. Epigenetic states have been observed in a variety of cellular processes, and are maintained by diverse mechanisms. Some of these involve alternative DNA methylation patterns that are stably transmitted to daughter molecules and can affect DNA-protein interactions (e.g., gene transcription). Other mechanisms exploit autocatalytic loops whereby proteins establish the proper conditions for their continued synthesis. Template polymers other than nucleic acids (e.g., components of the cell wall) may also propagate epigenetic states. Non-heritable memory is exemplified by parasitic organisms that bear a signature of their previous host, such as host-controlled modification of phage DNA or porin hitchhiking in predatory bacteria. The heterogeneous nature of the examples known may be indicative of widespread occurrence of memory mechanisms in bacteria and phage. However, the actual extent, variety and potential selective value of prokaryotic memory devices remain open questions, still to be addressed experimentally. [TOP OF PAGE]

  964. Isolation and characterization of virus infecting Emiliania huxleyi (Haptophyceae). Castberg,T., Thyrhaug,R., Larsen,A., Sandaa,R.-A., Heldal,M., Bratbak,G. (2002). Phycology 38:767-774. The isolation and characterization of a virus (designated EhV) that infects the marine coccolithophorid Emiliania huxleyi (Lohmann) Hay & Mohler are described. Three independent clones of EhV were isolated from Norwegian coastal waters in years 1999 and 2000. EhV is a double-stranded DNA-containing virus with a genome size of ~415 kilo-base pairs. The viral particle is an icosahedron with a diameter of 160-180 nm. The virus particle contains at least nine proteins ranging from 10 to 140 kDa; the major capsid protein weighs ~54 kDa. EhV has a latent period of 12-14 h and a burst size of 400-1000 (mean, 620) viral particles per cell. A phylogenetic tree based on DNA polymerase amino acid sequences indicates EhV should be assigned to the Phycodnaviridae virus family and that the virus is most closely related to viruses that infect Micromonas pusilla and certain Chlorella species. [TOP OF PAGE]

  965. Phage therapy of local and systemic disease caused by Vibrio vulnificus in iron-dextran-treated mice. Cerveny,K.E., Depaola,A., Duckworth,D.H., Gulig,P.A. (2002). Infect. Immun. 70:6251-6262. Vibrio vulnificus is a gram-negative bacterium that contaminates filter-feeding shellfish such as oysters. After ingestion of contaminated oysters, predisposed people may experience highly lethal septicemia. Contamination of wounds with the bacteria can result in devastating necrotizing fasciitis, which can progress to septicemia. The extremely rapid progression of these diseases can render antibiotic treatment ineffective, and death is a frequent outcome. In this study, we examined the potential use of bacteriophages as therapeutic agents against V. vulnificus in an iron-dextran-treated mouse model of V. vulnificus infection. Mice were injected subcutaneously with 10 times the lethal dose of V. vulnificus and injected intravenously, either simultaneously or at various times after infection, with phages. Treatment of mice with phages could prevent death; systemic disease, as measured by CFU per gram of liver and body temperature; and local disease, as measured by CFU per gram of lesion material and histopathologic analysis. Two different phages were effective against three different V. vulnificus strains with various degrees of virulence, while a third phage that required the presence of seawater to lyse bacteria in vitro was ineffective at treating mice. Optimum protection required that the phages be administered within 3 h of bacterial inoculation at doses as high as 108 PFU. One of the protective phages had a half-life in blood of over 2 h. These results demonstrate that bacteriophages have therapeutic potential for both localized and systemic infections caused by V. vulnificus in animals. This model should be useful in answering basic questions regarding phage therapy. [TOP OF PAGE]

  966. Isolation and genetic characterization of a novel filamentous bacteriophage, a deleted form of phage f237, from a pandemic Vibrio parahaemolyticus O4:K68 strain. Chan,B., Miyamoto,H., Taniguchi,H., Yoshida,S.I. (2002). Microbiology and Immunology 46:565-569. We isolated a filamentous bacteriophage, VfO4K68, from the pandemic Vibrio parahaemolyticus strain belonging to 04:K68 serovar. The VfO4K68 DNA lacked a 1,893-bp fragment present in that of the distinctive region of f237, a filamentous phage isolated from a pandemic 03:K6 strain (Nasu, H. et al., J. Clin. Microbiol., 38, 2156-2161, 2000). The deletion resulted in the formation of a novel open reading frame (ORF) that possesses homology to the ORF 27 of ETA phage and staphylococcal enterotoxin E (SEE) of Staphylococcus aureus. VfO4K68 was able to infect the recipient 03:K6 serovar strains. These results suggest that VfO4K68 might act as a genetic transmitter and play some roles in the pandemic V. parahaemolyticus infection. [TOP OF PAGE]

  967. Rapid identification of microorganisms. Chang,T.C., Chen,S., Ding,H.C. (2002). Food Industry Research and Development Institute. 655132(6,428,976). TW. A method of determining whether a test microorganism is a known microorganism, involving use of an agent that specifically affects the growth of the known microorganism. The invention also features a method of identifying E. coli O157:H7 that are based on following criteria: a test microorganism is E. coli O157:H7 if the microorganism is (i) E. coli, (ii) incapable of fermenting sorbitol, and (iii) susceptible to infection by AR1 phage. [TOP OF PAGE]

  968. A conductance method for the identification of Escherichia coli O157:H7 using bacteriophage AR1. Chang,T.C., Ding,H.C., Chen,S. (2002). J. Food Prot. 65:12-17. The feasibility of using a specific phage (AR1) in conjunction with a conductance method for the identification of Escherichia coli O157:H7 was evaluated. The multiplication of strains of E. coli O157:H7 was inhibited by AR1; therefore, a time point (detection time, DT) at which an accelerating change in conductance in the culture broth was not obtained. Bacterial strains were subcultured on sorbitol-MacConkey agar and incubated at 35°C for 24 h, and the ability of the bacteria to ferment sorbitol was recorded. An aliquot of 0.5 ml of the bacterial suspension (107 CFU/ml) and 0.5 ml of the phage suspension (108 PFU/ml) were added to the conductance tube of a Malthus analyzer containing 5 ml of culture broth. The tubes were incubated at 35°C, and conductance changes in the tubes were continuously monitored at 6-min intervals for 24 h by the instrument. A positive reaction was defined as an E. coli strain that could not utilize sorbitol and caused no conductance change (i.e., no DT) within an incubation period of 24 h. Of the 41 strains of E. coli O157:H7 tested, all produced positive reactions. When a total of 155 strains of non-O157:H7 E. coli were tested, 14 did not have a DT within 24 h. However, among these 14 strains, 13 were sorbitol fermenters, and the remaining one was a nonfermenter. Therefore, by definition, only one strain produced a false-positive reaction. The sensitivity and specificity of the present method were 100% (41 of 41) and 99.4% (154 of 155), respectively. The present method incorporating conductimetric measurement and phage AR1 for the identification of E. coli O157:H7 was simple and capable of automation. [TOP OF PAGE]

  969. Distribution of spontaneous mutants and inferences about the replication mode of the RNA bacteriophage f6. Chao,L., Rang,C.U., Wong,L.E. (2002). J. Virol. 76:3276-3281. When a parent virus replicates inside its host, it must first use its own genome as the template for replication. However, once progeny genomes are produced, the progeny can in turn act as templates. Depending on whether the progeny genomes become templates, the distribution of mutants produced by an infection varies greatly. While information on the distribution is important for many population genetic models, it is also useful for inferring the replication mode of a virus. We have analyzed the distribution of mutants emerging from single bursts in the RNA bacteriophage f6 and find that the distribution closely matches a Poisson distribution. The match suggests that replication in this bacteriophage is effectively by a stamping machine model in which the parental genome is the main template used for replication. However, because the distribution deviates slightly from a Poisson distribution, the stamping machine is not perfect and some progeny genomes must replicate. By fitting our data to a replication model in which the progeny genomes become replicative at a given rate or probability per round of replication, we estimated the rate to be very low and on the on the order of 10(-4). We discuss whether different replication modes may confer an adaptive advantage to viruses. [TOP OF PAGE]

  970. Effect of surfactants on the survival and sorption of viruses. Chattopadhyay,D., Chattopadhyay,S., Lyon,W.G., Wilson,J.T. (2002). Environ. Sci. Technol. 36:4017-4024. There is an increasing concern about the protection of groundwater from contamination by enteric viruses and the prevention of outbreaks of waterborne diseases. Knowledge of survivability and transport of viruses from their point of origin is necessary to determine their potential effects on the neighboring groundwater systems. The distribution of virus is, in turn, dependent on the physical and chemical compositions of the surrounding soil and subsurface systems. For the present study, we have determined the effects of different surfactants (cationic, anionic, nonionic, and biological) and natural organic matter (NOM) on bacteriophages. Results indicated that surfactants and NOM adversely affect phage survival in binary systems, with surfactants being the most harmful. Studies with ternary systems also showed that the presence of surfactants reduced sorption of phages on sorbents either by occupying available sorption sites on the sorbent material or by displacing the sorbed phages from the sorbent surface. Water contact angles of the selected phages and different sorbent surfaces have been measured. Experimental data demonstrated that the sorption of hydrophobic viruses was favored by hydrophobic sorbents, while the sorption of hydrophilic viruses was favored by hydrophilic sorbents. [TOP OF PAGE]

  971. Genomic sequence and evolution of marine cyanophage P60: a new insight on lytic and lysogenic phages. Chen,F., Lu,J. (2002). Appl. Environ. Microbiol. 68:2589-2594. The genome of cyanophage P60, a lytic virus which infects marine Synechococcus WH7803, was completely sequenced. The P60 genome contained 47,872 bp with 80 potential open reading frames that were mostly similar to the genes found in lytic phages like T7, fYeO3-12, and SIO1. The DNA replication system, consisting of primase-helicase and DNA polymerase, appeared to be more conserved in podoviruses than in siphoviruses and myoviruses, suggesting that DNA replication genes could be the critical elements for lytic phages. Strikingly high sequence similarities in the regions coding for nucleotide metabolism were found between cyanophage P60 and marine unicellular cyanobacteria. [TOP OF PAGE]

  972. Method of detecting a pathogen using a virus. Cherwonogrodzky,J.W., Lotfali,K. (2002). Her Majesty the Queen in right of Canada, as represented by the Minister of. 376288(6,436,652). Ottawa, CA. A bacteriophage linked to an enzyme can replace an antibody in a system for detecting the presence of a bacteria in a sample. Specifically Brucella abortus (a pathogen which causes brucellosis in cattle) can be detected using Brucella bacteriophage for the virus, urease for the enzyme linked to the bacteriophage, m-maleimidobenzoyl-N-hydrosysuccimide ester as a coupling reagent, sera from mice immunized with Brucella bacteriophage for a detector antibody, urease conjugated to anti-mouse sheep antibody for an indicator, and urea with bromcresol purple as the substrate. The materials can be used in indirect (sandwich) or direct enzyme-linked viral assays (ELVirA). [TOP OF PAGE]

  973. Method of detecting a pathogen using a virus. Cherwonogrodzky,J.W., Lotfali,K. (2002). Her Majesty the Queen in right of Canada, as represented by the Minister of. 514096(6,355,445). Ottawa, CA. A bacteriophage linked to an enzyme can replace an antibody in a system for detecting the presence of a bacteria in a sample. Specifically Brucella abortus (a pathogen which causes brucellosis in cattle) can be detected using Brucella bacteriophage for the virus, urease for the enzyme linked to the bacteriophage, m-maleimidobenzoyl-N-hydrosysuccimide ester as a coupling reagent, sera from mice immunized with Brucella bacteriophage for a detector antibody, urease conjugated to anti-mouse sheep antibody for an indicator, and urea with bromcresol purple as the substrate. The materials can be used in indirect (sandwich) or direct enzyme-linked viral assays (ELVirA). [TOP OF PAGE]

  974. [The antiviral activity of chitosan (review)]. Chirkov,S.N. (2002). Prikladnaia Biokhimiia I Mikrobiologiia 38:5-13. Data on the inhibitory effect of chitosan on viral infections in animals, plants, and microorganisms are reviewed. The effects of the physicochemical parameters and structure of chitosan on its antiviral activity are analyzed. Possible mechanisms of the inhibitory effect of chitosan on viral infections are discussed. [TOP OF PAGE]

  975. Filamentous phage active on the gram-positive bacterium Propionibacterium freudenreichii. Chopin,M.C., Rouault,A., Ehrlich,S.D., Gautier,M. (2002). J. Bacteriol. 184:2030-2033. We present the first description of a single-stranded DNA filamentous phage able to replicate in a gram-positive bacterium. Phage B5 infects Propionibacterium freudenreichii and has a genome consisting of 5,806 bases coding for 10 putative open reading frames. The organization of the genome is very similar to the organization of the genomes of filamentous phages active on gram-negative bacteria. The putative coat protein exhibits homology with the coat proteins of phages PH75 and Pf3 active on Thermus thermophilus and Pseudomonas aeruginosa, respectively. B5 is, therefore, evolutionarily related to the filamentous phages active on gram-negative bacteria. [TOP OF PAGE]

  976. The antibody response to bacteriophage is linked to the lymphopenia gene in congenic BioBreeding rats. Clark,L., Greenbaum,C., Jiang,J., Lernmark,A., Ochs,H. (2002). FEMS Immunol. Med. Microbiol. 32:205-209. Congenic BioBreeding (BB) rats, homozygous for the autosomal lymphopenia (Lyp) gene (Lyp/Lyp), heterozygous (Lyp/+), or wild-type (+/+), were immunized with the T cell-dependent bacteriophage fX174 to determine effects of Lyp on primary and secondary antibody responses. The primary fX174 antibody response did not differ between the three different genotypes. In contrast, the secondary immune response, expressed as the peak neutralizing titer, was markedly reduced in Lyp/Lyp (9.9+/-3.2; mean value+/-S.E.M. for seven rats) compared to both Lyp/+ (51+/-12; n=13; P=0.006) and +/+ (100+/-20; n=7; P=0.004) BB rats. We suggest that the secondary antibody response to the T cell-dependent neoantigen fX174 is linked in a recessive manner to genetic factor(s) in the Lyp gene region. [TOP OF PAGE]

  977. Bacteriophage-resistance systems in dairy starter strains: molecular analysis to application. Coffey,A., Ross,R.P. (2002). Antonie van Leeuwenhoek J. Microbiol. 82:303-321. Starter inhibition by bacteriophage infection in dairy fermentations can limit the usage of specific bacterial strains used in the manufacture of Cheddar, Mozzarella and other cheeses and can result in substantial economic losses. A variety of practical measures to alleviate the problem of phage infection have been adopted over the years but has invariably resulted in a very limited number of strains which can withstand intensive usage in industry. The application of genetic techniques to improve the phage-resistance of starter cultures for dairy fermentations has been intensively studied for the last 20 years to a point where this approach now has significant potential to alleviate the problem. This paper highlights the recent findings and developments that have been described in the literature that will have an impact on improvement of the phage-resistance of starter cultures. [TOP OF PAGE]

  978. Est-ce que les bactériophages pourraient être une thérapie antimicrobienne efficace pour résoudre le problème de la résistance bactérienne aux antibiotiques ? Colbert,M., Guilmette,M. (2002). [TOP OF PAGE]

  979. Occurrence and levels of indicator bacteriophages in bathing waters throughout Europe. Contreras-Coll,N., Lucena,F., Mooijman,K., Havelaar,A., Pierz,V., Boque,M., Gawler,A., Holler,C., Lambiri,M., Mirolo,G., Moreno,B., Niemi,M., Sommer,R., Valentin,B., Wiedenmann,A., Young,V., Jofre,J. (2002). Water Res. 36:4963-4974. Somatic coliphages, F-specific RNA bacteriophages, bacteriophages infecting Bacteroides fragilis, Escherichia coli and enterococci were counted in bathing waters in the late spring and summer. We tested fresh and marine bathing waters from North, South, East and West Europe expected to contain between 100 and 500 E. coli per 100 ml, although wider ranges were sometimes found. Bacteriophages were counted after concentration, since a preliminary study proved that this step was necessary to obtain positive counts. During monitoring, a first-line quality control with reference materials for bacteria and bacteriophages was performed by all the laboratories participating in the study. The same microbes were also counted in raw sewage samples from various areas in Europe, where the bacterial indicators and the three groups of bacteriophages were detected in roughly the same numbers. All groups of bacteriophages were detected in both fresh and marine bathing waters throughout Europe. Reliable and complete results from 147 samples showed that for log-transformed values, E. coli and bacteriophages were slightly correlated. However, the slope of the regression line changed according to E. coli concentration and the correlation diminished when this concentration was close to zero per 100 ml. The ratios between E. coli and phages in bathing waters differed significantly from those in sewage. The relative amounts of bacteriophages, mainly somatic coliphages and phages infecting Bact. fragilis RYC2056, increased in bathing waters with low E. coli concentration, especially in seawater samples containing < 100 E. coli per 100 ml. The relationship of bacteriophages with respect to enterococci paralleled that of bacteriophages with respect to E. coli. Somatic coliphages and bacteriophages infecting Bact. fragilis are useful to predict the presence of some pathogens with the same origin as present bacterial indicators but with higher survival rates. [TOP OF PAGE]

  980. Sequence analysis of the lactococcal bacteriophage bIL170: insights into structural proteins and HNH endonucleases in dairy phages. Crutz-Le Coq,A.-M., Cesselin,B., Commissaire,J., Anba,J. (2002). Microbiology 148:985-1001. The complete 31754 bp genome of bIL170, a virulent bacteriophage of Lactococcus lactis belonging to the 936 group, was analysed. Sixty-four ORFs were predicted and the function of 16 of them was assigned by significant homology to proteins in databases. Three putative homing endonucleases of the HNH family were found in the early region. An HNH endonuclease with zinc-binding motif was identified in the late cluster, potentially being part of the same functional module as terminase. Three putative structural proteins were analysed in detail and show interesting features among dairy phages. Notably, gpl12 (putative fibre) and gpl20 (putative baseplate protein) of bIL170 are related by at least one of their domains to a number of multi-domain proteins encoded by lactococcal or streptococcal phages. A 110- to 150-aa-long hypervariable domain flanked by two conserved motifs of about 20 aa was identified. The analysis presented here supports the participation of some of these proteins in host-range determination and suggests that specific adsorption to the host may involve a complex multi-component system. Divergences in the genome of phages of the 936 group, that may have important biological properties, were noted. Insertions/deletions of units of one or two ORFs were the main source of divergence in the early clusters of the two entirely sequenced phages, bIL170 and sk1. An exchange of fragments probably affected the regions containing the putative origin of replication. It led to the absence in bIL170 of the direct repeats recognized in sk1 and to the presence of different ORFs in the ori region. Shuffling of protein domains affected the endolysin (putative cell-wall binding part), as well as gpl12 and gpl20. [TOP OF PAGE]

  981. Autolysis and autoaggregation in Pseudomonas aeruginosa colony morphology mutants. D'Argenio,D.A., Calfee,M.W., Rainey,P.B., Pesci,E.C. (2002). J. Bacteriol. 184:6481-6489. Two distinctive colony morphologies were noted in a collection of Pseudomonas aeruginosa transposon insertion mutants. One set of mutants formed wrinkled colonies of autoaggregating cells. Suppressor analysis of a subset of these mutants showed that this was due to the action of the regulator WspR and linked this regulator (and the chemosensory pathway to which it belongs) to genes that encode a putative fimbrial adhesin required for biofilm formation. WspR homologs, related in part by a shared GGDEF domain, regulate cell surface factors, including aggregative fimbriae and exopolysaccharides, in diverse bacteria. The second set of distinctive insertion mutants formed colonies that lysed at their center. Strains with the most pronounced lysis overproduced the Pseudomonas quinolone signal (PQS), an extracellular signal that interacts with quorum sensing. Autolysis was suppressed by mutation of genes required for PQS biosynthesis, and in one suppressed mutant, autolysis was restored by addition of synthetic PQS. The mechanism of autolysis may involve activation of the endogenous prophage and phage-related pyocins in the genome of strain PAO1. The fact that PQS levels correlated with autolysis suggests a fine balance in natural populations of P. aeruginosa between survival of the many and persistence of the few. [TOP OF PAGE]

  982. Higher abundance of bacteria than of viruses in deep mediterranean sediments. Danovaro,R., Manini,E., Dell'Anno,A. (2002). Appl. Environ. Microbiol. 68:1468-1472. The interactions between viral abundance and bacterial density, biomass, and production were investigated along a longitudinal transect consisting of nine deep-sea stations encompassing the entire Mediterranean basin. The numbers of viruses were very low (range, 3.6 x 10(7) to 12.0 x 10(7) viruses g(-1)) and decreased eastward. The virus-to-bacterium ratio was always < 1.0, indicating that the deep-sea sediments of the Mediterranean Sea are the first example of a marine ecosystem not numerically dominated by viruses. The lowest virus numbers were found where the lowest bacterial metabolism and turnover rates and the largest cell size were observed, suggesting that bacterial doubling time might play an important role in benthic virus development. [TOP OF PAGE]

  983. A satellite phage-encoded antirepressor induces repressor aggregation and cholera toxin gene transfer. Davis,B.M., Kimsey,H.H., Kane,A.V., Waldor,M.K. (2002). EMBO J. 21:4240-4249. CTXphi is a filamentous bacteriophage whose genome encodes cholera toxin, the principal virulence factor of Vibrio cholerae. We have found that the CTXphi-related element RS1 is a satellite phage whose transmission depends upon proteins produced from a CTX prophage (its helper phage). However, unlike other satellite phages and satellite animal viruses, RS1 can aid the CTX prophage as well as exploit it, due to the RS1-encoded protein RstC. RstC, whose function previously was unknown, is an antirepressor that counteracts the activity of the phage repressor RstR. RstC promotes transcription of genes required for phage production and thereby promotes transmission of both RS1 and CTXphi. Antirepression by RstC also induces expression of the cholera toxin genes, ctxAB, and thus may contribute to the virulence of V.cholerae. In vitro, RstC binds directly to RstR, producing unusual, insoluble aggregates containing both proteins. In vivo, RstC and RstR are both found at the cell pole, where they again appear to form stable complexes. The sequestration/inactivation process induced by RstC resembles those induced by mutant polyglutamine-containing proteins implicated in human neurodegenerative disorders. [TOP OF PAGE]

  984. Transformation in aquatic environments. Day,M. (2002). pp. 63-80. In In Syvanen,M. and Kado,C.I. (eds.), Horizontal Gene Transfer. Academic Press, San Diego. [TOP OF PAGE]

  985. Comparative genomics of phages and prophages in lactic acid bacteria. Desiere,F., Lucchini,S., Canchaya,C., Ventura,M., Brüssow,H. (2002). Antonie van Leeuwenhoek J. Microbiol. 82:73-91. Comparative phage genomics has become possible due to the availability of more than 100 complete phage genome sequences and the development of powerful bioinformatics tools. This technology, profiting from classical molecular-biology knowledge, has opened avenues of research for topics, which were difficult to address in the past. Now, it is possible to retrace part of the evolutionary history of phage modules by comparative genomics. The diagnosis of relatedness is hereby not uniquely based on sequence similarity alone, but includes topological considerations of genome organization. Detailed transcription maps have allowed in silico predictions of genome organization to be verified and refined. This comparative knowledge is providing the basis for a new taxonomic classification concept for bacteriophages infecting low G + C-content Gram-positive bacteria based on the genetic organization of the structural gene module. An Sfi21-like and an Sfi11-like genus of Siphoviridae is proposed. The gene maps of many phages show remarkable synteny in their structural genes defining a lambda super-group within Siphoviridae. A hierarchy of relatedness within the lambda super-group suggests elements of vertical evolution in Siphoviridae. Tailed phages are the result of both vertical and horizontal evolution and are thus fascinating objects for the study of molecular evolution. Prophage sequences integrated into the genomes of their bacterial host present theoretical challenges for evolutionary biologists. Prophages represent up to 10% of the genome in some LAB. In pathogenic streptococci prophages confer genes of selective value for the lysogenic cell. The lysogenic conversion genes are located between the lysin gene and the right phage attachment site. Non-attributed genes were found at the same genome position of prophages from lactic streptococci. These genes belong to the few prophage genes transcribed in the lysogen. Prophages from dairy bacteria might therefore also contribute to the evolutionary fitness of non-pathogenic LAB. [TOP OF PAGE]

  986. Snapshot of the genome of the pseudo-T-even bacteriophage RB49. Desplats,C., Dez,C., Tetart,F., Eleaume,H., Krisch,H.M. (2002). J. Bacteriol. 184:2789-2804. RB49 is a virulent bacteriophage that infects Escherichia coli. Its virion morphology is indistinguishable from the well-known T-even phage T4, but DNA hybridization indicated that it was phylogenetically distant from T4 and thus it was classified as a pseudo-T-even phage. To further characterize RB49, we randomly sequenced small fragments corresponding to about 20% of the approximately 170-kb genome. Most of these nucleotide sequences lacked sufficient homology to T4 to be detected in an NCBI BlastN analysis. However, when translated, about 70% of them encoded proteins with homology to T4 proteins. Among these sequences were the numerous components of the virion and the phage DNA replication apparatus. Mapping the RB49 genes revealed that many of them had the same relative order found in the T4 genome. The complete nucleotide sequence was determined for the two regions of RB49 genome that contain most of the genes involved in DNA replication. This sequencing revealed that RB49 has homologues of all the essential T4 replication genes, but, as expected, their sequences diverged considerably from their T4 homologues. Many of the nonessential T4 genes are absent from RB49 and have been replaced by unknown sequences. The intergenic sequences of RB49 are less conserved than the coding sequences, and in at least some cases, RB49 has evolved alternative regulatory strategies. For example, an analysis of transcription in RB49 revealed a simpler pattern of regulation than in T4, with only two, rather than three, classes of temporally controlled promoters. These results indicate that RB49 and T4 have diverged substantially from their last common ancestor. The different T4-type phages appear to contain a set of common genes that can be exploited differently, by means of plasticity in the regulatory sequences and the precise choice of a large group of facultative genes. [TOP OF PAGE]

  987. Bacteriophages: potential treatment for bacterial infections. Duckworth,D.H., Gulig,P.A. (2002). BioDrugs 16:57-62. Bacteriophages (phages) are viruses of bacteria that can kill and lyse the bacteria they infect. After their discovery early in the 20th century, phages were widely used to treat various bacterial diseases in people and animals. After this enthusiastic beginning to phage therapy, problems with inappropriate use and uncontrolled studies and ultimately the development of antibacterials caused a cessation of phage therapy research in the West. However, a few institutions in Eastern Europe continued to study and use phages as therapeutic agents for human infections. The alarming rise in antibacterial resistance among bacteria has led to a review of the Eastern European studies and to the initiation of controlled experiments in animal models. These recent studies have confirmed that phages can be highly effective in treating many different types of bacterial infections. The lethality and specificity of phages for particular bacteria, the ability of phages to replicate within infected animal hosts, and the safety of phages make them efficacious antibacterial agents. Although there are still several hurdles to be overcome, it appears likely that phage therapy will regain a role in both medical and veterinary treatment of infectious diseases, especially in the scenario of emerging antibacterial resistance. [TOP OF PAGE]

  988. Removal and inactivation of indicator bacteriophages in fresh waters. Duran,A.E., Muniesa,M., Mendez,X., Valero,F., Lucena,F., Jofre,J. (2002). J. Appl. Microbiol. 92:338-347. Aims: The removal and inactivation of faecal coliform (FC) bacteria, enterococci (ENT), sulphite-reducing clostridia (SRC), somatic coliphages, F-specific RNA bacteriophages and bacteriophages infecting Bacteroides fragilis in fresh waters. Methods and Results: Removal was studied in two areas of a river. The results showed different removal of each group of microbes. Faecal coliform bacteria were removed faster than any other, whereas SRC and bacteriophages infecting Bact. fragilis were the most persistent. Inactivation was measured by 'in situ' experiments, which showed significant differences in survival of the different groups of bacterial and bacteriophage indicators. The SRC and bacteriophages were more resistant than faecal coliforms and enterococci, with the exception of F-specific RNA bacteriophages in the summer. Inactivation experiments with pure cultures of bacteriophages confirmed that phage B40-8 of Bact. fragilis was the most resistant. Conclusions: Bacteria and bacteriophages show different resistance to natural inactivation. The use of phages allows information to be obtained in addition to that provided by bacterial indicators. Somatic coliphages and phages infecting Bact. fragilis might supply that indicator function. Significance and Impact of the Study: Confirmation was obtained that bacteriophages provided additional information to that provided by bacterial indicators to monitor the natural inactivation of viruses and/or pathogens. [TOP OF PAGE]

  989. Combined antimicrobial effect of nisin and a listeriophage against Listeria monocytogenes in broth but not in buffer or on raw beef. Dykes,G.A., Moorhead,S.M. (2002). Int. J. Food Microbiol. 73:71-81. The effect of nisin and listeriophage LH7, alone and in combination, on the growth and survival of two strains of Listeria monocytogenes in broth and two model food systems, with appropriate controls, was determined. Growth curves for both bacterial strains in tryptic soy broth incubated at 7 or 30 degrees C, and with the addition of nisin and/or listeriophage at lag, mid-exponential or early stationary phase, were obtained by measuring absorbance at 550 nm. Numbers of mixed populations of both L. monocytogenes strains in phosphate buffered saline (pH 5.5) and on vacuum-packaged fresh beef, both stored for 4 weeks at 4 degrees C, and with the addition of nisin and/or listeriophage, were determined. This was achieved by plating appropriately diluted samples on both Tryptic Soy Agar and Modified Oxford Agar to determine both L. monocytogenes numbers and the presence of sub-lethal injury. In broth nisin alone, reduced levels or prevented growth of the two strains under the conditions studied, but regrowth to levels equivalent to those of untreated cells, occurred. Listeriophage LH7 alone, on the other hand, had no effect in broth under the conditions studied. Notably, however, a mixture of nisin and listeriophage displayed a combined effect in broth and reduced levels of cells substantially without regrowth under the conditions studied. In both model food systems only nisin appeared to be active, in a manner consistent with existing literature, and no combined action was apparent. The use of nisin and listeriophage has potential to control L. monocytogenes in foods but a further understanding of the interactions in this complex system needs to be achieved before it could be applied practically. [TOP OF PAGE]

  990. Isolation and characterization of two types of actinophages infecting Streptomyces scabies MR13. el Sayed,A.e., el Didamony,G., Mansour,K. (2002). Acta Microbiol. Immunol. Hungarica 49:469-482. Two types of actinophages, phi S and phi L, were isolated from soil samples by using Streptomyces scabies MR13, a potato scab pathogen, as an indicator strain. The phages were partially characterized according to their physicochemical properties, plaques and particles morphology and their host-range. The host-range of these phages was narrow for phi S and wide for phi L. The adsorption rate constants of the phi S and phi L were 3.44 x 10-9 and 3.18 x 10-9 ml/min, and their burst sizes were 1.61 and 3.75 virions, respectively. One-step growth indicated that phi S and phi L have a latent period of 30 min followed by a rise period of 30 min. The temperate character of these phages was tested in other isolates of Streptomyces. Four of the phages (phi SS3, phi SS12, phi SS13 and phi SS17) were identified as temperate phages, since they were able to lysogenize SS3, SS12, SS13 and SS17. phi SS3, phi SS12 and phi SS13 were homoimmune, and they were heteroimmune with respect to phi SS17. The restriction barriers of lysogenic isolates (SS12, SS13 and SS17) interfered with the blockage of plaques formation by phages (phi SS12, phi SS13 or phi SS17) propagated on them, about 75% of lysogenic isolates had restriction systems. The exposure of the lysogenic isolates (SS12, SS13 and SS17) to UV-irradiation prevented the possible restriction barriers of these isolates, and these barriers could be overcome. [TOP OF PAGE]

  991. Biological properties and cell tropism of Chp2, a bacteriophage of the obligate intracellular bacterium Chlamydophila abortus. Everson,J.S., Garner,S.A., Fane,B., Liu,B.L., Lambden,P.R., Clarke,I.N. (2002). J. Bacteriol. 184:2748-2754. A number of bacteriophages belonging to the Microviridae have been described infecting chlamydiae. Phylogenetic studies divide the Chlamydiaceae into two distinct genera, Chlamydia and Chlamydophila, containing three and six different species, respectively. In this work we investigated the biological properties and host range of the recently described bacteriophage Chp2 that was originally discovered in Chlamydophila abortus. The obligate intracellular development cycle of chlamydiae has precluded the development of quantitative approaches to assay bacteriophage infectivity. Thus, we prepared hybridomas secreting monoclonal antibodies (monoclonal antibodies 40 and 55) that were specific for Chp2. We demonstrated that Chp2 binds both C. abortus elementary bodies and reticulate bodies in an enzyme-linked immunosorbent assay. Monoclonal antibodies 40 and 55 also detected bacteriophage Chp2 antigens in chlamydia-infected eukaryotic cells. We used these monoclonal antibodies to monitor the ability of Chp2 to infect all nine species of chlamydiae. Chp2 does not infect members of the genus Chlamydia (C. trachomatis, C. suis, or C. muridarum). Chp2 can infect C. abortus, C. felis, and C. pecorum but is unable to infect other members of this genus, including C. caviae and C. pneumoniae, despite the fact that these chlamydial species support the replication of very closely related bacteriophages. [TOP OF PAGE]

  992. Giardia and Cryptosporidium removal from waste-water by a duckweed (Lemna gibba L.) covered pond. Falabi,J.A., Gerba,C.P., Karpiscak,M.M. (2002). Lett. Appl. Microbiol. 34:384-387. AIMS: To determine the ability of duckweed ponds used to treat domestic waste-water to remove Giardia and Cryptosporidium. METHODS AND RESULTS: The influent and effluent of a pond covered with duckweed with a 6 day retention time was tested for Giardia cysts, Cryptosporidium oocysts, faecal coliforms and coliphage. Giardia cysts and Cryptosporidium oocysts were reduced by 98 and 89%, respectively, total coliforms by 61%, faecal coliforms by 62% and coliphage by 40%. There was a significant correlation between the removal of Giardia cysts and Cryptospordium oocysts by the pond (P < 0.001). Influent turbidity and parasite removal were also significantly correlated (Cryptosporidium and turbidity, P=0.05; Giardia and turbidity, P=0.01). CONCLUSIONS: The larger organisms (parasites) probably settled to the bottom of the pond, while removal of smaller bacteria and coliphages in the pond was not as effective. SIGNIFICANCE AND IMPACT OF THE STUDY: Duckweed ponds may play an important role in wetland systems for reduction of Giardia and Cryptosporidium. [TOP OF PAGE]

  993. RS1 element of Vibrio cholerae can propagate horizontally as a filamentous phage exploiting the morphogenesis genes of CTXF. Faruque,S.M., Kamruzzaman,M., Nandi,R.K., Ghosh,A.N., Nair,G.B., Mekalanos,J.J., Sack,D.A. (2002). Infect. Immun. 70:163-170. In toxigenic Vibrio cholerae, cholera toxin is encoded by the CTX prophage, which consists of a core region carrying ctxAB genes and genes required for CTXF morphogenesis, and an RS2 region encoding regulation, replication, and integration functions. Integrated CTXF is often flanked by another genetic element known as RS1 which carries all open reading frames (ORFs) found in RS2 and an additional ORF designated rstC. We identified a single-stranded circularized form of the RS1 element, in addition to the CTXF genome, in nucleic acids extracted from phage preparations of 32 out of 83 (38.5%) RS1-positive toxigenic V. cholerae strains analyzed. Subsequently, the corresponding double-stranded replicative form (RF) of the RS1 element was isolated from a representative strain and marked with a kanamycin resistance (Km(r)) marker in an intergenic site to construct pRS1-Km. Restriction and PCR analysis of pRS1-Km and sequencing of a 300-bp region confirmed that this RF DNA was the excised RS1 element which formed a novel junction between ig1 and rstC. Introduction of pRS1-Km into a V. cholerae O1 classical biotype strain, O395, led to the production of extracellular Km(r) transducing particles, which carried a single-stranded form of pRS1-Km, thus resembling the genome of a filamentous phage (RS1-KmF). Analysis of V. cholerae strains for susceptibility to RS1-KmF showed that classical biotype strains were more susceptible to the phage compared to El Tor and O139 strains. Nontoxigenic (CTX(-)) O1 and O139 strains which carried genes encoding the CTXF receptor toxin-coregulated pilus (TCP) were also more susceptible (>1,000-fold) to the phage compared to toxigenic El Tor or O139 strains. Like CTXF, the RS1F genome also integrated into the host chromosomes by using the attRS sequence. However, only transductants of RS1-KmF which also harbored the CTXF genome produced a detectable level of extracellular RS1-KmF. This suggested that the core genes of CTXF are also required for the morphogenesis of RS1F. The results of this study showed for the first time that RS1 element, which encodes a site-specific recombination system in V. cholerae, can propagate horizontally as a filamentous phage, exploiting the morphogenesis genes of CTXF. [TOP OF PAGE]

  994. Legionella and Legionnaires' disease: 25 years of investigation. Fields,B.S., Benson,R.F., Besser,R.E. (2002). Clin. Microbiol. Rev. 15:506-526. There is still a low level of clinical awareness regarding Legionnaires' disease 25 years after it was first detected. The causative agents, legionellae, are freshwater bacteria with a fascinating ecology. These bacteria are intracellular pathogens of freshwater protozoa and utilize a similar mechanism to infect human phagocytic cells. There have been major advances in delineating the pathogenesis of legionellae through the identification of genes which allow the organism to bypass the endocytic pathways of both protozoan and human cells. Other bacteria that may share this novel infectious process are Coxiella burnetti and Brucella spp. More than 40 species and numerous serogroups of legionellae have been identified. Most diagnostic tests are directed at the species that causes most of the reported human cases of legionellosis, L. pneumophila serogroup 1. For this reason, information on the incidence of human respiratory disease attributable to other species and serogroups of legionellae is lacking. Improvements in diagnostic tests such as the urine antigen assay have inadvertently caused a decrease in the use of culture to detect infection, resulting in incomplete surveillance for legionellosis. Large, focal outbreaks of Legionnaires' disease continue to occur worldwide, and there is a critical need for surveillance for travel-related legionellosis in the United States. There is optimism that newly developed guidelines and water treatment practices can greatly reduce the incidence of this preventable illness. [TOP OF PAGE]

  995. High control of bacterial production by viruses in a eutrophic oxbow lake. Fischer,U.R., Velimirov,B. (2002). Aquat. Microb. Ecol. 27:1-12. The aim of the study was to test the hypothesis that the magnitude of viral control on bacterial production in a eutrophic oxbow lake of the River Danube would be higher than all average values reported so far in the literature. This assumption was based on the findings of low grazing of heterotrophic nanoflagellates (HNF) in this system, accounting on average for 5% of the bacterial mortality. Several approaches (viral decay method, estimation of the frequency of infected bacterial cells) to determine viral control of bacterial production were applied on a comparative basis. All system-specific parameters necessary to describe virus-bacteria interactions (burst size, bacterial production, contact rates) were monitored simultaneously. The average viral control of bacterial production determined by the different approaches was similar, ranging from 55.7 to 62.7 %, and prevailing over HNF grazing by a factor of more than 11. For individual events, however, we observed large variations between the methods, indicating that the use of one single method is not reliable to decide whether a detected trend is representative of a specific system. We discuss error sources of the applied methods and mathematical models, and accounted for them when calculating the contribution of viruses to bacterial mortality. We demonstrated that viruses could control more than 100 % of the bacterial production in the Alte Donau, which implies that occasionally up to 1.6 % h-1 of the bacterial standing stock was removed from the water column. High bacterial mortality due to viruses indicated that a large amount of dissolved organic carbon might be recycled from bacteria by phage-induced cell lysis. On average 15.2 mu g C l-1 d-1, corresponding to some 46 % of the bacterial secondary production (BSP), was released into the water column due to viral lysis of bacterial cells and again became available for microheterotrophic consumption. [TOP OF PAGE]

  996. Time-delayed spread of viruses in growing plaques. Fort,J., Méndez,V. (2002). Phys. Rev. Lett. 89:178101 The spread of viruses in growing plaques predicted by classical models is greater than that measured experimentally. There is a widespread belief that this discrepancy is due to biological factors. Here we show that the observed speeds can be satisfactorily predicted by a purely physical model that takes into account the delay time due to virus reproduction inside infected cells. No free or adjustable parameters are used. [TOP OF PAGE]

  997. Prokaryotic and viral diversity patterns in marine plankton. Fuhrman,J.A., Griffith,J., Schwalbach,M. (2002). Ecol. Res. 17:183-194. Prokaryotes and viruses play critical roles in marine ecosystems, where they are both highly abundant and active. Although early work on both prokaryotes and viruses revealed little of their diversity, molecular biological approaches now allow us to break apart these 'black boxes.' The most revealing methods have been cloning and sequencing of 16S rRNA genes, community fingerprinting (such as terminal restriction fragment length polymorphism; TRFLP), and fluorescent in situ hybridization. Viral diversity can now be analyzed by pulsed field gel electrophoresis (PFGE) of viral genomes. The present paper summarizes recent advances in bacterial and virus diversity studies, and presents examples of measurements from polar, tropical, and temperate marine waters. Terminal restriction fragment length polymorphism shows that many of the same operationally defined prokaryotic taxa are present in polar and tropical waters, but there are also some unique to each environment. By one measure, a sample from over a Philippine coral reef had about 100 operationally defined taxa, whereas one from the open tropical Atlantic had about 50 and from the icy Weddell Sea, about 60. Pulsed field gel electrophoresis of two depth profiles, to 500 m, from Southern California, measured 2 months apart, shows striking similarities in viral genome length diversity over time, and some distinct differences with depth. The euphotic zone samples had extremely similar apparent diversity, but samples from 150 m and 500 m were different. An obvious next step is to compare the bacterial and viral diversity patterns, because theory tells us they should be related. [TOP OF PAGE]

  998. Sunlight inactivation of human enteric viruses and fecal bacteria. Fujioka,R.S., Yoneyama,B.S. (2002). Water Sci. Technol. 46:291-295. Three human enteric viruses (poliovirus, echovirus, coxsackievirus) suspended in seawater or buffer were stable for 6 hr in the absence of sunlight but were inactivated at the same rate in the presence of sunlight. Under summer sunlight conditions, at least 3 logs of these viruses were inactivated by one-hit kinetics while under winter sunlight conditions only 1 log of these viruses was inactivated by two-hit kinetics. Under these same conditions, 6 logs of E. coli were inactivated within 1 hr by one-hit kinetics under summer and winter conditions. In comparison, E. faecalis was inactivated by two-hit kinetics and only 2.5 logs of inactivation were observed after 4 hr of exposure to winter sunlight. Since human enteric viruses are considerably more resistant to sunlight inactivation than E. coli and moderately more resistant than E. faecalis, marine recreational water quality standards should be based on concentrations of enterococci and not on coliform bacteria. Since the mechanism and rate of inactivation of coliphage and human enteric viruses are similar, coliphages appear to be the best indicator for the presence of human enteric viruses in recreational waters, especially coastal waters where abundant sunshine is available. [TOP OF PAGE]

  999. The cell surface protein Ag43 facilitates phage infection of Escherichia coli in the presence of bile salts and carbohydrates. Gabig,M., Herman-Antosiewicz,A., Kwiatkowska,M., Los,M., Thomas,M.S., Wegrzyn,G. (2002). Microbiology (Reading) 2002 May, 148:1533-1542. It was found that infection of Escherichia coli by bacteriophage lambda is inhibited in the presence of certain bile salts and carbohydrates when cells are in the "OFF" state for production of the phase-variable cell surface protein antigen 43 (Ag43). The inhibition of phage growth was found to be due to a significant impairment in the process of phage adsorption. Expression of the gene encoding Ag43 (agn43) from a plasmid or inactivation of the oxyR gene (encoding an activator of genes important for defence against oxidative stress) suppressed this inhibition. A mutation, rpoA341, in the gene encoding the alpha subunit of RNA polymerase also facilitated phage adsorption in the presence of bile salts and carbohydrates. The rpoA341 mutation promoted efficient production of Ag43 in a genetic background that would otherwise be in the "OFF" phase for expression of the agn43 gene. Analysis of a reporter gene fusion demonstrated that the promoter for the agn43 gene was more active in the rpoA341 mutant than in the otherwise isogenic rpoA(+) strain. The combined inhibitory action of bile salts and carbohydrates on phage adsorption and the abolition of this inhibition by production of Ag43 was not restricted to lambda, as a similar phenomenon was observed for the coliphages P1 and T4. [TOP OF PAGE]

  1000. Bacteriophage replication and reactivation in stationary phase hosts. Gallimore,W.H., Burgess,J.M., Kokjohn,T.A. (2002). Research Signpost 6:467-476. Bacteriophage dynamics in stationary phase or stressed bacterial hosts are poorly understood. Using one-step growth experiments we have demonstrated that stationary phase does not constitute an absolute block to phage multiplication, although latent periods are extended and burst sizes decreased substantially compared to exponential phase infections. Using infectious center assays to quantify lysogen responses to DNA damage revealed that while there was a range of sensitivity to ultraviolet (UV) radiation, no prophages were induced by sunlight exposure. Comparing the capacity of exponential phase and stationary phase cells to resist UV irradiation and reactivate UV-damaged phage revealed that cells maintained in the stationary phase at the time of infection expressed significantly higher levels of DNA repair. Photoreactivation experiments in stationary phase hosts revealed that light-mediated reversal of phage DNA damage definitely occurred in stationary phase bacterial hosts. Our experiments demonstrate that many bacteriophages multiply actively and are competent to reverse DNA damage in post exponential phase host cells. In order to establish the scope and significance of bacteriophages to aquatic ecosystem ecology a more complete understanding of virus dynamics in both growing and stationary phase hosts is essential. [TOP OF PAGE]

  1001. Bacteroides fragilis and Escherichia coli bacteriophages in human faeces. Gantzer,C., Henny,J., Schwartzbrod,L. (2002). International Journal of Hygiene and Environmental Health 205:325-328. Some bacteriophages found in human faeces are being evaluated as possible indicators of viral contamination of water. These bacteriophages include somatic coliphages and Bacteroides fragilis phages. The aims of this study were to determine the occurrence and concentrations of somatic coliphages and Bacteroides fragilis phages in the stools of a human population residing in eastern France (n = 193). Somatic coliphages were detected in 68% of the stools at a mean concentration of 4.3 x 10(3) PFU.g-1 and Bacteroides fragilis phages were detected in 11% of the stools at a mean concentration of 7 x 10(1) PFU.g-1. Statistical analysis showed no correlation between the phage concentration and the age or sex of the human subject. [TOP OF PAGE]

  1002. [The bacteriophages and their gene products as antimicrobial agents]. Garcia,E., Lopez,R. (2002). Rev. Espan. Quimioter. 15:306-312. The viruses that infect bacteria (bacteriophages or phages) were first isolated about 90 years ago. Phages have been fundamental tools in the development of molecular biology. Phages were early hypothesized as therapeutic agents for combatting pathogenic bacteria. However, the discovery and successful use of antibiotics to treat infectious diseases hindered this aim. the development of bacterial resistance to most available drugs has recently led researchers to test the possibilities of using phages as therapeutic agents. We review here recent achievements in this field, taking into consideration former bias in handling phages as well as previous achievements carried out in Eastern Europe where bacteriophages have been employed for decades as an alternative to antibiotics. [TOP OF PAGE]

  1003. Phages and other mobile virulence elements in gram-positive pathogens. Gentry-Weeks,C., Coburn,P.S., Gilmore,M.S. (2002). Curr. Top. Microbiol. Immunol. 264:79-94. [first paragraph] Over the past 30 years, gram-positive bacteria have re-emerged as leading agents of nosocomial and community-acquired infection. This resurgence correlates with the expansion of populations harboring virulence and multiple antibiotic resistance traits. Recent bacterial genome nucleotide sequence determinations show the heretofore unappreciated extent to which mobile genetic elements, including bacteriophages, conjugative plasmids, and transposons, have contributed to the horizontal dissemination of virulence factors and antibiotic resistance determinants. This chapter examines the role that phages, plasmids, and transposons have played in enhancing the ability of highly adapted gram-positive bacteria to cause human disease. [TOP OF PAGE]

  1004. Fluorescent dye labeled bacteriophages--a new tracer for the investigation of viral transport in porous media: 2. Studies of deep-bed filtration. Gitis,V., Adin,A., Nasser,A., Gun,J., Lev,O. (2002). Water Res. 36:4235-4242. Viral transport in deep-bed sand filters was studied by a new method that enables rapid and simple quantitation of labeled viruses. The residence time distribution (RTD) of viruses in the bed was compared to the RTD of a fluorescein dye under conditions that simulate a filter run. The characteristics of the RTD curves for the free dye and the labeled bacteriophages followed very different trends during the filter run. While the retention time of free dye was practically independent of the filtration stage, the average retention time of the labeled bacteriophage depended in a non-linear way on filtration time. Average virus retention time as well as virus-removal efficiency were minimal at the ripening stage, increased during the operational stage and then decreased again towards the turbidity breakthrough stage. This complex trend reflects two opposing mechanisms that dominate the behavior of the filter. During the ripening stage the accumulation of the kaolin-alum material in the filter increases the adsorption surface area and retards virus mobility. After sufficient kaolin-alum deposit is accumulated in the filter, aging and densification of the alum deposit induces size exclusion phenomenon giving faster apparent mobility of viruses in the filter bed. [TOP OF PAGE]

  1005. Fluorescent dye labeled bacteriophages--a new tracer for the investigation of viral transport in porous media: 1. Introduction and characterization. Gitis,V., Adin,A., Nasser,A., Gun,J., Lev,O. (2002). Water Res. 36:4227-4234. A new method for the study of pathogen transport in porous media is presented. The method is based on conjugation of fluorescent dyes to target bacteriophages and application of the modified bacteriophages for tracer studies. We demonstrate that the relevant transport determining properties of Rhodamine and several fluorescein-labeled phages are practically identical to those of the native bacteriophages. The advantages of the proposed method relative to direct enumeration of bacteriophages by plaque forming unit method, turbidity, fluorescent microspheres, and other alternative tracers are discussed. Notable advantages include simple quantitation by optical methods, unbiased signals even when virus aggregates are formed, and the ability to decouple inactivation kinetics from transport phenomena. Additionally, the signal reflects the removal and transport of the studied microorganism and not a surrogate. [TOP OF PAGE]

  1006. Observations on cyanobacterial population collapse in eutrophic lake water. Gons,H.J., Ebert,J., Hoogveld,H.L., van den Hove,L., Pel,R., Takkenberg,W., Woldringh,C.J. (2002). Antonie van Leeuwenhoek J. Microbiol. 81:319-326. In two laboratory-scale enclosures of water from the shallow, eutrophic Lake Loosdrecht (the Netherlands), the predominating filamentous cyanobacteria grew vigorously for 2 weeks, but then their populations simultaneously collapsed, whereas coccoid cyanobacteria and algae persisted. The collapse coincided with a short peak in the counts of virus-like particles. Transmission electron microscopy showed the morphotype Myoviridae phages, with isometric heads of about 90 nm outer diameter and > 100-nm long tails, that occurred free, attached to and emerging from cyanobacterial cells. Also observed were other virus-like particles of various morphology. Similar mass mortality of the filamentous cyanobacteria occurred in later experiments, but not in Lake Loosdrecht. As applies to lakes in general, this lake exhibits high abundance of virus-like particles. The share and dynamics of infectious cyanophages remain to be established, and it is as yet unknown which factors primarily stabilize the host-cyanophage relationship. Observations on shallow, eutrophic lakes elsewhere indicate that the cyanophage control may also fail in natural water bodies exhibiting predominance of filamentous cyanobacteria. Rapid supply of nutrients appeared to be a common history of mass mortality of cyanobacteria and algae in laboratory and outdoor enclosures as well as in highly eutrophic lakes. [TOP OF PAGE]

  1007. Conserved filamentous prophage in Escherichia coli O18:K1:H7 and Yersinia pestis biovar orientalis. Gonzalez,M.D., Lichtensteiger,C.A., Caughlan,R., Vimr,E.R. (2002). J. Bacteriol. 184:6050-6055. Microbial virulence is known to emerge by horizontal gene transfer mechanisms. Here we describe the discovery of a novel filamentous prophage, designated CUS-1, which is integrated into the chromosomal dif homologue of the high-virulence clone Escherichia coli O18:K1:H7. An homologous chromosomal element (CUS-2) in Yersinia pestis biovar orientalis is integrated at the same relative location as CUS-1; both lysogenic E. coli and Y. pestis strains produce particles with properties expected of single-stranded DNA virions. CUS(phi) is epidemiologically correlated with the emergence of K1 strains with increased virulence and with the Y. pestis biovar responsible for the current (third) plague pandemic. [TOP OF PAGE]

  1008. Reporter bacteriophage assays as a means to detect foodborne pathogenic bacteria. Goodridge,L., Griffiths,M. (2002). Food Res. Int. 35:863-870. Bacterial disease due to the consumption of contaminated food is a global problem that has necessitated the need for modern rapid bacterial detection techniques. There has been much recent interest in the use of reporter bacteriophages as a tool to aid in the detection of foodborne, and clinical bacterial pathogens. The reporter bacteriophage concept provides a sensitive method for bacterial detection and sensitivity to antimicrobial agents. This review presents the current status of reporter bacteriophage technology. The bacterial and eucaryotic luciferases, the ice nucleation protein, and the E. coli b-galactosidase reporter genes are discussed, along with many examples that demonstrate the usefulness of reporter bacteriophage as tools to detect foodborne bacterial contamination. [TOP OF PAGE]

  1009. [Action of Spirulina platensis on bacterial viruses]. Gorobets,O.B., Blinkova,L.P., Baturo,A.P. (2002). Zh. Mikrobiol. Epidemiol. Immunobiol. 18-21. The impact of the biomass of the blue-green microalga (cyanobacterium) S. platensis on bacteriophage T4 (bacterial virus) has been evaluated. The study revealed that the addition of S. platensis biomass into the agar nutrient medium, followed by sterilization with 2% chloroform and thermal treatment, produced an inhibiting or stimulating effect on the reproduction of the bacteriophage in Escherichia coli B cells, depending on the concentration of S. platensis and the multiplicity of phage infection, as well as on the fact whether the microalgae were added during the first cycle of the development of the virus. The reproduction of the bacteriophage in E. coli B was influenced by the method and duration of the sterilization of the nutrient medium with S. platensis. [TOP OF PAGE]

  1010. Characterization of phi12, a bacteriophage related to phi6: nucleotide sequence of the large double-stranded RNA. Gottlieb,P., Potgieter,C., Wei,H., Toporovsky,I. (2002). Virology 295:266-271. The isolation of additional bacteriophages besides phi6 containing segmented double-stranded RNA genomes (dsRNA) has expanded the Cystoviridae family to nine members. Comparing the genomic sequences of these viruses has allowed evaluation of important genetic as well as structural motifs. These comparative studies are resulting in greater understanding of viral evolution and the role played by genetic and structural variation in the assembly mechanisms of the cystoviruses. In this regard, the large double-stranded RNA genomic segment of bacteriophage phi12 was copied as cDNA and its nucleotide sequence determined. This genome's organization is similar to that of the large segment of bacteriophages phi6, phi8, and phi13. In the amino acid sequence of the viral RNA-dependent RNA polymerase (P2), similarity was found to the comparable proteins of phi6, phi8, and phi13. Amino acid sequence similarity was also noted in the nucleotide triphosphate phosphorylase (P4) to the comparable proteins of phi8 and phi13. [TOP OF PAGE]

  1011. Characterization of phi 12, a bacteriophage related to phi 6: nucleotide sequence of the small and middle double-stranded RNA. Gottlieb,P., Wei,H., Potgieter,C., Toporovsky,I. (2002). Virology 293:118-124. The isolation of additional bacteriophages containing segmented double-stranded RNA genomes has expanded the Cystoviridae family to nine members. Comparing the genomic sequences of these viruses has allowed evaluation of important genetic as well as structural motifs. These comparative studies are resulting in greater understanding of viral evolution and the role played by genetic and structural variation in the assembly mechanisms of the cystoviruses. In this regard, the small and middle double-stranded RNA genomic segments of bacteriophage phi 12 were copied as cDNA and their nucleotide sequences determined. This genome's organization is similar to that of the small and middle segments of bacteriophages phi 6, phi 8, and phi 13. Although there is little similarity in the nucleotide sequences, similarity exists in the amino acid sequence of the lysis cassette proteins to those of phi 6. The host cell attachment proteins are found to have marked similarity to the phi 13 attachment proteins. [TOP OF PAGE]

  1012. Control of Brochothrix thermosphacta spoilage of pork adipose tissue using bacteriophages. Greer,G.G., Dilts,B.D. (2002). J. Food Prot. 65:861-863. Adipose tissue discs were coinoculated with Brochothrix thermosphacta and homologous bacteriophages (phages) to determine the effects these had on phage multiplication, bacterial growth, and off-odor development during storage at 2 degrees C or under simulated retail display at 6 degrees C. In the presence of about 10(5) bacteria/cm2 and an equivalent number of phages, there was a 3-log increase in phage numbers and a 2-log decrease in bacterial numbers, and objectionable off-odors were suppressed during refrigerated storage. Up to 68% of the surviving bacterial population were resistant to phages. The storage life of adipose tissue could be increased from 4 days in controls to 8 days in phage-treated samples by preventing the development of off-odors associated with the growth of B. thermosphacta. Phages may provide a novel approach to extending the storage quality of chilled meats. [TOP OF PAGE]

  1013. Novel phage-based treatment effective against mycobacterial infections. Greer,M. (2002). TB & Outbreaks Week October 22, 8. Researchers in the United States have developed a novel technique for fighting tuberculosis and other mycobacterial infections. ¶ While effective treatment options are already available for afflicted patients, standard antimicrobial agents are "limited by the emergence of drug resistance and the inability of antibiotics to kill dormant organisms," according to Lawrence Broxmeyer and colleagues at Med-America Research in Whitestone, New York and other institutions in California, Nebraska, and Texas. ¶ To overcome these problems, nonpathogenic mycobacteria could be used to deliver bacteriophage viruses to attack infected cells, Broxmeyer and coauthors argued. ¶ The researchers assessed the antimicrobial efficacy of Mycobacterium smegmatis organisms carrying the lytic TM4 phage virus, introducing these virus-laden microbes into tuberculosis-infected macrophage cultures. The amount of viable intracellular pathogens dropped significantly after phage treatment, according to the report. ¶ Similar results were seen in cultures of cells infected with M. avium, study data showed. This organism is responsible for life-threatening opportunistic infections in HIV/AIDS patients although it is generally harmless in people with normal immune function. ¶ TM4-induced reductions in bacilli levels were both time- and dose-dependent (Killing of Mycobacterium avium and Mycobacterium tuberculosis by a mycobacteriophage delivered by a nonvirulent mycobacterium: a model for phage therapy of intracellular bacterial pathogens. Journal of Infectious Diseases, October 15, 2002;186(8):1155-1160). [TOP OF PAGE]

  1014. Viral distribution and activity in Antarctic waters. Guixa-Boixereu,N., Vaque,D., Gasol,J.M., Sanchez-Camara,J., Pedros-Alio,C. (2002). Deep-Sea Research II 49:827-845. Variability in abundance of virus-like particles (VLP), VLP decay rates and prokaryotic mortality due to viral infection were determined in three Antarctic areas: Bellingshausen Sea, Bransfield Strait and Gerlache Strait, during December 1995 and February 1996. VLP abundance showed very small spatial variability in the three areas (7 x 106-2 x 107 VLP ml-1). VLP abundance, on the other hand, decreased one order of magnitude from the surface to the bottom, in two stations where deep vertical profiles were sampled. Low seasonal variability in VLP abundance was found when comparing each area separately. Diel VLP variability was also very low. VLP abundance showed the lowest values when solar irradiation was maximal, in two of the three stations where diel cycles were examined. Viral decay rates (VDR) were determined using KCN in two kinds of experiments. Type 1 experiments were performed in 6 stations to determine viral decay. Type 2 experiments were carried out in 2 stations to examine the influence of temperature and organic matter concentration on viral decay. VDR was not influenced by these parameters. Prokaryotic mortality due to viral infection was always higher than that due to bacterivores in the stations where both factors of prokaryotic mortality were measured. Viral infection accounted for all the prokaryotic heterotrophic production in Bellingshausen Sea and Gerlache Strait and for half of the prokaryotic heterotrophic production in Bransfield Strait. These high values of prokaryotic mortality due to viral infection are difficult to reconcile in nature, and more work is necessary to determine the mechanisms involved in the disappearance of viruses. [TOP OF PAGE]

  1015. Distinguishing between selection and population expansion in an experimental lineage of bacteriophage T7. Hahn,M.W., Rausher,M.D., Cunningham,C.W. (2002). Genetics 161:11-20. Experimental evolution of short-lived organisms offers the opportunity to study the dynamics of polymorphism over time in a controlled environment. Here, we characterize DNA polymorphism data over time for four genes in bacteriophage T7. Our experiment ran for 2500 generations and populations were sampled after 500, 2000, and 2500 generations. We detect positive selection, purifying ("negative") selection, and population expansion in our experiment. We also present a statistical test that is able to distinguish demographic from selective events, processes that are hard to identify individually because both often produce an excess of rare mutations. Our "heterogeneity test" modifies common statistics measuring the frequency spectrum of polymorphism (e.g., Fu and Li's D) by looking for processes producing different patterns on nonsynonymous and synonymous mutations. Test results agree with the known conditions of the experiment, and we are therefore confident that this test offers a tool to evaluate natural populations. Our results suggest that instances of segregating deleterious mutations may be common, but as yet undetected, in nature. [TOP OF PAGE]

  1016. Use of signal-mediated amplification of RNA technology (SMART) to detect marine cyanophage DNA. Hall,M.J., Wharam,S.D., Weston,A., Cardy,D.L.N., Wilson,W.H. (2002). BioTechniques 32:604-611. Here, we describe the application of an isothermal nucleic acid amplification assay, signal-mediated amplification of RNA technology (SMART), to detect DNA extracted from marine cyanophages known to infect unicellular cyanobacteria from the genus Synechococcus. The SMART assay is based on the target-dependent production of multiple copies of an RNA signal, which is measured by an enzyme-linked oligosorbent assay. SMART was able to detect both synthetic oligonucleotide targets and genomic cyanophage DNA using probes designed against the portal vertex gene (g20). Specific signals were obtained for each cyanophage strain (S-PM2 and S-BnMI). Nonspecific genomic DNA did not produce false signals or inhibit the detection of a specific target. In addition, we found that extensive purification of target DNA may not be required since signals were obtained from crude cyanophage lysates. This is the first report of the SMART assay being used to discriminate between two similar target sequences. [TOP OF PAGE]

  1017. Microbiology. A tail of two specifi-cities. Hatfull,G.F. (2002). Science 295:2031-2032. In the world of microbial biowarfare, standing still in the face of a ferocious viral attack is a recipe for disaster. Various subspecies of Bordetella bacteria, which cause respiratory illness in mammals including whooping cough in humans, must withstand attack by bacteriophage viruses such as BPP-1. They accomplish this by switching between two distinct phases: One (Bvg+) expresses virulence genes and colonizes the respiratory tract of hosts, and the other (Bvg–) is better adapted for growth outside of a host (1). Among the genes whose expression is altered on switching between these two phases is prn. This gene encodes an adhesion protein, pertactin, which is the receptor for the bacteriophage BPP-1. Thus, BPP-1 infects only the Bvg+ strain of Bordetella that expresses pertactin and not the Bvg– strain. [TOP OF PAGE]

  1018. Effects of temperatures, pH-values, ultra-violet light, ethanol and chloroform on the growth of isolated thermophilic Bacillus phages. Hazem,A. (2002). New. Microbiol. 25:469-476. Seven thermophilic Bacillus phages were characterized with reference to their host range, time of appearance, morphology of plaques, thermal inactivation, stability, lipid presence and inactivation by ultraviolet irradiation. Response surface methodology was adapted to describe the response of growth parameters to environmental changes. Most phages are susceptible to temperatures above 60 degrees C and inactivated immediately at 103 degrees C. Most phages are resistant to pH ranges 5 to 9 and almost all to pH 7 to 8. Both phages 46 and 80 were highly resistance to UV exposure for 13 minutes and 20 minutes, respectively. The presence of chloroform or 75% ethanol showed no effect on almost all isolated phages that indicate of possibility of the absence of lipids. The isolated phages were slow in their growth, possibly due to the lower gross growth efficiency. [TOP OF PAGE]

  1019. Bacteriophages: evolution of the majority. Hendrix,R.W. (2002). Theor. Pop. Biol. 61:471-480. The dsDNA-tailed bacteriophages are probably the largest evolving group in the Biosphere and they are arguably very ancient. Comparative examination of genomes indicates that the hallmark of phage evolution is horizontal exchange of sequences. This is accomplished, first, by rampant non-homologous recombination between different genomes and, second, by reassortment of the variant sequences so created through homologous recombination. The comparative analysis suggests mechanisms by which new genes can be added to phage genomes and by which genes with novel functions may be assembled from parts. Horizontal exchange of sequences occurs most frequently among closely related phages, but it also extends across the entire global population at lower frequency. Bacteriophages also have probable ancestral connections with viruses of eukaryotes and archaea. [TOP OF PAGE]

  1020. Evolutionary relationships among diverse bacteriophages and prophages: All the world's a phage. Hendrix,R.W., Smith,M.C.M., Burns,R.N., Ford,M.E., Hatfull,G.F. (2002). pp. 133-140. In In Syvanen,M. and Kado,C.I. (eds.), Horizontal Gene Transfer. Academic Press, San Diego. [TOP OF PAGE]

  1021. Bacteriophage RM 378 of a thermophilic host organism. Hjorleifsdottir,S., Hreggvidsson,G.O., Fridjonsson,O.H., Aevarsson,A., Kristjansson,J.K. (2002). 585858(6,492,161). A novel bacteriophage RM 378 of Rhodothermus marinus, the nucleic acids of its genome, nucleic acids comprising nucleotide sequences of open reading frames (ORFs) of its genome, and polypeptides encoded by the nucleic acids, are described. [TOP OF PAGE]

  1022. Enteric bacteriophages as potential fecal indicators in ground beef and poultry meat. Hsu,F.-C., Shieh,Y.S.C., Sobsey,M.D. (2002). J. Food Prot. 65:93-99. Recovery efficiencies of enteric bacteriophages (F+ RNA coliphages, somatic coliphages, and Salmonella phages) as alternative fecal indicators were determined from ground beef and chicken breast meat using amino acid eluants (glycine and threonine) and a complex eluant (3% beef extract). Levels of F+ RNA coliphages (MS2, GA, Qbeta, FI, and SP), the somatic coliphage PHIX174, and three environmental isolates of Salmonella phages (isolated from raw sewage) were assayed using three respective hosts: Escherichia coli Famp, E. coli C, and Salmonella typhimurium. When 8% polyethylene glycol and 0.1 M NaCl were used to precipitate bacteriophages eluted with five different eluants, the highest recoveries of the three phage groups were with 0.5 M threonine and 0.25 M glycine-threonine. The average recoveries of F+ RNA coliphages, somatic coliphages, and the Salmonella phages from ground beef and chicken meat were 100, 69, and 65%, respectively, with threonine (0.5 M, pH 9.0) as the eluate. Of eight market food samples tested, F+ RNA coliphages were detected in five (63%) and somatic coliphages were detected in seven (88%). The overall detection sensitivity of the method was 3 PFU/100 g of ground beef or chicken meat. Levels of bacteriophages and bacterial indicators on chicken carcass surfaces were determined at identified critical control points at a poultry plant. Through the processing steps of evisceration, washing, and chilling, the levels of F+ RNA coliphages and fecal coliforms were reduced by 1.6 and 1.9 log10 PFU or CFU/100 g, respectively. F+ RNA coliphages and perhaps other enteric bacteriophages may be effective candidate indicators for monitoring the microbiological quality of meat, poultry, and perhaps other foods during processing. The bacteriophage concentration method developed provides a simple, rapid, and practical tool for the evaluation of fecal contamination levels in ground beef and processed chicken meat. [TOP OF PAGE]

  1023. Uneven growth and different susceptibility to viruses among bacteria increase estimates of virus production in the East Sea based on TEM observation. Huang,C.Y., Cho,B.C. (2002). Aquat. Microb. Ecol. 27:211-218. We developed a theory that uneven distribution of bacterial growth rates and different susceptibility to viral infection in bacterial community could result in higher estimates of virus production based on transmission electron microscopy (TEM) observation (VPTEM) compared to estimates obtained by neglecting uneven growth and different susceptibilities in the bacterial community. We tested this idea by classifying bacteria into 4 groups based on morphotypes (i.e. rods, cocci, curved shapes, and spirillae) and enumerating the frequency of visibly infected cells (FVIC) and the frequency of dividing cells (FDC; an indirect measure of the growth rates) in the 4 groups in the East Sea samples (n = 15). FVIC and FDC varied between different morphotypes and were highest in cocci. Further, FVIC and estimated growth rates of bacterial morphotypes were significantly correlated. The presence of a fast-growing bacterial group (apparently more susceptible to viral infection) in the bacterial community substantially increased (1.2 to 2.8-fold) the estimates of VPTEM in comparison to those obtained by using the mean growth rate of the bacterial community. Until now the differences between virus production measured by virus decay rates (VPD) and VPTEM have been thought to be explained by adsorption of viruses to particles, degradation of viruses due to enzymes and inactivation of viruses caused by sunlight. It seems that uneven growth and different susceptibility to viruses in the bacterial community might be another additional explanation for the discrepancy between VPD and VPTEM. [TOP OF PAGE]

  1024. Prevention of Escherichia coli infection in broiler chickens with a bacteriophage aerosol spray. Huff,W.E., Huff,G.R., Rath,N.C., Balog,J.M., Donoghue,A.M. (2002). Poult. Sci. 81:1486-1491. Bacteriophage to an Escherichia coli isolate that is pathogenic in poultry were isolated from municipal sewer treatment facilities or poultry processing plants. Three studies were conducted to determine the efficacy of aerosol administration of bacteriophage to prevent an E. coli respiratory infection in broiler chickens. In all three studies the experimental design consisted of nine treatments with three replicate pens of 10 birds. Three treatments were not challenged with E. coli and consisted of unsprayed birds, birds sprayed with a diluent control, and birds sprayed with a combination of two bacteriophages. Six treatments were challenged with E. coli by injecting 104 cfu into the thoracic air sac when birds were 7, 8, or 10 d of age after being sprayed at 7 d of age with either a diluent control or a combination of two bacteriophages. In Studies 1 and 2, BW at 2 wk of age of all the birds challenged with E. coli, regardless of spray treatment, were decreased significantly from the unchallenged controls, except in Study 2 for the birds sprayed with bacteriophage and challenged at 10 d of age. There was a significant decrease in mortality in Studies 1 and 2 when the birds were challenged with E. coli immediately after bacteriophage administration and in Study 2 in birds challenged at 10 d of age. In Study 3 a suspected pre-existing E. coli infection resulted in mortality in the unchallenged, unsprayed controls, and in the diluent sprayed controls of 20 and 27%, respectively. The mortality in the unchallenged bacteriophage sprayed birds was 3%, representing a significant decrease. Mortality in Study 3 was significantly decreased in the bacteriophage-sprayed birds challenged with E. coli immediately or 1 d later but not 3 d after bacteriophage administration. The decrease in BW at 2 wk of age in challenged birds indicates that bacteriophage treatment did not provide complete protection; however, in all three studies mortality was significantly decreased, indicating that aerosol spray of bacteriophage may be practical for administration of bacteriophage and may provide an alternative to the use of antibiotics in poultry production. [TOP OF PAGE]

  1025. Prevention of Escherichia coli respiratory infection in broiler chickens with bacteriophage (SPR02). Huff,W.E., Huff,G.R., Rath,N.C., Balog,J.M., Xie,H., Moore,P.A.J., Donoghue,A.M. (2002). Poult. Sci. 81:437-441. Bacteriophages are viruses that can infect and kill bacteria. Three studies were conducted to determine the efficacy of bacteriophage to prevent an Escherichia coli respiratory infection in broiler chickens. In the first study 3-d-old-birds were challenged with an air sac inoculation of 103 cfu of E. coli per mL mixed with either 103 or 106 pfu of bacteriophage, or 104 cfu E. coli mixed with 104 or 108 pfu of bacteriophage. In the second study, drinking water of birds to 1 wk of age was treated with 103 or 104 pfu of bacteriophage per mL and birds were air sac challenged with 103 cfu of E. coli, or water was treated with 104 or 106 pfu of bacteriophage per milliliter and birds were challenged with 104 cfu of E. coli. In the third study, birds were air sac challenged at 1 wk of age with 104 cfu of E. coli and given 105 or 106 pfu of bacteriophage per mL of water from 1 d of age to 2 wk of age. In Studies 1 and 2, there were two replicate pens per treatment with 10 birds per pen, and in Study 3, there were four replicate pens per treatment with 10 birds per pen. The studies were all concluded when the birds were 3 wk of age. In Study 1, BW was decreased at 1 and 2 wk of age in the birds that were challenged with 103 or 104 cfu of E. coli and was decreased at 2 wk of age in the birds challenged with 104 cfu of E. coli mixed with 104 pfu of the bacteriophage. Mortality was decreased from 80% in the birds challenged with 103 cfu of E. coli to 25 and 5% when mixed with 103 or 106 pfu of the bacteriophage, respectively. Mortality was decreased from 85% in birds challenged with 104 cfu of E. coli to 35% when mixed with 104 pfu of the bacteriophage, and no mortality occurred when mixed with 106 pfu of bacteriophage. There was essentially no protection observed in Studies 2 and 3 when the birds were challenged with 103 or 104 cfu of E. coli with bacteriophage present in their drinking water at any level. These data suggest that bacteriophage can protect birds from a respiratory challenge with E. coli, but that adding the bacteriophage to the drinking water offered no protection to the birds. The complete protection of the birds observed in Study 1 suggests that bacteriophage may possibly be developed as an alternative to antibiotic use in poultry. [TOP OF PAGE]

  1026. Comparative analysis of the genomes of the temperate bacteriophages phi 11, phi 12 and phi 13 of Staphylococcus aureus 8325. Iandolo,J.J., Worrell,V., Groicher,K.H., Qian,Y., Tian,R., Kenton,S., Dorman,A., Ji,H., Lin,S., Loh,P., Qi,S., Zhu,H., Roe,B.A. (2002). Gene 289:109-118. The genomes of the three temperate bacteriophages contained in the chromosome of Staphylococcus aureus 8325 have been extracted from the sequence database and analyzed. phi 11, phi 12 and phi 13 are members of the same lytic group but different serogroups and consequently co-habitate the same host cell. Their genomes are approximately 42 kb to 45 kb and contain about 90 ORFs of at least 50 codons. Of these, about 50 have similarities to known genes or to genes of other staphylococcal phages. Each of the phages clusters within a homology group that share large regions of sequence identity while intergroup homology is comparatively low. The arrangement of genes on the chromosomes of the three phages is similar and consistent with current modular theory of phage gene organization. The replicated genomes appear to be packaged by different mechanisms. Phage phi 11 and phi 12 have been found to contain sequences consistent with pac-site phages while phi 13 has sequences consistent with cos-site phages. The attBsite for phi 11 is located in an intergenic region of the S. aureus chromosome while phi 12 and phi 13 integrate into specific genes. The phi 12 att-site is within an unknown gene, but the phi 13 att-site is within the beta-toxin gene. In contrast to the other two phages, phi 13 also introduces the staphylokinase gene (sak) and a second gene related to expression of fib. [TOP OF PAGE]

  1027. Dissemination of the phage-associated novel superantigen gene speL in recent invasive and noninvasive Streptococcus pyogenes M3/T3 isolates in Japan. Ikebe,T., Wada,A., Inagaki,Y., Sugama,K., Suzuki,R., Tanaka,D., Tamaru,A., Fujinaga,Y., Abe,Y., Shimizu,Y., Watanabe,H. (2002). Infect. Immun. 70:3227-3233. In Japan, more than 10% of streptococcal toxic shock-like syndrome (TSLS) cases have been caused by Streptococcus pyogenes M3/T3 isolates since the first reported TSLS case in 1992. Most M3/T3 isolates from TSLS or severe invasive infection cases during 1992 to 2001 and those from noninvasive cases during this period are indistinguishable in pulsed-field gel electropherograms. The longest fragments of these recent isolates were 300 kb in size, whereas those of isolates recovered during or before 1973 were 260 kb in size. These 260- and 300-kb fragments hybridized to each other, suggesting the acquisition of an about 40-kb fragment by the recent isolates. The whole part of the acquired fragment was cloned from the first Japanese TSLS isolate, NIH1, and its nucleotide sequence was determined. The 41,796-bp fragment is temperate phage phiNIH1.1, containing a new superantigen gene speL near its right attachment site. The C-terminal part of the deduced amino acid sequence of speL has 48 and 46% similarity with well-characterized erythrogenic toxin SpeC and the most potent superantigen, SmeZ-2, respectively. None of 10 T3 isolates recovered during or before 1973 has speL, whereas all of 18 M3/T3 isolates recovered during or after 1992 and, surprisingly, Streptococcus equi subsp. equi ATCC 9527 do have this gene. Though plaques could not be obtained from phiNIH1.1, its DNA became detectable from the phage particle fraction upon mitomycin C induction, showing that this phage is not defective. A horizontal transfer of the phage carrying speL may explain the observed change in M3/T3 S. pyogenes isolates in Japan. [TOP OF PAGE]

  1028. Food additive petition for: Control of pathogens on meat by use of a mixture of bacteriophages. Intralytix Inc. (2002). Fed. Reg. 67:47823 SUMMARY: The Food and Drug Administration (FDA) is announcing that Intralytix, Inc., has filed a petition proposing that the food additive regulations be amended to provide for the safe use of a mixture of bacteriophages as an antimicrobial agent on foods, including fresh meat, meat products, fresh poultry, and poultry products. ¶ FOR FURTHER INFORMATION CONTACT: Raphael A. Davy, Center for Food Safety and Applied Nutrition (HFS-265), Food and Drug Administration, 5100 Paint Branch Pkwy., College Park, MD 20740, 202-418-3405. ¶ SUPPLEMENTARY INFORMATION: Under the Federal Food, Drug, and Cosmetic Act (sec. 409(b)(5) (21 U.S.C. 348(b)(5))), notice is given that a food additive petition (FAP 2A4738) has been filed by Intralytix, Inc., c/o Lewis & Harrison, 122 C St. NW., suite 740, Washington, DC 20001. The petition proposes to amend the food additive regulations to provide for the safe use of a mixture of bacteriophages as an antimicrobial agent on foods, including fresh meat, meat products, fresh poultry, and poultry products. ¶ The agency has determined under 21 CFR 25.32(r) that this action is of a type that does not individually or cumulatively have a significant effect on the human environment. Therefore, neither an environmental assessment nor an environmental impact statement is required. [TOP OF PAGE]

  1029. Characterization of serracin P, a phage-tail-like bacteriocin, and its activity against Erwinia amylovora, the fire blight pathogen. Jabrane,A., Sabri,A., Compere,P., Jacques,P., Vandenberghe,I., Van Beeumen,J., Thonart,P. (2002). Appl. Environ. Microbiol. 68:5704-5710. Serratia plymithicum J7 culture supernatant displayed activity against many pathogenic strains of Erwinia amylovora, the causal agent of the most serious bacterial disease of apple and pear trees, fire blight, and against Klebsiella pneumoniae, Serratia liquefaciens, Serratia marcescens, and Pseudomonas fluorescens. This activity increased significantly upon induction with mitomycin C. A phage-tail-like bacteriocin, named serracin P, was purified from an induced culture supernatant of S. plymithicum J7. It was found to be the only compound involved in the antibacterial activity against sensitive strains. The N-terminal amino acid sequence analysis of the two major subunits (23 and 43 kDa) of serracin P revealed high homology with the Fels-2 prophage of Salmonella enterica, the coliphages P2 and 168, the phiCTX prophage of Pseudomonas aeruginosa, and a prophage of Yersinia pestis. This strongly suggests a common ancestry for serracin P and these bacteriophages. [TOP OF PAGE]

  1030. Flow cytometric analysis of an Emiliania huxleyi bloom terminated by viral infection. Jacquet,S., Heldal,M., Iglesias-Rodriguez,D., Larsen,A., Wilson,W.H. (2002). Aquat. Microb. Ecol. 27:111-124. During a field mesocosm experiment conducted in coastal waters off western Norway, 11 m(3) enclosures were filled with unfiltered seawater and enriched daily with different nitrate and phosphate concentrations in order to induce a bloom of the coccolithophorid Emiliana huxleyi under different nutrient regimes. Flow cytometry (FCM) analysis was performed 5 times d(-1) in order to follow the initiation, development and termination of the bloom as well as the production of large virus-like particles (LVLPs) identified as E. huxleyi viruses (EhV). EhV production was observed first in the enclosure where N was in excess, and P limitation induced a lower burst size compared to nitrate-replete enclosures. These observations suggest a critical role for both P and N in E. huxleyi-EhV interactions. Concomitant to EhV production, a shift was observed between the original population (coccolith-bearing cells) towards a population characterized by the same chlorophyll a (chl a) fluorescence but with lower right angle light scatter values. This population is likely to correspond to either senescent cells losing their coccoliths or cells characterized by a lower production of coccoliths possibly due to viral infection. At the end of experiment, a significant proportion of E. huxleyi had survived after the end of the bloom. This suggests either the presence of a resistant form of the coccolithophorid or a change in the dominance of different host and/or viral strains during the bloom. A periodical pattern in virus production was recorded with virus number decreasing during the second part of the day suggesting that virus production may be synchronized to the daily light cycle. Our results provide new insights towards the understanding of the relationship between a key marine species and its specific virus. [TOP OF PAGE]

  1031. Bacteriophages as indicators. Jofre,J. (2002). pp. 354-363. In In G.Bitton (ed.), Enciclopedia of Environmental Microbiology. John Willey and Sons Inc., New York. [TOP OF PAGE]

  1032. Viral Trojan horse for combating tuberculosis. Johnston,N. (2002). Drug Discov. Today 7:333-335. The emergence of pathogenic bacteria resistant to one or more antibiotics has outpaced the development of new drugs. Using bacteriophage, Raul Barletta (Dept of Veterinary and Biomedical Sciences, University of Nebraska, Lincoln, NE, USA) and colleagues at the California Pacific Medical Center (San Francisco, CA, USA) have devised a promising new approach to killing the intracellular pathogens Mycobacterium avium, which commonly afflicts AIDS patients, and Mycobacterium tuberculosis, the causative agent of tuberculosis. Their findings were presented at the 41st Interscience Conference on Antimicrobial Agents and Chemotherapy hosted by the American Society for Microbiology in Chicago, IL, USA [1]. [TOP OF PAGE]

  1033. The effect of storage and lag time on MS2 bacteriophage susceptibility to ultraviolet radiation. Jolis,D. (2002). Water Environ Res 74:516-520. The susceptibility of MS2 bacteriophage suspensions to UV radiation was assessed using a collimated beam technique. Storage of MS2 bacteriophage cultures at 4 degrees C resulted in a decrease in phage susceptibility to UV radiation over time. After 18 days, the level of MS2 bacteriophage inactivation achieved for the range of UV doses tested decreased by 0.7 to 1.1 logs, but remained constant after that point. Changes in the protein coat of the bacteriophage, a decrease in viability over time, and an increase in coagulation may have played a role in the observed susceptibility decrease. A 2-hour lag time between the preparation of the MS2 suspension and the irradiation test also resulted in a decrease in phage susceptibility. [TOP OF PAGE]

  1034. Bacteriophages isolated from activated sludge process and their polyvalency. Kahn,M.A., Satoh,H., Katayama,H., Kurisu,F., Mino,T. (2002). Water Res. 36:3364-3370. In this study, bacteriophages were isolated from activated sludge and their host range was studied. Bacterial isolates were obtained from an activated sludge process treating urban sewage, and bacteriophages were obtained by plaque assay using the bacterial isolates obtained in this study as the host. Out of 15 bacteria isolated, 9 supported plaque formation. The host range test was conducted with a combination of 8 bacteriophage isolates and 9 bacterial isolates. All of the 8 bacteriophages tested were found to form plaques on more than 1 host, and 4 of them formed plaques on both Gram-positive and Gram-negative bacterial isolates. Three of the 8 bacteriophages failed to form plaques on their original bacterial host. The experimental result indicates that bacteriophages are an active part of the activated sludge microbial ecosystem, having a very close ecological relationship with their host bacteria. [TOP OF PAGE]

  1035. Vibrio cholerae phage K139: complete genome sequence and comparative genomics of related phages. Kapfhammer,D., Blass,J., Evers,S., Reidl,J. (2002). J. Bacteriol. 184:6592-6601. In this report, we characterize the complete genome sequence of the temperate phage K139, which morphologically belongs to the Myoviridae phage family (P2 and 186). The prophage genome consists of 33,106 bp, and the overall GC content is 48.9%. Forty-four open reading frames were identified. Homology analysis and motif search were used to assign possible functions for the genes, revealing a close relationship to P2-like phages. By Southern blot screening of a Vibrio cholerae strain collection, two highly K139-related phage sequences were detected in non-O1, non-O139 strains. Combinatorial PCR analysis revealed almost identical genome organizations. One region of variable gene content was identified and sequenced. Additionally, the tail fiber genes were analyzed, leading to the identification of putative host-specific sequence variations. Furthermore, a K139-encoded Dam methyltransferase was characterized. [TOP OF PAGE]

  1036. [Effect of bacteriophage on the lipid peroxidation process and antioxidant protective enzymes in experimental uveitis]. Karimova,M.K., Bakhritdinova,F.A. (2002). Vestn. Oftalmol. 118:38-40. Experimental uveitis features distinct hyperlipoperoxidation in damaged eye tissues, blood serum and the liver. The activity of antioxidant defense (AOD) enzymes decreases in tissues and blood of experimental animals whereas catalase compensatorily activates in hepatic tissue. Experimental therapy of uveitis with gentamycin and bacteriophage results in reducing hyperlipoperoxidation, increased activity of AOD enzymes but no complete normalization is observed. This manifested in preservation of inflammations to a certain degree. [TOP OF PAGE]

  1037. Overcoming the phage replication threshold: a mathematical model with implications for phage therapy. Kasman,L.M., Kasman,A., Westwater,C., Dolan,J., Schmidt,M.G., Norris,J.S. (2002). J. Virol. 76:5557-5564. Prior observations of phage-host systems in vitro have led to the conclusion that susceptible host cell populations must reach a critical density before phage replication can occur. Such a replication threshold density would have broad implications for the therapeutic use of phage. In this report, we demonstrate experimentally that no such replication threshold exists and explain the previous data used to support the existence of the threshold in terms of a classical model of the kinetics of colloidal particle interactions in solution. This result leads us to conclude that the frequently used measure of multiplicity of infection (MOI), computed as the ratio of the number of phage to the number of cells, is generally inappropriate for situations in which cell concentrations are less than 10(7)/ml. In its place, we propose an alternative measure, MOI(actual), that takes into account the cell concentration and adsorption time. Properties of this function are elucidated that explain the demonstrated usefulness of MOI at high cell densities, as well as some unexpected consequences at low concentrations. In addition, the concept of MOI(actual) allows us to write simple formulas for computing practical quantities, such as the number of phage sufficient to infect 99.99% of host cells at arbitrary concentrations. [TOP OF PAGE]

  1038. Deleterious impact of a virulent bacteriophage on survival and biocontrol activity of Pseudomonas fluorescens strain CHAO in natural soil. Keel,C., Ucurum,Z., Michaux,P., Adrian,M., Haas,D. (2002). Mol. Plant-Microbe Interact. 15:567-576. Many biotic and abiotic factors affect the persistence and activity of beneficial pseudomonads introduced into soil to suppress plant diseases. One such factor may be the presence of virulent bacteriophages that decimate the population of the introduced bacteria, thereby reducing their beneficial effect. We have isolated a lytic bacteriophage (phi)GP100) that specifically infects the biocontrol bacterium Pseudomonas fluorescens CHA0 and some closely related Pseudomonas strains. phiGP100 was found to be a double-stranded-DNA phage with an icosahedral head, a stubby tail, and a genome size of approximately 50 kb. Replication of phiGP100 was negatively affected at temperatures higher than 25 degrees C. phiGP100 had a negative impact on the population size and the biocontrol activity of P. fluorescens strain CHA0-Rif (a rifampicin-resistant variant of CHA0) in natural soil microcosms. In the presence of phiGP100, the population size of strain CHA0-Rif in soil and on cucumber roots was reduced more than 100-fold. As a consequence, the bacterium's capacity to protect cucumber against a root disease caused by the pathogenic oomycete Pythium ultimum was entirely abolished. In contrast, the phage affected neither root colonization and nor the disease suppressive effect of a phiDGP100-resistant variant of strain CHA0-Rif. To our knowledge, this study is the first to illustrate the potential of phages to impair biocontrol performance of beneficial bacteria released into the natural soil environment. [TOP OF PAGE]

  1039. Degree of ultraviolet radiation damage and repair capabilities are related to G+C content in marine vibriophages. Kellogg,C.A., Paul,J.H. (2002). Aquat. Microb. Ecol. 27:13-20. A key issue in the ecology of viruses in the marine environment is the rate of viral production and decay. The ultraviolet (UV) radiation in sunlight has been found to cause loss of infectivity in marine bacteriophages at rates nearly equal to all other decay mechanisms combined. There are 2 main host-mediated mechanisms that can repair UV-damaged phage DNA: photoreactivation and excision repair. Both these mechanisms were investigated in 2 marine Vibrio parahaemolyticus hosts as they catalyzed the reactivation of 7 phages. Photoreactivation was the dominant repair mode in all but one case. A significant correlation was found between G+C content of the phage DNAs (16 to 70 %) and degree of DNA damage (r = 0.955), indicating a strong relationship between the number of thymine dimer targets and the capability to photoreactive DNA damage. Evolution of high G+C content may be a strategy for protection from UV damage in marine phages. [TOP OF PAGE]

  1040. Bacteriophages isolated from activated sludge processes and their polyvalency. Khan,M.A., Satoh,H., Katayama,H., Kurisu,F., Mino,T. (2002). Water Res. 36:3364-3370. In this study, bacteriophages were isolated from activated sludge and their host range was studied. Bacterial isolates were obtained from an activated sludge process treating urban sewage, and bacteriophages were obtained by plaque assay using the bacterial isolates obtained in this study as the host. Out of 15 bacteria isolated, 9 supported plaque formation. The host range test was conducted with a combination of 8 bacteriophage isolates and 9 bacterial isolates. All of the 8 bacteriophages tested were found to form plaques on more than 1 host, and 4 of them formed plaques on both gram-positive and gram-negative bacterial isolates. Three of the 8 bacteriophages failed to form plaques on their original bacterial host. The experimental result indicates that bacteriophages are an active part of the activated sludge microbial ecosystem, having a very close ecological relationship with their host bacteria. [TOP OF PAGE]

  1041. Bacteriophage-host interaction in the enhanced biological phosphate removing activated sludge system. Khan,M.A., Satoh,H., Mino,T., Katayama,H., Kurisu,F., Matsuo,T. (2002). Water Sci. Technol. 46:39-43. Bacteriophages were isolated from a laboratory scale enhanced biological phosphate removing (EBPR) activated sludge process, and their host range was examined. Bacterial isolates to host the bacteriophages were isolated from the EBPR activated sludge process. Bacteriophages were eluted from the EBPR activated sludge, enriched by incubation with the bacterial isolates, and then tested for plaque formation on each of the bacterial isolates. Out of 12 bacterial isolates isolated, 4 supported plaque formation. Four bacteriophages were obtained from the plaques. The host range test was conducted with the combination of the bacteriophage isolates and the bacterial isolates. Three of the bacteriophages were found to form plaques on more than one host, and one of them formed plaques on both gram +ve and gram -ve bacterial isolates. Two of the four bacteriophages failed to form plaques on their original bacterial host, indicating the existence of mutation on either both or one of the host and the bacteriophage. This study strongly suggests that bacteriophages are an active part of the activated sludge microbial ecosystem, having very complex interaction with their host bacteria. [TOP OF PAGE]

  1042. Nucleotide sequence of a ssRNA phage from Acinetobacter: kinship to coliphages. Klovins,J., Overbeek,G.P., van,d.W., Ackermann,H.-W., van,D. (2002). J. Gen. Virol. 83:1523-1533. The complete nucleotide sequence of ssRNA phage AP205 propagating in Acinetobacter species is reported. The RNA has three large ORFs, which code for the following homologues of the RNA coliphage proteins: the maturation, coat and replicase proteins. Their gene order is the same as that in coliphages. RNA coliphages or Leviviridae fall into two genera: the alloleviviruses, like Qb, which have a coat read-through protein, and the leviviruses, like MS2, which do not have this coat protein extension. AP205 has no read-through protein and may therefore be classified as a levivirus. A major digression from the known leviviruses is the apparent absence of a lysis gene in AP205 at the usual position, overlapping the coat and replicase proteins. Instead, two small ORFs are present at the 5' terminus, preceding the maturation gene. One of these might encode a lysis protein. The other is of unknown function. Other new features concern the 3'-terminal sequence. In all ssRNA coliphages, there are always three cytosine residues at the 3' end, but in AP205, there is only a single terminal cytosine. Distantly related viruses, like AP205 and the coliphages, do not have significant sequence identity; yet, important secondary structural features of the RNA are conserved. This is shown here for the 3' UTR and the replicase-operator hairpin. Interestingly, although AP205 has the genetic map of a levivirus, its 3' UTR has the length and RNA secondary structure of an allolevivirus. Sharing features with both MS2 and Q(beta) suggests that, in an evolutionary sense, AP205 should be placed between Q(beta) and MS2. A phylogenetic tree for the ssRNA phages is presented. [TOP OF PAGE]

  1043. Engineering a reduced Escherichia coli genome. Kolisnychenko,V., Plunkett,G., Herring,C.D., Feher,T., Posfai,J., Blattner,F.R., Posfai,G. (2002). Genome Res. 12:640-647. Our goal is to construct an improved Escherichia coli to serve both as a better model organism and as a more useful technological tool for genome science. We developed techniques for precise genomic surgery and applied them to deleting the largest K-islands of E. coli, identified by comparative genomics as recent horizontal acquisitions to the genome. They are loaded with cryptic prophages, transposons, damaged genes, and genes of unknown function. Our method leaves no scars or markers behind and can be applied sequentially. Twelve K-islands were successfully deleted, resulting in an 8.1% reduced genome size, a 9.3% reduction of gene count, and elimination of 24 of the 44 transposable elements of E. coli. These are particularly detrimental because they can mutagenize the genome or transpose into clones being propagated for sequencing, as happened in 18 places of the draft human genome sequence. We found no change in the growth rate on minimal medium, confirming the nonessential nature of these islands. This demonstration of feasibility opens the way for constructing a maximally reduced strain, which will provide a clean background for functional genomics studies, a more efficient background for use in biotechnology applications, and a unique tool for studies of genome stability and evolution. [TOP OF PAGE]

  1044. [The resistance conferred by the R/M system LlaBIII against bacteriophages]. Kong,J., Josephsen,J., Ma,G.R. (2002). Acta Microbiol. Sin. 42:246-250. LlaBIII, isolated from the naturally occurring plasmid pJW566 from Lactococcus lactis subsp. cremoris W56, encoded a restriction and modification (R/M) system. The plasmid pJK1 carrying the R/M system LlaBIII was transformed into L. lactis IL1403 with type I R/M system located on chromosome and the strain MG1614[pAW601] with AbiS gene on plasmid pAW601, respectively. The transformants obtained showed stacking resistance against bacteriophages. The plasmid pJK1 was transformed into industrial strains L. lactis SMQ86 and CHCC2281, the transformants showed the EOP of the bacteriophages decreased by 10(-3) and 10(-5), respectively. The results indicated that the R/M system LlaBIII could protect strains from bacteriophages in dairy fermentation. [TOP OF PAGE]

  1045. The ability of the plasmid-encoded restriction and modification system LlaBIII to protect Lactococcus lactis against bacteriophages. Kong,J., Josephsen,J. (2002). Lett. Appl. Microbiol. 34:249-253. AIMS: To investigate the potential of the plasmid-encoded restriction and modification (R/M) system LlaBIII to protect Lactococcua lactis against bacteriophages during milk fermentations. METHODS AND RESULTS: The R/M system LlaBIII on plasmid pJW566 was cloned with a chloramphenicol cassette, resulting in plasmid pJK1. When introduced into L. lactis strains, pJK1 conferred increased phage resistance against the three most common lactococcal phage species 936, c2, and P335 and three unclassified industrial phages. The growth of the strains in RSM was not affected by the presence of plasmid pJK1. CONCLUSIONS: The plasmid-encoded R/M system LlaBIII has great ability to protect L. lactis strains against bacteriophages in milk fermentations. SIGNIFICANCE AND IMPACT OF THE STUDY: This study evaluates the ability of the LlaBIII R/M system to function as a phage defence mechanism which is an essential step prior to considering utilizing it for improving starter cultures. [TOP OF PAGE]

  1046. The activity of chosen bacteriophages on Yersinia enterocolitica strains. Kot,B., Bukowski,K., Jakubczak,A., Kaczorek,I. (2002). Pol. J. Vet. Sci. 5:47-50. The aim of the present study was to evaluate the lytic activity of three bacteriophages on Yersinia enterocolitica strains isolated from humans and pigs. The Y. enterocolitica strains tested belonged to 0:3, 0:9 and 0:2 serogroups. The ZD5 phage was obtained from a water sample, but remaining phages were obtained from the lysogenic Y. frederiksenii 7291 and Y. enterocolitica 8684 strains. All the Y. enterocolitica strains tested which belonged to 0:9 serogroup did not show any susceptibility to the bacteriophages used. The bacteriophages tested showed different lytic activity on the Y. enterocolitica 0:3 strains investigated. The phage susceptibility of Y. enterocolitica 0:3 strains revealed 9 different phage patterns. ZD5 phage showed the highest lytic activity, because it produced confluent lysis of the most Y. enterocolitica 0:3 strains tested. The Y. enterocolitica 0:2 strains isolated from pigs showed the similar phage susceptibility. The Y. kristensenii and Y. pseudotuberculosis strains tested were not sensitive to the bacteriophages used. [TOP OF PAGE]

  1047. Phagotherapy: myths and realities. Krylov,V. (2002). Rus. Acad. Sci. Pres. 4:40-46. The current situation of uncontrolled uses of medicinal preparations, pollution of nature with toxic wastes and other adverse phenomena of this kind have produced what experts call another spiral in the eovlution of bacteria—the development of their multiple-resistant strains. As often as not, many expensive antibiotics of the last generation (vancomycin, imipenem, etc.) turn out to be powerless. And the way medical experts see it—the way out of this situation—consists in pioneering some alternative therapies. The most promising of these is believed to be phagotherapy—the use of specific bacterial viruses (bacteriophages or phages). [TOP OF PAGE]

  1048. Detection of bacteria using foreign DNA: the development of a bacteriophage reagent for Salmonella. Kuhn,J., Suissa,M., Wyse,J., Cohen,I., Weiser,I., Reznick,S., Lubinsky-Mink,S., Stewart,G., Ulitzur,S. (2002). Int. J. Food Microbiol. 74:229-238. A phage-based reagent was developed for the detection of Salmonella in food samples. The parental phage was Felix 01, which lyses practically all Salmonella. Using data obtained about the molecular biology of the phage, a recombinant phage that carried the bacterial genes specifying luciferase was produced. The method involved the isolation of amber nonsense mutations and subsequent crosses to render doubly mutant phage with a very low reversion rate on strains lacking an amber suppressor. A plasmid was constructed that contained a segment of Felix 01 DNA with two adjacent genes, one dispensable and the other essential, and their flanking sequences. Recombinant DNA technology was used to remove the two genes and the luxA and luxB genes for luciferase, and a gene specifying a tRNA that recognizes amber codons (supF=tyrT) was put in their stead. This region could be transferred into the genome of the phage by homologous recombination. The recombinant phage cannot grow because it lacks an essential gene. However, it can grow in a host that synthesizes the missing protein. This technique allows the construction of "locked" recombinant phages that carry foreign DNA but which cannot propagate themselves in nature. [TOP OF PAGE]

  1049. A bacteriophage reagent for Salmonella: molecular studies on Felix 01. Kuhn,J., Suissa,M., Chiswell,D., Azriel,A., Berman,B., Shahar,D., Reznick,S., Sharf,R., Wyse,J., Bar-On,T., Cohen,I., Giles,R., Weiser,I., Lubinsky-Mink,S., Ulitzur,S. (2002). Int. J. Food Microbiol. 74:217-227. Felix 01 (F01) is a bacteriophage originally isolated by Felix and Callow which lyses almost all Salmonella strains and has been widely used as a diagnostic test for this genus. Molecular information about this phage is entirely lacking. In the present study, the DNA of the phage was found to be a double-stranded linear molecule of about 80 kb. 11.5 kb has been sequenced and in this region A + T content is 60%. There are relatively few restriction endonuclease cleavage sites in the native genome and clones show this is due to their absence rather than modification. A restriction map of the genome has been constructed. The ends of the molecule cannot be ligated although they contain 5' phosphates. At least 60% of the genome must encode proteins. In the sequenced portion, many open reading frames exist and these are tightly packed together. These have been examined for homology to published proteins but only 1 to 17 shows similarity to known proteins. F01 is therefore the prototype of a new phage family. On the basis of restriction sites, codon usage and the distribution of nonsense codons in the unused reading frames, a strong case can be made for natural selection that reacts to mRNA structure and function. [TOP OF PAGE]

  1050. Complete genomic sequence of bacteriophage ul36: demonstration of phage heterogeneity within the P335 quasi-species of lactococcal phages. Labrie,S., Moineau,S. (2002). Virology 296:308-320. The complete genomic sequence of the Lactococcus lactis virulent phage ul36 belonging to P335 lactococcal phage species was determined and analyzed. The genomic sequence of this lactococcal phage contained 36,798 bp with an overall G+C content of 35.8 mol %. Fifty-nine open reading frames (ORFs) of more than 40 codons were found. N-terminal sequencing of phage structural proteins as well as bioinformatic analysis led to the attribution of a function to 24 ORFs (41%). A lysogeny module was found within the genome of this virulent phage. The putative integrase gene seems to be the product of a horizontal transfer because it is more closely related to Streptococcus pyogenes phages than it is to L. lactis phages. Comparative genome analysis with six complete genomes of temperate P335-like phages confirmed the heterogeneity among phages of P335 species. A dUTPase gene is the only conserved gene among all P335 phages analyzed as well as the phage BK5-T. A genetic relationship between P335 phages and the phage-type of the BK5-T species was established. Thus, we proposed that phage BK5-T be included within the P335 species and thereby reducing the number of lactococcal phage species to 11. [TOP OF PAGE]

  1051. Viruses causing lysis of the toxic bloom-forming alga, Heterosigma akashiwo (Raphidophyceae), are widespread in coastal sediments of British Columbia, Canada. Lawrence,J.E., Chan,A.M., Suttle,C.A. (2002). Limnol. Oceanogr. 47:545-550. Viruses that infect and cause lysis of the toxic alga Heterosigma akashiwo are abundant and widespread in the Strait of Georgia, Canada, and adjacent inlets during the summer months when blooms of this alga occur. Because viruses are subjected to many mechanisms of removal and their host is intermittently dormant, the persistence of viruses may be dependent on environmental reservoirs. We extracted pore water from sediments collected in the Strait of Georgia and screened for the presence of infectious agents that cause lysis of H. akashiwo. Lytic agents were widespread throughout the study region, being detected in 17 of 20 sites surveyed. Lytic agents were present in sediments ranging from highly organic to clay-rich and were retrieved from cores taken at water depths of 25-285 m. The highest concentration of lytic agents was found at the sediment-water interface; however, lytic agents were found as deep as 40 cm below the sediment-water interface. Examination of agents isolated from various sites revealed virus-like particles similar to50 nm in diameter. These are similar to other virus-like particles that have been isolated that infect this alga. This suggests that the most abundant lytic agents in the sediments are viruses and that these viruses may be long-lived once buried in the sediments. The widespread presence of viral-size lytic agents that infect H. akashiwo is consistent with viral infection being a mortality agent of this alga in the overlying waters and suggests that they may play in important role in regulating their population dynamics. [TOP OF PAGE]

  1052. Imbroglios of viral taxonomy: genetic exchange and failings of phenetic approaches. Lawrence,J.G., Hatfull,G.F., Hendrix,R.W. (2002). J. Bacteriol. 184:4891-4905. The practice of classifying organisms into hierarchical groups originated with Aristotle and was codified into nearly immutable biological law by Linnaeus. The heart of taxonomy is the biological species, which forms the foundation for higher levels of classification. Whereas species have long been established among sexual eukaryotes, achieving a meaningful species concept for prokaryotes has been an onerous task and has proven exceedingly difficult for describing viruses and bacteriophages. Moreover, the assembly of viral "species" into higher-order taxonomic groupings has been even more tenuous, since these groupings were based initially on limited numbers of morphological features and more recently on overall genomic similarities. The wealth of nucleotide sequence information that catalyzed a revolution in the taxonomy of free-living organisms necessitates a reevaluation of the concept of viral species, genera, families, and higher levels of classification. Just as microbiologists discarded dubious morphological traits in favor of more accurate molecular yardsticks of evolutionary change, virologists can gain new insight into viral evolution through the rigorous analyses afforded by the molecular phylogenetics of viral genes. For bacteriophages, such dissections of genomic sequences reveal fundamental flaws in the Linnaean paradigm that necessitate a new view of viral evolution, classification, and taxonomy. [TOP OF PAGE]

  1053. Preventing phage lysis of Lactococcus lactis in cheese production using a neutralizing heavy-chain antibody fragment from llama. Ledeboer,A.M., Bezemer,S., de Hiaard,J.J.W., Schaffers,I.M., Verrips,C.T., van Vliet,C., Dusterhoft,E.M., Zoon,P., Moineau,S., Frenken,L.G.J. (2002). J. Dairy Sci. 85:1376-1382. Bacteriophage infection is still a persistent problem in large dairy processes despite extensive studies over the last decades. Consequently, new methods are constantly sought to prevent phage infection. In this paper, we show that phage neutralizing heavy-chain antibody fragments, obtained from Camelidae and produced at a large scale in the generally regarded as safe microorganism Saccharomyces cerevisiae, can effectively be used to impede phage induced lysis during a cheese process. The growth inhibition of the cheese starter culture by 105 pfu/ml cheese-milk of the small isometric-headed 936-type phage p2 was prevented by the addition of only 0.1 microg/ml (7 nM) of the neutralizing antibody fragment. The use of such antibody fragments in cheese manufacturing are a realistic and interesting option because of the small amount of antibody fragments that are needed. Moreover the antibodies are produced in a food grade microorganism and can easily be isolated from the fermentation liquid in a pure and DNA free form. [TOP OF PAGE]

  1054. Evolution of bacteriophage in continuous culture: a model system to test antiviral gene therapies for the emergence of phage escape mutants. Lindemann,B.F., Klug,C., Schwienhorst,A. (2002). J. Virol. 76:5784-5792. The emergence of viral escape mutants is usually a highly undesirable phenomenon. This phenomenon is frequently observed in antiviral drug applications for the treatment of viral infections and can undermine long-term therapeutic success. Here, we propose a strategy for evaluating a given antiviral approach in terms of its potential to provoke the appearance of resistant virus mutants. By use of Q beta RNA phage as a model system, the effect of an antiviral gene therapy, i.e., a virus-specific repressor protein expressed by a recombinant Escherichia coli host, was studied over the course of more than 100 generations. In 13 experiments carried out in parallel, 12 phage populations became resistant and 1 became extinct. Sequence analysis revealed that only two distinct phage mutants emerged in the 12 surviving phage populations. For both escape mutants, sequence variations located in the repressor binding site of the viral genomic RNA, which decrease affinity for the repressor protein, conferred resistance to translational repression. The results clearly suggest the feasibility of the proposed strategy for the evaluation of antiviral approaches in terms of their potential to allow resistant mutants to appear. In addition, the strategy proved to be a valuable tool for observing virus-specific molecular targets under the impact of antiviral drugs. [TOP OF PAGE]

  1055. Reverse transcriptase-mediated tropism switching in Bordetella bacteriophage. Liu,M., Deora,R., Doulatov,S.R., Gingery,M., Eiserling,F.A., Preston,A., Maskell,D.J., Simons,R.W., Cotter,P.A., Parkhill,J., Miller,J.F. (2002). Science 295:2091-2094. Host-pathogen interactions are often driven by mechanisms that promote genetic variability. We have identified a group of temperate bacteriophages that generate diversity in a gene, designated mtd (major tropism determinant), which specifies tropism for receptor molecules on host Bordetella species. Tropism switching is the result of a template-dependent, reverse transcriptase-mediated process that introduces nucleotide substitutions at defined locations within mtd. This cassette-based mechanism is capable of providing a vast repertoire of potential ligand-receptor interactions. [TOP OF PAGE]

  1056. Prevalence of Escherichia coli O157:H7 prophage-like sequences among German Salmonella enterica serotype Typhimurium phage types and their use in detection of phage type DT104 by the polymerase chain reaction. Malorny,B., Schroeter,A., Bunge,C., Helmuth,R. (2002). Vet Microbiol 87:253-265. A 1.6kb DNA fragment identified by random amplifiable polymorphic DNA differentiation (RAPD) from a Salmonella enterica serotype Typhimurium phage type DT104 isolate was used to investigate the prevalence of the region in 160 DT104 isolates, 83 other epidemiological important S. Typhimurium phage types and 20 strains selected from 17 other Salmonella serotypes. PCR screening tests using two different primer-sets derived from the RAPD fragment's nucleotide sequence showed that 76% of the 160 DT104 isolates investigated, including subtypes DT104A, DT104B, DT104B low, DT104H and DT104L, reacted positively. High sensitivity was shown for DT104 strains expressing at least the penta-resistance pattern ACSSuT (97% of 104 strains tested). DT104 susceptible strains showed only a sensitivity of 35% (17 strains tested). In contrast, 83% of the 83 strains from the other S. Typhimurium phage types reacted negatively. Strains from five out of the 17 other serotypes showed a positive signal with one primer-set. The other primer-set exhibited only a positive reaction with one S. Dublin isolate. The analysis of a 2415bp extended sequence revealed homologies to genes encoded by Escherichia coli O157:H7 prophages, suggesting that the described region contains genes of a prophage specific for DT104 and related phage types. [TOP OF PAGE]

  1057. Recombinant phages. Mardh,S. (2002). 603153(6,497,874). The present invention relates to bacteriophages for use in the treatment or prophylaxis of bacterial infections, especially mucosal bacterial infections such as Heliobacter pylori infections, in particular, it relates to modified filamentous bacteriophages, e.g. M13 phages, for such use, which bacteriophages present at the surface a recombinant protein comprising (i) a first component derived from a bacteriophage surface protein; and (ii) a second component comprising variable region sequences of an antibody to provide a bacterial antigen binding site, said second component rendering said bacteriophage capable of binding to and thereby inhibiting growth of bacterial cells involved in the etiology of said infection. [TOP OF PAGE]

  1058. Predatory prokaryotes: an emerging research opportunity. Martin,M.O. (2002). J. Mol. Microbiol. Biotechnol. 4:467-477. Predatory prokaryotes have evolved a unique strategy of obtaining energy and biosynthetic materials from their surroundings: acquiring them from other living bacterial cells. These types of microbes have been found in a diverse variety of environments, and may play an important role in modulating microbial population structure and dynamics, as has been hypothesized for marine viruses and possibly protists. Only one genus of predatory bacterium, Bdellovibrio, has been extensively described and studied, though several other examples have been reported in the literature. In this review, the four basic strategies used by currently described predatory prokaryotes will be discussed: "wolfpack" group predation, epibiotic attachment, direct cytoplasmic invasion, and periplasmic invasion. Special adaptations to each approach will be considered, and compared overall to the genetic and biochemical characteristics of symbiotic or pathogenic prokaryotes living within eukaryotic cells. Two specific examples of predatory microbes, Bdellovibrio and Ensifer, will be described in terms of predation strategy, association with host cells, and host range. The prospects for bringing to bear the tools of molecular microbial genetics to the study of predatory prokaryotes will be explored, using current research with Bdellovibrio and Ensifer as examples. [TOP OF PAGE]

  1059. ??? Matsumoto,T., Okabe,N. (2002). J. Soc. Trop. Agri 9:205-213. [TOP OF PAGE]

  1060. Plankton blooms: Lysogeny in marine Synechococcus. McDaniel,L., Houchin,L.A., Williamson,S.J., Paul,J.H. (2002). Nature 415:496 Viral infection of bacteria can be lytic, causing destruction of the host cell, or lysogenic, in which the viral genome is instead stably maintained as a prophage within its host. Here we show that lysogeny occurs in natural populations of an autotrophic picoplankton (Synechococcus) and that there is a seasonal pattern to this interaction. Because lysogeny confers immunity to infection by related viruses, this process may account for the resistance to viral infection seen in common forms of autotrophic picoplankton. We undertook a seasonal study in Tampa Bay, Florida, of prophage induction in cyanobacteria over the year ending in October 2000 to find out whether lysogeny occurs in natural Synechococcus populations and, if so, how it is affected by changing environmental conditions. [TOP OF PAGE]

  1061. Identification and characterization of phage-resistance genes in temperate lactococcal bacteriophages. McGrath,S., Fitzgerald,G.F., van Sinderen,D. (2002). Mol. Microbiol. 43:509-520. The sie2009 gene, which is situated between the genes encoding the repressor and integrase, on the lysogeny module of the temperate lactococcal bacteriophage Tuc2009, was shown to mediate a phage-resistance phenotype in Lactococcus lactis against a number of bacteriophages. The Sie2009 protein is associated with the cell membrane and its expression leaves phage adsorption, transfection and plasmid transformation unaffected, but interferes with plasmid transduction, as well as phage replication. These observations indicate that this resistance is as a result of DNA injection blocking, thus representing a novel superinfection exclusion system. A polymerase chain reaction (PCR)-based strategy was used to screen a number of lactococcal strains for the presence of other prophage-encoded phage-resistance systems. This screening resulted in the identification of two such systems, without homology to sie2009, which were shown to mediate a phage-resistance phenotype similar to that conferred by sie2009. To our knowledge, this is the first description of a phage-encoded superinfection exclusion/injection blocking mechanism in the genus Lactococcus. [TOP OF PAGE]

  1062. Influence of flow rate on transport of bacteriophage in shale saprolite. McKay,L.D., Harton,A.D., Wilson,G.V. (2002). J. Environ. Qual. 31:1095-1105. The objective of this study was to investigate the influence of flow rate on transport and retention of bacteriophage tracers in a fractured shale saprolite, which is a highly weathered, fine-grained subsoil that retains much of the fabric of the parent bedrock. Synthetic ground water containing PRD-1, MS-2, and bromide was passed through a saturated column of undisturbed shale saprolite at rates ranging from 0.0075 to 0.96 m d '. First arrival of the bacteriophage tracers in effluent samples in each of the experiments occurred within 0.01 to 0.04 pore volumes (PV) of the start of injection, indicating that bacteriophage were advectively transported mainly through fractures or macropores. Bacteriophage transport velocities, based on first arrival in the effluent, were very similar to fracture flow velocities calculated using the cubic law for flow in a fractured material. For MS-2, maximum concentration and mass of tracer recovered both increased steadily as flow rate increased. For PRD-1, these values initially increased, but were nearly constant at flow rates above 0.039 m d-1, indicating that approximately 50% of the observed losses were independent of flow rate. Evaluation of the data indicates that physical straining and electrostatic or hydrophobic attachment to fracture or macropore walls were the dominant retention processes. Inactivation and gravitational settling playing secondary roles, except at the slowest flow rates. The study suggests that microbial contamination from sources such as septic fields and sewage ponds may pose a threat to the quality of ground water and surface water in areas with saprolitic subsoils. [TOP OF PAGE]

  1063. Application of actinomycetes to soil to ameliorate water repellency. McKenna,F., El Tarabily,K.A., Petrie,S., Chen,C., Dell,B. (2002). Lett. Appl. Microbiol. 35:107-112. AIMS: The aim of this study was to develop a novel isolation technique using a mixture of Bacillus and Streptomyces phages to selectively isolate wax-utilizing non-streptomycete actinomycetes effective in ameliorating water repellency in a problem soil. METHODS AND RESULTS: Phages added to a soil suspension reduced the dominance of Bacillus and Streptomyces isolates and significantly increased the number of non-streptomycete actinomycetes on isolation plates. Promising isolates, grown on a medium containing beeswax as sole carbon source, were selected for application to water repellent soil. Their addition significantly reduced water repellency. CONCLUSIONS: Phage application significantly increased the isolation of non-streptomycete actinomycetes. Wax-utilizing isolates were found to significantly reduce water repellency in a problem soil. SIGNIFICANCE AND IMPACT OF THE STUDY: The phage technique can be used for the routine isolation of non-streptomycete actinomycetes. Beeswax medium can be used to selectively isolate wax-utilizing micro-organisms with the potential to ameliorate water repellency in soil. [TOP OF PAGE]

  1064. Phage tests for diagnosis and drug susceptibility testing. McNerney,R. (2002). Int. J. Tuberc. Lung Dis. 6:1129-1130. [TOP OF PAGE]

  1065. Conservation of phage reference materials and water samples containing bacteriophages of enteric bacteria. Mendez,J., Jofre,J., Lucena,F., Contreras,N., Mooijman,K., Araujo,R. (2002). J. Virol. Meth. 106:215-224. The survival was determined in different conservation conditions of: somatic coliphages, F-specific RNA bacteriophages and phages infecting proposed as model micro-organisms for water quality control. Titres of phages of all groups either in pure culture phage suspensions or in naturally occurring phage suspensions were stable at (-70+/-10) degrees C and at (-20+/-5) degrees C when protected with glycerol. Moreover, phage analysis of stored suspensions demonstrated that their numbers were homogeneous, both between vials and within vials, and consequently they can be used as reference materials. Furthermore, changes in the storage temperature of the vials cause unpredictable changes in the numbers of bacteriophages. Consequently, phage reference materials and samples containing a quantitative number of phages must be maintained and dispatched at a constant temperature. Consequently, the results indicate that bacteriophages should be packed in dry ice during transport and storage. Finally, the number of phages in water samples stored at (5+/-3) degrees C in the dark does not decrease significantly during the first 72 h of storage. In addition, phage concentrates from natural samples obtained by adsorption-elution to cellulose nitrate filters and mixed with 10% glycerol were stable at least for 2 months at (-70+/-10) degrees C and at (-20+/-5) degrees C. [TOP OF PAGE]

  1066. The genome of bacteriophage phiKZ of Pseudomonas aeruginosa. Mesyanzhinov,V.V., Robben,J., Grymonprez,B., Kostyuchenko,V.A., Bourkaltseva,M.V., Sykilinda,N.N., Krylov,V.N., Volckaert,G. (2002). J. Mol. Biol. 317:1-19. Bacteriophage phiKZ is a giant virus that efficiently infects Pseudomonas aeruginosa strains pathogenic to human and, therefore, it is attractive for phage therapy. We present here the complete phiKZ genome sequence and a preliminary analysis of its genome structure. The 280,334 bp genome is a linear, circularly permutated and terminally redundant, A+T-rich double-stranded DNA molecule. The phiKZ DNA has no detectable sequence homology to other viruses and microorganisms, and it does not contain NotI, PstI, SacI, SmaI, XhoI, and XmaIII endonuclease restriction sites. The genome has 306 open reading frames (ORFs) varying in size from 50 to 2237 amino acid residues. According to the orientation of transcription, ORFs are apparently organized into clusters and most have a clockwise direction. The phiKZ genome also encodes six tRNAs specific for Met (AUG), Asn (AAC), Asp (GAC), Leu (TTA), Thr (ACA), and Pro (CCA). A putative promoter sequence containing a TATATTAC block was identified. Most potential stem-loop transcription terminators contain the tetranucleotide UUCG loops. Some genes may be assigned as phage-encoded RNA polymerase subunits. Only 59 phiKZ gene products exhibit similarity to proteins of known function from a diversity of organisms. Most of these conserved gene products, such as dihydrofolate reductase, ribonucleoside diphosphate reductase, thymidylate synthase, thymidylate kinase, and deoxycytidine triphosphate deaminase are involved in nucleotide metabolism. However, no virus-encoded DNA polymerase, DNA replication-associated proteins, or single-stranded DNA-binding protein were found based on amino acid homology, and they may therefore be strongly divergent from known homologous proteins. Fifteen phiKZ gene products show homology to proteins of pathogenic organisms, including Mycobacterium tuberculosis, Haemophilus influenzae, Listeria sp., Rickettsia prowazakeri, and Vibrio cholerae that must be considered before using this phage as a therapeutic agent. The phiKZ coat contains at least 40 polypeptides, and several proteins are cleaved during virus assembly in a way similar to phage T4. Eleven phiKZ-encoded polypeptides are related to proteins of other bacteriphages that infect a variety of hosts. Among these are four gene products that contain a putative intron-encoded endonuclease harboring the H-N-H motif common to many double-stranded DNA phages. These observations provide evidence that phages infecting diverse hosts have had access to a common genetic pool. However, limited homology on the DNA and protein levels indicates that bacteriophage phiKZ represents an evolutionary distinctive branch of the Myoviridae family. [TOP OF PAGE]

  1067. Viral and bacterial production in the North Water polynya: In situ measurements, batch culture experiments, and characterization and distribution of a virus-host system. Middelboe,M., Nielson,T.G., Bjørnsen,P.K. (2002). Deep-Sea Research II 49:5063-5079. Growth and viral lysis of bacterioplankton at subzero temperatures were measured in the North Water polynya in July 1998. In situ measurements of bacterial carbon consumption in surface waters ranged from 15 to 63mgCl-1d-1 in the eastern and 6 to 7mgCl-1d-1 in the northern part of the polynya. Both bacterial abundance and activity appeared to increase in response to the decay of the phytoplankton bloom that developed in the North Water. Organic carbon was the limiting substrate for bacteria in the polynya since addition of glucose, but not inorganic nutrients, to batch cultures increased both the carrying capacity of the substrate and the growth rate of the bacteria. Bacterial growth rates ranged from 0.11 to 0.40d-1, corresponding to bacterial generation times of 1.7-6.3d. The in situ viral production rate was estimated both from the frequency of visibly infected cells and from the rate of viral production in batch cultures; it ranged from 0.04 to 0.52d-1 and from 0.25 to 0.47d-1, respectively. From 6% to 28% of bacterial production was found to be lost due to viral lysis. The average virus-bacteria ratio was 5.1±3.1, with the abundance of viruses being correlated positively with bacterial production. A Pseudoalteromonas sp. bacterial host and an infective virus were isolated from the polynya; characteristics and distribution of the virus-host system were examined. The Pseudoalteromonas sp. showed psychrotolerant growth and sustained significant production of viruses at 0°C. The virus-host system was found throughout the polynya. Overall the results suggested that a large amount of organic carbon released during the development and breakdown of the spring phytoplankton bloom was consumed by planktonic bacteria and that the microbial food web was an important and dynamic component of the planktonic food web in the North Water. [TOP OF PAGE]

  1068. Regeneration of dissolved organic matter by viral lysis in marine microbial communities. Middelboe,M., Lyck,P.G. (2002). Aquat. Microb. Ecol. 27:187-194. The influence of viruses on bacterial net growth and respiration was investigated in batch cultures with natural assemblages of marine bacterioplankton, which were manipulated with respect to abundance of natural virioplankton. In 1 set of cultures (-virus), a virus-free water sample (0.02 mu m filtered) was inoculated with a bacterioplankton concentrate, and in a parallel set of cultures (control) a virus-containing water sample (0.2 mu m filtered) was inoculated with the bacterioplankton concentrate. The 0.02 mu m filtration procedure reduced viral abundance by 62 to 92% in the -virus cultures relative to the parallel control cultures with the natural density of viruses (i.e. the fraction of natural viruses < 0.2 mu m). This approach allowed us to examine the effects of reduced viral densities on the production of natural assemblages of bacteria and viruses and on the distribution of added 3H-thymidine into size fractions (the bacterial size fraction, viral size fraction, dissolved size fraction and respired fraction). The results showed significantly higher bacterial net growth and growth efficiency in cultures with a reduced abundance of viruses relative to control cultures with natural viral abundance, and indicated viral regulation of bacterial abundance in the control cultures. We suggest that viral lysis significantly affected the bacterial carbon cycling in the cultures by liberating a fraction of the organic matter already taken up by the bacteria, thus stimulating recycling of bacterial carbon and reducing the net bacterial production. The implications of such regeneration of dissolved organic matter by viral lysis for pelagic carbon cycling and for measurements of bacterial production are discussed. [TOP OF PAGE]

  1069. Evaluation of bacteriophages during the treatment of sludge. Mignotte-Cadiergues,B., Gantzer,C., Schwartzbrod,L. (2002). Water Sci. Technol. 46:189-194. The aim of this work was to determine the effect of liming and composting on the fate of three bacteriophages (somatic coliphages, F-RNA phages, phages) considered as potential indicators of viral contamination. It was shown that the three bacteriophages studied exhibited variable densities in sludge. Somatic coliphages were most abundant (104 to 105 x 10 g-1 DM) then F-RNA bacteriophages (102 to 104 x 10 g-1 DM) and Bacteroides fragilis phages (101 to 102 x 10 g-1 DM). The efficacy of liming was found to be pH dependent but also sludge dependent. The pH allowing 99% elimination of somatic coliphage is close to 9 for solid sludges and close to 13.5 for liquid sludges. For composting, our findings clearly demonstrated that phage inactivation is very clearly temperature-dependent. For temperatures reaching 70 degrees, there is a 5 log reduction in somatic coliphages while for temperature in the 50-55 degrees C range, the drop off is only 2 log. Considering the efficacy of the treatment methods, it is clear that the well-established industrial procedures that reach temperatures in the 60-70 degrees C range totally inactivate all 3 phages tested and present in sludge before composting. [TOP OF PAGE]

  1070. Pseudolysogeny: A bacteriophage strategy for increasing longevity in situ. Miller,R.V., Ripp,S.A. (2002). pp. 81-91. In In Syvanen,M. and Kado,C.I. (eds.), Horizontal Gene Transfer. Academic Press, San Diego. [TOP OF PAGE]

  1071. Use of lacticin 481 to facilitate delivery of the bacteriophage resistance plasmid, pCBG104 to cheese starters. Mills,S., Coffey,A., O'Sullivan,L., Stokes,D., Hill,C., Fitzgerald,G.F., Ross,R.P. (2002). J. Appl. Microbiol. 92:238-246. AIMS: Use of lacticin 481 to facilitate the conjugal transfer of the bacteriophage resistance plasmid pCBG104 to various starter cultures. METHODS AND RESULTS: A raw milk isolate of Lactococcus was found to harbour determinants for lacticin 481 production and immunity and phage resistance on a plasmid designated pCBG104. The lacticin 481 was successfully used to mobilize the phage resistance determinant to a variety of cheese starters enabling the formation of highly phage resistant starters. In addition, it facilitated the stacking of a number of phage resistance genes, namely a type I restriction modification system, a phage abortive infection system and a phage adsorption blocking system in a single Lactococcus strain without the use of recombinant techniques. The transconjugants were all shown to produce lacticin 481 and to contain the entire 481 operon. Subsequently one transconjugant was selected and successfully used for large-scale cheddar cheese manufacture. CONCLUSIONS: Lacticin 481 could be used as a food-grade selectable marker to facilitate the introduction of advantageous traits to starter cultures for industrial food fermentations. SIGNIFICANCE AND IMAPCT OF THE STUDY: Food-grade selectable markers greatly facilitate the introduction of various advantageous traits to starter cultures for industrial food fermentation. Indeed self-cloning which is becoming increasingly important for strain improvement has a requirement for the identification and demonstration of the utility of tools such as lacticin 481. [TOP OF PAGE]

  1072. Microbial genome evolution: sources of variability. Mira,A., Klasson,L., Andersson,S.G.E. (2002). Curr. Opin. Mirobiol. 5:506-512. Comparative genome analyses of close relatives have yielded exciting insight into the sources of microbial genome variability with respect to gene content, gene order and evolution of genes with unknown functions. The genomes of free-living bacteria often carry phages and repetitive sequences that mediate genomic rearrangements in contrast to the small genomes of obligate host-associated bacteria. This suggests that genomic stability correlates with the genomic content of repeated sequences and movable genetic elements, and thereby with bacterial lifestyle. Genes with unknown functions present in a single species tend to be shorter than conserved, functional genes, indicating that the fraction of unique genes in microbial genomes has been overestimated. [TOP OF PAGE]

  1073. Temperate phages in Salmonella enterica serovar Typhimurium: implications for epidemiology. Mmolawa,P.T., Willmore,R., Thomas,C.J., Heuzenroeder,M.W. (2002). Int. J. Med. Microbiol. 291:633-644. [TOP OF PAGE]

  1074. Phages of lactic acid bacteria: From genomics to industrial applications. Moineau,S., Tremblay,D., Labrie,S. (2002). ASM News 68:388-393. Dairy microbiologists seek new ways to protect milk-fermenting microorganisms against damaging phages. Dairy microbiologists have been trying for more than 70 years to eleminate, or at least bring under better control, the bacteriophages that interfere with the manufacture of many fermented milk products. Products such as yogurt and cheese are manufactured through use of nonsporulating gram-positive microorganisms know as the lactic acid bacteria (LAB) (see p. 369). Among them are members of serveral genera, including Lactococcus, Streptococcus, Lactobacillus, and Leuconostoc. Large-scale fermentations of dairy products typically begin following inoculation with carefully seleted started cultures containing a mixture of 107 LAB per ml of milk. The choice of the LAB strains in starter cultures serves to control the fermentation and to yield high-quality produts. ¶ Annually, perhaps 1023 cells of LAB are used globally for such fermentations. Wtih this extensive commercial use, interest in studying LAB has increased to an unprecedented level in recent years. In fact, some researchers have even enthusiastically dubbed LAB "the bug of the new millenium." However, these commercial fermentative processes are vulnerable to specialized bacteriophages. A keen interest in overcoming this vulnerability is bolstering an established fascination for these microorganisms, and dairy microobiologists are learning to control phages in numerous ways. The phage diversity has led such researchers to a wide range of fundamental and applied studies, engaging them in the "ome" era of genome, proteome, and transcriptomes with the hope of developing novel anti-phage mechanisms. [= first two paragraphs]. [TOP OF PAGE]

  1075. Uptake and processing of modified bacteriophage M13 in mice: implications for phage display. Molenaar,T.J.M., Michon,I., de Haas,S.A.M., van Berkel,T.J.C., Kuiper,J., Biessen,E.A.L. (2002). Virology 293:182-191. Internalization and degradation of filamentous bacteriophage M13 by a specific target cell may have major consequences for the recovery of phage in in vivo biopanning of phage libraries. Therefore, we investigated the pharmacokinetics and processing of native and receptor-targeted phage in mice. 35S-radiolabeled M13 was chemically modified by conjugation of either galactose (lacM13) or succinic acid groups (sucM13) to the coat protein of the phage to stimulate uptake by galactose recognizing hepatic receptors and scavenger receptors, respectively. Receptor-mediated endocytosis of modified phage reduced the plasma half-life of native M13 (t(1/2) = 4.5 h) to 18 min for lactosylated and 1.5 min for succinylated bacterophage. Internalization of sucM13 was complete within 30 min after injection and resulted in up to 5000-fold reduction of bioactive phage within 90 min. In conclusion, these data provide information on the in vivo behavior of wild-type and receptor-targeted M13, which has important implications for future in vivo phage display experiments and for the potential use of M13 as a viral gene delivery vehicle. [TOP OF PAGE]

  1076. Regrowth and survival of indicator microorganisms on the surfaces of household containers used for the storage of drinking water in rural communities of South Africa. Momba,M.N.B., Kaleni,P. (2002). Water Res. 36:3023-3028. The present study covered two rural communities of South Africa: Ncera and Ntselamanzi villages. Raw water from Ncera river is used by the community of Ncera village for drinking, while the community of Ntselamanzi receives their drinking water from Alice purification system. Treated water is supplied to the community by a public standpipe system. In rural communities of South Africa, many households use polyethylene (PE) and galvanized steel (GS) containers for the storage of their drinking water. To investigate the regrowth and survival of indicator microorganisms on the surface of household containers during the storage of drinking water, PE and GS slides were suspended in the appropriate household containers for a period of 48 h. This period of 48 h was chosen as the study period because results from the questionnaire indicated that the largest percentage (62%) of households store their water for that length of time. The experiment was performed to test drinking water as it is collected and stored by rural communities. No disinfection of household containers or slides was done during the study period. Attached coliphages (F-RNA (FP) and somatic phage (SP), coliform bacteria (total coliform (TC), presumptive Escherichia coli (EC), Salmonella (Sal) and Clostridium perfringens (CP) were measured during the study period. With the exception of CP, attached indicator microorganisms consisted of TC, presumptive E. coli and Salmonella, somatic and F-RNA coliphages, although the yield (average count) for the last four groups (EC: < 1-3 cfu cm-2, Sal: < 1-15 cfu cm-2, FP: < 1-7 pfu cm-2, SP: < 1-7pfu cm-2) was lower than that of TC (3-183 cfu cm-2). However, the lowest yield of indicator microorganisms was noted for presumptive E. coli. Whereas the occurrence and survival of TC was noted on the surface of household containers during the entire period of the experimental study, other indicator microorganisms occurred from time to time. The regrowth of indicator microorganisms occurred 48 h after the exposure of slides to both types of test waters. This length of time mostly resulted in the regrowth of TC (with an increase in bacterial counts) while the persistence of other indicator organism groups on the surface of the slides was apparent. A comparison between PE and GS containers showed that more TC (average count) regrew on PE than on GS containers (for river water, PE: from 36 to 55 cfu cm-2, GS: from 25 to 26 cfu cm-2; for standpipe water, PE: from 147 to 183 cfu cm-2, GS from 3 to 4 cfu cm-2). This study revealed that both types of household containers supported the growth and survival of indicator microorganisms due to the bad quality of the intake water before storage. The storage of drinking water for 48 h mainly resulted in the regrowth of TC. Nevertheless, the persistence of other indicator microorganisms was observed on the surface of household containers. [TOP OF PAGE]

  1077. Virus-like particle analysis in a seston-rich coastal pond using transmission electron microscopy. Montanie,H., Hartmann,H.J., Crottereau,C., Trichet,C. (2002). Aquat. Microb. Ecol. 28:105-115. A method was developed to analyse virus-like particles (VLPs) in seston-rich waters and to quantify their dynamics in a coastal marsh of the Bay of Biscay, French Atlantic coast. The method combined clarification and concentration steps with electron microscopy to obtain information on particle abundance, type and size distribution (e.g. presence of tailed phages, Fuselloviridae, etc.). The mean recovery rates of T2-phages using this method were 71 to 79%, higher than other published rates. The transmission electron microscopy (TEM) counts were validated with T2 plaque lysis assay and epifluorescent (DAPI-stained) particle counting: the TEM method was valid for environmental particle concentrations above 1 to 2 x 106 VLP ml-1; TEM counts were lower than T2-plaque counts (TEM/lysis median = 0.293) but higher than DAPI counts (TEM/DAPI median = 2.39). The method was used to evaluate the coupling between viral and bacterial dynamics in a marsh pond during 2 months. The VLP abundance varied from 1 to 30 x 106 ml super(-1) and the viral population was dominated by small particles (20 to 64 nm). Tailed phages, identified as bacteriophages, were always less abundant than non-tailed VLPs (4 to 23% of total virus), yet their dynamics were better linked with bacterial development than those of total virus. Our results demonstrate that the best way to characterise bacterial lysis from virus in seston-rich coastal environments would be to study the dynamics of tailed phages and virus size-classes rather than the commonly applied total VLPs. [TOP OF PAGE]

  1078. Optimisation of ISO 10705-1 on enumeration of F-specific bacteriophages. Mooijman,K.A., Bahar,M., Muniesa,M., Havelaar,A.H. (2002). J. Virol. Meth. 103:129-136. During the European project 'Bacteriophages in bathing waters' (January 1996-June 1999), research was carried out to optimise the method for detection and enumeration of F-specific (RNA) phages in water. It was evaluated whether further optimisation would be possible/needed for the procedure as described in the standard method of the International Organisation for Standardisation (ISO) 10705-1. The research focused mainly on optimisation of the different steps for culturing the host strain WG49 Salmonella Typhimurium. It was concluded that all steps described in ISO 10705-1 are necessary and, if followed carefully, using a culture of host strain WG49 Salmonella Typhimurium of good quality, reliable results could be obtained for the enumeration of F-specific RNA phages. [TOP OF PAGE]

  1079. Microbial quality of wastewater: detection of hepatitis A virus by reverse transcriptase-polymerase chain reaction. Morace,G., Aulicino,F.A., Angelozzi,C., Costanzo,L., Donadio,F., Rapicetta,M. (2002). J. Appl. Microbiol. 92:828-836. AIMS: The persistent circulation of hepatitis A virus (HAV) in the Mediterranean area suggests the need for monitoring its presence in the environment. A reverse transcriptase-polymerase chain reaction (RT-PCR) was used to detect the presence of HAV in several consecutive raw sewage and final effluent samples, collected over an 8-month period from an activated sludge treatment plant in southern Italy. METHODS AND RESULTS: Two distinct purification protocols, either based on antigen-capture with monoclonal antibody (AC) or RNA extraction, were compared. The possible influence of the antibody used in the AC phase was evaluated in preliminary experiments on HAV-spiked samples, using two different monoclonal antibodies. Hepatitis A virus RNA was detected in all but one sewage environmental sample examined. The contemporary presence of enteroviruses, reoviruses and phages was observed, while HAV growth in cell culture was hampered. CONCLUSIONS: The RT-PCR technique was confirmed to be a valuable tool for the rapid monitoring of HAV in sewage samples. In addition, this study demonstrated that application of different sample purification methods can result in different levels of sensitivity of the assay and that, in the antigen-capture method, the choice of antibody can have a crucial role. SIGNIFICANCE AND IMPACT OF THE STUDY: This work underlines the need for technical uniformity in environmental studies from different laboratories for a correct and useful comparison of the results. [TOP OF PAGE]

  1080. Bacteriophage Mu genome sequence: analysis and comparison with Mu-like prophages in Haemophilus, Neisseria and Deinococcus. Morgan,G.J., Hatfull,G.F., Casjens,S., Hendrix,R.W. (2002). J. Mol. Biol. 317:337-359. We report the complete 36,717 bp genome sequence of bacteriophage Mu and provide an analysis of the sequence, both with regard to the new genes and other genetic features revealed by the sequence itself and by a comparison to eight complete or nearly complete Mu-like prophage genomes found in the genomes of a diverse group of bacteria. The comparative studies confirm that members of the Mu-related family of phage genomes are genetically mosaic with respect to each other, as seen in other groups of phages such as the phage lambda-related group of phages of enteric hosts and the phage L5-related group of mycobacteriophages. Mu also possesses segments of similarity, typically gene-sized, to genomes of otherwise non-Mu-like phages. The comparisons show that some well-known features of the Mu genome, including the invertible segment encoding tail fiber sequences, are not present in most members of the Mu genome sequence family examined here, suggesting that their presence may be relatively volatile over evolutionary time.The head and tail-encoding structural genes of Mu have only very weak similarity to the corresponding genes of other well-studied phage types. However, these weak similarities, and in some cases biochemical data, can be used to establish tentative functional assignments for 12 of the head and tail genes. These assignments are strongly supported by the fact that the order of gene functions assigned in this way conforms to the strongly conserved order of head and tail genes established in a wide variety of other phages. We show that the Mu head assembly scaffolding protein is encoded by a gene nested in-frame within the C-terminal half of another gene that encodes the putative head maturation protease. This is reminiscent of the arrangement established for phage lambda. [TOP OF PAGE]

  1081. Characterization of a virulent bacteriophage specific for Escherichia coli O157:H7 and analysis of its cellular receptor and two tail fiber genes. Morita,M., Tanji,Y., Mizoguchi,K., Akitsu,T., Kijima,N., Unno,H. (2002). FEMS Microbiol. Lett. 211:77-83. A virulent phage, named PP01, specific for Escherichia coli O157:H7 was isolated from swine stool sample. The phage concentration in a swine stool, estimated by plaque assay on E. coli O157:H7 EDL933, was 4.2x10(7) plaque-forming units per g sample. PP01 infects strains of E. coli O157:H7 but does not infect E. coli strains of other O-serogroups and K-12 strains. Infection of an E. coli O157:H7 culture with PP01 at a multiplicity of infection of two produced a drastic decrease of the optical density at 600 nm due to cell lysis. The further incubation of the culture for 7 h produced phage-resistant E. coli O157:H7 mutant. One PP01-resistant E. coli O157:H7 mutant had lost the major outer membrane protein OmpC. Complementation by ompC from a O157:H7 strain but not from a K-12 strain resulted in the restoration of PP01 susceptibility suggesting that the OmpC protein serves as the PP01 receptor. DNA sequences and homology analysis of two tail fiber genes, 37 and 38, responsible for the host cell recognition revealed that PP01 is a member of the T-ev en bacteriophages, especially the T2 family. [TOP OF PAGE]

  1082. Horizontal gene transfer in bacteriophages. Mosig,G., Calendar,R. (2002). pp. 141-146. In In Syvanen,M. and Kado,C.I. (eds.), Horizontal Gene Transfer. Academic Press, San Diego. [TOP OF PAGE]

  1083. Effect of denture cleaner using ozone against methicillin-resistant Staphylococcus aureus and E. coli T1 phage. Murakami,H., Mizuguchi,M., Hattori,M., Ito,Y., Kawai,T., Hasegawa,J. (2002). Dental Mat. J. 21:53-60. We examined the bactericidal and virucidal effectiveness of a denture cleaner that uses ozone (ozone concentration, 10 ppm) against methicillin-resistant Staphylococcus aureus (MRSA) and T1 phage, respectively. In the bactericidal activity test, with the ozone supply turned on, the number of bacteria was 3.1 x 10(3) CFU/mL at the beginning of the experiment, fell to 1.0 x 10(0) CFU/mL 10 min later, and was 1.0 x 10(0) CFU/mL or less afterwards. In contrast, when the ozone supply was cut off (air bubble only), the number of bacteria was 3.4 x 10(3) CFU/mL at the beginning of the experiment, and had fallen to 3.0 x 10(3) CFU/mL 60 min later (no statistically significant difference). In the virucidal activity test, the number of phages was 1.2 x 10(6) PFU/mL before ozone treatment, fell to about 1/10 of that number 10 min later, and was 6.1 x 10(0) PFU/mL 40 min later. These results indicate that the use of ozone in this denture cleaner is effective against MRSA and viruses. [TOP OF PAGE]

  1084. Bacteriophage therapy of infectious disease in aquaculture. Nakai,T., Park,S.C. (2002). Res. Microbiol. 153:13-18. Bacteriophages may be candidates as therapeutic agents in bacterial infections. Here we describe the protective effects of phages against experimentally induced bacterial infections of cultured fish and discuss the potential for phage therapy in aquaculture. [TOP OF PAGE]

  1085. The effect of an agglutogen on virus infection: biotinylated filamentous phages and avidin as a model. Nakamura,M., Tsumoto,K., Ishimura,K., Kumagai,I. (2002). FEBS Lett. 520:77-80. To address the effect of an agglutogen on virus infection, we studied the avidin-associated inhibition of infection by biotinylated M13 phages (BIO-phages). Microscopic observation of mixtures of BIO-phages and avidin-fluorescein conjugates revealed many aggregates. Even at low phage concentrations, avidin induced inhibition of infection significantly. Anti-M13 phage antibody also made aggregates and inhibited the infection but in a different manner from avidin. The inhibition by avidin was at > or = 2 microg/ml, time dependent and marked until 10 min after the mixing of the BIO-phages and Escherichia coli. On the other hand, antibody inhibited the infection at > or = 0.1 microg/ml dose dependently, and the inhibition was time dependent and marked until 45 min after the mixing at moderate and low phage concentrations. These results indicate that avidin against BIO-phages and antibodies are agglutogens, and the inhibition of the BIO-phages by avidin is closely related to the tetramerization of avidin. Agglutogens may be novel alternative antiviral drugs. [TOP OF PAGE]

  1086. Contribution of microbial activity to virus reduction in saturated soil. Nasser,A.M., Glozman,R., Nitzan,Y. (2002). Water Res. 36:2589-2595. Application of wastewater to soil may result in the contamination of groundwater and soil with pathogenic microorganisms and other biological and chemical agents. This study was performed to determine the antiviral microbial activity of soil saturated with secondary effluent. Low concentrations (0.05mg/ml) of protease pronase resulted in the inactivation of more than 90% of seeded Cox-A9 virus, whereas Poliovirus type 1, Hepatitis A virus (HAV) and MS2 bacteriophages were found to be insensitive to the enzyme activity. Exposure of Cox A9 virus to P. aeruginosa extracellular enzymes resulted in 99% inactivation of the seeded virus. Hepatitis A virus was found to be as sensitive as the Cox A9 virus, whereas Poliovirus 1 and MS2 were found to be insensitive to P. aeruginosa extracellular enzymatic activity. Furthermore, the time required for 99% reduction (T99) of Cox A9 and MS-2 Bacteriophage, at 15 degrees C, in soil saturated with secondary effluent was found to be 7 and 21 days, respectively. Faster inactivation was observed for MS2 and Cox A9 in soil saturated with secondary effluent incubated at 30 degrees C, T99 of 2 and 0.3 days, respectively. Although the concentration of the total bacterial count in the soil samples increased from 103 cfu/g to 105 cfu/g after 20 days of incubation at 30 degrees C, the proteolytic activity was below the detection level. The results of this study indicate that the virucidal effect of microbial activity is virus type dependent. Furthermore microbial activity in the soil material can be enhanced by the application of secondary effluent at higher temperature. The results also showed that MS2 bacteriophage can be used to predict viral contamination of soil and groundwater. [TOP OF PAGE]

  1087. Combined phage typing and amperometric detection of released enzymatic activity for the specific identification and quantification of bacteria . Neufeld,T., Schwartz-Mittelmann,A., Biran,D., Ron,E.Z., Rishpon,J. (2002). Analyt. Chem. 75:580-585. Here, we describe a novel electrochemical method for the rapid identification and quantification of pathogenic and polluting bacteria. The design incorporates a bacteriophage, a virus that recognizes, infects, and lyses only one bacterial species among mixed populations, thereby releasing intracellular enzymes that can be monitored by the amperometric measurement of enzymatic activity. As a model system, we used virulent phage typing and cell-marker enzyme activity (-D-galactosidase), a combination that is specific for the bacterial strain Escherichia coli (K-12, MG1655). Filtration and preincubation before infecting the bacteria with the phage enabled amperometric detection at a wide range of concentrations, reaching as low as 1 colony-forming unit/100 mL within 6-8 h. In principle, this electrochemical method can be applied to any type of bacterium-phage combination by measuring the enzymatic marker released by the lytic cycle of a specific phage. [TOP OF PAGE]

  1088. Evolution and spread of antibiotic resistance. Normark,B.H., Normark,S. (2002). J. Internal Med. 252:91-106. Antibiotic resistance is a clinical and socioeconomical problem that is here to stay. Resistance can be natural or acquired. Some bacterial species, such as Pseudomonas aeruginosa, show a high intrinsic resistance to a number of antibiotics whereas others are normally highly antibiotic susceptible such as group A streptococci. Acquired resistance evolve via genetic alterations in the microbes own genome or by horizontal transfer of resistance genes located on various types of mobile DNA elements. Mutation frequencies to resistance can vary dramatically depending on the mechanism of resistance and whether or not the organism exhibits a mutator phenotype. Resistance usually has a biological cost for the microorganism, but compensatory mutations accumulate rapidly that abolish this fitness cost, explaining why many types of resistances may never disappear in a bacterial population. Resistance frequently occurs stepwise making it important to identify organisms with low level resistance that otherwise may constitute the genetic platform for development of higher resistance levels. Self-replicating plasmids, prophages, transposons, integrons and resistance islands all represent DNA elements that frequently carry resistance genes into sensitive organisms. These elements add DNA to the microbe and utilize site-specific recombinases/integrases for their integration into the genome. However, resistance may also be created by homologous recombination events creating mosaic genes where each piece of the gene may come from a different microbe. The selection with antibiotics have informed us much about the various genetic mechanisms that are responsible for microbial evolution. [TOP OF PAGE]

  1089. Mobilization of the Vibrio pathogenicity island between Vibrio cholerae isolates mediated by CP-T1 generalized transduction. O'Shea,Y.A., Boyd,E.F. (2002). FEMS Microbiol. Lett. 214:153-157. Pathogenicity islands are large chromosomal regions encoding virulence genes that were acquired by horizontal gene transfer and are found in a wide range of pathogenic bacteria. In toxigenic Vibrio cholerae isolates the receptor for the cholera toxin encoding filamentous phage CTXf, the toxin-coregulated pilus, is part of the Vibrio pathogenicity island (VPI). In this paper, we show that the VPI can be transferred between O1 serogroup strains, the predominant cause of epidemic cholera, via a generalized transducing phage CP-T1. [TOP OF PAGE]

  1090. Effects of Azithromycin on Shiga Toxin Production by Escherichia coli and Subsequent Host Inflammatory Response . Ohara,T., Kojio,S., Taneike,I., Nakagawa,S., Gondaira,F., Tamura,Y., Gejyo,F., Zhang,H.-M., Yamamoto,T. (2002). Antimicrob. Agents Chemother. 46:3478-3483. Shiga toxin (Stx)-producing Escherichia coli (STEC) colonizes the human intestinal mucosa, produces Stx from phage, and causes the development of hemolytic-uremic syndrome via Stx-induced inflammatory cytokine production. Azithromycin exhibited strong in vitro activity against STEC without inducing Stx-converting phage, in marked contrast to norfloxacin. Azithromycin decreased the tumor necrosis factor alpha (TNF-), interleukin-1ß (IL-1ß), and IL-6 production from Stx-treated human peripheral mononuclear cells or monocytes to a greater extent than did clarithromycin. In Stx-injected mice, azithromycin significantly suppressed Stx-induced TNF-, IL-1ß, and IL-6 levels in serum and improved the outcome as assessed by survival rate. In the STEC oral infection experiment using immature mice immediately after weaning (weaned immature-mouse model), all mice died within 7 days postinfection. Azithromycin administration gave the mice 100% protection from killing, while ciprofloxacin administration gave them 67% protection. The data suggest that azithromycin (at least at higher concentrations) has a strong effect on Stx production by STEC and on the Stx-induced inflammatory host response and prevents death in mice. Azithromycin may have a beneficial effect on STEC-associated disease. [TOP OF PAGE]

  1091. Lysogeny and lytic viral production during a bloom of the cyanobacterium Synechococcus spp. Ortmann,A.C., Lawrence,J.E., Suttle,C.A. (2002). Microb. Ecol. 43:225-231. Lytic viral production and lysogeny were investigated in cyanobacteria and heterotrophic bacteria during a bloom of Synechococcus spp. in a pristine fjord in British Columbia, Canada. Triplicate seawater samples were incubated with and without mitomycin C and the abundances of heterotrophic bacteria, cyanobacteria, total viruses and infectious cyanophage were followed over 24 h. Addition of mitomycin C led to increases in total viral abundance as well as the abundance of cyanophages infecting Synechococcus strain DC2. Given typical estimates of burst size, these increases were consistent with 80% of the heterotrophic bacteria and 0.6% of Synechococcus cells being inducible by the addition of mitomycin C. This is the highest percentage of lysogens reported for a natural microbial community and demonstrates induction in a marine Synechococcus population. It is likely that the cyanophage production following the addition of mitomycin C was much higher than that titered against a single strain of Synechococcus; hence this estimate is a minimum. In untreated seawater samples, lytic viral production was estimated to remove ca. 27% of the gross heterotrophic bacterial production, and a minimum of 1.0% of the gross cyanobacterial production. Our results demonstrate very high levels of lysogeny in the heterotrophic bacterial community, outside of an oligotrophic environment, and the presence of inducible lysogens in Synechococcus spp. during a naturally occurring bloom. These data emphasize the need for further examination of the factors influencing lytic and lysogenic viral infection in natural microbial communities. [TOP OF PAGE]

  1092. When phage, plasmids, and transposons collide: genomic islands, and conjugative- and mobilizable-transposons as a mosaic continuum. Osborn,A.M., Boltner,D. (2002). Plasmid 48:202-212. Plasmids and bacteriophage represent the classical vectors for gene transfer within the horizontal gene pool. However, the more recent discovery of an increasing array of other mobile genetic elements (MGE) including genomic islands (GIs), conjugative transposons (CTns), and mobilizable transposons (MTns) which each integrate within the chromosome, offer an increasingly diverse assemblage contributing to bacterial adaptation and evolution. Molecular characterisation of these elements has revealed that they are comprised of functional modules derived from phage, plasmids, and transposons, and further that these modules are combined to generate a continuum of mosaic MGE. In particular, they are comprised of any one of three distinct types of recombinase, together with plasmid-derived transfer and mobilisation gene functions. This review highlights both the similarities and distinctions between these integrating transferable elements resulting from combination of the MGE toolbox. [TOP OF PAGE]

  1093. Complete nucleotide sequence and likely recombinatorial origin of bacteriophage T3. Pajunen,M.I., Elizondo,M.R., Skurnik,M., Kieleczawa,J., Molineux,I.J. (2002). J. Mol. Biol. 319:1115-1132. We report the complete genome sequence (38,208 bp) of bacteriophage T3 and provide a bioinformatic comparative analysis with other completely sequenced members of the T7 group of phages. This comparison suggests that T3 has evolved from a recombinant between a T7-like coliphage and a yersiniophage. To assess this, recombination between T7 and the Yersinia enterocolitica serotype O:3 phage phiYeO3-12 was accomplished in vivo; coliphage progeny from this cross were selected that had many biological properties of T3. This represents the first experimentally observed recombination between lytic phages whose normal hosts are different bacterial genera. [TOP OF PAGE]

  1094. A filterable lytic agent obtained from a red tide bloom that caused lysis of Karenia brevis (Gymnodinum breve) cultures. Paul,J.H., Houchin,L., Griffin,D., Slifko,T., Guo,M., Richardson,B., Steidinger,K. (2002). Aquat. Microb. Ecol. 27:21-27. A filterable lytic agent (FLA) was obtained from seawater in the southeastern Gulf of Mexico during a red tide bloom that caused lysis of Karenia brevis (formerly Gymnodinium breve) Piney Island. This agent was obtained from <0.2 mu m filtrates that were concentrated by ultrafiltration using a 100 kDa filter. The FLA was propagated by passage on K. brevis cultures, and the filtered supernatants of such cultures resulted in K. brevis lysis when added to such cultures. The lytic activity was lost upon heating to 65 degree C or by 0.02 mu m filtration. Epifluorescence and transmission electron microscopy (TEM) of supernatants of K. brevis cultures treated with the lytic agent indicated a high abundance of viral particles (4 x 109 to 7 x 109 virus-like particles [VLPs] ml-1) compared to control cultures ( similar to 107 ml-1). However, viral particles were seldom found in TEM photomicrograph thin sections of lysing K. brevis cells. Although a virus specific for K. brevis may have been the FLA, other explanations such as filterable bacteria or bacteriophages specific for bacteria associated with the K. brevis cultures cannot be discounted. [TOP OF PAGE]

  1095. Marine phage genomics. Paul,J.H., Sullivan,M.B., Segall,A.M., Rohwer,F. (2002). Comparative Biochemistry and Physiology 133:463-476. Marine phages are the most abundant biological entities in the oceans. They play important roles in carbon cycling through marine food webs, gene transfer by transduction and conversion of hosts by lysogeny. The handful of marine phage genomes that have been sequenced to date, along with prophages in marine bacterial genomes, and partial sequencing of uncultivated phages are yielding glimpses of the tremendous diversity and physiological potential of the marine phage community. Common gene modules in diverse phages are providing the information necessary to make evolutionary comparisons. Finally, deciphering phage genomes is providing clues about the adaptive response of phages and their hosts to environmental cues. [TOP OF PAGE]

  1096. The Brucella suis genome reveals fundamental similarities between animal and plant pathogens and symbionts. Paulsen,I.T., et al (2002). Proc. Natl. Acad. Sci. USA 99:13148-13153. The 3.31-Mb genome sequence of the intracellular pathogen and potential bioterrorism agent, Brucella suis, was determined. Comparison of B. suis with Brucella melitensis has defined a finite set of differences that could be responsible for the differences in virulence and host preference between these organisms, and indicates that phage have played a significant role in their divergence. Analysis of the B. suis genome reveals transport and metabolic capabilities akin to soil/plant-associated bacteria. Extensive gene synteny between B. suis chromosome 1 and the genome of the plant symbiont Mesorhizobium loti emphasizes the similarity between this animal pathogen and plant pathogens and symbionts. A limited repertoire of genes homologous to known bacterial virulence factors were identified. [TOP OF PAGE]

  1097. Evidence for a phage proliferation threshold? Payne,R.J.H., Jansen,V.A.A. (2002). J. Virol. 76:13123 [first paragraph] Both experiments (5) and theory (3, 4) have suggested that for a population of phage to increase in numbers requires the host cell population to surpass a critical density termed the "replication threshold" or the "proliferation threshold." However, recently in the Journal of Virology, Kasman et al. (1) argued that no such threshold exists. Why this discrepancy? For a population of phage to increase in numbers, not only must phage from the initial dose replicate but also progeny phage must survive long enough to sustain further replication. This in turn depends on the density of remaining uninfected cells and on the rate of loss of free phage. The proliferation threshold is that cell density above which the probability of a progeny phage replicating is greater than the probability of that phage being lost (4). From this we identify three ways to reconcile the apparent inconsistencies between Kasman et al. (1) and Wiggins and Alexander (5). [TOP OF PAGE]

  1098. From Russia with gloves Ex-Soviet Union viruses could fill antibiotic gap. Pearson,H. (2002). Nature . Russian remedies could take out hardy US bacteria. Long-abandoned by Western medicine, viruses that naturally kill microbes are being imported as a potential substitute for antibiotics. ¶ The emergence of multi-drug-resistant bacteria is intensifying the search for antibiotic replacements. Bemoaning the problem, clinician Glenn Morris of the University of Maryland in College Park got an idea from a colleague from the former Soviet republic of Georgia. Morris explains: "He said, 'why don't you use 'phage therapy?'; I said, 'what's 'phage therapy?'." ¶ 'Phages - more properly, bacteriophages - are viruses that are harmless to humans but kill bacteria. They were widely researched as a means to tackle disease until the 1940s. When potent antibiotics appeared on the scene, the West discarded them. ¶ Eastern Europe and the former Soviet Union pursued 'phage therapy, so 'phage creams, pills and plasters are commonly available there. Now Morris and his colleagues are carrying out basic tests to update the treatments for US product licenses. ¶ Worktops contaminated with the foodborne bacteria Listeria are clean within 24 hours of 'phage treatment, he told the Experimental Biology 2002 meeting in New Orleans on Sunday. Salmonella and Escherichia coli are similarly wiped out. 'Phages could be used in food production or packaging, Morris suggests. ¶ Unlike antibiotics, 'phages kill only a specific bacterial type, leaving other, beneficial bugs intact. For example, antibiotic resistant strains of the gut bacteria Enterococcus, which can cause dangerous infections after surgery or in chemotherapy patients, are also being tackled. ¶ We are naturally surrounded by 'phages. The type that Morris is using attack and multiply inside bacteria then split them apart to escape. The 'phages keep killing until their victims run out, and then quietly die. ¶ Cold science ¶ Part of the reason that the West dropped 'phages was that bacteria might evade them, says Richard Young, who studies pathogenic microbes at the Whitehead Institute in Cambridge, Massachusetts. A single change in the bacterial receptor to which they bind could render it resistant to the virus: "It was viewed as its Achilles heel," he says. ¶ A mixture of 30-40 different 'phages all aimed at the same bug should get around this problem. "A cocktail is important," agrees Heidi Kaplan, who studies antibiotic-resistant bacteria at the University of Texas Medical School in Houston. ¶ "US science tends to have a prejudice against Soviet science," adds Morris, who now collaborates with the Eliava Institute of Bacteriophage, Microbiology and Virology in Tbilisi, Georgia. But Morris is not alone in trying to bring down the scientific cold wall - two small biotech companies besides his are also on the case. [TOP OF PAGE]

  1099. Bacteriophage resistance of a deltathyA mutant of Lactococcus lactis blocked in DNA replication. Pedersen,M.B., Jensen,P.R., Janzen,T., Nilsson,D. (2002). Appl. Environ. Microbiol. 68:3010-3023. The thyA gene, which encodes thymidylate synthase (TS), of Lactococcus lactis CHCC373 was sequenced, including the upstream and downstream regions. We then deleted part of thyA by gene replacement. The resulting strain, MBP71 deltathyA, was devoid of TS activity, and in media without thymidine, such as milk, there was no detectable dTTP pool in the cells. Hence, DNA replication was abolished, and acidification by MBP71 was completely unaffected by the presence of nine different phages tested at a multiplicity of infection (MOI) of 0.1. Nonreplicating MBP71 must be inoculated at a higher level than CHCC373 to achieve a certain pH within a specified time. For a pH of 5.2 to be reached in 6 h, the inoculation level of MBP71 must be 17-fold higher than for CHCC373. However, by adding a limiting amount of thymidine this could be lowered to just 5-fold the normal amount, while acidification was unaffected with MBP71 up to an MOI of 0.01. It was found that nonreplicating MBP71 produced largely the same products as CHCC373, though the acetaldehyde production of the former was higher. [TOP OF PAGE]

  1100. The dilemma of phage taxonomy illustrated by comparative genomics of Sfi21-like Siphoviridae in lactic acid bacteria. Proux,C., van Sinderen,D., Suarez,J., Garcia,P., Ladero,V., Fitzgerald,G.F., Desiere,F., Brüssow,H. (2002). J. Bacteriol. 184:6026-6036. The complete genome sequences of two dairy phages, Streptococcus thermophilus phage 7201 and Lactobacillus casei phage A2, are reported. Comparative genomics reveals that both phages are members of the recently proposed Sfi21-like genus of Siphoviridae, a widely distributed phage type in low-GC-content gram-positive bacteria. Graded relatedness, the hallmark of evolving biological systems, was observed when different Sfi21-like phages were compared. Across the structural module, the graded relatedness was represented by a high level of DNA sequence similarity or protein sequence similarity, or a shared gene map in the absence of sequence relatedness. This varying range of relatedness was found within Sfi21-like phages from a single species as demonstrated by the different prophages harbored by Lactococcus lactis strain IL1403. A systematic dot plot analysis with 11 complete L. lactis phage genome sequences revealed a clear separation of all temperate phages from two classes of virulent phages. The temperate lactococcal phages share DNA sequence homology in a patchwise fashion over the nonstructural gene cluster. With respect to structural genes, four DNA homology groups could be defined within temperate L. lactis phages. Closely related structural modules for all four DNA homology groups were detected in phages from Streptococcus or Listeria, suggesting that they represent distinct evolutionary lineages that have not uniquely evolved in L. lactis. It seems reasonable to base phage taxonomy on data from comparative genomics. However, the peculiar modular nature of phage evolution creates ambiguities in the definition of phage taxa by comparative genomics. For example, depending on the module on which the classification is based, temperate lactococcal phages can be classified as a single phage species, as four distinct phage species, or as two if not three different phage genera. We propose to base phage taxonomy on comparative genomics of a single structural gene module (head or tail genes). This partially phylogeny-based taxonomical system still mirrors some aspects of the current International Committee on Taxonomy in Virology classification system. In this system the currently sequenced lactococcal phages would be grouped into five genera: c2-, sk1, Sfi11-, r1t-, and Sfi21-like phages. [TOP OF PAGE]

  1101. Bacteriophage T4 development in Escherichia coli is growth rate-dependent. Rabinovitch,A., Fishov,I., Hadas,H., Einav,M., Zaritsky,A. (2002). J. Theor. Biol. 216:1-4. Three independent parameters (eclipse and latent periods, and rate of ripening during the rise period) are essential and sufficient to describe bacteriophage development in its bacterial host. A general model to describe the classical "one-step growth" experiment [Rabinovitch et al. (1999a) J. Bacteriol.181, 1687-1683] allowed their calculations from experimental results obtained with T4 in Escherichia coli B/r under different growth conditions [Hadas et al. (1997) Microbiology143, 179-185]. It is found that all three parameters could be described by their dependence solely on the culture doubling time tau before infection. Their functional dependence on tau, derived by a best-fit analysis, was used to calculate burst size values. The latter agree well with the experimental results. The dependence of the derived parameters on growth conditions can be used to predict phage development under other experimental manipulations. [TOP OF PAGE]

  1102. The dual role of wild phages for horizontal gene transfer among Salmonella strains. Rabsch,W., Mirold,S., Hardt,W.D., Tschape,H. (2002). Berl Munch Tierarztl Wochenschr 115:355-359. Salmonella bacteriophages seem to mediate horizontal transfer of virulence functions among Salmonella strains in two different ways: by general transduction and also by lysogenic conversion. The majority of wild phages isolated from Salmonella strains belong to the P22 like phages and were able to transduce. Our data show that the lysogenic conversion is generally accompanied by changes in the susceptibility to the typing phages used for epidemiological purposes. Similar phage type conversions to S. Typhimurium DT104 could be detected upon lysogenization with two other S. Typhimurium strains. For some S. Typhimurium strains the typical phage pattern is actually associated with alterations of virulence characteristics. For example, all tested wild type isolates of phage types DT49 and DT204 were found to be SopE phi-lysogens. The Anderson typing phages interfere with the prophages and/or cryptic phages and so the complex genetic short-term evolution can be demonstrated in the lab. This is one reason for the successful application of phage typing in Salmonella epidemiology since the 50s. [TOP OF PAGE]

  1103. Remarkable morphological diversity of viruses and virus-like particles in hot terrestrial environments. Rachel,R., Bettstetter,M., Hedlund,B.P., Haring,M., Kessler,A., Stetter,K.O., Prangishvili,D. (2002). Arch. Virol. 147:2419-2429. Electron microscopic studies of the viruses in two hot springs (85 degrees C, pH 1.5-2.0, and 75-93 degrees C, pH 6.5) in Yellowstone National Park revealed particles with twelve different morphotypes. This diversity encompassed known viruses of hyperthermophilic archaea, filamentous Lipothrixviridae, rod-shaped Rudiviridae, and spindle-shaped Fuselloviridae, and novel morphotypes previously not observed in nature. Two virus types resembled head-and-tail bacteriophages from the families Siphoviridae and Podoviridae, and constituted the first observation of these viruses in a hydrothermal environment. Viral hosts in the acidic spring were members of the hyperthermophilic archaeal genus Acidianus. [TOP OF PAGE]

  1104. The nucleotide sequence of shiga toxin (Stx) 2e-encoding phage fP27 is not related to other Stx phage genomes, but the modular genetic structure is conserved. Recktenwald,J., Schmidt,H. (2002). Infect. Immun. 70:1896-1908. In this study we determined the complete nucleotide sequence of Shiga toxin 2e-encoding bacteriophage fP27, isolated from the Shiga toxin-producing Escherichia coli patient isolate 2771/97. fP27 is integrated as a prophage in the chromosomal yecE gene. This integration generates identity segments of attL and attR sites with lengths of 11 nucleotides. The integrated prophage genome has a size of 42,575 bp. We identified 58 open reading frames (ORFs), each with a length of >150 nucleotides. The deduced proteins of 44 ORFs showed significant homologies to other proteins present in sequence databases, whereas 14 putative proteins did not. For 29 proteins, we could deduce a putative function. Most of these are related to the basic phage propagation cycle. The fP27 genome represents a mosaic composed of genetic elements which are obviously derived from related and unrelated phages. We identified five short linker sequences of 22 to 151 bp in the fP27 sequence which have also been detected in a couple of other lambdoid phages. These linkers are located between functional modules in the phage genome and are thought to play a role in genetic recombination. Although the overall DNA sequence of fP27 is not highly related to other known phages, the data obtained demonstrate a typical lambdoid genome structure. [TOP OF PAGE]

  1105. Les bactériophages, nouvelle perspective dans le traitement des maladies infectieuses? Resch,G., Meyer,J. (2002). Rev. Mens. Suisse Odontostomatol. 112:643-645. De nombreuses bactéries ont été identifiées, et cela depuis des décennies, comme étant des agents responsables de nombreuses maladies infectieuses de l'homme. Ainsi, il a été mis en évidence que certaines bactéries buccales jouent un rôle primordial dans l'étiologie de la carie et des pathologies du parodontium. Ces bactéries peuvent être, à leur tour, infectées par des virus appelés bactériophages. Ces bactériophages, qui sont des parasites obligatoires, sont capables d'altérer profondément les caractéristiques de leur hôte. Nous verrons, dans la suite, quelques aspects de la biologie de ces virus et de leur importance. [TOP OF PAGE]

  1106. Spatial stability of bacterial and viral community compositions in Danish coastal waters as depicted by DNA fingerprinting techniques. Riemann,L., Middelboe,M. (2002). Aquat. Microb. Ecol. 27:219-232. [TOP OF PAGE]

  1107. Viral lysis of marine bacterioplankton: Potential implications for organic matter cycling and bacterial clonal composition. Riemann,L., Middelboe,M. (2002). Ophelia 56:57-68. [TOP OF PAGE]

  1108. The Phage Proteomic Tree: a genome-based taxonomy for phage. Rohwer,F., Edwards,R. (2002). J. Bacteriol. 184:4529-4535. There are approximately 1031 phage in the biosphere, making them the most abundant biological entities on the planet. Despite their great numbers and ubiquitous presence, very little is known about phage biodiversity, biogeography, or phylogeny. Information is limited, in part, because the current ICTV taxonomical system is based on culturing phage and measuring physical parameters of the free virion. No sequence-based taxonomic systems have previously been established for phage. We present here the "Phage Proteomic Tree," which is based on the overall similarity of 105 completely sequenced phage genomes. The Phage Proteomic Tree places phage relative to both their near neighbors and all other phage included in the analysis. This method groups phage into taxa that predicts several aspects of phage biology and highlights genetic markers that can be used for monitoring phage biodiversity. We propose that the Phage Proteomic Tree be used as the basis of a genome-based taxonomical system for phage. [TOP OF PAGE]

  1109. Experimental genomic evolution: extensive compensation for loss of DNA ligase activity in a virus. Rokyta,D., Badgett,M.R., Molineux,I.J., Bull,J.J. (2002). Mol. Biol. Evol. 19:230-238. Deletion of the viral ligase gene drastically reduced the fitness of bacteriophage T7 on a ligase-deficient host. Viral evolution recovered much of this fitness during long-term passage, but the final fitness remained below that of the intact virus. Compensatory changes occurred chiefly in genes involved in DNA metabolism: the viral endonuclease, helicase, and DNA polymerase. Two other compensatory changes of unknown function also occurred. Using a method to distinguish compensatory mutations from other beneficial mutations, five additional substitutions from the recovery were shown to enhance adaptation to culture conditions and were not compensatory for the deletion. In contrast to the few previous studies of viral recovery from deletions, the compensatory changes in T7 did not restore the deletion or duplicate major regions of the genome. The ability of this deleted genome to recover much of the lost fitness via mutations in its remaining genes reveals a considerable evolutionary potential to modify the interactions of its elements in maintaining an essential set of functions. [TOP OF PAGE]

  1110. Field and laboratory investigations of inactivation of viruses (PRD1 and MS2) attached to iron oxide-coated quartz sand. Ryan,J.N., Harvey,R.W., Metge,D., Elimelech,M., Navigato,T., Pieper,A.P. (2002). Environ. Sci. Technol. 36:2403-2413. Field and laboratory experiments were conducted to investigate inactivation of viruses attached to mineral surfaces. In a natural gradient transport field experiment, bacteriophage PRD1, radiolabeled with 32P, was injected into a ferric oxyhydroxide-coated sand aquifer with bromide and linear alkylbenzene sulfonates. In a zone of the aquifer contaminated by secondary sewage infiltration, small fractions of infective and 32P-labeled PRD1 broke through with the bromide tracer, followed by the slow release of 84% of the 32P activity and only 0.011% of the infective PRD1. In the laboratory experiments, the inactivation of PRD1, labeled with 35S (protein capsid), and MS2, dual radiolabeled with 35S (protein capsid) and 32P (nucleic acid), was monitored in the presence of groundwater and sediment from the contaminated zone of the field site. Release of infective viruses decreased at a much faster rate than release of the radiolabels, indicating that attached viruses were undergoing surface inactivation. Disparities between 32P and 35S release suggest that the inactivated viruses were released in a disintegrated state. Comparison of estimated solution and surface inactivation rates indicates solution inactivation is approximately 3 times as fast as surface inactivation. The actual rate of surface inactivation may be substantially underestimated owing to slow release of inactivated viruses. [TOP OF PAGE]

  1111. Distribution of genotypes of F-specific RNA bacteriophages in human and non-human sources of faecal pollution in South Africa and Spain. Schaper,M., Jofre,J., Uys,M., Grabow,W.O.K. (2002). J. Appl. Microbiol. 92:657-667. AIMS: To assess whether the distribution of genotypes of F-specific RNA bacteriophages reflects faecal pollution of human and animal origin in water environments. METHODS AND RESULTS: Stool samples, animal feedlot waste slurries and a wide variety of faecally polluted waters were studied in South Africa and Spain. Genotyping was performed by plaque and spot hybridization with genotype-specific probes. Only genotypes II and III were detected in human stool. Animal faeces contained predominantly, but not exclusively, genotypes I and IV. Raw hospital and municipal sewage contained mostly genotypes II and III, whereas genotypes I and II prevailed in settled sewage, secondary treated sewage and non-point diffuse effluents from developing communities. Abattoir wastewaters contained mostly genotypes I and IV. No differences were observed between the distribution of genotypes in Spain and South Africa. CONCLUSIONS: Although the association of genotypes II and III with human excreta and I and IV with animal excreta was statistically significant, the results suggest that the association cannot be used for absolute distinction between faecal pollution of human and animal origin. SIGNIFICANCE AND IMPACT OF THE STUDY: This study contributes greatly to understanding the usefulness of genotypes of F-specific RNA bacteriophages in source tracking of faecal wastes. [TOP OF PAGE]

  1112. Comparative resistance of phage isolates of four genotypes of f-specific RNA bacteriophages to various inactivation processes. Schaper,M., Duran,A.E., Jofre,J. (2002). Appl. Environ. Microbiol. 68:3702-3707. The effect of natural inactivation in freshwater, chlorination, ammonia, extreme pHs, temperature, and salt content on phage inactivation was evaluated on mixtures of F-specific RNA bacteriophage isolates belonging to genotypes I, II, III, and IV. The bacteriophages studied were previously but recently isolated from natural samples, characterized as F-specific RNA bacteriophages and genotyped by plaque hybridization with genotype-specific probes. Natural inactivation in river water was modeled by in situ incubation of bacteriophages inside submerged dialysis tubes. After several days bacteriophages of genotype I showed the highest persistence, which was significantly different from that of bacteriophages of genotype II, IV, or III. The pattern of resistance of phages belonging to the various genotypes to extreme pHs, ammonia, temperature, salt concentration, and chlorination was similar. In all cases, phages of genotype I showed the highest persistence, followed by the phages of genotypes II, III, and IV. The phages of genotypes III and IV were the least resistant to all treatments, and resistance of genotypes III and IV to the treatments was similar. Bacteriophages of genotype II showed intermediate resistance to some of the treatments. The resistance of four phages of genotype I to natural inactivation and chlorination did not differ significantly. These results indicate that genotypes III and IV are much more sensitive to environmental stresses and to treatments than the other genotypes, especially than genotype I. This should be taken into consideration in future studies aimed at using genotypes of F-specific RNA bacteriophages to fingerprint the origin of fecal pollution. [TOP OF PAGE]

  1113. Column experiments to study nonlinear removal of bacteriophages by passage through saturated dune sand. Schijven,J.F., Hassanizadeh,S.M., de Bruin,H.A.M. (2002). J. Contam. Hydrol. 58:243-259. In a recent field study on dune recharge, bacteriophages MS2 and PRD1 were found to be removed 3 log10 over the first 2.4 m and only 5 log10 over the next 27 m. To understand the causes of this nonlinear removal, column experiments were carried out under conditions similar to the field: same recharge water, temperature (5 +/- 3 degrees C) and pore water velocity (1.5 m day(-1)). Soil samples were taken along a streamline between the recharge canal and the first monitoring well. Bacteriophage phiX174 was included for comparison. The high initial removal in the field was found not to be due to heterogeneity of phage suspensions but to soil heterogeneity. Phage removal rates correlated strongly positively with soil organic carbon content, and relatively strongly positively with silt content and the presence of ferric oxyhydroxides. Soil organic carbon content, silt content and the presence of ferric oxyhydroxides were found to decrease exponentially with travel distance. Removal rates of phiX174 were found to be 3-10 times higher than those of MS2 and PRD1 due to the lower electrostatic repulsion that the less negatively charged phiX174 experiences. It is suggested that the high initial removal in the field is due to the presence of favorable sites for attachment formed by ferric oxyhydroxides that decrease exponentially with travel distance. Similar removal rates may be found at both laboratory and field scale. However, due to local variations at field scale detailed knowledge on soil heterogeneity may be needed to enable a reliable prediction of removal. [TOP OF PAGE]

  1114. Two-site kinetic modeling of bacteriophages transport through columns of saturated dune sand. Schijven,J.F., Hassanizadeh,S.M., de Bruin,R.H.A.M. (2002). J. Contam. Hydrol. 57:259-279. Breakthrough curves, on a semi-log scale, from tests in porous media with block-input of viruses, bacteria, protozoa and colloidal particles often exhibit a typical skewness: a rather slowly rising limb and a smooth transition of a declining limb to a very long tail. One-site kinetic models fail to fit the rising and declining limbs together with the tail satisfactorily. Inclusion of an equilibrium adsorption site does not seem to improve simulation results. This was encountered in the simulation of breakthrough curves from a recent field study on the removal of bacteriophages MS2 and PRD1 by passage through dune sand. In the present study, results of laboratory experiments for the study of this issue are presented. Breakthrough curves of salt and bacteriophages MS2, PRDI, and phiX174 in 1 D column experiments have been measured. One- and two-site kinetic models have been applied to fit and predict breakthrough curves from column experiments. The two-site model fitted all breakthrough curves very satisfactorily, accounting for the skewness of the rising limb as well as for the smooth transition of the declining limb to the tail of the breakthrough curve. The one-site model does not follow the curvature of the breakthrough tail, leading to an overestimation of the inactivation rate coefficient for attached viruses. Interaction with kinetic site 1 is characterized by relatively fast attachment and slow detachment, whereas attachment to and detachment from kinetic site 2 is fast. Inactivation of viruses and interaction with kinetic site 2 provide only a minor contribution to removal. Virus removal is mainly determined by the attachment to site 1. Bacteriophage phiX174 attached more than MS2 and PRD1, which can be explained by the greater electrostatic repulsion that MS2 and PRD1 experience compared to the less negatively charged phiX174. [TOP OF PAGE]

  1115. Bacteriophage SP6 is closely related to phages K1-5, K5, and K1E but encodes a tail protein very similar to that of the distantly related P22. Scholl,D., Adhya,S., Merril,C.R. (2002). J. Bacteriol. 184:2833-2836. The lytic salmonella phage SP6 encodes a tail protein with a high degree of sequence similarity to the tail protein of the biologically unrelated lysogenic salmonella phage P22. The SP6 tail gene is flanked by an upstream region that contains a promoter and a downstream region that contains a putative Rho-independent transcription terminator, giving it a cassette or modular structure almost identical to the structure of the tail genes of coliphages K1E, K5, and K1-5. It now appears that SP6, K1-5, K5, and K1E are very closely related but have different tail fiber proteins, giving them different host specificities. [TOP OF PAGE]

  1116. Coccolithovirus (Phycodnaviridae): characterisation of a new large dsDNA algal virus that infects Emiliania huxleyi. Schroeder,D., Oke,J., Malin,G., Wilson,W.H. (2002). Arch. Virol. 147:1685-1698. Emiliania huxleyi-specific viruses (EhV) were isolated from E. huxleyi blooms off the coast of Plymouth, UK, in July 1999 and July/August 2001, and from an E. huxleyi bloom induced during a mesocosm experiment in a fjord off Bergen, Norway, during June 2000. Transmission electron microscopy revealed that all 10 virus isolates are 170-200 nm in diameter with an icosahedral symmetry. Their density is approximately 1.2 in CsCl gradients and they have large double stranded DNA genomes approximately 410 kb in size. Phylogenetic analysis of the DNA polymerase genes of these viruses suggests that EhV belongs to a new genus within the family of algal viruses, Phycodnaviridae. We propose to name this new virus genus Coccolithovirus. Differences within members of the Coccolithovirus were elucidated by host range analysis of the virus isolates and sequence analysis of a gene fragment encoding part of their putative major capsid protein. All 10 virus isolates within this new genus only infected E. huxleyi strains that have previously been shown to exhibit low dimethylsulphoniopropionate lyase (DMSP-lyase) activity (CCMP1516, CCMP374 and L), while E. huxleyi strains with high DMSP-lyase activity (CCMP373 and CCMP379) were resistant to infection. [TOP OF PAGE]

  1117. Improved method for recovery of bacteriophage from large volumes of water using negatively charged microporous filters. Scott,T.M., Lukasik,J., Farrah,S.R. (2002). Can. J. Microbiol. 48:305-310. Current virus-recovery procedures using negatively charged microporous filters provide an inexpensive, reliable method for the recovery and detection of enteroviruses from water and wastewater; however, adjustment of the test samples to pH 3.5 to promote enterovirus adsorption results in significant inactivation of bacteriophage and an inability to simultaneously recover them from large volumes of water using this procedure. Procedures specifically designed for the detection of bacteriophage are currently in use but generally are only effective for small volumes of water. Positively charged filters can be used to recover both enteroviruses and bacteriophage from large volumes of water at neutral pH; however, the filters are expensive. The addition of manganese chloride to test solutions at pH 3.5 prior to filtration through negatively charged Filterite filters allowed for sampling of larger volumes of water by reducing the inactivation of bacteriophage and increasing the recovery of PRD1, MS2, and naturally isolated bacteriophage by a factor of four or five when compared with recoveries from solutions without MnCl2. This method provides an inexpensive, reliable alternative to large-volume bacteriophage recovery procedures that use positively charged filters at neutral pH. [TOP OF PAGE]

  1118. Microbial source tracking: current methodology and future directions. Scott,T.M., Rose,J.B., Jenkins,T.M., Farrah,S.R., Lukasik,J. (2002). Appl. Environ. Microbiol. 68:5796-5803. [first paragraph] Maintenance of the microbiological quality and safety of water systems used for drinking, for recreating, and in the harvesting of seafood is imperative, as contamination of these systems can exact high risks to human health as well as result in significant economic losses due to closures of beaches and shellfish harvesting areas. Waters contaminated with human feces are generally regarded as a greater risk to human health, as they are more likely to contain human-specific enteric pathogens, including Salmonella enterica serovar Typhi, Shigella spp., hepatitis A virus, and Norwalk-group viruses. Animals can also serve as reservoirs for a variety of enteric pathogens (e.g., various serotypes of Salmonella, Escherichia coli, and Cryptosporidium spp.). Understanding the origin of fecal pollution is paramount in assessing associated health risks as well as the actions necessary to remedy the problem while it still exists. Traditional and alternative indicator microorganisms have been used for many years to predict the presence of fecal pollution in water; however, it is well established that the majority of these organisms are not limited to humans but also exist in the intestines of many other warm-blooded animals (55). Due to the ubiquitous nature of these organisms, the effectiveness of using traditional indicators to predict the presence of human or animal waste impact and subsequent health risks is limited. The usefulness of the microbial indicators as tools for risk assessment can be significantly enhanced by the development of testing methods and analysis techniques that can define specific sources of these organisms. [TOP OF PAGE]

  1119. The adaptation and survival of phages in nature. Semchuk,L.I., Ignatenko,T., Romashev,S.A., Andriychuk,O., Yatskovska,L. (2002). Bulletin of the University of Kiev, series Biology 38:54-56. The short analyse of the phage's properties changing, their evolution and survival adaptation in the environment is given. [TOP OF PAGE]

  1120. Site-specific integrative elements of rhizobiophage 16-3 can integrate into proline tRNA (CGG) genes in different bacterial genera. Semsey,S., Blaha,B., Koles,K., Orosz,L., Papp,P.P. (2002). J. Bacteriol. 184:177-182. The integrase protein of the Rhizobium meliloti 41 phage 16-3 has been classified as a member of the Int family of tyrosine recombinases. The site-specific recombination system of the phage belongs to the group in which the target site of integration (attB) is within a tRNA gene. Since tRNA genes are conserved, we expected that the target sequence of the site-specific recombination system of the 16-3 phage could occur in other species and integration could take place if the required putative host factors were also provided by the targeted cells. Here we report that a plasmid (pSEM167) carrying the attP element and the integrase gene (int) of the phage can integrate into the chromosomes of R. meliloti 1021 and eight other species. In all cases integration occurred at so-far-unidentified, putative proline tRNA (CGG) genes, indicating the possibility of their common origin. Multiple alignment of the sequences suggested that the location of the att core was different from that expected previously. The minimal attB was identified as a 23-bp sequence corresponding to the anticodon arm of the tRNA. [TOP OF PAGE]

  1121. E.coli cell-cycle regulation by bacteriophage lambda. Sergueev,K., Court,D., Reaves,L., Austin,S. (2002). J. Mol. Biol. 324:297-307. We re-examined the old but surprising claim of Kourilsky and Knapp that transient expression of genes located downstream of the p(L) promoter of bacteriophage lambda can induce cell-cycle synchrony in a population of Escherichia coli cells. Although we were unable to reproduce a lasting synchrony, a cessation of division, followed by one or two fairly synchronous cell divisions was observed. This line up of the cell cycle was found to be due to two genetically separable events: a temporary block of cell division and, at the same time, a block to the initiation of new rounds of DNA replication. These blocks then release after about one mass doubling so that chromosome replication and cell division occur during a short time interval in all the cells in the population. The cell division block is a result of the transient expression of the lambda kil gene. The block to initiation of DNA replication requires a region that we term bin (blocks initiation) immediately upstream of the xis gene. The region consists of ea22 and ea8.5 and two small open reading frames (ORFs) that flank them. Deletion-substitution mutagenesis suggests that all four ORFs may be required for the initiation block. The ability of the phage to modify two aspects of the host cell cycle presumably reflects a stratagem that provides the phage with an advantage for lysogeny or lytic growth. [TOP OF PAGE]

  1122. Use of bacteriophage Ba1 to identify properties associated with Bordetella avium virulence. Shelton,C.B., Temple,L.M., Orndorff,P.E. (2002). Infect. Immun. 70:1219-1224. Bordetella avium causes bordetellosis, an upper respiratory disease of birds. Commercially raised turkeys are particularly susceptible. We report here on the use of a recently described B. avium bacteriophage, Ba1, as a tool for investigating the effects of lysogeny and phage resistance on virulence. We found that lysogeny had no effect on any of the in vivo or in vitro measurements of virulence we employed. However, two-thirds (six of nine) spontaneous phage-resistant mutants of our virulent laboratory strain, 197N, were attenuated. Phage resistance was associated, in all cases, with an inability of the mutants to bind phage. Further tests of the mutants revealed that all had increased sensitivities to surfactants, and increased amounts of incomplete (O-antigen-deficient) lipopolysaccharide (LPS) compared to 197N. Hot phenol-water-extracted 197N LPS inactivated phage in a specific and dose-dependent manner. Acid hydrolysis and removal of lipid A had little effect upon the ability of isolated LPS to inactivate Ba1, suggesting that the core region and possibly the O antigen were required for phage binding. All of the mutants, with one exception, were significantly more sensitive to naive turkey serum and, without exception, significantly less able to bind to tracheal rings in vitro than 197N. Interestingly, the three phage-resistant mutants that remained virulent appeared to be O antigen deficient and were among the mutants that were the most serum sensitive and least able to bind turkey tracheal rings in vitro. This observation allowed us to conclude that even severe defects in tracheal ring binding and serum resistance manifested in vitro were not necessarily indicative of attenuation and that complete LPS may not be required for virulence. [TOP OF PAGE]

  1123. Sequence analysis of marine virus communities reveals groups of related algal viruses are widely distributed in nature. Short,S.M., Suttle,C.A. (2002). Appl. Environ. Microbiol. 68:1290-1296. Algal-virus-specific PCR primers were used to amplify DNA polymerase (pol) gene fragments from geographically isolated natural virus communities. Natural algal virus communities were obtained from coastal sites in the Pacific Ocean in British Columbia, Canada, and the Southern Ocean near the Antarctic peninsula. Genetic fingerprints of algal virus communities were generated using denaturing gradient gel electrophoresis (DGGE). Sequencing efforts recovered 33 sequences from the gradient gel. Of the 33 sequences examined, 25 encoded a conserved amino acid motif indicating that the sequences were pol gene fragments. Furthermore, the 25 pol sequences were related to pol gene fragments from known algal viruses. In addition, similar virus sequences (>98% sequence identity) were recovered from British Columbia and Antarctica. Results from this study demonstrate that DGGE with degenerate primers can be used to qualitatively fingerprint and assess genetic diversity in specific subsets of natural virus communities and that closely related viruses occur in distant geographic locations. DGGE is a powerful tool for genetically fingerprinting natural virus communities and may be used to examine how specific components of virus communities respond to experimental manipulations. [TOP OF PAGE]

  1124. Sunlight inactivation of fecal indicator bacteria and bacteriophages from waste stabilization pond effluent in fresh and saline waters. Sinton,L.W., Hall,C.H., Lynch,P.A., Davies-Colley,R.J. (2002). Appl. Environ. Microbiol. 68:1122-1131. Sunlight inactivation in fresh (river) water of fecal coliforms, enterococci, Escherichia coli, somatic coliphages, and F-RNA phages from waste stabilization pond (WSP) effluent was compared. Ten experiments were conducted outdoors in 300-liter chambers, held at 14C (mean river water temperature). Sunlight inactivation (k(S)) rates, as a function of cumulative global solar radiation (insolation), were all more than 10 times higher than the corresponding dark inactivation (k(D)) rates in enclosed (control) chambers. The overall k(S) ranking (from greatest to least inactivation) was as follows: enterococci > fecal coliforms greater-than-or-equal E. coli > somatic coliphages > F-RNA phages. In winter, fecal coliform and enterococci inactivation rates were similar but, in summer, enterococci were inactivated far more rapidly. In four experiments that included freshwater-raw sewage mixtures, enterococci survived longer than fecal coliforms (a pattern opposite to that observed with the WSP effluent), but there was little difference in phage inactivation between effluents. In two experiments which included simulated estuarine water and seawater, sunlight inactivation of all of the indicators increased with increasing salinity. Inactivation rates in freshwater, as seen under different optical filters, decreased with the increase in the spectral cutoff (50% light transmission) wavelength. The enterococci and F-RNA phages were inactivated by a wide range of wavelengths, suggesting photooxidative damage. Inactivation of fecal coliforms and somatic coliphages was mainly by shorter (UV-B) wavelengths, a result consistent with photobiological damage. Fecal coliform repair mechanisms appear to be activated in WSPs, and the surviving cells exhibit greater sunlight resistance in natural waters than those from raw sewage. In contrast, enterococci appear to suffer photooxidative damage in WSPs, rendering them susceptible to further photooxidative damage after discharge. This suggests that they are unsuitable as indicators of WSP effluent discharges to natural waters. Although somatic coliphages are more sunlight resistant than the other indicators in seawater, F-RNA phages are the most resistant in freshwater, where they may thus better represent enteric virus survival. [TOP OF PAGE]

  1125. Fates of bacteriophages and bacterial indicators in the Moselle river (France). Skraber,S., Gantzer,C., Maul,A., Schwartzbrod,L. (2002). Water Res. 36:3629-3637. It has been suggested that bacteriophages can provide useful information about the pathogenic microorganisms, particularly enteric viruses, present in water. This information is complementary to that obtained from bacterial indicators of faecal contamination, which would be of great value for evaluating the risks associated with the use of certain types of water. Before bacteriophages can be used as indicators of faecal contamination, we need to confirm that bacteriophages give a different response to that given by the well-known bacteria indicators and to determine what happens to bacteriophages in river water. Indeed, drinking water is often produced from river water, either by natural filtration through the soil or after undergoing various treatments. We collected 96 river water samples from six different sites between February and November 2000. The samples were analysed for three faecal indicator bacteria (thermotolerant coliforms, enterococci and spores of sulphite-reducing anaerobes) and three types of bacteriophages (somatic coliphages, F-specific phages and Bacteroides fragilis phages). The densities of thermotolerant coliforms and enterococci depended mainly on physical factors such as flow rate and water temperature. High temperature and low flow rate led to a decrease in the density of these microorganisms, especially in the absence of a major input of faecal pollution. Conversely, the densities of somatic coliphages, F-specific phages and spores of sulphite-reducing anaerobes remained constant regardless of the flow rate and temperature. The density of Bacteroides fragilis phages was too low for unambiguous determination of their fate in river water. [TOP OF PAGE]

  1126. Rex-centric mutualism. Slavcev,R.A., Hayes,S. (2002). J. Bacteriol. 184:857-858. We asked whether Rex exclusion encoded by a lambda prophage confers a protective or a cell-killing phenotype. We found that the Rex system can channel lysogenic cells into an arrested growth phase that gives an overall protective ability to the host despite some associated killing. [TOP OF PAGE]

  1127. The prevalence and diversity of mobile genetic elements in bacterial communities of different environmental habitats: Insights gained from different methodological approaches. Smalla,K., Sobecky,P.A. (2002). FEMS Microbiol. Ecol. 42:165-175. The pool of mobile genetic elements (MGE) in microbial communities consists of plasmids, bacteriophages and other elements that are either self-transmissible or use mobile plasmids and phages as vehicles for their dissemination. By facilitating horizontal gene exchange, the horizontal gene pool (HGP) promotes the evolution and adaptation of microbial communities. Efforts to characterise MGE from bacterial populations resident in a variety of ecological habitats have revealed a surprisingly vast and seemingly untapped diversity. MGE, conferring such selectable traits as mercury or antibiotic resistance and degradative functions, have been readily acquired from diverse microbial communities. To circumvent the need to isolate microbial hosts, polymerase chain reaction (PCR)-based detection methods have frequently been used to assess the prevalence of MGE-specific sequences resident in the 'microbial community' HGP. As studies continue to reveal novel and distinct MGE, sequencing of newly isolated MGE from diverse habitats is essential for the continued development of DNA probes, PCR primers as well as for gene array and proteomics-based approaches. This minireview highlights insight gained from different methodological approaches, biased albeit largely toward plasmids in Gram-negative bacteria, used to study the HGP of naturally occurring microbial communities from various aquatic and terrestrial habitats. [TOP OF PAGE]

  1128. Three recently acknowledged Escherichia species strikingly differ in the incidence of bacteriocinogenic and lysogenic strains. Smarda,J., Smajs,D., Lhotova,H. (2002). J. Basic Microbiol. 42:429-433. The incidence of bacteriocinogeny and lysogeny was followed in bacteria of 3 recently acknowledged species of the genus Escherichia: E. hermanii, E. vulneris and E. fergusonii. Almost all of the strains examined were of human origin. In 30 strains of E. hermanii no one was found bacteriocinogenic while 57% were lysogenic, in 30 strains of E. vulneris none was found to be bacteriocinogenic and only 10% were lysogenic, and in 50 strains E. fergusonii 12% were bacteriocinogenic and 40% lysogenic. From the 6 bacteriocinogenic strains of E. fergusonii, 3 were producers of colicin E1, 2 of colicin Ib and 1 of colicin Ia. In addition, 3 E. fergusonii strains produced aerobactin. [TOP OF PAGE]

  1129. Otlichiia v sostave genov virulentnosti v shtammakh Vibrio cholerae eltor, vydelennykh iz raznykh istochnikov na territorii Turkmenistana [Differences in virulence genes in Vibrio cholerae eltor strains isolated from different sources in Turkmenistan territory]. Smirnova,N.I., Kostromitina,E.A., Cheldyshova,N.B., Kutyrev,V.V. (2002). Molekuliarnaia genetika 2002,12-18. Polymerase chain reaction (PCR) detected the presence of various genes associated with virulence in genome of strains V. cholerae eltor isolated in Turkmenistan territory during epidemic and epidemic-free perios. It was found that a complete set of virulence genes (ctxA+, tcpA+ and toxR+) contained strains isolated from patients, carriers and environment only in cholera epidemics. Strains isolated from the environment in the period free of epidemics did not contain ctxA and tcpA in 78.2% of cases, but 5.2% of the strains carried a complete set of virulence genes. There were also nontoxigenic strains containing genes tcpA and toxR. Such strains were isolated from the environment (16.6%) and vibrion carriers (42.9%). Isolated were also strains V.cholerae eltor carrying bacteriophage CTX phi with incomplete set of virulence genes and having genotype ctxA-, ace+ and zot+. Almost all the strains ctxA-, tcpA+ carry attRS1-site in genome. This shows that such strains may transform into toxigenic as a result of infection with bacteriophage CTX phi. [TOP OF PAGE]

  1130. Legionella pneumophila: an aquatic microbe goes astray. Steinert,M., Hentschel,U., Hacker,J. (2002). FEMS Microbiol. Rev. 26:149-162. Legionella pneumophila is naturally found in fresh water were the bacteria parasitize within protozoa. It also survives planctonically in water or biofilms. Upon aerosol formation via man-made water systems, L. pneumophila can enter the human lung and cause a severe form of pneumonia, called Legionnaires' disease. The pathogenesis of Legionnaires' disease is largely due to the ability of L. pneumophila to invade and grow within macrophages. An important characteristic of the intracellular survival strategy is the replication within the host vacuole that does not fuse with endosomes or lysosomes. In recent times a great number of bacterial virulence factors which affect growth of L. pneumophila in both macrophages and protozoa have been identified. The ongoing Legionella genome project and the use of genetically tractable surrogate hosts are expected to significantly contribute to the understanding of bacterium-host interactions and the regulation of virulence traits during the infection cycle. Since person-to-person transmission of legionellosis has never been observed, the measures for disease prevention have concentrated on eliminating the pathogen from water supplies. In this respect detection and analysis of Legionella in complex environmental consortia become increasingly important. With the availability of new molecular tools this area of applied research has gained new momentum. [TOP OF PAGE]

  1131. Bacteriophage therapy. Stalin's forgotten cure. Stone,R. (2002). Science 298:728-731. Bacteriophage therapy, pioneered in Stalin-era Russia, is attracting renewed attention in the West as a potential weapon against drug-resistant bugs and hard-to-treat infections. [first paragraph] TBILISI-Last December, three woodsmen in the mountains of Georgia stumbled upon a pair of canisters that were, oddly, hot to the touch. The men lugged the objects back to their campsite to warm themselves on a bitterly cold night. That turned out to be a terrible mistake: The canisters, Soviet relics once used to power remote generators, were intensely radioactive and burned two of the men severely. The victims were rushed to the capital, Tbilisi, where doctors plied them with antibiotics but failed to prevent staphylococcus bacteria from invading the deep wounds. Septic shock seemed just around the corner. Then a kinder legacy of the Soviet Union came to the rescue. [TOP OF PAGE]

  1132. Bacteriophage therapy. Food and agriculture: testing grounds for phage therapy. Stone,R. (2002). Science 298:730 [first paragraph] Last month, the U.S. Food and Drug Administration tightened another screw in its effort to curb the spread of antibiotic resistance from the burgeoning use of agricultural drugs. The agency aired draft regulations requiring manufacturers to test potential livestock pharmaceuticals for their ability to help pathogens acquire resistance to human drugs. But farmers are concerned that they could be left with fewer weapons to combat Listeria and other foodborne pathogens that cause several hundred deaths each year in the United States alone."When farmers are told they can't use any antibiotics used in humans, they say, 'What do we use?'" says Toney Ilenchuk. His firm, Biophage Pharma in Montreal, Canada, believes it has part of the answer: bacteriophages against Salmonella and pathogenic strains of Escherichia coli. [TOP OF PAGE]

  1133. Expression of antisense RNA targeted against Streptococcus thermophilus bacteriophages. Sturino,J.M., Klaenhammer,T.R. (2002). Appl. Environ. Microbiol. 68:588-596. Antisense RNA complementary to a putative helicase gene (hel3.1) of a cos-type Streptococcus thermophilus bacteriophage was used to impede the proliferation of a number of cos-type S. thermophilus bacteriophages and one pac-type bacteriophage. The putative helicase gene is a component of the Sfi21-type DNA replication module, which is found in a majority of the S. thermophilus bacteriophages of industrial importance. All bacteriophages that strongly hybridized a 689-bp internal hel3.1 probe were sensitive to the expression of antisense hel3.1 RNA. A 40 to 70% reduction in efficiency of plaquing (EOP) was consistently observed, with a concomitant decrease in plaque size relative to that of the S. thermophilus parental strain. When progeny were released, the burst size was reduced. Growth curves of S. thermophilus NCK1125, in the presence of variable levels of bacteriophage kappa3, showed that antisense hel3.1 conferred protection, even at a multiplicity of infection of approximately 1.0. When the hel3.1 antisense RNA cassette was expressed in cis from the kappa3-derived phage-encoded resistance (PER) plasmid pTRK690::ori3.1, the EOP for bacteriophages sensitive to PER and antisense targeting was reduced to between 10-7 and 10-8, beyond the resistance conferred by the PER element alone (less than 10-6). These results illustrate the first successful applications of antisense RNA and explosive delivery of antisense RNA to inhibit the proliferation of S. thermophilus bacteriophages. [TOP OF PAGE]

  1134. Thermophilic lactic acid bacteria phages isolated from Argentinian dairy industries. Suarez,V.B., Quiberoni,A., Binetti,A.G., Reinheimer,J.A. (2002). J. Food Prot. 65:1597-1604. Sixty-one natural phages (59 of Streptococcus thermophilus and 2 of Lactobacillus delbrueckii subsp. bulgaricus) were isolated from Argentinian dairy plants from November 1994 to July 2000. Specifically, 17 yogurt samples (18% of all samples) and 26 cheese samples (79%) contained phages lytic to S. thermophilus strains. The number of viral particles found in samples ranged from 102 to 109 PFU/ml. The phages belonged to Bradley's group B or the Siphoviridae family (morphotype B1). They showed high burst size values and remarkably short latent periods. The results of this study show that phages were found more frequently in cheesemaking processes than in yogurt-making processes. The commercial streptococcus strains appeared to propagate more phages, whereas the natural strains propagated fewer phage strains. These results suggest that the naturally occurring cultures are inherently more phage resistant. [TOP OF PAGE]

  1135. Effectiveness of thermal treatments and biocides in the inactivation of Argentinian Lactococcus lactis phages. Suarez,V.B., Reinheimer,J.A. (2002). J. Food Prot. 65:1756-1759. The thermal and chemical resistance levels of four autochthonal bacteriophages of Lactococcus lactis subsp. lactis, isolated from cheese processes, was investigated. The times required to obtain 99% inactivation of phages (T99) at 63 and 72 degrees C in three suspension media (M17 broth, reconstituted commercial nonfat skim milk, and Tris magnesium gelatin buffer) were determined. Thermal resistance was dependent on the phage studied, and the results of this study demonstrate that pasteurization treatments used in dairy industries may leave viable viral particles in milk. It was possible to determine that M17 broth was generally the least protective medium, while phosphate buffer was the most protective one. Peracetic acid (0.15%, vol/vol) was the most effective viricidal agent, with exposures of 5 min being sufficient to inactivate high-titer phage suspensions (>10(6) PFU/ml). To achieve total inactivation (<10 PFU/ml) of viral suspensions, sodium hypochlorite was effective at 100 ppm for only two phages, while the other two phages needed concentrations of 200 and 300 ppm. Ethanol at concentrations of 100 and 75% proved to be very efficient in inactivating phages, but isopropanol was not effective against them. [TOP OF PAGE]

  1136. Genetics of the phage growth limitation (Pgl) system of Streptomyces coelicolor A3(2). Sumby,P., Smith,M.C.M. (2002). Mol. Microbiol. 44:489-500. The phage growth limitation (Pgl) system, encoded by Streptomyces coelicolor A3(2), confers protection against the temperate bacteriophage phiC31 and its homoimmune relatives. The Pgl phenotype is characterized by the ability of Pgl+ hosts to support a phage burst on initial infection but subsequent cycles are severely attenuated. Previously, two adjacent genes pglY and pglZ were shown to be required for Pgl. It had been shown by Southern blotting that Streptomyces lividans, a close relative of S. coelicolor and naturally Pgl-, does not contain homologues of pglYZ and that introduction of pglYZ into S. lividans is not sufficient to confer a Pgl+ phenotype. Moreover, the mechanism of the Pgl+<--> Pgl- phase variation associated with this phenotype is also not understood. Here we describe two novel genes, pglW and pglX, that were shown to be part of this system by complementation of Pgl- mutants and by insertional mutagenesis. pglW encodes a 169 kDa protein that includes putative motifs for both serine/threonine protein kinase activity and DNA binding. pglX encodes a 136 kDa protein with putative adenine-specific DNA methyltransferase activity. pglW and pglX have overlapping stop-start codons suggesting transcriptional and translational coupling. S1 mapping of transcripts initiating up-stream of pglW indicated that, like pglYZ, pglWX is expressed in uninfected cultures. A homologue of pglX with 76% amino acid identity was identified in S. coelicolor, and insertional mutagenesis indicated that this gene was not required for the Pgl+ phenotype. Southern blots indicated that S. lividans does not contain homologues of pglW or pglX. A plasmid encoding pglWXYZ was able to confer the Pgl+ phenotype to S. lividans implying that these four genes constitute the whole system. [TOP OF PAGE]

  1137. Community Structure: Viruses. Suttle,C.A. (2002). pp. 364-370. In In Hurst,C.J., Knudson,G.R., McInerney,M.J., Stezenbach,L.D., and Walter,M.V. (eds.), Manual of Environmental Microbiology (2nd Edition). ASM Press, Washington, DC. [TOP OF PAGE]

  1138. Mutations in bacteriophage T4 genome. Switala-Jelen,K., Dabrowska,K., Gorski,A., Sliwa,L. (2002). Acta virologica. English ed 46:57-62. Bacteriophage (phage) T4 belonging to T-even phages is one of the best known phages with a completely deciphered genome sequence. As a model of living systems, T4 phage has many technical advantages. It can be very easily grown in large quantities, manipulated by classical genetics, and engineered by site-directed mutagenesis. Many substances have been first tested for mutagenicity in T-even phages. The results of these tests were very often applicable to higher organisms due to similar mechanisms of mutagenesis. T4 phage is also important in phage therapy, which represents an alternative treatment of bacterial infections since the bacterial resistance to antibiotics has become a serious medical problem. The site-directed mutagenesis is a method that enables to introduce mutations which can influence phage affinity to bacteria and can be a practical technique for enriching phage collections and for widening specificity of phages for new bacterial strains now insensitive to phage therapy. [TOP OF PAGE]

  1139. The efficacy of bacteriophage as a method of biofilm eradication. Tait,K., Skilmman,L.C., Sutherland,I.W. (2002). Biofouling 18:305-311. The ability of bacteriophage and their associated polysaccharide depolymerases to control enteric biofilm formation was investigated. Bacteriophages specific for Enterobacter strains were isolated from primary effluent sewage. Combinations of three phages were required before complete eradication of single species biofilms of Enterobacter cloace occurred. Attempts to eliminate a susceptible bacterial population within a dual species biofilm were unsuccessful. It was thought that the structural heterogeneity of the biofilm produced pockets of unattainable, susceptible bacteria. These results suggest that phage and bacteria can co-exist stably within a biofilm. Bacteriophage, would, therefore, make poor tools for the control of biofilm formation. However, the results suggest that combined treatment with bacteriophage polysaccharide depolymerases and disinfectant may provide an alternative control strategy. [TOP OF PAGE]

  1140. Rapid detection of phylloplane bacterium Enterobacter cloacae based on chitinase gene transformation and lytic infection by specific bacteriophages. Takikawa,Y., Mori,H., Otsu,Y., Matsuda,Y., Nonomura,T., Kakutani,K., Tosa,Y., Mayama,S., Toyoda,H. (2002). J. Appl. Microbiol. 93:1042-1050. AIMS: To establish a rapid and efficient method for detecting Enterobacter cloacae based on chitinase gene transformation and lytic infection by virulent bacteriophages. METHODS AND RESULTS: A phylloplane strain of E. cloacae was isolated from tomato leaves and transformed with a chitinase gene. Transformed bacteria were collected from single colonies and infected with newly isolated, virulent bacteriophages in the presence of the chitinase substrate 4-methylumbelliferon (4MU)-(GlcNac)3. To assay chitinase activity in the lysates, the product 4MU was measured spectrofluorophotometrically or visibly detected under u.v. irradiation. Chitinase gene-transformed bacteria obtained from single colonies could be specifically identified in 30 min by the emission of 4MU fluorescence following lysis caused by phage infection. CONCLUSIONS: The chitinase gene was used as a reporter gene to construct a new system for easy and rapid monitoring of transgenic strains of E. cloacae released in the environment, in combination with specific recognition by virulent bacteriophages. SIGNIFICANCE AND IMPACT OF THE STUDY: The assay is simple, rapid, inexpensive, easy to perform and applicable to other strains. The system can be used for the routine monitoring of bacteria, which is important because of the increased use of transgenic strains of E. cloacae as an antagonistic biological control agent for plant diseases. [TOP OF PAGE]

  1141. 50 million years of genomic stasis in endosymbiotic bacteria. Tamas,I., Klasson,L., Canback,B., Naslund,A.K., Eriksson,A.S., Wernegreen,J.J., Sandstrom,J.P., Moran,N.A., Andersson,S.G.E. (2002). Science 296:2376-2379. Comparison of two fully sequenced genomes of Buchnera aphidicola, the obligate endosymbionts of aphids, reveals the most extreme genome stability to date: no chromosome rearrangements or gene acquisitions have occurred in the past 50 to 70 million years, despite substantial sequence evolution and the inactivation and loss of individual genes. In contrast, the genomes of their closest free-living relatives, Escherichia coli and Salmonella spp., are more than 2000-fold more labile in content and gene order. The genomic stasis of B. aphidicola, likely attributable to the loss of phages, repeated sequences, and recA, indicates that B. aphidicola is no longer a source of ecological innovation for its hosts. [TOP OF PAGE]

  1142. Metal ion-induced lateral aggregation of filamentous viruses fd and M13. Tang,J.X., Janmey,P.A., Lyubartsev,A., Nordenskiold,L. (2002). Biophys. J. 83:566-581. We report a detailed comparison between calculations of inter-filament interactions based on Monte-Carlo simulations and experimental features of lateral aggregation of bacteriophages fd and M13 induced by a number of divalent metal ions. The general findings are consistent with the polyelectrolyte nature of the virus filaments and confirm that the solution electrostatics account for most of the experimental features observed. One particularly interesting discovery is resolubilization for bundles of either fd or M13 viruses when the concentration of the bundle-inducing metal ion Mg2+ or Ca2+ is increased to large (>100 mM) values. In the range of Mg2+ or Ca2+ concentrations where large bundles of the virus filaments are formed, the optimal attractive interaction energy between the virus filaments is estimated to be on the order of 0.01kT per net charge on the virus surface when a recent analytical prediction to the experimentally defined conditions of resolubilization is applied. We also observed qualitatively distinct behavior between the alkali-earth metal ions and the divalent transition metal ions in their action on the charged viruses. The understanding of metal ions-induced reversible aggregation based on solution electrostatics may lead to potential applications in molecular biology and medicine. [TOP OF PAGE]

  1143. Effectiveness of the lactococcal abortive infection systems AbiA, AbiE, AbiF and AbiG against P335 type phages. Tangney,M., Fitzgerald,G.F. (2002). FEMS Microbiol. Lett. 210:67-72. Four lactococcal abortive infection mechanisms were introduced into strains which were sensitive hosts for P335 type phages and plaque assay experiments performed to assess their effect on five lactococcal bacteriophages from this family. Results indicate that AbiA inhibits all five P335 phages tested, while AbiG affects phiP335 itself and phiQ30 but not the other P335 species phages. AbiA was shown to retard phage Q30 DNA replication as previously reported for other phages. It was also demonstrated that AbiG, previously shown to act at a point after DNA replication in the cases of c2 type and 936 type phages, acts at the level of, or prior to phage Q30 DNA replication. AbiE and AbiF had no effect on the P335 type phages examined. [TOP OF PAGE]

  1144. Fate of coliphage in waste water treatment process and detection of phages carrying the Shiga toxin type 2 gene. Tanji,Y., Mizoguchi,K., Akitsu,T., Morita,M., Hori,K., Unno,H. (2002). Water Sci. Technol. 46:285-289. Abundances of phages specific to Escherichia coli in the wastewater treatment process were analyzed. Relatively abundant coliphages were detected in sewage influent. Phages in the influent were found both suspended in liquid phase and attached on the solid particles. Phage concentration was not reduced in the settling tank without chemical agglutination. Anaerobic followed by aerobic treatment of the sewage reduced concentration of suspended phages. Almost no phage was detected as a suspended form in the aerobic tank. Most of the phages were detected as attaching form and were excluded by aggregation with sludge. Using an experimental approach based on the detection of Shiga toxin 2 (Stx 2) gene by a phage enrichment culture followed by nested PCR, bacteriophages carrying Stx 2 gene were detected in the influent, settling tank, and anaerobic tank. It was revealed that the presence of phages carrying Stx 2 gene is common in sewage and these phages are effectively eliminated through sewage treatment process. [TOP OF PAGE]

  1145. Emerging foodborne pathogens. Tauxe,R.V. (2002). Int. J. Food Microbiol. 78:31-41. The broad spectrum of foodborne infections has changed dramatically over time, as well-established pathogens have been controlled or eliminated, and new ones have emerged. The burden of foodborne disease remains substantial: one in four Americans is estimated to have a significant foodborne illness each year. The majority of these illnesses are not accounted for by known pathogens, so more must remain to be discovered. Among the known foodborne pathogens, those more recently identified predominate, suggesting that as more and more is learned about pathogens, they come under control. In addition to the emergence or recognition of new pathogens, other trends include global pandemics of some foodborne pathogens, the emergence of antimicrobial resistance, the identification of pathogens that are highly opportunistic, affecting only the most high-risk subpopulations, and the increasing identification of large and dispersed outbreaks. New pathogens can emerge because of changing ecology or changing technology that connects a potential pathogen with the food chain. They also can emerge de novo by transfer of mobile virulence factors, often through bacteriophage. Though this is rarely observed, it can be reconstructed. Better understanding of the ecology and dynamics of phage transmission among bacteria will help us to understand the appearance of new pathogens in the future. One may look for emerging foodborne pathogens among the silent zoonoses, and among the severe infections affecting the immunocompromised humans. We should expect the unexpected. In the past, separating human sewage and animal manure from human food and water supplies was critical to improving public health. Now, our health depends increasingly on the safety of the feed and water supplies for the animals themselves. The successes of the 20th century and the new challenges we face mean that public health vigilance, careful investigation of new problems, responsible attention to food safety from farm to table, and partnerships to bring about new foodborne disease control measures will be needed for the foreseeable future. [TOP OF PAGE]

  1146. New ways to treat bacterial infections. Taylor,P.W., Stapleton,P.D., Paul L.J. (2002). Drug Discov. Today 7:1086-1091. There is an urgent need for fresh approaches to the treatment of bacterial infections because of the changing patterns of infectious disease and the emergence of bacterial strains resistant to current antibiotics. Modification of the cell phenotype to sensitize bacteria to components of the hosts' immune system or to previously ineffective antibiotics could prevent the emergence of the resistant genotype. In addition, the use of light-activated antibacterial agents and lytic bacteriophage specific for key pathogens should be considered as safe and inexpensive alternatives to conventional treatment regimens for certain non-systemic infections. [TOP OF PAGE]

  1147. One of two copies of the gene for the activatable shiga toxin type 2d in Escherichia coli O91:H21 strain B2F1 is associated with an inducible bacteriophage. Teel,L.D., Melton-Celsa,A.R., Schmitt,C.K., O'Brien,A.D. (2002). Infect. Immun. 70:4282-4291. Shiga toxin (Stx) types 1 and 2 are encoded within intact or defective temperate bacteriophages in Stx-producing Escherichia coli (STEC), and expression of these toxins is linked to bacteriophage induction. Among Stx2 variants, only stx(2e) from one human STEC isolate has been reported to be carried within a toxin-converting phage. In this study, we examined the O91:H21 STEC isolate B2F1, which carries two functional alleles for the potent activatable Stx2 variant toxin, Stx2d, for the presence of Stx2d-converting bacteriophages. We first constructed mutants of B2F1 that produced one or the other Stx2d toxin and found that the mutant that produced only Stx2d1 made less toxin than the Stx2d2-producing mutant. Consistent with that result, the Stx2d1-producing mutant was attenuated in a streptomycin-treated mouse model of STEC infection. When the mutants were treated with mitomycin C to promote bacteriophage induction, Vero cell cytotoxicity was elevated only in extracts of the Stx2d1-producing mutant. Additionally, when mice were treated with ciprofloxacin, an antibiotic that induces the O157:H7 Stx2-converting phage, the animals were more susceptible to the Stx2d1-producing mutant. Moreover, an stx(2d1)-containing lysogen was isolated from plaques on strain DH5alpha that had been exposed to lysates of the mutant that produced Stx2d1 only, and supernatants from that lysogen transformed with a plasmid encoding RecA were cytotoxic when the lysogen was induced with mitomycin C. Finally, electron-microscopic examination of extracts from the Stx2d1-producing mutant showed hexagonal particles that resemble the prototypic Stx2-converting phage 933W. Together these observations provide strong evidence that expression of Stx2d1 is bacteriophage associated. We conclude that despite the sequence similarity of the stx(2d1)- and stx(2d2)-flanking regions in B2F1, Stx2d1 expression is repressed within the context of its toxin-converting phage while Stx2d2 expression is independent of phage induction. [TOP OF PAGE]

  1148. Fighting Foam with Phages? Thomas,J.A., Soddell,J.A., Kurtboke,D.I. (2002). Water Sci. Technol. 46:511-553. Seventeen(17) phages infective for the mycolata were isolated from six samples of activated sludge using 21 prospective hosts from the genera Dietzia, Nocardia, Rhodococcus, Tsukamurella and Mycobacterium. Their morphology indicated that they were all members of the viral family Siphoviridae, but they varied in the size of the icosahedral head and length of non-contractile tail, suggesting they were different. This was confirmed by host-range studies with 47 strains of mycolata, which showed that each phage had a unique host-range, and this was polyvalent in the majority (15/17) of cases, with 12 infective for hosts representing two or three of the genera Gordonia, Nocardia and Rhodococcus. The potential for use of these phages in the control of foaming and other applications is discussed. [TOP OF PAGE]

  1149. Mobile elements as a combination of functional modules. Toussaint,A., Merlin,C. (2002). Plasmid 47:26-35. Prokaryotic mobile elements have traditionally been classified as bacteriophages, plasmids, and transposons. We propose here a global classification of these and other bacterial and archaeal mobile elements based on their modular structure. This would allow for setting up interconnected databases where mobile elements could be stored as combinations of functional modules. Such a database would be very helpful. It would, for instance, allow for analyzing the phylogeny of individual blocks within an element, to understand how modules get associated and properly express the functions they carry in various bacterial hosts. Modules of practical importance, as for instance those that encode toxins or other virulence factors, could be identified and compared, and probes devised to test bacterial populations for the presence of such modules. [TOP OF PAGE]

  1150. [Study of Erwinia carotovora phage resistance with the use of temperate bacteriophage ZF40]. Tovkach,F.I. (2002). Mikrobiologiia 71:82-88. The causes of the unique phage resistance of the pectinolytic phytopathogenic strains of Erwinia carotovora were studied with the use of temperate bacteriophage ZF40. It was shown that, in these bacteria, the bacteriophage-cell interaction can be substantially blocked at the adsorption level. An adequate indicator for studying the temperate bacteriophages of erwinias was developed on the basis of mutants resistant to macromolecular bacteriocins. Various restriction-modification systems, which influence cell resistance to bacteriophages, were revealed for the first time in E. carotovora. The phage resistance was shown to be determined by the wide occurrence of homoimmune temperate viruses in pectinolytic erwinias. [TOP OF PAGE]

  1151. [Comparative study of properties of temperate erwiniophages 49 and 59]. Tovkach,F.I., Shevchenko,T.V., Gorb,T.E., Mukvich,N.S., Romaniuk,L.V. (2002). Mikrobiol. Zh. 64:65-81. Molecular-biological properties of two relative temperate erwiniophages 49 and 59 have been comparatively studied. The both phages are highly specific with respect to sensitive bacteria and lyse only inconsiderable quantity of amylovora-like strains of Erwinia horticola. It has been established that erwiniophages are distinguished by the basic parameters of a single reproduction cycle in the cells of common host E. horticola 450. Considerable differences between phages have been also found in the areas of genomes responsible for the establishment and maintenance of lysogenic state in the cells of the bacterium-host. Study of structure polypeptides has confirmed the identity of capsids and tails of phages 49 and 59. It has been shown that phage 49 has another, as compared to phage 59, basal plate, which availability destabilises the phage tail and leads to virion destruction under various physical effects. Virion DNA of phages 49 and 59 are of the same size--47.9 kbp, but differ as to GC-content. Using the restriction analysis it has been shown that genome of phage 49, as well as the genome of phage 59, is permuted, but its permutation is of discrete character. The fact of recombination interaction between erwiniophages 49 and 59 has been established. It is supposed that phage 49 is the recombination (hybrid) derivative of phage 59 and unknown phage, or prophage, genetic module. The given recombination, probably, took place under the persistence of different phages in the general polylysogenic system of E. horticola. [TOP OF PAGE]

  1152. [Temperate bacteriophage ZF40 of Erwinia carotovora: phage particle structure and DNA restriction analysis]. Tovkach,F.I. (2002). Mikrobiologiia 71:75-81. Structural organization of the temperate bacteriophage ZF40 of Erwinia carotovora was studied. Phage ZF40 proved to be a typical member of the Myoviridae family (morphotype A1). Phage particles consist of an isometric head 58.3 nm in diameter and a contractile 86.3-nm-long tail with a complex basal plate and short tail fibers (31.5 nm). Phage tail sheath, a truncated cone in shape, is characterized by specific packaging of structural subunits. The ZF40 phage genome is 45.8 kb in size, as determined by restriction analysis, and contains DNA cohesive ends. The ZF40 phage of Erwinia carotovora is assumed to be a new species of bacteriophages specific for enterobacteria. [TOP OF PAGE]

  1153. Defektnaia lizogenia Erwinia carotovora [Defective lysogeny in Erwinia carotovora]. Tovkach,F.I. (2002). Mikrobiologiia 71:359-367. The electron microscopic study of several Erwinia carotovora strains showed that the SOS-induced cells of this pectolytic phytopathogenic bacterium produce particular phage parts (tails, heads, and baseplates) but do not assemble them into fully functional phage particles. E. carotovora cells produced several times greater amounts of phage tails in response to induction by mitomycin C than in response to induction by nalidixic acid. The tails were 128-192 nm in length and 13-21 nm in diameter. Phage heads were characterized by four discrete ranges of diameters: 18, 55-59, 66-75, and 92-98 nm. The diameters of phage baseplates varied from 39 to 53 nm, depending on the particular strain. It was shown that cells of the same species may contain several different types of phage tails and heads. The structural organization of phage tails and baseplates in the nalidixic acid-induced lysate of E. carotovora J2 was studied in more detail. The data obtained suggest that pectolytic phytopathogenic erwinia are characterized by defective polylysogeny. [TOP OF PAGE]

  1154. Viruses stop antibiotic-resistant bacteria. Travis,J. (2002). Science News 161(2), ??? Nearly a century ago, biologists discovered viruses that prey upon bacteria. When penicillin and other antibiotics emerged a few decades later, however, physicians largely abandoned their efforts to use these bacteriophages, or phages, to thwart infectious diseases. ¶ As more bacteria develop resistance to antibiotics, there's renewed interest in phages (SN: 6/3/00, p. 358). Scientists now report that these viruses can prevent mice from dying after being infected with an antibiotic-resistant bacterium. [TOP OF PAGE]

  1155. ??? Tsujita,M., Matsui,C. (2002). Proc. Jpn. Acad. 31:180-185. [TOP OF PAGE]

  1156. Role of bacteriophage MAV1 as a mycoplasmal virulence factor for the development of arthritis in mice and rats. Tu,A.H., Lindsey,J.R., Schoeb,T.R., Elgavish,A., Yu,H., Dybvig,K. (2002). J. Infect. Dis. 186:432-435. The lysogenic bacteriophage MAV1 has been shown to be a virulence factor for the development of arthritis in rats infected with Mycoplasma arthritidis. In the present study, arthritis was evaluated by histopathologic examination to demonstrate that MAV1 is a virulence factor not only in the rat but also in the mouse. Specifically, the MAV1 lysogen 158L3-1 was more virulent than the nonlysogen strain 158 in DBA/2NCr, C3H/HeNCr, C3H/HeJ, and C3Smn.CB17-Prkdc(scid)/J mice, as well as in LEW rats. [TOP OF PAGE]

  1157. Ecological and molecular maintenance strategies of mobile genetic elements. Turner,S.L., Bailey,M.J., Lilley,A.K., Thomas,C.M. (2002). FEMS Microbiol. Ecol. 42:177-185. This review considers the influence of selection pressure, fitness and population structures on the evolution of mobile genetic elements (including plasmids, phage, pathogenicity islands, transposons and insertion sequences) that constitute the horizontal gene pool of bacteria. These are considered at different scales using examples from in vitro evolutionary studies of Escherichia coli and associated bacteriophage, detailed molecular analyses of the broad host-range IncP-1 plasmids, population surveys of pseudomonad plasmids and genomic comparisons of members of the Rhizobiaceae. All biological systems show genetic redundancy (the existence of allelic variation) at some population level, i.e. within a cell, a clone, population or community. We consider the level(s) at which redundancy is expressed and how this will affect and has influenced the evolution of mobile genetic elements. [TOP OF PAGE]

  1158. Removal of indigenous coliphages and fecal coliforms by a novel sewage treatment system consisting of UASB and DHS units. Uemura,S., Takahashi,K., Takaishi,A., Machdar,I., Ohashi,A., Harada,H. (2002). Water Sci. Technol. 46:303-309. A novel sewage treatment system, which consists of an upflow anaerobic sludge blanket (UASB) pre-treatment unit and the following downflow hanging sponge (DHS) unit for polishing up the UASB effluent, was developed as a cost-effective and easy-maintenance sewage treatment system for developing countries. A long-term experiment with actual sewage was conducted in order to evaluate its treatment efficiency of organic substances, nutrients, and pathogen indicator microorganisms such as total coliphages, F+-specific RNA coliphages (RNA coliphages), and fecal coliforms. The main objective of this paper is to investigate the removal efficiency of those indicator microorganisms by the UASB-DHS combined system. The results obtained from the continuous flow experiment indicated a fairly promising removal of the indicator microorganisms, i.e., the log10 reductions of total coliphages, RNA coliphages, and fecal coliforms (based on sewage and DHS effluent) achieved were 2.01 log, 2.02 log, and 2.57 log, respectively. The UASB-DHS combined system was superior to the conventional activated sludge process in the reduction of fecal coliforms, but in the reductions of total and RNA coliphages, the system showed somewhat less removal efficiency. The vertical reducing patterns of the indicator microorganisms along the DHS reactor were also discussed. [TOP OF PAGE]

  1159. In vivo generation of hybrids between different species of RNA phages. van Meerten,D., Groeneveld,H., Miller,D.M.J., Marechal,G.B., Tsareva,N.V., Olsthoorn,R.C.L., de la Pena,M., van Duin,J. (2002). J. Gen. Virol. 83:1223-1235. Hybrids between different species or genera of the single-stranded RNA coliphages have not been found in nature. Here, it has been shown that viable hybrids between different phage species can easily be generated in the laboratory by in vivo recombination. cDNA of species I phage MS2 located on a plasmid and lacking part of its 5' untranslated leader (5' UTR) was complemented with another plasmid carrying the 5' half of the genome of fr, a species I phage, or of KU1, a species II representative with low sequence similarity. When the two plasmids were present in the same cell there was spontaneous production of hybrid phages. Interestingly, these hybrids did not arise by a double or single crossover that would replace the missing MS2 sequences with those of fr or KU1. Rather, hybrids arose by attaching the complete 5' UTR of fr or KU1 to the 5' terminus of the defective MS2 phage. Several elements of the 5' UTR then occurred twice, one from KU1 (or fr) and the other from MS2. These redundant elements are in most cases deleted upon evolution of the hybrids. As a result, the 5' UTR of KU1 (or fr) then replaced that of MS2. It was earlier shown that this 5' UTR could assume two alternating structures that facilitated transient translation of the proximal maturation gene. Apparently, this timer function of the 5' UTR was exchangeable and could function independently of the rest of the genome. When hybrids were competed against wild-type, they were quickly outgrown, probably explaining their absence from natural isolates. [TOP OF PAGE]

  1160. Use of bacteriophages for control of Escherichia coli O157. Waddell,T.E., Mazzocco,A., Pacan,J., Ahmed,R., Johnson,R., Poppe,C., Khakhria,R. (2002). 873949(6,485,902). A method of reducing levels of E. coli O157 strains within the gastrointestinal tract of a ruminant animal using specific bacteriophage(s) is herein described. Also described is a pharmaceutical composition comprising at least one of said bacteriophages and a method for isolating or selecting bacteriophages useful in reducing E. coli O157 levels as described above. [TOP OF PAGE]

  1161. Bacteriophage control of bacterial virulence. Wagner,P.L., Waldor,M.K. (2002). Infect. Immun. 70:3985-3993. [LAST PARAGRAPH] In summary, bacteriophages are intimately involved in bacterial pathogenesis, including adhesion and invasion, evasion of immunity, and exotoxin production, largely by transducing a diverse set of virulence genes. In some cases, the expression of these genes may be driven in part by induction of the prophages that encode them. Since prophage induction contributes to virulence gene expression and since the human body is replete with prophage-inducing chemicals, prophage induction in the human body may be an important, underrecognized mechanism of bacterial pathogenesis. [TOP OF PAGE]

  1162. Bacteriophage control of Shiga toxin 1 production and release by Escherichia coli. Wagner,P.L., Livny,J., Neely,M.N., Acheson,D.W.K., Friedman,D.I., Waldor,M.K. (2002). Mol. Microbiol. 44:957-970. The stx genes of many Shiga toxin-encoding Escherichia coli (STEC) strains are encoded by prophages of the l bacteriophage family. In the genome of the Stx1-encoding phage H-19B, the stx(1)AB genes are found approximately 1 kb downstream of the late phage promoter, p(R)', but are known to be regulated by the associated iron-regulated promoter, p(Stx1). Growth of H-19B lysogens in low iron concentrations or in conditions that induce the prophage results in increased Stx1 production. Although the mechanism by which low iron concentration induces Stx1 production is well understood, the mechanisms by which phage induction enhances toxin production have not been extensively characterized. The studies reported here identify the factors that contribute to Stx1 production after induction of the H-19B prophage. We found that replication of the phage genome, with the associated increase in stx(1)AB copy number, is the most quantitatively important mechanism by which H-19B induction increases Stx1 production. Three promoters are shown to be involved in stx(1)AB transcription after phage induction, the iron-regulated p(Stx1) and the phage-regulated p(R) and p(R)' promoters, the relative importance of which varies with environmental conditions. Late phage transcription initiating at the p(R)' promoter, contrary to previous findings in the related Stx2-encoding phage f361, was found to be unnecessary for high-level Stx1 production after phage induction. Finally, we present evidence that phage-mediated lysis regulates the quantity of Stx1 produced by determining the duration of Stx1 accumulation and provides a mechanism for Stx1 release. By amplifying stx(1)AB copy number, regulating stx(1)AB transcription and allowing for Stx1 release, the biology of the Stx-encoding phages contributes greatly to the production of Stx, the principal virulence factor of STEC. [TOP OF PAGE]

  1163. ??? Wakimoto,S. (2002). Ann. Phytopathol. Soc. Japan 25:205-208. [TOP OF PAGE]

  1164. Effect of phage therapy on the turnover and function of peripheral neutrophils. Weber-Dabrowska,B., Zimecki,M., Mulczyk,M., Gorski,A. (2002). FEMS Immunol. Med. Microbiol. 34:135-138. The aim of this investigation was to establish the impact of phage therapy on the turnover and function of circulating neutrophils in 37 patients with suppurative bacterial infections. We determined the levels of circulating neutrophils and their precursors before therapy, after 3 weeks of therapy, and at a distant time interval (3 months) following the beginning of therapy. In addition, we measured the ability of neutrophils to phagocytize Staphylococcus aureus in vitro. Eight healthy blood donors served as a control group. The results showed that, among the studied parameters, the significant changes involved neutrophil precursor count and the ability of neutrophils to phagocytize bacteria. The percentage of neutrophils in patients before therapy was lower than in healthy donors (mean 58.0, versus 61.4). This value dropped further in patients after 3 months of following the therapy (mean 55.6). The content of neutrophil precursors, on the other hand, was lower in healthy donors than in patients before therapy (mean 2.5, versus 3.8). After 3 weeks of the therapy and after 3 months, the levels of neutrophil precursors were significantly higher (mean 4.8 and 4.9, respectively) than in control donors. The phagocytic index was lower in patients before therapy than in control donors (mean 66.3, versus 70.1) and decreased further after 3 weeks of therapy (mean 59.0) and after 3 months (mean 59.6). The results of this investigation indicate that successful phage therapy accelerates the turnover of neutrophils, accompanied by a decrease in their ability to phagocytize bacteria. [TOP OF PAGE]

  1165. Modulation of the susceptibility of intestinal bacteria to bacteriophages in response to Ag43 phase variation -- a hypothesis. Wegrzyn,G., Thomas,M.S. (2002). Medical Science Monitor 8:HY15-HY18 Escherichia coli is a Gram-negative bacterium which colonizes the intestinal tract of man and other animals. In addition to being a part of the normal bacterial flora of the human intestine, there are a number of enteropathogenic strains of E. coli which cause infections ranging in consequence from diarrhoea to colitis. Antigen 43 (Ag43) is the major phase-variable protein in the outer membrane of E. coli. One benefit for bacteria resulting from phase variation of surface antigens is usually ascribed to evasion of host defences. However, results of recent studies indicate that infection of E. coli by different bacteriophages is inhibited in the presence of certain bile salts and carbohydrates (components present in the human intestine but absent in standard bacteriological media) when cells are in the 'OFF' state for production of Ag43. The inhibition of bacteriophage development was found to be due to a significant impairment in the process of phage adsorption and evidence was presented for the binding of phage to Ag43. Here we present a hypothesis that in the case of Ag43, phase-variation might benefit the host bacterium by modulating the susceptibility to phage infection in the gut. If this hypothesis is true, it may have important implications not only for basic research but also for development of bacteriophage therapy, a re-discovered method of treatment of patients with infectious diseases. [TOP OF PAGE]

  1166. Reconsidering transmission electron microscopy based estimates of viral infection of bacterio-plankton using conversion factors derived from natural communities. Weinbauer,M.G., Winter,C., Hofle,M.G. (2002). Aquat. Microb. Ecol. 27:103-110. The frequency of virus infected bacterial cells (FIC) was estimated in surface waters of the Mediterranean Sea, the Baltic Sea and the North Sea using the frequency of visibly infected cells (FVIC) as determined by transmission electron microscopy (TEM) and published average conversion factors (average 5.42, range 3.7 to 7.14) to relate FVIC to FIC. A virus dilution approach was used to obtain an independent estimation of FIC in bacterioplankton, and we provide evidence for the reliability of this approach. Across all investigated environments, FIC ranged from 2.4 to 43.4 %. FIC data using both approaches were well correlated; however, the values were higher using the virus dilution approach, This indicates that the TEM approach has the potential to reveal spatiotemporal trends of viral infection; however, it may underestimate the significance of viral infection of bacteria when average conversion factors are used. Using data from the virus dilution approach and the TEM approach, we calculated new conversion factors for relating FVIC to FIC (average 7.11, range 4.34 to 10.78). Virally caused mortality of bacteria estimated from published FVIC data of marine and freshwater systems and using the new conversion factors ranged from not detectable to 129 %, thus confirming that viral infection is a significant and spatiotemporally variable cause of bacterial cell death. [TOP OF PAGE]

  1167. Pseudoalteromonas spp. phages, a significant group of marine bacteriophages in the North Sea. Wichels,A., Gerdts,G., Schuett,C. (2002). Aquat. Microb. Ecol. 27:233-239. The occurrence and distribution of specific bacteriophages of marine Pseudoalteromonas spp. in the North Sea (North Sea phages) and their genetic relationship to several previously isolated marine phage species from waters of the Helgoland Roads (German Bight, Helgoland phages) were investigated. During 3 cruises from the Elbe estuary to western Norwegian waters, phages were concentrated by ultrafiltration. Detection and isolation of North Sea phages were performed by plaque assay, with 70 host bacteria of the genus Pseudoalteromonas. The genetic relationship between North Sea phages from different stations and Helgoland phages, formerly described as Pseudoalteromonas spp. phages, was assessed by DNA-DNA hybridization. DNA probes were prepared using whole phage DNA derived from 13 Helgoland phages. This approach provides the first information on the distribution of specific Pseudoalteromonas spp. phage-host systems (PHS) in the North Sea. The occurrence of Pseudoalteromonas spp. phages, which are specific for the tested Pseudoalteromonas spp. host bacteria, was restricted to a narrow geographical region of the German Bight between 53 degree 30' and 57 degree 00' N latitude. Most of the previously isolated Helgoland phages were highly host specific (54%), whereas this was true for only some of the 39 North Sea phages (16%). The most common Pseudoalteromonas spp. phage species found in the North Sea belong to the virus family Siphoviridae (species H103/1). Several phage strains within this phage species displayed different host sensitivity patterns. [TOP OF PAGE]

  1168. Estimation of biologically damaging UV levels in marine surface waters with DNA and viral dosimeters. Wilhelm,S.W., Jeffrey,W.H., Suttle,C.A., Mitchell,D.L. (2002). Photochem Photobiol 76:268-273. We have surveyed the biologically harmful radiation penetrating the water column along a transect in the western Gulf of Mexico using dosimeters consisting of intact viruses or naked calf-thymus DNA (ctDNA). The indigenous marine bacteriophage PWH3a-P1, which lytically infects the heterotrophic bacterium Vibrio natriegens (strain PWH3a), displayed decay rates for infectivity approaching 1.0 h(-1) in surface waters when deployed in a seawater-based dosimeter. The accumulation of pyrimidine dimers in ctDNA dosimeters provided a strong correlation to these results, with pyrimidine dimers representing more than 0.3% (up to ca 3800 dimers Mb(-1) DNA) of the total DNA in dosimeters exposed to sea surface levels of solar radiation. The results demonstrate a strong correlation between the dimer formation in the DNA dosimeters, the decay rates of viral infectivity and the penetration of UVB radiation into the water column. The decay of viral infectivity attenuated with depth in a manner similar to the decay of solar radiation and was still significant at 10 m in offshore oligotrophic water and at dimer frequencies less than 0.1% (ca 200-300 dimers Mb(-1) DNA). [TOP OF PAGE]

  1169. Direct measurements of viral production in stratified and tidally mixed waters in the Strait of Georgia. Wilhelm,S.W., Brigden,S.M., Suttle,C.A. (2002). Microb. Ecol. 43:168-173. The abundance of heterotrophic bacteria and viruses, as well as rates of viral production and virus-mediated mortality, were measured in Discovery Passage and the Strait of Georgia (British Columbia, Canada) along a gradient of tidal mixing ranging from well mixed to stratified. The abundances of bacteria and viruses were approximately 106 and 107 mL-1, respectively, independent of mixing regime. Viral production estimates, monitored by a dilution technique, demonstrated that new viruses were produced at rates of 106 to 107 mL-1 h-1 across the different mixing regimes. Using an estimated burst size of 50 viruses per lytic event, ca. 19 to 27% of the standing stock of bacteria at the stratified stations and 46 to 137% at the deep-mixed stations were removed by viruses. The results suggest that mixing of stratified waters during tidal exchange enhances virus-mediated bacterial lysis. Consequently, viral lysis recycled a greater proportion of the organic carbon required for bacterial growth under non-steady-state compared to steady-state conditions. [TOP OF PAGE]

  1170. Bacteriophage HP2 of Haemophilus influenzae. Williams,B.J., Golomb,M., Phillips,T., Brownlee,J., Olson,M.V., Smith,A.L. (2002). J. Bacteriol. 184:6893-6905. Temperate bacteriophages effect chromosomal evolution of their bacterial hosts, mediating rearrangements and the acquisition of novel genes from other taxa. Although the Haemophilus influenzae genome shows evidence of past phage-mediated lateral transfer, the phages presumed responsible have not been identified. To date, six different H. influenzae phages are known; of these, only the HP1/S2 group, which lyosogenizes exclusively Rd strains (which were originally encapsulated serotype d), is well characterized. Phages in this group are genetically very similar, with a highly conserved set of genes. Because the majority of H. influenzae strains are nonencapsulated (nontypeable), it is important to characterize phages infecting this larger, genetically more diverse group of respiratory pathogens. We have identified and sequenced HP2, a bacteriophage of nontypeable H. influenzae. Although related to the fully sequenced HP1 (and even more so to the partially sequenced S2) and similar in genetic organization, HP2 has a few novel genes and differs in host range; HP2 will not infect or lysogenize Rd strains. Genomic comparisons between HP1/S2 and HP2 suggest recent divergence, with new genes completely replacing old ones at certain loci. Sequence comparisons suggest that H. influenzae phages evolve by recombinational exchange of genes with each other, with cryptic prophages, and with the host chromosome. [TOP OF PAGE]

  1171. Method to detect bacteria. Wilson,S.M. (2002). Biotec Laboratories Limited. 594918(6,461,833). Ipswich, GB. The present invention relates to a method for enhancing the time of response of an assay for a first bacterium, wherein: a) the first bacterium is exposed to infection by phage particles to which the first bacterium is permissive; b) the infected bacterium is treated to inactivate exogenous phage particles; c) the treated bacterium is cultivated in the presence of a second bacterium which is permissive to infection by the phage or its replicand and which has a doubling rate greater than the effective doubling rate of the first bacterium; and d) assessing the extent of plaque formation and/or of second bacterium growth in the cultivated second bacterium cells. The method can be used to assess the presence of first bacterium in a sample, notably where the first bacterium is a slow growing bacterium, such as Mycobacterium tuberculosis, where the method enables an operator to detect the presence of low amounts of the bacterium in sample within days instead of weeks as required by conventional cultivation techniques. The invention can also be used to assess the effect of a drug or other treatment on a bacterium or on a virus. The invention also provides a diagnostic kit for use in the method of the invention. [TOP OF PAGE]

  1172. Virus dynamics in a coccolithophore-dominated bloom in the North Sea. Wilson,W.H., Tarran,G., Zubkoy,M.V. (2002). Deep Sea Research Part II: Tropical Studies in Oceanography 49:2951-2963. We used analytical flow cytometry (AFC) to determine virus concentrations through vertical profiles in a coccolithophore-dominated bloom in the northern North Sea during June 1999. We present the first high-intensity sampling data of viruses from a lagrangian survey to gain a unique insight into the temporal and spatial dynamics of viruses in an open-water sight. Virus abundances ranged from 2.6×10(5) to 5.4×10(6)ml(-1), which is within the range expected for open-water environments. The highest concentrations were invariably observed in surface waters. During the lagrangian experiment there was a net decrease in virus numbers, suggesting that they were actively infecting hosts. Large viruses could be easily discriminated from small viruses since there was at least an order of magnitude difference in their AFC side-scatter values. Large viruses, assumed to infect DMS-producing algae, did not appear to influence DMS/DMSP production. It is likely that microzooplankton out-competed viruses for coccolithophore prey/hosts. Lower small virus to bacteria ratios (VBR) were observed in a subsurface layer compared to the more productive surface layers. The subsurface layer was dominated by a species of a-proteobacteria related to the genus Roseobacter, and the low VBR may indicate that viruses were infecting Roseobacter in this layer. Application of AFC is an excellent technique for high-definition sampling of virus communities, although it is recognised that we are working at the limit of detection for many small viruses using currently available nucleic acid stains. [TOP OF PAGE]

  1173. Isolation of viruses responsible for the demise of an Emiliania huxleyi bloom in the English Channel. Wilson,W.H., Tarran,G.A., Schroeder,D., Cox,M., Oke,J., Malin,G. (2002). Journal of the Marine Biological Association of the UK 82:369-377. This study used analytical flow cytometry (AFC) to monitor the abundance of phytoplankton, coccoliths, bacteria and viruses in a transect that crossed a high reflectance area in the western English Channel. The high reflectance area, observed by satellite, was caused by the demise of an Emiliania huxleyi bloom. Water samples were collected from depth profiles at four stations, one station outside and three stations inside the high reflectance area. Plots of transect data revealed very obvious differences between Station 1, outside, and Stations 2-4, inside the high reflectance area. Inside, concentrations of viruses were higher; E. huxleyi cells were lower; coccoliths were higher; bacteria were higher and virus:bacteria ratio was lower than at Station 1, outside the high reflectance area. This data can simply be interpreted as virus-induced lysis of E. huxleyi cells in the bloom causing large concentrations of coccoliths to detach, resulting in the high reflectance observed by satellite imagery. This interpretation was supported by the isolation of two viruses, EhV84 and EhV86, from the high reflectance area that lysed cultures of E. huxleyi host strain CCMP1516. Basic characterization revealed that they were lytic viruses approximately 170 nm-190 nm in diameter with an icosahedral symmetry. Taken together, transect and isolation data suggest that viruses were the major contributor to the demise of the E. huxleyi population in the high reflectance area. Close coupling between microalgae, bacteria and viruses contributed to a large organic carbon input. Consequent cycling influenced the succession of an E. huxleyi-dominated population to a more characteristic mixed summer phytoplankton community. [TOP OF PAGE]

  1174. Burkholderia thailandensis E125 harbors a temperate bacteriophage specific for Burkholderia mallei. Woods,D.E., Jeddeloh,J.A., Fritz,D.L., DeShazer,D. (2002). J. Bacteriol. 184:4003-4017. Burkholderia thailandensis is a nonpathogenic gram-negative bacillus that is closely related to Burkholderia mallei and Burkholderia pseudomallei. We found that B. thailandensis E125 spontaneously produced a bacteriophage, termed phiE125, which formed turbid plaques in top agar containing B. mallei ATCC 23344. We examined the host range of phiE125 and found that it formed plaques on B. mallei but not on any other bacterial species tested, including B. thailandensis and B. pseudomallei. Examination of the bacteriophage by transmission electron microscopy revealed an isometric head and a long noncontractile tail. B. mallei NCTC 120 and B. mallei DB110795 were resistant to infection with phiE125 and did not produce lipopolysaccharide (LPS) O antigen due to IS407A insertions in wbiE and wbiG, respectively. wbiE was provided in trans on a broad-host-range plasmid to B. mallei NCTC 120, and it restored LPS O-antigen production and susceptibility to phiE125. The 53,373-bp phiE125 genome contained 70 genes, an IS3 family insertion sequence (ISBt3), and an attachment site (attP) encompassing the 3' end of a proline tRNA (UGG) gene. While the overall genetic organization of the phiE125 genome was similar to lambda-like bacteriophages and prophages, it also possessed a novel cluster of putative replication and lysogeny genes. The phiE125 genome encoded an adenine and a cytosine methyltransferase, and purified bacteriophage DNA contained both N6-methyladenine and N4-methylcytosine. The results presented here demonstrate that phiE125 is a new member of the lambda supergroup of Siphoviridae that may be useful as a diagnostic tool for B. mallei. [TOP OF PAGE]

  1175. Evolution of lambdoid replication modules. Wrobel,B., Wegrzyn,G. (2002). Virus Genes 24:163-171. Comparison of the putative iteron-binding proteins of lambdoid phages allows us to propose that in the case of lambdoid replication modules, the units on which natural selection acts do not coincide with the open reading frames. Rather, the first replication gene is split into two segments, and its 3' part (corresponding to the C-terminal domain of the iteron-binding protein) forms one unit with the second gene. We also propose from the phylogenetic analysis of phage-encoded homologs of E. coli DnaB and DnaC, that the recombination with the host sequences is not frequent. Accessory ATP-ases for helicase loading (E. coli DnaC homologs) may not be universal replication proteins. Our analysis may suggest that the bacterial helicase loaders might be of phage origin. The comparison of DnaC homologs of enterobacteria and enterobacterial phages supports the experimental data on residues important in interaction with DnaB. We propose that construction of plasmids carrying the replication origins of lambdoid prophages could be useful not only in further research on DNA replication but also on the role of these prophages in shuttling genes for bacterial virulence. The phage replication sequences could be also useful for identification of clinical enterobacterial isolates. [TOP OF PAGE]

  1176. Isolation and characterization of bacteriophages from fermenting sauerkraut. Yoon,S.S., Barrangou-Poueys,R., Breidt,F., Jr., Klaenhammer,T.R., Fleming,H.P. (2002). Appl. Environ. Microbiol. 68:973-976. This paper presents the first report of bacteriophage isolated from commercial vegetable fermentations. Nine phages were isolated from two 90-ton commercial sauerkraut fermentations. These phages were active against fermentation isolates and selected Leuconostoc mesenteroides and Lactobacillus plantarum strains, including a starter culture. Phages were characterized as members of the Siphoviridae and Myoviridae families. All Leuconostoc phages reported previously, primarily of dairy origin, belonged to the Siphoviridae family. [TOP OF PAGE]

  1177. Effects of Escherichia coli physiology on growth of phage T7 in vivo and in silico. You,L., Suthers,P.F., Yin,J. (2002). J. Bacteriol. 184:1888-1894. Phage development depends not only upon phage functions but also on the physiological state of the host, characterized by levels and activities of host cellular functions. We established Escherichia coli at different physiological states by continuous culture under different dilution rates and then measured its production of phage T7 during a single cycle of infection. We found that the intracellular eclipse time decreased and the rise rate increased as the growth rate of the host increased. To develop mechanistic insight, we extended a computer simulation for the growth of phage T7 to account for the physiology of its host. Literature data were used to establish mathematical correlations between host resources and the host growth rate; host resources included the amount of genomic DNA, pool sizes and elongation rates of RNA polymerases and ribosomes, pool sizes of amino acids and nucleoside triphosphates, and the cell volume. The in silico (simulated) dependence of the phage intracellular rise rate on the host growth rate gave quantitatively good agreement with our in vivo results, increasing fivefold for a 2.4-fold increase in host doublings per hour, and the simulated dependence of eclipse time on growth rate agreed qualitatively, deviating by a fixed delay. When the simulation was used to numerically uncouple host resources from the host growth rate, phage growth was found to be most sensitive to the host translation machinery, specifically, the level and elongation rate of the ribosomes. Finally, the simulation was used to follow how bottlenecks to phage growth shift in response to variations in host or phage functions. [TOP OF PAGE]

  1178. Phage mediated horizontal transfer of the sopE1 gene increases enteropathogenicity of Salmonella enterica serotype Typhimurium for calves. Zhang,S., Santos,R.L., Tsolis,R.M., Mirold,S., Hardt,W.D., Adams,L.G., Baumler,A.J. (2002). FEMS Microbiol. Lett. 217:243-247. Epidemiological evidence shows that the sopE1 gene is associated with Salmonella Typhimurium phage types causing epidemics in cattle. In this study we demonstrate that horizontal transfer of the sopE1 gene by lysogenic conversion with the SopEphi increased enteropathogenicity of S. Typhimurium in the bovine ligated ileal loop model. These data support the hypothesis that phage mediated horizontal transfer of the sopE1 gene contributes to the emergence of epidemic cattle-associated S. Typhimurium clones. [TOP OF PAGE]

  1179. Genomic analysis of Clostridium perfringens bacteriophage phi3626, which integrates into guaA and possibly affects sporulation. Zimmer,M., Scherer,S., Loessner,M.J. (2002). J. Bacteriol. 184:4359-4368. Two temperate viruses, phi3626 and phi8533, have been isolated from lysogenic Clostridium perfringens strains. Phage phi3626 was chosen for detailed analysis and was inspected by electron microscopy, protein profiling, and host range determination. For the first time, the nucleotide sequence of a bacteriophage infecting Clostridium species was determined. The virus belongs to the Siphoviridae family of the tailed phages, the order Caudovirales. Its genome consists of a linear double-stranded DNA molecule of 33,507 nucleotides, with invariable 3'-protruding cohesive ends of nine residues. Fifty open reading frames were identified, which are organized in three major life cycle-specific gene clusters. The genes required for lytic development show an opposite orientation and arrangement compared to the lysogeny control region. A function could be assigned to 19 gene products, based upon bioinformatic analyses, N-terminal amino acid sequencing, or experimental evidence. These include DNA-packaging proteins, structural components, a dual lysis system, a putative lysogeny switch, and proteins that are involved in replication, recombination, and modification of phage DNA. The presence of genes encoding a putative sigma factor related to sporulation-dependent sigma factors and a putative sporulation-dependent transcription regulator suggests a possible interaction of phi3626 with onset of sporulation in C. perfringens. We found that the phi3626 attachment site attP lies in a noncoding region immediately downstream of int. Integration of the viral genome occurs into the bacterial attachment site attB, which is located within the 3' end of a guaA homologue. This essential housekeeping gene is functionally independent of the integration status, due to reconstitution of its terminal codons by phage sequence. [TOP OF PAGE]

  1180. Mutant bacteriophage with non-catalytic endosialidase binds to both bacterial and eukaryotic polysialic acid and can be used as probe for its detection. Aalto,J., Pelkonen,S., Kalimo,H., Finne,J. (2001). Glycoconj. J. 18:751-758. There is a molecular mimicry between the polysialic acid polysaccharide of bacterial pathogens causing sepsis and meningitis, and the carbohydrate units of the neural cell adhesion molecule NCAM. We investigated whether bacteriophage mutants with catalytically disabled endosialidase, which bind but do not cleave polysialic acid, could recognise and bind to bacterial and eukaryotic polysialic acid. In nitrocellulose dot blot assay the mutant bacteriophages, but not the wild-type phages, remained specifically bound to polysialic acid-containing bacteria including Escherichia coli K1 and K92, group B meningococci, Mannheimia (Pasteurella) haemolytica A2, and Moraxella nonliquefaciens. A minimum binding requirement was determined to be 10 sialyl residues in the polysialic acid chain. In Western blots the mutant phages specifically bound to the embryonic polysialylated form of NCAM, but not to the adult less sialylated form of the molecule. The mutant phages together with secondary anti-phage antibodies were subsequently successfully used in fluorescence microscopy of cultured cells and light microscopy of paraffin-embedded tissue sections as a probe for the eukaryotic polysialic acid. Thus, mutant bacteriophages of meningitis causing bacteria bind to and detect the molecularly mimicked polysialic acid of the neural cell adhesion molecule in host tissues. [TOP OF PAGE]

  1181. Bacteriophage latent-period evolution as a response to resource availability. Abedon,S.T., Herschler,T.D., Stopar,D. (2001). Appl. Environ. Microbiol. 67:4233-4241. Bacteriophages (phages) modify microbial communities by lysing hosts, transferring genetic material, and effecting lysogenic conversion. To understand how natural communities are affected it is important to develop predictive models. Here we consider how variation between models in eclipse period, latent period, adsorption constant, burst size, the handling of differences in host quantity and host quality, and in modeling strategy can affect predictions. First we compare two published models of phage growth, which differ primarily in terms of how they model the kinetics of phage adsorption; one is a computer simulation and the other is an explicit calculation. At higher host quantities (~108 cells/ml), both models closely predict experimentally determined phage population growth rates. At lower host quantities (107 cells/ml), the computer simulation continues to closely predict phage growth rates, but the explicit model does not. Next we concentrate on predictions of latent-period optima. A latent-period optimum is the latent period that maximizes the population growth of a specific phage growing in the presence of a specific quantity and quality of host cells. Both models predict similar latent-period optima at higher host densities (e.g., 17 min at 108 cells/ml). At lower host densities, however, the computer simulation predicts latent-period optima that are much shorter than those suggested by explicit calculations (e.g., 90 versus 1,250 min at 105 cells/ml). Finally, we consider the impact of host quality on phage latent-period evolution. By taking care to differentiate latent-period phenotypic plasticity from latent-period evolution, we argue that the impact of host quality on phage latent-period evolution may be relatively small. [TOP OF PAGE]

  1182. Le matin des bactériophages. Ackermann,H.-W. (2001). Virologie 5:35-43. With about 5 150 electron microscopic observations, phages contstitute the larges of all viral groups. The International Committee on Taxonomy of Viruses (ICTV) persently recognized one order, 13 families, and 30 genera. The order Caudovirales includes three families of tailed phages and about 5 000 members (96.4%). The 10 families of icosahedral, filamentous, or pleomorphic phages totalize 186 viruses. Phages are found in all the bacterial world and, with up to 1010 particles/ml in seawater, seem to be the most frequent microbes of Earth. Phages are polyphyletic in origin. Tailed phages appear to be the msot ancient viruses and recombination with exchange of genes or gene blocs (modular evolution) seems to be their preferred way of evolution. Tailed phages and herpesviruses present multiple analogies. Harmful phages can create havoc in bacterial fermentations, especially in the dairy industry. By contrast, phages are very useful in general bacteriology, therapy of infectious diseases is making a come-back and phages are likely to have a brilliant future in research. [TOP OF PAGE]

  1183. Frequency of morphological phage descriptions in the year 2000. Brief Review. Ackermann,H.-W. (2001). Arch. Virol. 146:843-857. Over 5100 bacteria viruses have been examined in the electron microscope since 1959. About 4950 phages (96%) are tailed and only 186 phages (3.6%), are cubic, filamentous, or pleomorphic. Phages belong to 13 virus families and occur in over 140 bacterial genera. Phages are listed by morphotypes and host genera. Siphoviridae or phages with long, noncontractile tails compromise 61% of tailed phages. The distribution of phages in different bacterial phylogenetic divisions is shown. [TOP OF PAGE]

  1184. Vibrio cholerae VPIPHI/CTXPHI/TCP: Interactions of phage-phage-bacterium. Ai,Y.-C., Meng,F. (2001). Acta Microbiol. Sin. 41:510-512. [TOP OF PAGE]

  1185. Distribution of virus-like particles in an oligotrophic marine environment (Alboran Sea, Western Mediterranean). Alonso,M.C., Jimenez-Gomez,F., Rodriguez,J., Borrego,J.J. (2001). Microb. Ecol. 42:407-415. Viruses are abundant in a variety of aquatic environments, often exceeding bacterial abundance by one order of magnitude. In the present study, the spatial distribution of viruses in offshore waters of the Alboran Sea (Western Mediterranean) have been studied to determine the relationships between viruses and host communities in this oligotrophic marine environment. Viral abundance was determined using two methods: (i) epifluorescence light microscopy using the dsDNA binding fluorochrome DAPI, and (ii) direct counts by transmission electron microscopy (TEM). The results obtained were significantly different; the highest viral counts were obtained by mean of TEM analyses. In all the samples tested the number of viruses was exceeded by the bacterial concentrations, with a ratio between viral and bacterial titers varying between 1.4 and 20. VLP (virus-like particle) counts were not significantly correlated (p>0.001) with chlorophyll a concentration or the abundance of cyanobacteria. However, there was a positive and significant correlation with bacterial abundance (p<0.001). The analysis of size and morphology of viral particles by TEM and the correlation obtained between the numbers of VLP and bacteria suggest that the majority of the viral particles in the Alboran Sea are bacteriophages. None of the indirect evidence suggested that eukaryotic algae or cyanobacteria were important host organisms in these waters. [TOP OF PAGE]

  1186. The bacteriophages of ruminal prevotellas. Ambrozic,J., Ferme,D., Grabnar,M., Ravnikar,M., Avgustin,G. (2001). Folia Microbiol (Praha) 46:37-39. Rumen bacteriophage-lyzed bacterial strains of the genus Prevotella were isolated and preliminarily characterized. The strain TCl-1 the species P. bryantii was the only prevotella strain successfully infected with filter sterilized rumen fluid from a black-and-white Holstein cow. Two types of plaques were observed, both rather small and turbid. Preliminary electron microscopy observation showed that several morphologically different bacteriophages were present in these plaques. The plaque eluates were further used for the infection of other prevotella strains. The plaques produced by the bacteriophages were observed with two strains, i.e. P. bryantii B(1)4 and P. brevis GA33. The bacteriophages from both strains were examined by transmission electron microscopy and several morphologically different bacteriophages were observed, among others also a large virion with an icosahedral head with the diameter of approximately 120 nm. The bacteriophage was identified in plaques of bacterial cells of the strain GA33 and has an approximately 800 nm long helical tail, which places it among the largest ruminal bacteriophages described to date. Other bacteriophages from the same indicator strain as well as from P. bryantii B(1)4 strain were smaller and tail structures were not observed in all of them. [TOP OF PAGE]

  1187. Optimisation and standardisation of a method for detecting and enumerating bacteriophages infecting Bacteroides fragilis. Araujo,R., Muniesa,M., Mendez,J., Puig,A., Queralt,N., Lucena,F., Jofre,J. (2001). J. Virol. Meth. 93:127-136. A method for the detection and enumeration of bacteriophages infecting Bacteroides fragilis has been standardised. The recommended host strain is RYC2056 (ATCC 700786) because of the relatively high counts (10(4)-10(5) PFU/100ml) that it recovers in sewage from very different geographical areas. The addition of 0.25% bile to the culture and assay media and the manipulation of the host strain under strict anaerobic conditions resulted in a significant increase (more than 100%) in the number of phages detected. No other changes in the media and culture conditions resulted in changes in the phage counts detected. However, these increases do not justify changing the culture conditions and media described, taking into consideration that bile renders the media cloudy making it difficult to follow the host growth and that most laboratories do not have the facilities to work under strict anaerobic conditions. Nalidixic acid (100 microg/ml) and kanamycin (100 microg/ml) in the assay medium significantly reduce the background flora from polluted samples without affecting the phage counts. Freezing cultures just before the end of the log-phage growth at (-70+/-10) degrees C with BSA-sucrose as cryoprotector, storing of 1-2 ml in glass vials at (-70+/-10) degrees C and using them directly to inoculate fresh broth allows the obtention of cultures ready for phage enumeration in about 2.5 h. All these developments have been incorporated into a procedure that makes the method for detecting phages infecting B. fragilis as workable as the standardised methods available for the detection of coliphages. [TOP OF PAGE]

  1188. Isolation and characterization of bacteriophage-resistant mutants of Vibrio cholerae O139. Attridge,S.R., Fazeli,A., Manning,P.A., Stroeher,U.H. (2001). Microb. Pathog. 30:237-246. Vibrio cholerae O139 strains produce a capsule which is associated with complement resistance and is used as a receptor by bacteriophage JA1. Spontaneous JA1-resistant mutants were found to have several phenotypes, with loss of capsule and/or O-antigen from the cell surface. Determination of the residual complement resistance and infant mouse colonization potential of each mutant suggested that production of O-antigen is of much greater significance than the presence of capsular material for both of these properties. Two different in vitro assays of complement resistance were compared and the results of one shown to closely reflect the comparative recoveries of bacteria from the colonization experiments. Preliminary complementation studies implicated two rfb region genes, wzz and wbfP, as being essential for the biosynthesis of capsule but not O-antigen. [TOP OF PAGE]

  1189. Methods for isolation and titration of bacteriophages from Lactobacillus. Auad,L., Raya,R.R. (2001). pp. 203-208? In In Spencer,J.F.T. and Ragout de Spencer,A.L. (eds.), Food Microbiology Protocols. Humana Press, Totowa, New Jersey. [TOP OF PAGE]

  1190. Bacteriological and virological quality of seawater bathing areas along the Tyrrhenian coast. Aulicino,F.A., Orsini,P., Carere,M., Mastrantonio,A. (2001). International Journal of Environmental Health Research 11:5-11. Monitoring was carried out during summer 1997 along a selected area of the Tyrrhenian coast near the Tiber river mouth. Fifty-eight seawater samples, collected from 19 stations, were examined for coliforms, streptococci, Enteroviruses, Salmonellae, coliphages, Bacteroides fragilis phages, Pseudomonas, alophilic Vibrios, Aeromonas and yeasts. Salmonellae and coliphages were isolated in 3 and 12 out of 58 samples, respectively. Enteroviruses and Bacteroides fragilis phages were not isolated. Reoviruses were isolated only from 2 out of 58 samples. A limited number of samples of the northern stations located near the Tiber and other river mouths exceeded the guide values for bathing water by the EU Directive. All the southern stations, located near canals, were of very good microbiological quality. Pseudomonas, Vibrio, Aeromonas and yeasts were isolated from all stations and their values in 100 ml of seawater were 10-106, 10-106, 0-106 and 1-103, respectively. An extensive disinfection practice carried out on domestic wastes, which are discharged in rivers and canals, probably brought pollution levels of most stations to values within the bacterial standards. The spread of Pseudomonas, Aeromonas, etc. showed that all the coastal area studied was characterized by the presence of organic matter coming from land that can support the presence of opportunistic pathogens and other microbial flora. [TOP OF PAGE]

  1191. Persistence of viral pathogens and bacteriophages during sewage treatment: Lack of correlation with indicator bacteria. Baggi,F., Demarta,A., Peduzzi,R. (2001). Res. Microbiol. 152:743-751. The effects of different sewage treatments on the viral contamination in rivers which receive water from treatment plants without a final sand filtration step were investigated. They were all heavily contaminated with bacteriophages and human enteric viruses (detected by single step reverse transcription amplification followed by a nested polymerase chain reaction). Bacteriophages, but not faecal indicator organisms, were correlated with viral contamination. [TOP OF PAGE]

  1192. Luciferase reporter mycobacteriophages for detection, identification, and antibiotic susceptibility testing of Mycobacterium tuberculosis in Mexico. Banaiee,N., Bobadilla-del-Valle,M., Bardarov,S., Jr., Riska,P.F., Small,P.M., Ponce-de-Leon,A., Jacobs,W.R., Jr., Hatfull,G.F., Sifuentes-Osornio,J. (2001). J. Clin. Microbiol. 39:3883-3888. The utility of luciferase reporter mycobacteriophages (LRPs) for detection, identification, and antibiotic susceptibility testing of Mycobacterium tuberculosis was prospectively evaluated in a clinical microbiology laboratory in Mexico City, Mexico. Five hundred twenty-three consecutive sputum samples submitted to the laboratory during a 5-month period were included in this study. These specimens were cultivated in Middle-brook 7H9 (MADC), MGIT, and Lowenstein-Jensen (LJ) media. Of the 71 mycobacterial isolates recovered with any of the three media, 76% were detected with the LRPs, 97% were detected with the MGIT 960 method, and 90% were detected with LJ medium. When contaminated specimens were excluded from the analysis, the LRPs detected 92% (54 of 59) of the cultures. The median time to detection of bacteria was 7 days with both the LRPs and the MGIT 960 method. LRP detection of growth in the presence of p-nitro-alpha-acetylamino-beta-hydroxypropiophenone (NAP) was used for selective identification of M. tuberculosis complex (MTC) and compared to identification with BACTEC 460. Using the LRP NAP test, 47 (94%) out of 50 isolates were correctly identified as tuberculosis complex. The accuracy and speed of LRP antibiotic susceptibility testing with rifampin, streptomycin, isoniazid, and ethambutol were compared to those of the BACTEC 460 method, and discrepant results were checked by the conventional proportion method. In total, 50 MTC isolates were tested. The overall agreement between the LRP and BACTEC 460 results was 98.5%. The median LRP-based susceptibility turnaround time was 2 days (range, 2 to 4 days) compared to 10.5 days (range, 7 to 16 days) by the BACTEC 460 method. Phage resistance was not detected in any of the 243 MTC isolates tested. Mycobacteriophage-based approaches to tuberculosis diagnostics can be implemented in clinical laboratories with sensitivity, specificity, and rapidity that compare favorably with those of the MGIT 960 and BACTEC 460 methods. The phages currently provide the fastest phenotypic assay for susceptibility testing. [TOP OF PAGE]

  1193. Microbial indicator removal in onsite constructed wetlands for wastewater treatment in the southeastern U.S. Barrett,E.C., Sobsey,M.D., House,C.H., White,K.D. (2001). Water Sci. Technol. 44:177-182. Seven onsite constructed wetlands for wastewater treatment in the coastal plains of Alabama and North Carolina were studied from September 1997 to July 1998. Each site was examined for its ability to remove a range of fecal contamination indicators from settled wastewater. Indicator organisms include total and fecal coliforms, enterococci, Clostridium perfringens, and somatic and male-specific (F+) coliphages. Four identical domestic wastewater treatment sites in Alabama were evaluated. In these sites the Log10 geometric mean reductions ranged between 0.5 and 2.6 for total and fecal coliforms, 0.1 and 1.5 for enterococci, 1.2 to 2.7 for C. perfringens, -0.3 and 1.2 for somatic coliphages, and -0.2 and 2.2 for F+ coliphages. Three unique designs were examined in North Carolina. Log10 geometric mean reductions ranged between 0.8 to 4.2 for total and fecal coliforms, 0.3 to 2.9 for enterococci, 1.6 to 2.9 for C. perfringens, -0.2 and 2.8 for somatic coliphages, and -0.1 and 1.5 for F+ coliphages. Somatic and F+ coliphage detection was highly variable from month to month. [TOP OF PAGE]

  1194. The use of bacteriophages for treatment and prevention of bacterial disease in animals and animal models of human infection. Barrow,P.A. (2001). J. Chem. Technol. Biotechnol. 76:677-682. A brief history of the use of lytic bacteriophages in bacterial disease therapy is presented. After early disillusionment with the idea following poor experimental work, control of phages and field trials, studies were set up in the 1980's in the UK to study their use in farm animal infections. Work with E. coli septicaemia and diarrhoea has shown that phages can be highly effective prophylactically and therapeutically and more effective than antibiotics. There is considerable potential for their use in a limited number of infection types in both man and animals. [TOP OF PAGE]

  1195. Proteins PblA and PblB of Streptococcus mitis, which promote binding to human platelets, are encoded within a lysogenic bacteriophage. Bensing,B.A., Siboo,I.R., Sullam,P.M. (2001). Infect. Immun. 69:6186-6192. The binding of platelets by bacteria is a proposed central mechanism in the pathogenesis of infective endocarditis. Platelet binding by Streptococcus mitis strain SF100 (an endocarditis isolate) was recently shown to be mediated in part by the surface proteins PblA and PblB. The genes encoding PblA and PblB are clustered with genes nearly identical to those of streptococcal phages r1t, 01205, and Dp-1, suggesting that pblA and pblB might reside within a prophage. To address this possibility, cultures of SF100 were exposed to either mitomycin C or UV light, both of which are known to induce the lytic cycle of many temperate phages. Both treatments caused a significant increase in the transcription of pblA. Treatment with mitomycin C or UV light also caused a substantial increase in the expression of PblA and PblB, as detected by Western blot analysis of proteins in the SF100 cell wall. By electron microscopy, phage particles were readily visible in the supernatants from induced cultures of SF100. The phage, designated SM1, had a double-stranded DNA genome of approximately 35 kb. Southern blot analysis of phage DNA indicated that pblA and pblB were contained within the SM1 genome. Furthermore, Western blot analysis of phage proteins revealed that both PblA and PblB were present in the phage particles. These findings indicate that PblA and PblB are encoded by a lysogenic bacteriophage, which could facilitate the dissemination of these potential virulence determinants to other bacterial pathogens. [TOP OF PAGE]

  1196. Phage treatment: can we utilise it for certain infective diseases in India? Bhatia,R.S. (2001). Journal of the Association of Physicians of India 49:590 [TOP OF PAGE]

  1197. Bacteriophages transducing antibiotic resistance from a cluster of lysogenic strains of Pseudomonas aeruginosa isolated from patients. Blahová,J., Kralikova,K., Krcmery,V., Sr., Schafer,V. (2001). J. Chemother. 13:331-333. [TOP OF PAGE]

  1198. The genome of the archael virus SIRV1 has features in common with genomes of eukaryal viruses. Blum,H., Zillig,W., Mallock,S., Domdey,H., Prangishvili,D. (2001). Virology 281:6-9. The virus SIRV1 of the extremely thermophilic archaeon Sulfolobus has a double-stranded DNA genome similar in architecture to the genomes of eukaryal viruses of the families Poxviridae, Pycodnaviridae, and Asfarviridae: the two strands of the 32,301 bp long linear genome are covalently connected forming a continuous polynucleotide chain and 2029 kb long inverted repeats are present at the termini. Very likely it also shares with these viruses mechanisms of initiation of replication and resolution of replicative intermediates. [TOP OF PAGE]

  1199. Phylogeny, genome evolution, and host specificity of single-stranded RNA bacteriophage (family Leviviridae). Bollback,J.P., Huelsenbeck,J.P. (2001). J. Mol. Evol. 52:117-128. Bacteriophage of the family Leviviridae have played an important role in molecular biology where representative species, such as Qbeta and MS2, have been studied as model systems for replication, translation, and the role of secondary structure in gene regulation. Using nucleotide sequences from the coat and replicase genes we present the first statistical estimate of phylogeny for the family Leviviridae using maximum-likelihood and Bayesian estimation. Our analyses reveal that the coliphage species are a monophyletic group consisting of two clades representing the genera Levivirus and Allolevivirus. The Pseudomonas species PP7 diverged from its common ancestor with the coliphage prior to the ancient split between these genera and their subsequent diversification. Differences in genome size, gene composition, and gene expression are shown with a high probability to have changed along the lineage leading to the Allolevivirus through gene expansion. The change in genome size of the Allolevivirus ancestor may have catalyzed subsequent changes that led to their current genome organization and gene expression. [TOP OF PAGE]

  1200. Phages of Lactococcus lactis: an ecological and economical equilibrium. Boucher,I., Moineau,S. (2001). Rec. Res. Dev. Virol. 3:243-256. Lactic acid bacteria (LAB) are a group of organisms widely used in food fermentation. Interests in these microorganisms have increased sharply in the last decade; these organisms have even been dubbed the bugs of the new millennium. One distinctive fact about LAB-fermented foods is that they are produced in non-sterile conditions. Thus, LAB are susceptible to infection by lytic bacteriophages naturally present in these environments. Recent developments (by our group and others) in the field of bacteriophages of Lactococcus, the most studied LAB, are investigated and presented in the review. [TOP OF PAGE]

  1201. Faecal bacteria and bacteriophage inactivation in a full-scale UV disinfection system used for wastewater reclamation. Bourrouet,A., Garcia,J., Mujeriego,R., Penuelas,G. (2001). Water Sci. Technol. 43:187-194. A study was carried out to compare the inactivation of faecal bacteria and one type of bacteriophage in a full-scale UV disinfection system. The system is part of a water reclamation facility for effluent reuse in golf course and agricultural irrigation. Influent and effluent samples were taken over two sampling periods (three consecutive days in July and one day in August), with three different UV doses applied each day (ranging from 10 to 40 mWcntdots/cm2 and 20 to 80 mWcntdots/cm2 in July and August, respectively). Effluent samples were also taken from a chlorine disinfection channel (5 mg Cl2/L dose) operating in parallel to the UV system. Total coliforms (TC), faecal coliforms (FC), faecal streptoccoci (FS) and somatic coliphages (SC) were measured in each sample. F-specific RNA bacteriophages and bacteriophages of Bacteroides fragilis were also measured one day in July. The decay ratio observed for all the microorganisms was similar when UV doses applied were low (July), ranging from 1.15 to 1.25 log-units. This suggests that bacterial indicators may be suitable for virus inactivation control when low UV doses are applied; however, such low doses are inadequate to achieve effluent quality requirements for unrestricted irrigation. At higher UV doses (August), decay ratios for TC and FC were 3.1 and 2.8 log-units respectively, indicating that they were more susceptible to UV exposure than SC and FS, with decay ratios of 2.6 and 1.0 log-units, respectively. Nevertheless, these higher doses were also inadequate to achieve water quality requirements for unrestricted irrigation. The decay ratio of SC during chlorine disinfection was clearly lower than that of the other microorganisms. Bacteriophages of Bacteroides fragilis were more resistant to UV disinfection than SC and F-specific RNA. In fact, bacteriophages of Bacteroides fragilis were not affected during UV exposure. A UV dose ranging from 40 to 80 mWcntdots/cm2 marks the borderline beyond which inactivation rates of SC are clearly lower than those of bacterial indicators. [TOP OF PAGE]

  1202. Bacteriophage-bacteriophage interactions in the evolution of pathogenic bacteria. Boyd,E.F., Davis,B.M., Hochhut,B. (2001). Trends Microbiol. 9:137-144. Many bacteriophages carry virulence genes encoding proteins that play a major role in bacterial pathogenesis. Recently, investigators have identified bacteriophage-bacteriophage interactions in the bacterial host cell that also contribute significantly to the virulence of bacterial pathogens. The relationships between the bacteriophages pertain to one bacteriophage providing a helper function for another, unrelated bacteriophage in the host cell. Accordingly, these interactions can involve the mobilization of bacteriophage DNA by another bacteriophage, for example in Escherichia coli, Vibrio coli and Staphylococcus aureus; the host receptor for one bacteriophage being encoded by another, as found in V. cholerae; and the presence of one bacteriophage potentiating the virulence properties of another bacteriophage, as found in V. cholerae and Salmonella enterica. [TOP OF PAGE]

  1203. Detection of populations of phages of phytopathogenic bacterias and their biological properties. Boyko,A.L., Semchuk,L.I., Romashev,S.A., Andriychuk,L.N. (2001). Bulletin of the Agroscience (8), 51-53. Excreted bacteriophages to X. axonopodis pv. beticola, E. carÏtovora, P. syringae pv. atrofaciens in the Kiev area. The bacteriophages showed specific specificity, were look-alike on morphology, sizes, are stable at long-term storage and in range pH 4-10. The role of populations of phages in biocenoses and capability of indication of pathogens with their help is discussed. [TOP OF PAGE]

  1204. An error-prone T7 RNA polymerase mutant generated by directed evolution. Brakmann,S., Grzeszik,S. (2001). Chembiochem : a European journal of chemical biology 2:212-219. Viruses replicate their genomes at exceptionally high mutation rates. Their offspring evolve rapidly and therefore, are able to evade common immunological and chemical antiviral agents. In parallel, virus genomes cannot tolerate a further increase in mutation rate: Experimental evidence exists that even few additional mutations are sufficient for the extinction of a viral population. A future antiviral strategy might therefore aim at increasing the error-producing capacity of viral replication enzymes. We employed the principles of directed evolution and developed a scheme for the stringent positive selection of error-prone polymerase activity. A mutant T7 RNA polymerase with a nucleotide substitution error rate at least 20-fold greater than that of the wild-type was selected. This enzyme synthesized highly heterogeneous RNA products in vitro or in vivo and also decreased the replication efficiency of wild-type bacteriophage T7 during infection. [TOP OF PAGE]

  1205. Phage-related DNA polymorphism in dairy and probiotic Lactobacillus. Brandt,K., Tilsala-Timisjarvi,A., Alatossava,T. (2001). Micron 32:59-65. Various DNA-based methods are presently being applied for identification of industrial bacterial cultures including dairy starter and probiotic strains of Lactobacillus. The success of strain-specific identification depends on the power of the DNA-based methods to reveal intraspecies DNA polymorphism. This study reveals that all eleven arbitrarily chosen Lactobacillus rhamnosus starter, laboratory and probiotic strains contain Lb. rhamnosus phage Lc-Nu related nucleotide sequences. One of these highly homologous regions in the genome of phage Lc-Nu was the 2.4 kb HindIII fragment, which has been sequenced. Nucleotide sequence analysis suggested that one side of the 2.4 kb HindIII fragment encodes a phage Lc-Nu helicase and accordingly represents an early gene region of phage Lc-Nu genome. Five forward and five reverse primers were derived from the nucleotide sequence of the 2.4 kb HindIII fragment of phage Lc-Nu DNA for PCR-based identification of the eleven Lb. rhamnosus strains included in this study. Six different types of PCR product patterns were obtained. Among the patterns three were unique to particular Lb. rhamnosus strains. The results suggest that phage-related DNA sequences are, surprisingly, distributed widely among the Lb. rhamnosus strains, and that these sequences could also be a source of DNA polymorphism to apply for DNA-based identification of bacterial strains. Phage Lc-Nu related DNA homology was also found in the chromosome of Lb. casei, the species closely related to Lb. rhamnosus. [TOP OF PAGE]

  1206. First evidence for a restriction-modification system in Leptospira sp. Brenot,A., Werts,C., Ottone,C., Sertour,N., Charon,N.W., Postic,D., Baranton,G., Saint Girons,I. (2001). FEMS Microbiol. Lett. 201:139-143. The LE1 leptophage exhibited a host range restricted to the saprophytic Leptospira biflexa [Saint Girons et al., Res. Microbiol. 141 (1990) 1131-1133] and mainly to the Patoc 1 strain (hereafter called PFRA) kept in the Paris, France collection. Results of titration of LE1 lysates indicated the presence of a host-controlled modification and restriction system within PUSA (Patoc 1 strain maintained in the Morgantown, WV, USA collection) that was absent in PFRA. Because genomic DNA of PITAL (Patoc 1 strain maintained in Trieste, Italy) appeared smeared in pulsed field gel electrophoresis (PFGE), this strain is likely to contain nucleases that are activated upon DNA isolation. Moreover, comparative NotI digestions of PUSA and PFRA DNAs, as visualized by PFGE, indicated that PUSA belonged to a different serovar than PFRA. Finally, 16S ribosomal sequence analysis indicated that PUSA belonged to the saprophytic Leptospira meyeri species, while PITAL and PFRA appertained to L. biflexa. The evolutionary significance and the importance of the restriction and modification enzymes or non-specific nucleases within strains for genetic experiments are discussed. [TOP OF PAGE]

  1207. Transport of biologically active substances through gravel strata. Bricelj,M., Sedmak,B. (2001). pp. 25-29. In In Seiler,K.-P. and Wohnlich,S. (eds.), New approaches characterizing grounfwater flow : proceedings of the XXXI International Association of hydrogeologists Congress, Munich, Germany, 10-14 September 2001. Balkema, Lisse. [TOP OF PAGE]

  1208. Selecting a sensitive bacteriophage assay for evaluation of a prototype water recycling system. Brion,G.M., Silverstein,J. (2001). Life Support Biosph. Sci. 8:9-14. A rapid, simple, and direct (RSD) assay of eluate from filter concentration was developed to enumerate low numbers of MS2 bacteriophage, used as a surrogate for enteric viruses, from samples collected from a prototype-sized water recycling system. The RSD assay utilized a 50-ml eluate volume in a modified single-layer assay, neutralizing eluate pH by buffered, double-strength agar. The RSD assay developed was simpler and minimized sample-handling steps compared with another published method. The RSD assay method showed greater sensitivity than the other published method for recovering phage from filter eluate while avoiding pH shifts, which can inactivate phage. Grant numbers: NAGW 2356. [TOP OF PAGE]

  1209. Analysis of the complete DNA sequence of the temperate bacteriophage TP901-1: Evolution, structure, and genome organization of lactococcal bacteriophages. Brondsted,L., Ostergaard,S., Pedersen,M., Hammer,K., Vogensen,F.K. (2001). Virology 283:93-109. A complete analysis of the entire genome of the temperate lactococcal bacteriophage TP901-1 has been performed and the function of 21 of 56 TP901-1-encoded ORFs has been assigned. This knowledge has been used to propose 10 functional modules each responsible for specific functions during bacteriophage TP901-1 proliferation. Short regions of microhomology in intergenic regions present in several lactococcal bacteriophages and chromosomal fragments of Lactococcus lactis are suggested to be points of exchange of genetic material through homologous recombination. Our results indicate that TP901-1 may have evolved by homologous recombination between the host chromosome and a mother phage and support the observation that phage remnants as well as prophages located in the Lactococcus chromosome contribute significantly to bacteriophage evolution. Some proteins encoded in the early transcribed region of the TP901-1 genome were more homologous to proteins encoded by phages infecting gram-positive hosts other than L. lactis. This protein homology argues for the occurrence of horizontal genetic exchange among these bacteriophages and indicates that they have access to a common gene pool. [TOP OF PAGE]

  1210. Induction of lysogenic bacteriophage and phage-associated toxin from group A streptococci during coculture with human pharyngeal cells. Broudy,T.B., Pancholi,V., Fischetti,V.A. (2001). Infect. Immun. 69:1440-1443. We found that when group A streptococci are cocultured with human pharyngeal cells, they upregulate and secrete a 25-kDa toxin, determined to be the bacteriophage-encoded streptococcal pyrogenic exotoxin C (SpeC). This prompted us to determine if the bacteriophage themselves are induced during coculture conditions. We found that bacteriophage induction does occur, resulting in the release of ~105 phage particles during the 3-h coculture. Furthermore, we show that the bacteriophage induction event is mediated by a pharyngeal cell soluble factor for which we provide an initial characterization. [TOP OF PAGE]

  1211. Collective action in an RNA virus. Brown,S.P. (2001). J. Evol. Biol. 14:821-828. A recent empirical study by Turner and Chao on the evolution of competitive interactions among phage virus strains revealed that a strain grown at high rates of co-infection evolved towards lowered fitness relative to an ancestral strain. The authors went on to show that the fitness pay-off matrix between the evolved and ancestral strain conforms to the prisoners' dilemma. In this paper, I use Turner and Chao's data to parameterize a simple model of parasite collective action. The prisoners' dilemma is based on pairwise interactions of a discrete cooperate/defect nature. In contrast, the collective action model explicitly deals with individual-group interactions where the extent of cooperation is a continuous variable. I argue here that the 'collective action' modelling approach is more appropriate than the prisoners' dilemma for the biology of virus evolution, and hence better able to form a predictive framework for further work on related strains of virus, linking mixing ecology, cooperative phenotype and fitness. Furthermore, the collective action model is used to motivate discussion on the evolutionary ecology of viruses, with a focus on the 'levels of selection' debate and the evolution of virulence. [TOP OF PAGE]

  1212. Phages of dairy bacteria. Brüssow,H. (2001). Ann. Rev. Microbiol. 55:283-303. Bacteriophages of lactic acid bacteria are a threat to industrial milk fermentation. Owing to their economical importance, dairy phages became the most thoroughly sequenced phage group in the database. Comparative genomics identified related cos-site and pac-site phages, respectively, in lactococci, lactic streptococci and lactobacilli. Each group was represented with closely related temperate and virulent phages. Over the structural genes their gene maps resembled that of lambdoid coliphages, suggesting distant evolutionary relationships. Despite a lack of sequence similarity, a number of biochemical characteristics of these dairy phages are l-like (genetic switch, DNA packaging, head and tail morphogenesis, and integration, but not excision). These dairy phages thus provide interesting variations to the phage l paradigm. The structural gene cluster of Lactococcus phage r1t resembled that of phages from mycobacteria. Virulent lactococcal phages with prolate heads (c2-like genus of Siphoviridae), in contrast, have no known counterparts in other bacterial genera. [TOP OF PAGE]

  1213. Comparative phage genomics and the evolution of Siphoviridae: Insights from dairy phages. Brüssow,H., Desiere,F. (2001). Mol. Microbiol. 39:213-222. Comparative phage genomics can retrace part of the evolutionary history of phage modules encoding phage-specific functions such as capsid building or establishment of the lysogenic state. The diagnosis of relatedness is not based exclusively on sequence similarity, but includes topological considerations of genome organization. The gene maps from the lambda-, psiM2-, L5-, Sfi21-, Sfi11-, phiC31-, sk1- and TM4-like phages showed a remarkable synteny of their structural genes defining a lambda supergroup within Siphoviridae (Caudovirales with long non-contractile tails). A hierarchy of relatedness within the lambda supergroup suggested elements of vertical evolution in the capsid module of Siphoviridae. Links to P22-like Podoviridae and P2-like Myoviridae were also detected. Numerous cases of horizontal gene transfer were observed, but recent transfers were limited to interbreeding phage populations. We suggest that tailed phages are the result of both vertical and horizontal evolution and are thus a good model system for web-like phylogenies. [TOP OF PAGE]

  1214. A general mechanism for viral resistance to suicide gene expression. Bull,J.J., Badgett,M.R., Molineux,I.J. (2001). J. Mol. Evol. 53:47-54. Bacteriophage T7 was challenged with either of two toxic genes expressed from plasmids. Each plasmid contained a different gene downstream of a T7 promoter; cells harboring each plasmid caused an infection by wild-type T7 to abort. T7 evolved resistance to both inhibitors by avoidance of the plasmid expression system rather than by blocking or bypassing the effects of the specific toxic gene product. Resistance was due to a combination of mutations in the T7 RNA polymerase and other genes expressed at the same time as the polymerase. Mutations mapped to sites that are unlikely to alter polymerase specificity for its cognate promoter but the basis for discrimination between phage and plasmid promoters in vivo was not resolved. A reporter assay indicated that, relative to wild-type phage, gene expression from the plasmid was diminished several-fold in cells infected by the evolved phages. A recombinant phage, derived from the original mutant but lacking a mutation in the gene for RNA polymerase, exhibited in-termediate activity in the reporter assay and intermediate resistance to the toxic gene cassettes. Alterations in both RNA polymerase and a second gene are thus responsible for resistance. These findings have broad evolutionary parallels to other systems in which viral inhibition is activated by viral regulatory signals such as defective-interfering particles, and they may have mechanistic parallels to the general phenomena of position effects and gene silencing. [TOP OF PAGE]

  1215. Generalized transduction in Streptomyces coelicolor. Burke,J., Schneider,D., Westpheling,J. (2001). Proc. Natl. Acad. Sci. USA 98:6289-6294. We report the isolation of generalized transducing phages for Streptomyces species able to transduce chromosomal markers or plasmids between derivatives of Streptomyces coelicolor, the principal genetic model system for this important bacterial genus. We describe four apparently distinct phages (DAH2, DAH4, DAH5, and DAH6) that are capable of transducing multiple chromosomal markers at frequencies ranging from 10-5 to 10-9 per plaque-forming unit. The phages contain DNA ranging in size from 93 to 121 kb and mediate linked transfer of genetic loci at neighboring chromosomal sites sufficiently close to be packaged within the same phage particle. The key to our ability to demonstrate transduction by these phages was the establishment of conditions expected to severely reduce superinfection killing during the selection of transductants. The host range of these phages, as measured by the ability to form plaques, extends to species as distantly related as Streptomyces avermitilis and Streptomyces verticillus, which are among the most commercially important species of this genus. Transduction of plasmid DNA between S. coelicolor and S. verticillus was observed at frequencies of approximately 10-4 transductants per colony-forming unit. [TOP OF PAGE]

  1216. The origins and evolution of viruses. Campbell,A. (2001). Trends Microbiol. 9:61 [TOP OF PAGE]

  1217. Chemical and microbial characterization of household graywater. Casanova,L.M., Gerba,C.P., Karpiscak,M. (2001). J. Environ. Sci. Health Part A Tox. Hazard. Subst. Environ. Eng. 36:395-401. In arid areas, the search for efficient methods to conserve water is of paramount importance. One of the methods of water conservation available today is graywater recycling--the reuse of water from the sinks, showers, washing machine, and dishwasher in a home. The purpose of this project was to characterize the chemical and microbial quality of graywater from a single-family home with two adults. Water samples from a graywater holding tank were analyzed over a seven-month period for total coliforms, fecal coliforms, fecal streptococci, Staphylococcus aureus (S. aureus), Pseudomonas aeruginosa (P. aeruginosa), and coliphages. The pH, turbidity, biological oxygen demand (BOD), suspended solids (SS), electrical conductivity (EC), sulfates (SO4), and chlorides (Cl) were also measured. The mean numbers of total coliforms, fecal coliforms, fecal streptococci, and P. aeruginosa were 8.03 x 107, 5.63 x 105, 2.38 x 102, and 1.99 x 104 CFU/100 mL, respectively. S. aureus and coliphages were not detected. In the chemical analysis, mean values of 7.47 for pH, 43 nephelometric turbidity units (NTU) for turbidity, 64.85 mg/L for BOD, 35.09 mg/L for SS, 0.43 mS/cm for EC, 59.59 mg/L for SO4, and 20.54 mg/L for Cl were measured. These data were compared to data taken in 1986 and 1987, when two adults and one child lived in the household. Analysis showed no statistically significant difference in levels of total coliforms and suspended solids between the two data sets. There were statistically significant differences in levels of fecal coliforms, pH, turbidity, chlorides, sulfates, and BOD between the two households. Fecal coliforms, turbidity, and BOD were higher in the household with two adults and one child. Levels of Cl, SO4, and pH were higher in the household with two adults. [TOP OF PAGE]

  1218. Microbial population dynamics and diversity during a bloom of the marine coccolithophorid Emiliania huxleyi (Haptophyta). Castberg,T., Larsen,A., Sandaa,R.-A., Brussaard,C.P.D., Egge,J.K., Heldal,M., Thyrhaug,R., van Hannen,E.J., Bratbak,G. (2001). Mar. Ecol. Prog. Ser. 221:39-46. Several previous studies have shown that Emiliania huxleyi blooms and terminations have been succeeded by an increase in large virus-like particles (LVLP), strongly suggesting the bloom collapse was caused by viral lysis. However, due to methodological limitations, knowledge of how such blooms affect the rest of the microbial community is limited. In the current study we induced a bloom of E. huxleyi in seawater enclosures and applied methods enabling us to describe the algae, bacteria and virus communities with greater resolution than has been done previously, The development of the dominating algal, viral and bacterial populations in the nutrient-amended seawater enclosures was followed by flow cytometry (FCM). Light microscopy (LM), PCR-denaturing gradient gel electrophoresis (PCR-DGGE) and pulsed-field gel electrophoresis (PFGE) were used to describe the changes in community composition in greater detail. The algal community was dominated by E. huxleyi until termination of the bloom by viral lysis, After bloom termination the additional algal populations present in the enclosures increased in abundance. A marked increase in viruses other than the one infecting E. huxleyi was also observed. Total bacterial number and community composition were also greatly influenced by the bloom and its collapse. [TOP OF PAGE]

  1219. Water quality improvement of treated wastewater by intermittent soil percolation. Castillo,G., Mena,M.P., Dibarrart,F., Honeyman,G. (2001). Water Sci. Technol. 43:187-190. Our research aimed to evaluate intermittent soil infiltration of treated sewage for reuse in the north of Chile. Aerated lagoon effluent was infiltrated in columns packed with native soils (sandy-lime, lime-gravel and limey-sand). Columns were operated for more than a year under different cycles of filling and drying, depths and load pressures depending on soil characteristics. The efficiency of the system was determined through influent-effluent microbiological indicators level (faecal coliforms, E. coli, Salmonella spp, MS2 phage, and protozoan cysts), physicochemical characterisation (TOC, COD, BOD, nitrogen), and hydraulic flow measurement. Results showed: (a) high reduction of enteric bacteria (5-7 log10), some inactivation of phage (2-4 log10) and complete removal of intestinal cyst; (b) stable removal of organic matter (80-90% reduction of TOC, COD, BOD); and (c) partial ammonia reduction through adsorption and nitrification with denitrification mainly occurring in sandy soil. Preliminary data from pilot plant working in the field showed better results that those obtained in the laboratory especially removal of microbiological indicators. Microbiological quality of effluent met Class A regulations for agricultural reuse (WHO, 1989) and the system looks like an attractive alternative to cope with water shortage in the region. [TOP OF PAGE]

  1220. Rapid Identification of Escherichia coli O157:H7. Chang,T.C., Chen,S., Ding,H.C. (2001). Food Industry Research and Development Institute. 907696(6,210,911). Hsinchu, TW. A method of determining whether a test microorganism is a known microorganism, involving use of an agent that specifically affects the growth of the known microorganism. The invention also features a method of identifying E. coli O157:H7 that are based on the following criteria: a test microorganism is E. coli O157:H7 if the microorganism is (i) E. coli, (ii) incapable of fermenting sorbitol, and (iii) susceptible to infection by AR1 phage. [TOP OF PAGE]

  1221. Phages and their application against drug-resistant bacteria. Chanishvili,N., Chanishvili,T., Tediashvili,M., Barrow,P.A. (2001). J. Chem. Technol. Biotechnol. 76:689-699. At the beginning of the 20th century the phenomenon of spontaneous bacterial lysis was discovered independently by Twort and d'Herelle. Despite the suggestion at that time by d'Herelle that these agents might be applied to the control of bacterial diseases in the west this idea was explored in a desultory fashion only and was eventually discarded largely due to the advent of extensive antibiotic usage. However, interest was maintained in countries of the former Soviet Union where bacteriophage therapy has been applied extensively since that time. Central to this work was the Eliava Institute of Bacteriophage, Microbiology and Virology in Tbilisi, Georgia, which was founded in 1923 through the joint efforts of d'Herelle and the Georgian George Eliava. Ironically, given his contributions to public health in the Soviet Union, Eliava was branded as an enemy of the people in 1937 and executed. d'Herelle never again returned to Georgia. In spite of these tragic events this institute remained the focus for phage therapy in the world and despite being continuously active in this field for 75 years, now struggles for its financial life. In the Eliava Institute, phages were sought for bacterial pathogens implicated in disease outbreaks in different parts of the Soviet Union and were dispatched for use in hospitals throughout the country. Although infections caused by a wide variety of bacterial pathogens have been treated, much of this has been published in Russian and is not readily available in the west. Work has also been carried out in Poland over many years and this has only recently been published in English. By contrast, interest in the west has been limited to a small number of enthusiasts and academics and until very recently little interest has been shown. The main reason that the medical and scientific communities are now beginning to take notice, is the continuing world-wide rise in the incidence of multiply-antibiotic-resistant bacterial pathogens and the absence of effective means for their control. Recent publicity over the work of the Eliava Institute has concentrated the minds of the western world on the potential for infectious disease control that bacteriophage offer, a procedure that is biologically more acceptable than antibiotic use and which has been in use for several decades already. [TOP OF PAGE]

  1222. Application of digital image analysis and flow cytometry to enumerate marine viruses stained with SYBR gold. Chen,F., Lu,J.R., Binder,B.J., Liu,Y.C., Hodson,R.E. (2001). Appl. Environ. Microbiol. 67:539-545. A novel nucleic acid stain, SYBR Gold, was used to stain marine viral particles in various types of samples. Viral particles stained with SYBR Gold yielded bright and stable fluorescent signals that could be detected by a cooled charge-coupled device camera or by flow cytometry. The fluorescent signal strength of SYBR Gold-stained viruses was about twice that of SYBR Green I-stained viruses. Digital images of SYBR Gold-stained viral particles were processed to enumerate the concentration of viral particles by using digital image analysis software. Estimates of viral concentration based on digitized images were 1.3 times higher than those based on direct counting by epifluorescence microscopy. Direct epifluorescence counts of SYBR Gold-stained viral particles were in turn about 1.34 times higher than those estimated by the transmission electron microscope method. Bacteriophage lysates stained with SYBR Gold formed a distinct population in flow cytometric signatures. Flow cytometric analysis revealed at least four viral subpopulations for a Lake Erie sample and two subpopulations for a Georgia coastal sample. Flow cytometry-based viral counts for various types of samples averaged 1.1 times higher than direct epifluorescence microscopic counts. The potential application of digital image analysis and flow cytometry for rapid and accurate measurement of viral abundance in aquatic environments is discussed. [TOP OF PAGE]

  1223. Analysis of six prophages in Lactococcus lactis IL1403: Different genetic structure of temperate and virulent phage populations. Chopin,A., Bolotin,A., Sorokin,A., Ehrlich,S.D., Chopin,M.C. (2001). Nucleic Acids Research 29:644-651. We report the genetic organisation of six prophages present in the genome of Lactococcus lactis IL1403. The three larger prophages (36-42 kb), belong to the already described P335 group of temperate phages, whereas the three smaller ones (13-15 kb) are most probably satellites relying on helper phage(s) for multiplication. These data give a new insight into the genetic structure of lactococcal phage populations. P335 temperate phages have variable genomes, sharing homology over only 10-33% of their length. In contrast, virulent phages have highly similar genomes sharing homology over >90% of their length. Further analysis of genetic structure in all known groups of phages active on other bacterial hosts such as Escherichia coli, Bacillus subtilis, Mycobacterium and Streptococcus thermophilus confirmed the existence of two types of genetic structure related to the phage way of life. This might reflect different intensities of horizontal DNA exchange: low among purely virulent phages and high among temperate phages and their lytic homologues. We suggest that the constraints on genetic exchange among purely virulent phages reflect their optimal genetic organisation, adapted to a more specialised and extreme form of parasitism than temperate/lytic phages. [TOP OF PAGE]

  1224. Nucleotide sequence of coliphage HK620 and the evolution of lambdoid phages. Clark,A.J., Inwood,W., Cloutier,T., Dhillon,T.S. (2001). J. Mol. Biol. 311:657-679. HK620 is a temperate lambdoid bacteriophage that adsorbs to the O-antigen of its host, Escherichia coli H. The genome of a temperature-sensitive clear-plaque mutant consists of 38,297 nucleotides in which we recognize 60 open reading frames (orfs). Eighteen of these lie in a region of the genome that we call the virion structure domain. The other 42 orfs lie in what we call the metabolic domain.Virions of HK620 resemble those of phage P22. The virion structural orfs encode three kinds of putative proteins relative to the virion proteins of P22: (1) those that are nearly (about 90 %) identical; (2) those that are weakly (about 30 %) identical; and (3) those composed of nearly and weakly identical segments. We hypothesize that these composite proteins form bridges between the virion proteins of the other two kinds.Three of the putative virion proteins that are only weakly identical to P22 proteins are 71, 60 and 79 % identical to proteins encoded by the phage APSE-1, whose virions also resemble those of P22. Because the hosts of APSE-1 and HK620 have been separated from each other by an estimated 200 My, we propose using the amino acid differences that have accumulated in these proteins to estimate a biological clock for temperate lambdoid phages.The putative transcriptional regulatory gene circuitry of HK620 seems to resemble that of phage lambda. Integration, on the other hand, resembles that of satellite phage P4 in that the attP sequence lies between the leftward promoter and int rather than downstream of int.Comparing the metabolic domains of several lambdoid phage genomes reveals seven short conserved sequences roughly defining boundaries of functional modules. We propose that these boundary sequences are foci of genetic recombination that serve to assort the modules and make the metabolic domain highly mosaic genetically. [TOP OF PAGE]

  1225. Traditional and molecular approaches to improving bacteriophage resistance of Cheddar and Mozzarella starters. Coffey,A., Stokes,D., Fitzgerald,G.F., Ross,R.P. (2001). Irish Journal of Agriculture and Food Research 40:239-271. [TOP OF PAGE]

  1226. Bacteriophage T4 multiplication in a glucose-limited Escherichia coli biofilm. Corbin,B.D., McLean,R.J.C., Aron,G.M. (2001). Can. J. Microbiol. 47:680-684. An Escherichia coli K-12 biofilm was grown at a dilution rate of 0.028 h-1 for 48 h in a glucose-limited chemostat coupled to a modified Robbins' device to determine its susceptibility to infection by bacteriophage T4. Bacteriophage T4 at a multiplicity of infection (MOI) of 10 caused a log reduction in biofilm density (expressed as colony forming units (CFU) per cm2) at 90 min postinfection. After 6 h, a net decrease and equilibrium in viral titer was seen. When biofilms were exposed to T4 phage at a MOI of 100, viral titer doubled after 90 min. After 6 h, viral titers (expressed as plaque forming units (PFU) per cm2) stabilized at levels approximately one order of magnitude higher than seen at a MOI of 10. Scanning confocal laser microscopy images also indicated disruption of biofilm morphology following T4 infection with the effects being more pronounced at a MOI of 100 than at a MOI of 10. These results imply that biofilms under carbon limitation can act as natural reservoirs for bacteriophage and that bacteriophage can have some influence on biofilm morphology. [TOP OF PAGE]

  1227. Inhibition of spontaneous induction of lambdoid prophages in Escherichia coli cultures: simple procedures with possible biotechnological applications. Czyz,A., Los,M., Wrobel,B., Wegrzyn,G. (2001). BMC Biotechnol. 1:1 BACKGROUND: Infections of bacterial cultures by bacteriophages are serious problems in biotechnological laboratories. Apart from such infections, prophage induction in the host cells may also be dangerous. Escherichia coli is a commonly used host in biotechnological production, and many laboratory strains of this bacterium harbour lambdoid prophages. These prophages may be induced under certain conditions leading to phage lytic development. This is fatal for further cultivations as relatively low, though still significant, numbers of phages may be overlooked. Thus, subsequent cultures of non-lysogenic strains may be infected and destroyed by such phage. RESULTS: Here we report that slow growth of bacteria decreases deleterious effects of spontaneous lambdoid prophage induction. Moreover, replacement of glucose with glycerol in a medium stimulates lysogenic development of the phage after infection of E. coli cells. A plasmid was constructed overexpressing the phage 434 cI gene, coding for the repressor of phage promoters which are necessary for lytic development. Overproduction of the cI repressor abolished spontaneous induction of the limm434 prophage. CONCLUSIONS: Simple procedures that alleviate problems with spontaneous induction of lambdoid prophage and subsequent infection of E. coli strains by these phages are described. Low bacterial growth rate, replacement of glucose with glycerol in a medium and overproduction of the cI repressor minimise the risk of prophage induction during cultivation of lysogenic bacteria and subsequent infection of other bacterial strains. [TOP OF PAGE]

  1228. [Current clinical application of bacteriophages and perspectives for their genetic modifications]. Dabrowska,K., Bus,R., Mazur,A., Weber-Dabrowska,B., Mulczyk,M., Gorski,A. (2001). Polskie Archiwum Medycyny Wewnetrznej 105:85-90. [TOP OF PAGE]

  1229. Clinical and environmental isolates of Vibrio cholerae serogroup O141 carry the CTX phage and the genes encoding the toxin-coregulated pili. Dalsgaard,A., Serichantalergs,O., Forslund,A., Lin,W., Mekalanos,J., Mintz,E., Shimada,T., Wells,J.G. (2001). J. Clin. Microbiol. 39:4086-4092. We report sporadic cases of a severe gastroenteritis associated with Vibrio cholerae serogroup O141. Like O1 and O139 serogroup strains of V. cholerae isolated from cholera cases, the O141 clinical isolates carry DNA sequences that hybridize to cholera toxin (CT) gene probes. The CT genes of O1 and O139 strains are carried by a filamentous bacteriophage (termed CTX phage) which is known to use toxin-coregulated pili (TCP) as its receptor. In an effort to understand the mechanism of emergence of toxigenic O141 V. cholerae, we probed a collection of O141 clinical and environmental isolates for genes involved in TCP production, toxigenicity, virulence regulation, and other phylogenetic markers. The collection included strains isolated between 1964 and 1995 from diverse geographical locations, including eight countries and five U.S. states. Information collected about the clinical and environmental sources of these isolates suggests that they had no epidemiological association. All clinical O141 isolates hybridized to probes specific for genes encoding CT (ctx), zonula occludens toxin (zot), repetitive sequence 1 (RS1), RTX toxin (rtxA), the major subunit of TCP (tcpA), and the essential regulatory gene that controls expression of both CT and TCP (toxR). In contrast, all but one of the nonclinical O141 isolates were negative for ctx, zot, RS1, and tcpA, although these strains were positive for rtxA and toxR. The one toxigenic environmental O141 isolate was also positive for tcpA. Ribotyping and CT typing showed that the O141 clinical isolates were indistinguishable or closely related, while a toxigenic water isolate from Louisiana showed a distantly related ribotype. Nonclinical O141 isolates displayed a variety of unrelated ribotypes. These data support a model for emergence of toxigenic O141 that involves acquisition of the CTX phage sometime after these strains had acquired the pathogenicity island encoding TCP. The clonal nature of toxigenic O141 strains isolated from diverse geographical locations suggests that the emergence is a rare event but that once it occurs, toxigenic O141 strains are capable of regional and perhaps even global dissemination. This study stresses the importance of monitoring V. cholerae non-O1, non-O139 serogroup strains for their virulence gene content as a means of assessing their epidemic potential. [TOP OF PAGE]

  1230. A link between virulence and ecological abundance in natural populations of Staphylococcus aureus. Day,N.P., Moore,C.E., Enright,M.C., et al. (2001). Science 292:59-60. Staphylococcus aureus is a major cause of severe infection in humans and yet is carried without symptoms by a large proportion of the population. We used multilocus sequence typing to characterize isolates of S. aureus recovered from asymptomatic nasal carriage and from episodes of severe disease within a defined population. We identified a number of frequently carried genotypes that were disproportionately common as causes of disease, even taking into account their relative abundance among carriage isolates. The existence of these ecologically abundant hypervirulent clones suggests that factors promoting the ecological fitness of this important pathogen also increase its virulence. [TOP OF PAGE]

  1231. Estimation of the average burst size of Phix174 am3, cs70 for use in mutation assays with transgenic mice. Delongchamp,R.R., Valentine,C.R., Malling,H.V. (2001). Environmental and Molecular Mutagenesis 37:356-360. In mutation assays using transgenic mice, with recoverable vectors such as PhiX174 am3, cs70, mutations originate from two sources: (1) in vivo mutations, that is, mutations that were fixed in the mouse, or (2) ex vivo mutations, that is, mutations that were fixed during recovery or plating. When a bacteriophage infects a bacterium, it multiplies and bursts the cell, releasing a number of phages referred to as the burst size. Our method for distinguishing between in vivo mutations and ex vivo mutations estimates the average number of bursts, the denominator of in vivo mutant frequencies, by dividing the total plaque-forming units (PFU) by the average number of phages in a burst. Herein, we outline a probability model relating observed plaque counts to the burst size and present the statistical method used to estimate the burst size. The average size of a single burst from nonrevertant phages was estimated in eight studies under the conditions of our mutation assay. The average burst size was stable across studies at 182.5 plaques per burst (standard error, 14.25). The probability that a burst is a specific size was approximated by a negative binomial distribution, which implies a Poisson-Pascal distribution for the observed plaque counts. The observed plaque counts were adequately fit by this approximation. [TOP OF PAGE]

  1232. Use of bacteriophage for elimination of Vibrio vulnificus from oysters. Depaola,A., Gulig,P.A., Smith,J.G., et al. (2001). 570 [p67]. Orlando, FL, American Society for Microbology 101st General Meeting. 5-24-0001.[TOP OF PAGE]

  1233. Comparative genomics of lactococcal phages: insight from the complete genome sequence of Lactococcus lactis phage BK5-T. Desiere,F., Mahanivong,C., Hillier,A.J., Chandry,P.S., Davidson,B.E., Brüssow,H. (2001). Virology 283:240-252. Lactococcus lactis phage BK5-T and Streptococcus thermophilus phage Sfi21, two cos-site temperate Siphoviridae with 40-kb genomes, share an identical genome organization, sequence similarity at the amino acid level over about half of their genomes, and nucleotide sequence identity of 60% over the DNA packaging and head morphogenesis modules. Siphoviridae with similarly organized genomes and substantial protein sequence similarity were identified in several genera of low-GC-content Gram-positive bacteria. These phages demonstrated a gradient of relatedness ranging from nucleotide sequence similarity to protein sequence similarity to gene map similarity over the DNA packaging and head morphogenesis modules. Interestingly, the degree of relatedness was correlated with the evolutionary distance separating their bacterial hosts. These observations suggest elements of vertical evolution in phages. The structural genes from BK5-T shared no sequence relationships with corresponding genes/proteins from lactococcal phages belonging to distinct lactococcal phage species, including phage sk1 (phage species 936) that showed a closely related gene map. Despite a clearly distinct genome organization, lactococcal phages sk1 and c2 showed nine sequence-related proteins. Over the early gene cluster phage BK5-T shared nine regions of high nucleotide sequence similarity, covering at most two adjacent genes, with lactococcal phage r1t (phage species P335). Over the structural genes, the closest relatives of phage r1t were not lactococcal phages belonging to other phage species, but Siphoviridae from Mycobacteria (high-GC-content Gram-positive bacteria). Evidence for recent horizontal gene transfer between distinct phage species was obtained for dairy phages, but these transfers were limited to phages infecting the same bacterial host species. [TOP OF PAGE]

  1234. Comparative genomics reveals close genetic relationships between phages from dairy bacteria and pathogenic streptococci: evolutionary implications for prophage-host interactions. Desiere,F., McShan,W.M., van,S., Ferretti,J.J., Brüssow,H. (2001). Virology 288:325-341. The genome of the highly pathogenic M1 serotype Streptococcus pyogenes isolate SF370 contains eight prophage elements. Only prophage SF370.1 could be induced by mitomycin C treatment. Prophage SF370.3 showed a 33.5-kb-long genome that closely resembled the genome organization of the cos-site temperate Siphovirus r1t infecting the dairy bacterium Lactococcus lactis. The two-phage genomes shared between 60 and 70% nucleotide sequence identity over the DNA packaging, head and tail genes. Analysis of the SF370.3 genome revealed mutations in the replisome organizer gene that may prevent the induction of the prophage. The mutated phage replication gene was closely related to a virulence marker identified in recently emerged M3 serotype S. pyogenes strains in Japan. This observation suggests that prophage genes confer selective advantage to the lysogenic host. SF370.3 encodes a hyaluronidase and a DNase that may facilitate the spreading of S. pyogenes through tissue planes of its human host. Prophage SF370.2 showed a 43-kb-long genome that closely resembled the genome organization of pac-site temperate Siphoviridae infecting the dairy bacteria S. thermophilus and L. lactis. Over part of the structural genes, the similarity between SF370.2 and S. thermophilus phage O1205 extended to the nucleotide sequence level. SF370.2 showed two probable inactivating mutations: one in the replisome organizer gene and another in the gene encoding the portal protein. Prophage SF370.2 also encodes a hyaluronidase and in addition two very likely virulence factors: prophage-encoded toxins acting as superantigens that may contribute to the immune deregulation observed during invasive streptococcal infections. The superantigens are encoded between the phage lysin and the right attachment site of the prophage genome. The genes were nearly sequence identical with a DNA segment in S. equi, suggesting horizontal gene transfer. The trend for prophage genome inactivation was even more evident for the remaining five prophage sequences that showed massive losses of prophage DNA. In these prophage remnants only 13-0.3 kb of putative prophage DNA was detected. We discuss the genomics data from S. pyogenes strain SF370 within the framework of Darwinian coevolution of prophages and lysogenic bacteria and suggest elements of genetic cooperation and elements of an arms race in this host-parasite relationship. [TOP OF PAGE]

  1235. Progeny of the phage school. Dixon,B. (2001). ASM News 69:432-433. Frederick Twort, the eccentric polymath who discovered bacterial viruses, would have robustly welcomed the applications of bacteriophages now emergy, from therapeutics to environmental protection. [TOP OF PAGE]

  1236. Direct and quantitative detection of bacteriophage by "hearing" surface detachment using a quartz crystal microbalance. Dultsev,F.N., Speight,R.E., Fiorini,M.T., Blackburn,J.M., Abell,C., Ostanin,V.P., Klenerman,D. (2001). Analyt. Chem. 73:3935-3939. We show that it is possible to detect specifically adsorbed bacteriophage directly by breaking the interactions between proteins displayed on the phage coat and ligands immobilized on the surface of a quartz crystal microbalance (QCM). This is achieved through increasing the amplitude of oscillation of the QCM surface and sensitively detecting the acoustic emission produced when the bacteriophage detaches from the surface. There is no interference from nonspecifically adsorbed phage. The detection is quantitative over at least 5 orders of magnitude and is sensitive enough to detect as few as 20 phage. The method has potential as a sensitive and low-cost method for virus detection. [TOP OF PAGE]

  1237. Identification of a genetic determinant responsible for host specificity in Streptococcus thermophilus bacteriophages. Duplessis,M., Moineau,S. (2001). Mol. Microbiol. 41:325-336. Phage-host interactions remain poorly understood in lactic acid bacteria and essentially in all Gram-positive bacteria. The aim of this study was to identify the phage genetic determinant (anti-receptor) involved in the recognition of Streptococcus thermophilus hosts. The complete genomic sequence of the lytic S. thermophilus phage DT1 was determined previously, and bioinformatic analysis indicated that orf18 might be the anti-receptor gene. The orf18 of six additional S. thermophilus phages was determined (DT2, DT4, MD1, MD2, MD4 and Q5) and compared with the orf18 of DT1. The deduced ORF18 was divided into three domains. The first domain, which contains the N-terminal part of the protein, was conserved in all seven phages. The second domain was detected in only two phages and flanked by a motif called collagen-like repeats. The second domain also contained a variable region (VR1). All seven phages had a third domain that consisted of the C-terminal section of the protein as well as another variable region (VR2). Chimeric DT1 phages were constructed by recombination; a portion of its orf18 was replaced by the corresponding section in orf18 of the phage MD4. All DT1 chimeric phages acquired the host range of phage MD4. Analysis of the orf18 in the chimeric phages revealed that host specificity in phages DT1 and MD4 resulted from VR2. This is the first report on the identification and characterization of a phage gene involved in the host recognition process of Gram-positive bacteria. [TOP OF PAGE]

  1238. Transduction by phiBB-1, a bacteriophage of Borrelia burgdorferi. Eggers,C.H., Kimmel,B.J., Bono,J.L., Elias,A.F., Rosa,P., Samuels,D.S. (2001). J. Bacteriol. 183:4771-4778. We previously described a bacteriophage of the Lyme disease agent Borrelia burgdorferi designated phiBB-1. This phage packages the host complement of the 32-kb circular plasmids (cp32s), a group of homologous molecules found throughout the genus Borrelia. To demonstrate the ability of phiBB-1 to package and transduce DNA, a kanamycin resistance cassette was inserted into a cloned fragment of phage DNA, and the resulting construct was transformed into B. burgdorferi CA-11.2A cells. The kan cassette recombined into a resident cp32 and was stably maintained. The cp32 containing the kan cassette was packaged by phiBB-1 released from this B. burgdorferi strain. phiBB-1 has been used to transduce this antibiotic resistance marker into naive CA-11.2A cells, as well as two other strains of B. burgdorferi. This is the first direct evidence of a mechanism for lateral gene transfer in B. burgdorferi. [TOP OF PAGE]

  1239. Bacteriophages of Borrelia burgdorferi and other spirochetes. Eggers,C.H., Casjens,S., Samuels,D.S. (2001). pp. 35-44. In In Saier,M.H., Jr. and García-Lara,J. (eds.), The Spirochetes Molecular and Cellular Biology. A number of bacteriophage-like particles have been observed in association with members of the bacterial order Spirochetales, the spirochetes. Several spirochete bacteriophages have been isolated and characterized at the molecular level. We have characterized a bacteriophage, designated fBB-1, of the Lyme disease agent, Borrelia burgdorferi. Here we review the history of the association between the spirochetes and their bacteriophages, with a particular emphasis on fBB-1 and its prophage, the 32-kb circular plasmid family of B. burgdorferi. [TOP OF PAGE]

  1240. Isolation and characterization of two types of actinophage infecting Streptomyces scabies. el Sayed,S.A., el Didamony,G., Mansour,K. (2001). Folia Microbiol (Praha) 46:519-526. Two types of actinophages, phi S and phi L, were isolated from soil samples by using Streptomyces scabies, a potato scab pathogen, as indicator strain. The phages were partially characterized according to their physicochemical properties, plaques and particles morphology, and their host range; this varied from narrow (for phi S) to wide (for phi L). The adsorption rate constants of the phi S and phi L were 3.44 and 3.18 pL/min, and their burst sizes were 1.61 and 3.75 virions per mL, respectively. One-step growth indicated that phi S and phi L have a latent period of 1/2 h followed by a rise period of 1/2 h. The temperate character of these phages was tested in other isolates of Streptomyces. Four of the phages (phi SS3, phi SS12, phi SS13 and phi SS17) were identified as temperate phages, since they were able to lysogenize SS3, SS12, SS13 and SS17. phi SS3, phi SS12 and phi SS13 were homoimmune, and they were heteroimmune with respect to phi SS17. The restriction barriers of lysogenic isolates (SS12, SS13 and SS17) interfered with the blockage of plaque formation by phages (phi SS12, phi SS13 or phi SS17) propagated on them, about 75% of lysogenic isolates had restriction systems. The exposure of the lysogenic isolates (SS12, SS13 and SS17) to UV-irradiation prevented the possible restriction barriers of these isolates so that these barriers could be overcome. [TOP OF PAGE]

  1241. Rapid methods for detection, identification, and enumeration. Entis,P., Fung,D.Y.C., Griffiths,M.W., McIntyre,L., Russell,S., Sharpe,A.N., Tortello,M.L. (2001). pp. 89-126. In In Downes,F.P. and Ito,K. (eds.), Compendium of Methods for the Microbiological Examination of Foods. American Public Health Association, Washington, DC. [first paragraph of section 10.7: phage probes] The existence of bacterial viruses known as bacteriophages-eaters of bacteria-was first reported by the British scientist F.W. Twort in 1915, and somewhat controversially in 1917 by the Canadian bacteriologist F d'Herelle. Twort's publication in the Lancet documented the phenomenon of "glassy transformation" observed in colonies of micrococci grown on agar in the presence of small pox vaccine. He postulated that the "transparent dissolving material" might be caused by: i) an enzyme; ii) could be part of the life cycle of the bacterium; or iii) was caused by a virus, a term first used vaguely by Jenner in 1796 and used more specifically in 1898 by the Dutch botanist Martinus Beijerinck in his study of Tobacco Mosaic Virus.114. [TOP OF PAGE]

  1242. Diminished diarrheal response to Vibrio cholerae strains carrying the replicative form of the CTXf genome instead of CTXf lysogens in adult rabbits. Faruque,S.M., Rahman,M.M., Hasan,A.K., Nair,G.B., Mekalanos,J.J., Sack,D.A. (2001). Infect. Immun. 69:6084-6090. Toxigenic Vibrio cholerae strains are lysogens of CTXf, a filamentous bacteriophage which encodes cholera toxin (CT). Following infection of recipient V. cholerae cells by CTX(Phi), the phage genome either integrates into the host chromosome at a specific attachment site (attRS) or exists as a replicative-form (RF) plasmid. We infected naturally occurring attRS-negative nontoxigenic V. cholerae or attenuated (CTX(-) attRS negative) derivatives of wild-type toxigenic strains with CTX(Phi) and examined the diarrheagenic potential of the strains carrying the RF of the CTXf genome using the adult rabbit diarrhea model. Under laboratory conditions, strains carrying the RF of CTX(Phi) produced more CT than corresponding lysogens as assayed by a G(M1)-based enzyme-linked immunosorbent assay and by fluid accumulation in ligated ileal loops of rabbits. However, when tested for diarrhea in rabbits, the attRS-negative strains (which carried the CTXf genome as the RF) were either negative or produced mild diarrhea, whereas the attRS-positive strains with integrated CTXf produced severe fatal diarrhea. Analysis of the strains after intestinal passage showed that the attRS-negative strains lost the phage genome at approximately a fivefold higher frequency than under in vitro conditions, and 75 to 90% of cells recovered from challenged rabbits after 24 h were CT negative. These results suggested that strains carrying the RF of CTXf are unable to cause severe disease due to rapid loss of the phage in vivo, and the gastrointestinal environment thus provides selection of toxigenic strains with an integrated CTXf genome. These results may have implications for the development of live V. cholerae vaccine candidates impaired in chromosomal integration of CTX(Phi). These findings may also contribute to understanding of the etiology of diarrhea occasionally associated with nontoxigenic V. cholerae strains. [TOP OF PAGE]

  1243. Development and optimization of a novel immunomagnetic separation- bacteriophage assay for detection of Salmonella enterica serovar enteritidis in broth. Favrin,S.J., Jassim,S.A., Griffiths,M.W. (2001). Appl. Environ. Microbiol. 67:217-224. Salmonella is the second-leading cause of food-borne illness in most developed countries, causing diarrhea, cramps, vomiting, and often fever. Many rapid methods are available for detection of Salmonella in foods, but these methods are often insensitive or expensive or require a high degree of technical ability to perform. In this paper we describe development and characterization of a novel assay that utilizes the normal infection cycle of bacteriophage SJ2 for detection of Salmonella enterica serovar Enteritidis in broth. The assay consists of four main stages: (i) capture and concentration of target cells by using immunomagnetic separation (IMS); (ii) infection of the target bacterium with phage; (iii) amplification and recovery of progeny phage; and (iv) assay of progeny phage on the basis of their effect on a healthy population of host cells (signal-amplifying cells). The end point of the assay can be determined by using either fluorescence or optical density measurements. The detection limit of the assay in broth is less than 104 CFU/ml, and the assay can be performed in 4 to 5 h. The results of this study demonstrate that the IMS-bacteriophage assay is a rapid, simple, and sensitive technique for detection of Salmonella serovar Enteritidis in broth cultures which can be applied to preenriched food samples. [TOP OF PAGE]

  1244. Variable assortment of prophages provides a transferable repertoire of pathogenic determinants in Salmonella. Figueroa-Bossi,N., Uzzau,S., Maloriol,D., Bossi,L. (2001). Mol. Microbiol. 39:260-271. Gene transfer between separate lineages of a bacterial pathogen can promote recombinational divergence and the emergence of new pathogenic variants. Temperate bacteriophages, by virtue of their ability to carry foreign DNA, are potential key players in this process. Our previous work has shown that representative strains of Salmonella typhimurium (LT2, ATCC14028 and SL1344) are lysogenic for two temperate bacteriophages: Gifsy-1 and Gifsy-2. Several lines of evidence suggested that both elements carry genes that contribute to Salmonella virulence. One such gene, on the Gifsy-2 prophage, codes for the (Cu, Zn) superoxide dismutase SodCI. Other putative pathogenicity determinants were uncovered more recently. These include genes for known or presumptive type III-translocated proteins and a locus, duplicated on both prophages, showing sequence similarity to a gene involved in Salmonella enteropathogenesis (pipA). In addition to Gifsy-1 and Gifsy-2, each of the above strains was found to harbour a specific set of prophages also carrying putative pathogenicity determinants. A phage released from strain LT2 and identified as phage Fels-1 carries the nanH gene and a novel sodC gene, which was named sodCIII. Strain ATCC14028 releases a lambdoid phage, named Gifsy-3, which contains the phoP/phoQ-activated pagJ gene and the gene for the secreted leucine-rich repeat protein SspH1. Finally, a phage specifically released from strain SL1344 was identified as SopEPHI. Most phage-associated loci transferred efficiently between Salmonella strains of the same or different serovars. Overall, these results suggest that lysogenic conversion is a major mechanism driving the evolution of Salmonella bacteria. [TOP OF PAGE]

  1245. H-mutant bacteriophages as a potential biocontrol of bacterial blight of geranium. Flaherty,J.E., Harbaugh,B.K., Jones,J.B., Somodi,G.C., Jackson,L.E. (2001). Hortscience 36:98-100. Bacteriophages specific to Xanthomonas campestris pv. pelargonii (Xcp), the causal agent of bacterial blight of geranium, Pelargonium Xhortorum L.H. Bailey, were isolated from soil and sludge samples from Florida, California, Minnesota, and Utah. Sixteen phages were evaluated for their potential to lyse 21 Xcp strains collected from around the world. The Xcp strains varied in their susceptibility to the phage isolates with 4 to 14 phages producing a lytic or highly virulent reaction. A mixture of five h-mutants was developed from phages that exhibited the broadest host-ranges and tested against the same Xcp strains. The h-mutant phage mixture lysed all 21 Xcp strains. Three experiments were designed to determine the efficacy of using a mixture of four h-mutant phages to control the spread of the bacterial blight pathogen on potted and seedling geraniums under greenhouse conditions. Plants surrounding diseased inoculated plants were treated with a phage mixture at 5 X 108 pfu/mL daily, biweekly, or triweekly, or treated with Phyton-27(R), at 2.0 mLcntdotL-1 every 10 or 14 days. In potted geraniums, daily foliar sprays of the phage mixture had reduced disease incidence and severity by 50% and 75%, respectively, relative to control plants after 6 weeks. In two plug experiments, the phage mixture applied daily also had reduced disease incidence and severity by 69% and 86%, and 85% and 92%, respectively, when compared with controls after 5 weeks. In all three experiments, disease incidence and severity were less for plants treated daily with phages than for those treated less frequently with phages or with Phyton-27(R). Chemical name used: copper sulfate pentahydrate (Phyton-27(R)). [TOP OF PAGE]

  1246. Comparative study of nine Lactobacillus fermentum bacteriophages. Foschino,R., Picozzi,C., Galli,A. (2001). J. Appl. Microbiol. 91:394-403. AIMS: To investigate the basic properties of six temperate and three virulent phages, active on Lactobacillus fermentum, on the basis of morphology, host ranges, protein composition and genome characterization. METHODS AND RESULTS: All phages belonged to the Siphoviridae family; two of them showed prolate heads. The host ranges of seven phages contained a common group of strains. SDS-PAGE protein profiles, restriction analysis of DNA and Southern blot hybridization revealed a high degree of homology between four temperate phages; partial homologies were also detected among virulent and temperate phages. Clustering derived from host range analysis was not related to the results of the DNA hybridizations. CONCLUSION: The phages investigated have common characteristics with other known phages active on the genus Lactobacillus. Sensitivity to viral infection is apparently enhanced by the presence of a resident prophage. SIGNIFICANCE AND IMPACT OF THE STUDY: These relationships contribute to the explanation for the origin of phage infection in food processes where Lact. fermentum is involved, such as sourdough fermentation. [TOP OF PAGE]

  1247. Bacteriophage lambda: Alive and well and still doing its thing. Friedman,D.I., Court,D.L. (2001). Curr. Opin. Mirobiol. 4:201-207. The lambda (lambda) family of bacteriophages continues to provide significant insights into the understanding of basic biological processes, as well as useful technological innovations. Areas in which recent advances have occurred include transcription elongation, repressor interactions, genomics and post-transcriptional regulation. The homologous lambda recombination functions have been exploited as an efficient in vivo recombinant engineering system for functional genomic studies. The virulence of some pathogenic strains of Escherichia coli is enhanced by the expression of Shiga toxin (stx) genes encoded on a resident lambdoid prophage. Recent work suggests that the phage regulatory network may be a significant contributor to toxin production and release by these pathogenic E. coli. [TOP OF PAGE]

  1248. Adsorption and survival of faecal coliforms, somatic coliphages and F-specific RNA phages in soil irrigated with wastewater. Gantzer,C., Gillerman,L., Kuznetsov,M., Oron,G. (2001). Water Sci. Technol. 43:117-124. This study was carried out to compare the adsorption and survival of faecal coliforms, somatic coliphages and F-specific RNA phages in soil irrigated with wastewater. Adsorption isotherms showed that 3-10X more faecal coliforms than somatic coliphages were adsorbed from wastewater onto soil. The adsorption behavior of F-specific RNA phages was intermediate between those of these two microorganisms. In wastewater, the inactivation factor of somatic coliphages at 8-22°C was 5-7 lower than those of faecal coliforms. F-specific RNA phages have a decrease close to faecal coliforms. In soil, at temperatures of 8-22°C and at moistures of 15-35%, somatic coliphages survived longer than the two other microorganisms. These results seemed to be confirmed by the soil column experiments. The rate of inactivation of all microorganisms was lower in soil than in wastewater and depended extensively on soil temperature and moisture content. Survival was optimal at low temperature (8°C) and low moisture content (15%). Thus, somatic coliphages seemed to be a better indicator of faecal contamination than faecal coliforms under our experimental conditions and based only on the two criteria tested (survival and adsorption). Somatic coliphages were able to contaminate the soil over greater distances and survive better in both wastewater and soil than faecal coliforms. These results need to be confirmed by studies on several soil columns using different kinds of soil and different kinds of wastewater. [TOP OF PAGE]

  1249. Bacteriophages: Update on application as models for viruses in water. Grabow,W.O.K. (2001). Water SA (Pretoria) 27:251-268. Phages are valuable models or surrogates for enteric viruses because they share many fundamental properties and features. Among these are structure, composition, morphology, size and site of replication. Even though they use different host cells, coliphages and Bacteroides fragilis phages predominantly replicate in the gastro-intestinal tract of humans and warm-blooded animals where enteric viruses also replicate. A major advantage of phages is that, compared to viruses, they are detectable by simple, inexpensive and rapid techniques. In view of these features, phages are particularly useful as models to assess the behaviour and survival of enteric viruses in the environment, and as surrogates to assess the resistance of human viruses to water treatment and disinfection processes. Since there is no direct correlation between numbers of phages and viruses, phages cannot to a meaningful extent be used to indicate numbers of viruses in polluted water. The presence of phages typically associated with human and animal excreta indicates the potential presence of enteric viruses. However, the absence of these phages from water environments is generally a meaningful indication of the absence of enteric viruses. This is because phages such as somatic coliphages, F-RNA coliphages and B. fragilis phages generally outnumber enteric viruses in water environments, and they are at least as resistant to unfavourable conditions including those in water treatment and disinfection processes. However, using highly sensitive molecular techniques viruses have been detected in drinking water supplies which yielded negative results in conventional tests for phages. Initially, data on phages were rather confusing because a wide variety of techniques was used. However, techniques for the detection of phages are being standardised internationally. This applies in particular to somatic and F-RNA coliphages, and B. fragilis phages, which are most commonly used in water quality assessment. Reliable and practical techniques now available include direct quantitative plaque assays on samples of water up to 100 ml, and qualitative tests on 500 ml or more using highly sensitive enrichment procedures. [TOP OF PAGE]

  1250. Holins kill without warning. Gründling,A., Manson,M.D., Young,R. (2001). Proc. Natl. Acad. Sci. USA 98:9348-9352. Holins comprise the most diverse functional group of proteins known. They are small bacteriophage-encoded proteins that accumulate during the period of late-protein synthesis after infection and cause lysis of the host cell at a precise genetically programmed time. It is unknown how holins achieve temporal precision, but a conserved feature of their function is that energy poisons subvert the normal scheduling mechanism and instantly trigger membrane disruption. On this basis, timing has been proposed to involve a progressive decrease in the energized state of the membrane until a critical triggering level is reached. Here, we report that membrane integrity is not compromised after the induction of holin synthesis until seconds before lysis. The proton motive force was monitored by the rotation of individual cells tethered by a single flagellum. The results suggest an alternative explanation for the lysis "clock," in which holin concentrations build to a critical level that leads to formation of an oligomeric complex that disrupts the membrane. [TOP OF PAGE]

  1251. Reviewing efficacy of alternative water treatment techniques. Hambidge,A. (2001). Health Estate 55:23-25. This section is designed to provide a brief summary of some of the findings. A good deal of work has been conducted by Mr N. L. Pavey and the team at BSRIA, Bracknell. The BSRIA publications are an excellent source of further information. Ultraviolet radiation: UV radiation of wavelength 254 nm destroys bacteria by a mechanism of damaging nucleic acids by producing thymine dimers which disrupt DNA replication [Gavdy and Gavdy, 1980]. L. pneumophila has been reported as sensitive to UV dosages of 2,500-7,000 uW.s/cm2 [Antopol & Ellner, 1979; Knudson, 1985]. Antopol and Ellner [1979] examined the susceptibility of L. pneumophila to UV dosage. Their results indicated that 50% of the organisms were killed by 380 uWs/cm2 and 90% were killed by 920 uWs/cm2. Kills of 99 and 99.9% were obtained using 1,840 and 2,760 uWs/cm2 respectively. Muraca et al [1987] showed that continuous UV irradiation resulted in a 5 logarithm decrease in waterborne L. pneumophila in a circulating system. Gilpin [1984] reported that in laboratory buffer solutions, exposure to 1 uW of UV radiation per cm2 achieved a 50% kill of L longbeachae in 5 minutes, L. gormanii in 2-30 minutes and L pneumophila in 17 minutes. Exposure times for 99% kills for L. longbeachae, L pneumophila and L. Gormanii were 33, 48 and 63 minutes respectively. The same research worker conducted experiments using a 3 litre circulating water system, connected to a stainless steel housing containing a UV source. The UV lamp output was 7 ergs/mm2 per second per 100 cm at 254 nm. L. pneumophila was killed within 15 seconds, that is within their first pass through the system. Continuous disinfection with UV has the advantages of imparting no taste, odour or harmful chemical by-products and requires minimal operation and maintenance [Muraca et al 1988]. Keevil et al [1989] state that UV irradiation fails to clear systems of biofilm because of poor penetration into microflocs of the micro-organisms. Copper/silver ionisation: A recent study of full scale hot water test rigs incorporating copper-silver ionisation systems has been reported by Pavey, 1996. Copper and silver ions were introduced into the water by electrolysis. One of the principal mechanisms of biocidal action of these ions is thought to be cell penetration. The positively charged copper ions form electrostatic bonds with negatively charged sites on the cell wall. The cell membrane is thus distorted, allowing ingress of silver ions which attack the cell by binding at specific sites to DNA, RNA, respiratory enzymes and cellular protein, causing catastrophic failure of the life support systems of the cell. Silver and copper ion concentrations of 40 and 400 ug/L respectively were effective against planktonic Legionellae in cold water systems and hot water systems containing soft water. In hard water, the ionisation was ineffective due to the inability to control silver ion concentrations. This was caused by scaling of the electrodes and silver ion complexation by the high concentration of dissolved solids. Bosch et al [1993] had earlier extended the application of copper-silver disinfection to human enteric viruses in water, such as adenovirus, rotavirus, hepatitis A virus, and poliovirus. Their work showed that copper and silver ions in the presence of reduced levels of free chlorine did not ensure the total elimination of viral pathogens from water. In the case of an amoeba, Naegleria fowleria [responsible for primary amoebic meningoencephalitis], Cassells et al [1995] have demonstrated that a combination of silver and copper ions were ineffective at inactivating the amoebae at 80 and 800 ug/L respectively. However addition of 1.0 mg/L free chlorine produced a synergistic effect, with superior inactivation relative to either chlorine or silver-copper in isolation. A similar synergy was reported by Yahya et al [1989] in their study of Staphylococcus sp. and Pseudomonas aeruginosa. Yahya et al [1992] also suggested an additive or synergistic effect in the inactivation of coliphage MS-2 and poliovirus. Other techniques: There are a number of other techniques. We have conducted trials of most of these in the control of Legionella sp., but these fall out of the scope of this article, and as such less emphasis has been placed on them here. Ozonation: Ozone [O3] is an oxidising gas, generated electrically from oxygen [O2]. L. pneumophila can be killed at < 1 mg/L of ozone [Edelstien et al 1982]. Muraca et al [1987] found that 1-2 mg/L of continuous ozone over a six hour contact time, produced a 5 logarithm decrease of L. pneumophila. The effectiveness of ozone treatment against a range of bacteria and coliphages has been studied Botzenhart et al [1993]. E. coli was least resistant to ozone, followed by MS 2-coliphage and PhiX 174-coliphage, with L. pneumophila and Bacillus subtilis spores being the most resistant. (ABSTRACT TRUNCATED). [TOP OF PAGE]

  1252. A conserved genetic module that encodes the major virion components in both the coliphage T4 and the marine cyanophage S-PM2. Hambly,E., Tétart,F., Desplats,C., Wilson,W.H., Krisch,H.M., Mann,N.H. (2001). Proc. Natl. Acad. Sci. USA 98:11411-11416. Sequence analysis of a 10-kb region of the genome of the marine cyanomyovirus S-PM2 reveals a homology to coliphage T4 that extends as a contiguous block from gene (g)18 to g23. The order of the S-PM2 genes in this region is similar to that of T4, but there are insertions and deletions of small ORFs of unknown function. In T4, g18 codes for the tail sheath, g19, the tail tube, g20, the head portal protein, g21, the prohead core protein, g22, a scaffolding protein, and g23, the major capsid protein. Thus, the entire module that determines the structural components of the phage head and contractile tail is conserved between T4 and this cyanophage. The significant differences in the morphology of these phages must reflect the considerable divergence of the amino acid sequence of their homologous virion proteins, which uniformly exceeds 50%. We suggest that their enormous diversity in the sea could be a result of genetic shuffling between disparate phages mediated by such commonly shared modules. These conserved sequences could facilitate genetic exchange by providing partially homologous substrates for recombination between otherwise divergent phage genomes. Such a mechanism would thus expand the pool of phage genes accessible by recombination to all those phages that share common modules. [TOP OF PAGE]

  1253. Reduction in exopolysaccharide viscosity as an aid to bacteriophage penetration through Pseudomonas aeruginosa biofilms. Hanlon,G.W., Denyer,S.P., Olliff,C.J., Ibrahim,L.J. (2001). Appl. Environ. Microbiol. 67:2746-2753. To cause an infection, bacteriophages must penetrate the alginate exopolysaccharide of Pseudomonas aeruginosa to reach the bacterial surface. Despite a lack of intrinsic motility, phage were shown to diffuse through alginate gels at alginate concentrations up to 8% (wt/vol) and to bring about a 2-log reduction in the cell numbers in 20-day-old biofilms of P. aeruginosa. The inability of alginate to act as a more effective diffusional barrier suggests that phage may cause a reduction in the viscosity of the exopolysaccharide. Samples (n = 5) of commercial alginate and purified cystic fibrosis (CF) alginate were incubated with 2 x 108 purified phage per ml for 24 h at 37 degrees C. After incubation the samples and controls were subjected to rheological analysis with a Carrimed controlled stress rheometer. The viscosities of phage-treated samples were reduced by up to 40% compared to those of controls incubated in the absence of phage. The experiment was repeated by using phage concentrations of 1010 and 1012 phage per ml and samples taken for analysis at intervals up to 4 h. The results indicated that there was a time- and concentration-dependent reduction in viscosity of up to 40% compared to the viscosities of the controls. Commercial and purified CF alginate samples, both phage treated and untreated, were subjected to gel filtration chromatography by using Sephacryl High Resolution S-400 medium in order to obtain evidence of degradation. The results demonstrated that alginate treated with phage had a lower molecular weight than untreated alginate. The data suggest that bacteriophage migration through P. aeruginosa biofilms may be facilitated by a reduction in alginate viscosity brought about by enzymic degradation and that the source of the enzyme may be the bacterial host itself. [TOP OF PAGE]

  1254. Isolation and characterization of a temperature-sensitive generalized transducing bacteriophage for Vibrio cholerae. Hava,D.L., Camilli,A. (2001). J. Microbiol. Meth. 46:217-225. CP-T1 is the only described generalized transducing bacteriophage for the intestinal pathogen Vibrio cholerae, yet many of its basic biological parameters remain unknown. Due to low frequencies of transduction and pseudolysogen formation, CP-T1 has not been widely used as a genetic tool. To overcome these limitations, we have isolated a conditional mutant of CP-T1 that exhibits temperature-sensitive plaque formation. Several biological properties of CP-T1ts were determined, including its restrictive temperature, adsorbance profile to host cells, burst time, and burst size. Based on these properties, an optimized transduction protocol was designed which resulted in several fold higher transduction frequencies for a variety of genetic markers from a number of chromosomal loci. Generalized transduction was also demonstrated between classical and E1 Tor biotype strains of V. cholerae. [TOP OF PAGE]

  1255. First evidence of lysogeny in Propionibacterium freudenreichii subsp. shermanii. Herve,C., Coste,A., Rouault,A., Fraslin,J.M., Gautier,M. (2001). Appl. Environ. Microbiol. 67:231-238. Dairy propionic acid bacteria, particularly the species Propionibacterium freudenreichii, play a major role in the ripening of Swiss type cheese. Isometric and filamentous bacteriophages infecting P. freudenreichii have previously been isolated from cheese. In order to determine the origin of these bacteriophages, lysogeny of P. freudenreichii was determined by isometric bacteriophage type analysis. The genomic DNA of 76 strains were hybridized with the DNA of nine bacteriophages isolated from Swiss type cheeses, and the DNA of 25 strains exhibited strong hybridization. Three of these strains released bacteriophage particules following UV irradiation (254 nm) or treatment with low concentrations of mitomycin C. A prophage-cured derivative of P. freudenreichii was readily isolated and subsequently relysogenized. Lysogeny was therefore formally demonstrated in P. freudenreichii. [TOP OF PAGE]

  1256. Effects of concentrated viral communities on photosynthesis and community composition of co-occurring benthic microalgae and phytoplankton. Hewson,I., O'Neil,J.M., Heil,C.A., Bratbak,G., Dennison,W.C. (2001). Aquat. Microb. Ecol. 25:1-10. Marine viruses have been shown to affect phytoplankton productivity; however, there are no reports on the effect of viruses on benthic microalgae (microphytobenthos). Hence, this study investigated the effects of elevated concentrations of virus-like particles on the photosynthetic physiology and community composition of benthic microalgae and phytoplankton. Virus populations were collected near the sediment surface and concentrated by tangential flow ultrafiltration, and the concentrate was added to benthic and water column samples that were obtained along a eutrophication gradient in the Brisbane River/Moreton Bay estuary, Australia. Photosynthetic and community responses of benthic microalgae, phytoplankton and bacteria were monitored over 7 d in aquaria and in situ. Benthic microalgal communities responded to viral enrichment in both eutrophic and oligotrophic sediments. In eutrophic sediments, Euglenophytes (Euglena sp.) and bacteria decreased in abundance by 20 to 60 and 26 to 66%, respectively, from seawater controls. In oligotrophic sediments, bacteria decreased in abundance by 30 to 42% from seawater controls but the dinoflagellate Gymnodinium sp. increased in abundance by 270 to 3600% from seawater controls, The increased abundance of Gymnodinium sp. may be related to increased availability of dissolved organic matter released from lysed bacteria. Increased (140 to 190% from seawater controls) initial chlorophyll a fluorescence measured with a pulse-amplitude modulated fluorometer was observed in eutrophic benthic microalgal incubations following virus enrichment, consistent with photosystem II damage. Virus enrichment in oligotrophic water significantly stimulated carbon fixation rates, perhaps due to increased nutrient availability by bacterial lysis. The interpretation of data from virus amendment experiments is difficult due to potential interaction with unidentified bioactive compounds within seawater concentrates. However, these results show that viruses are capable of influencing microbial dynamics in sediments. [TOP OF PAGE]

  1257. Virus-like particle distribution and abundance in sediments and overlying waters along eutrophication gradients in two subtropical estuaries. Hewson,I., O'Neil,J.M., Fuhrman,J.A., Dennison,W.C. (2001). Limnol. Oceanogr. 46:1734-1746. Viruses are recognized as ubiquitous components of marine ecosystems; however, there has been limited study of viral abundance and its ecological role in sediments. Viral abundance was determined in both the water column and sediments of a eutrophic (Brisbane River/Moreton Bay; 27º25'S, 153º5'E) and oligotrophic (Noosa River; 26º15'S, 153º0'E) estuary in subtropical Queensland, Australia. Viruses, bacteria, and microalgae from both water column and extracted sediment samples were enumerated using SYBR Green I staining and epifluorescence microscopy. Sediment viral abundance ranged from 107 to 109 particles cm-3 of sediment, bacterial abundance ranged from 107 to 108 cells cm-3 of sediment, and microalgal abundance ranged from 104 to 105 cells cm-3 sediment. Pelagic abundances for all microorganisms were 10-1,000-fold lower than sediment abundances. Correlations between viral abundances and suspended solids suggest that viruses sorbed to suspended material in the water column may settle out and contribute to the benthic viral population. Virus production was measured by a time course increase of viral abundance in seawater using a dilution technique. Virus production was highest in eutrophic waters of the Brisbane River, and addition of inorganic nutrients (NO3- + NH4+ + PO4-3 + SiO3) stimulated viral production rates at all stations by 14-52% above ambient, suggesting that inorganic nutrient availability may play a key role in aquatic viral abundance. [TOP OF PAGE]

  1258. Cheese making with bacteriophage resistant bacteria. Hicks,C.L. (2001). University of Kentucky Research Foundation. 440734(6,297,042 ). Lexington, KY. A method is provided for reducing or preventing bacteriophage attack on bacteria used in a cheese making process. The method includes (a) treating a blocker peptide precursor with a protease enzyme that hydrolyzes the precursor to produce blocker peptides; (b) collecting the blocker peptides so produced; (c) formulating a starter media with the blocker peptides; (d) growing bulk cultures of cheese making bacteria in the inoculated starter media; and (e) adding bacteria grown in the inoculated starter media to a fermentation medium for producing cheese. The present invention also includes a method of making cheese and cheese produced by the method. [TOP OF PAGE]

  1259. Removal of Salmonella and microbial indicators in constructed wetlands treating swine wastewater. Hill,V.R., Sobsey,M.D. (2001). Water Sci. Technol. 44:215-222. Reductions of Salmonella bacteria and enteric microbial indicator organisms were measured in swine wastewater treated by a field-scale surface flow (SF) constructed wetland at a commercial hog nursery in North Carolina and in laboratory-scale SF and subsurface flow (SSF) constructed wetland reactors. Overall reductions of Salmonella, fecal coliforms and E. coli were 96, 98 and 99%, respectively, in the two-cell field-scale wetland. Somatic and F-specific coliphage viral indicators were reduced by 99 and 98%, respectively. Reductions of Salmonella, fecal coliforms and E. coli were similar in the first cell of the field system and in the laboratory-scale SF wetland operated at a TKN loading of 25 kg ha(-1) d(-1) and 30 degrees C (approximately 70, 90 and 90%, respectively). In the SSF wetland reactor, Salmonella and fecal coliform reductions were 80 and 98%, respectively, at a 40 kg TKN ha(-1) d(-1) loading and 99.8 and 99.99%, respectively, at a 10 kg TKN ha(-1) d(-1) loading. These results show that SF constructed wetlands can be effective for reducing enteric pathogens in swine wastewater and that greater removals can be achieved using SSF designs and lower TKN loading rates. [TOP OF PAGE]

  1260. Bacteriophage therapy for bacterial infections: Rekindling a memory from the pre-antibiotics era. Ho,K. (2001). Perspect. Biol. Med. 44:1-16. [LAST PARAGRAPH] Bacteriophage therapy for bacterial infections has thus evolved from a promising, yet flawed treatment in the pre-antibiotics era into a potentially powerful solution for a worldwide problem of bacteria with far more evasive abilities. Its colorful history is highlighted by a variety of contrasts: between unbridled enthusiasm and sobering realism; between artful application and rigorous experimental clarification; between an affinity for natural cure and for one decidedly more laboratory-derived. Although the future of bacteriophage therapy rests within speculative terrain, a revisitation of its roots-with close attention to the tensions and ambiguities-may offer those presently as enamored by its tantalizing allure as their counterparts 70 years ago instructive insights into recapturing and redefining its potential. [TOP OF PAGE]

  1261. Seasonal dynamics of viruses in an alpine lake: Importance of filamentous forms. Hofer,J.S., Sommaruga,R. (2001). Aquat. Microb. Ecol. 26:1-11. Viruses are an important component of the planktonic food web in freshwater and marine systems, but most studies have been done in the ocean and in lowland lakes. In this work, the seasonal dynamics and structure of the virioplankton as well as their impact on bacteria during a day/night cycle were studied in an alpine lake located 2417 m above sea level. The abundance of virus-like particles (VLP) was determined at 5 discrete depths (0.5 to 8 m) by direct counts with a TEM in samples collected from May to November 1998 at weekly to bi-weekly intervals. Viruses reached the highest abundances under ice (4.6 X 106 VLP ml-1) with a second maximum in autumn. After ice-break, the VLP abundance decreased to undetectable values (<2 X 104 VLP ml-1) probably because of the negative effect of solar radiation that was negatively correlated with the viral abundance in the upper 2 m of the water column (Spearman rank correlation, rs = -0.773, p < 0.01). The virioplankton was morphologically diverse, consisting of forms commonly found in other aquatic systems, but unlike other studies, we found filamentous VLP (FVLP) 450 to 730 nm long that attained abundances of up to 1.3 X 106 ml-1 and accounted for 7 to 100% of the total viral abundance. These FVLP were found occasionally inside filamentous heterotrophic bacteria (> 10 mum) and their respective abundances were positively correlated (rs = 0.728, p < 0.01). The absence of these conspicuous forms in other aquatic ecosystems suggests that FVLP are well adapted to the harsh environmental conditions or are specific to bacterial hosts found in alpine lakes. Finally, between 5 and 28% of the newly produced bacteria were killed by non-filamentous viruses, which therefore are a modest cause of bacterial mortality in this lake. [TOP OF PAGE]

  1262. Profiles of adaptation in two similar viruses. Holder,K.K., Bull,J.J. (2001). Genetics 159:1393-1404. The related bacteriophages variant phiX174 and G4 were adapted to the inhibitory temperature of 44° and monitored for nucleotide changes throughout the genome. Phage were evolved by serial transfer at low multiplicity of infection on rapidly dividing bacteria to select genotypes with the fastest rates of reproduction. Both phage showed overall greater fitness effects per substitution during the early stages of adaptation. The fitness of variant phiX174 improved from -0.7 to 5.6 doublings of phage concentration per generation. Five missense mutations were observed. The earliest two mutations accounted for 85% of the ultimate fitness gain. In contrast, G4 required adaptation to the intermediate temperature of 41.5degree before it could be maintained at 44°. Its fitness at 44° increased from -2.7 to 3.2, nearly the same net gain as in variant phiX174, but with three times the opportunity for adaptation. Seventeen mutations were observed in G4: 14 missense, 2 silent, and 1 intergenic. The first 3 missense substitutions accounted for over half the ultimate fitness increase. Although the expected pattern of periodic selective sweeps was the most common one for both phage, some mutations were lost after becoming frequent, and long-term polymorphism was observed. This study provides the greatest detail yet in combining fitness profiles with the underlying pattern of genetic changes, and the results support recent theories on the range of fitness effects of substitutions fixed during adaptation. [TOP OF PAGE]

  1263. [Application of rapid detection for Mycobacterium tuberculosis with phage splitting assay]. Hu,Z., Pang,M., Jin,A. (2001). Zhonghua Jiehe He Huxi Zazhi 24:611-613. Objective: To study the significance of rapid identification for Mycobacterium tuberculosis with phage splitting assay. Methods: Strains of Mycobacterium tuberculosis, non-tuberculosis mycobacterium, non-mycobacterium and samples of sputum with pulmonary tuberculosis were rapidly detected by phage spot technique. Results: The strains of Mycobacterium tuberculosis H37Rv, bovis and africanum were all positive. The results of 10 strains of non-tuberculosis mycobacterium and 7 strains of non-mycobacterium were negative. All of 30 clinical isolates from the patients of the pulmonary tuberculosis were positive. 19 of 20 sputum specimen of pulmonary tuberculosis, which were all positive detected by smear and culture, were positive. There were 15 specimen positive in 21 sputum with negative tested by smear and positive by culture. Besides, 5 of 19 sputum specimen with negative by smear and culture were positive detected by this method. Conclusion: The phage splitting assay can be used for rapid identification of Mycobacterium tuberculosis, which possesses high specificity and sensitivity for detection of Mycobacterium tuberculosis in sputum specimen. [TOP OF PAGE]

  1264. Strategies for analysis of the evolution of bacteriophages. Huang,S., Hayes,S.J., Lieman,K., Griess,G.A., Serwer,P. (2001). Recent Res. Dev. Virol. 3:1-12. [TOP OF PAGE]

  1265. Filamentous phage associated with recent pandemic strains of Vibrio parahaemolyticus. Iida,T., Hattori,A., Tagomori,K., Nasu,H., Naim,R., Honda,T. (2001). Emerg. Infect. Dis. 7:477-478. A group of pandemic strains of Vibrio parahaemolyticus has recently appeared in Asia and North America. We demonstrate that a filamentous phage is specifically associated with the pandemic V. parahaemolyticus strains. An open reading frame unique to the phage is a useful genetic marker to identify these strains. [TOP OF PAGE]

  1266. Lytic and lysogenic infection of diverse Escherichia coli and Shigella strains with a verocytotoxigenic bacteriophage. James,C.E., Stanley,K.N., Allison,H.E., Flint,H.J., Stewart,C.S., Sharp,R.J., Saunders,J.R., McCarthy,A.J. (2001). Appl. Environ. Microbiol. 67:4335-4337. A verocytotoxigenic bacteriophage isolated from a strain of enterohemorrhagic Escherichia coli O157, into which a kanamycin resistance gene (aph3) had been inserted to inactivate the verocytotoxin gene (vt2), was used to infect Enterobacteriaceae strains. A number of Shigella and E. coli strains were susceptible to lysogenic infection, and a smooth E. coli isolate (O107) was also susceptible to lytic infection. The lysogenized strains included different smooth E. coli serotypes of both human and animal origin, indicating that this bacteriophage has a substantial capacity to disseminate verocytotoxin genes. A novel indirect plaque assay utilizing an E. coli recA441 mutant in which phage-infected cells can enter only the lytic cycle, enabling detection of all infective phage, was developed. [TOP OF PAGE]

  1267. Practical evaluation of molecular subtyping and phage typing in outbreaks of infection due to Salmonella enterica serotype typhimurium. Jeoffreys,N.J., James,G.S., Chiew,R., Gilbert,G.L. (2001). Pathology 33:66-72. Identification and control of food-poisoning outbreaks due to salmonellosis depend on prompt microbiological diagnosis and subtyping to identify the causative strain. In Australia, Salmonella enterica subspecies enterica serotype typhimurium (S. typhimurium) is responsible for 40-70% of cases of human salmonellosis. Phage typing is the usual method of subtyping S. typhimurium, but on its own, has limitations. We compared it with three molecular subtyping methods using 100 isolates of S. typhimurium, representing four different phage types (PT 1, 9, 126 and 135) and comprising 74 isolates from three presumed outbreaks, 25 isolates from sporadic cases of salmonellosis and S. typhimurium ATCC 10428 (phage type 126). The isolates were divided into 11 subtypes by IS200 restriction fragment length polymorphism (RFLP) typing, four each by ribotyping and pulsed-field gel electrophoresis (PFGE) and 17 distinct strains using a combination of phage and molecular typing. Isolates from two presumed outbreaks were resolved into multiple strains, possibly explaining the failure to identify a common source for either during the original investigations. IS200 RFLP analysis was the most discriminatory and reproducible typing method. Several strains were identifiable within and shared between phage types 1, 9 and 126. Phage and IS200 RFLP typing together, would provide improved definition of S. typhimurium outbreaks. [TOP OF PAGE]

  1268. Molecular Biology of Bacteriophage. Jia,P.X. (2001). Science Press, Beijing.[TOP OF PAGE]

  1269. Human adenoviruses and coliphages in urban runoff-impacted coastal waters of Southern California. Jiang,S., Noble,R., Chu,W. (2001). Appl. Environ. Microbiol. 67:179-184. A nested-PCR method was used to detect the occurrence of human adenovirus in coastal waters of Southern California. Twenty- to forty-liter water samples were collected from 12 beach locations from Malibu to the border of Mexico between February and March 1999. All sampling sites were located at mouths of major rivers and creeks. Two ultrafiltration concentration methods, tangential flow filtration (TFF) and vortex flow filtration (VFF), were compared using six environmental samples. Human adenoviruses were detected in 4 of the 12 samples tested after nucleic acid extraction of VFF concentrates. The most probable number of adenoviral genomes ranged from 880 to 7,500 per liter of water. Coliphages were detected at all sites, with the concentration varying from 5.3 to 3332 PFU/liter of water. F-specific coliphages were found at 5 of the 12 sites, with the concentration ranging from 5.5 to 300 PFU/liter. The presence of human adenovirus was not significantly correlated with the concentration of coliphage (r = 0.32) but was significantly correlated (r = 0.99) with F-specific coliphage. The bacterial indicators (total coliforms, fecal coliforms, and enterococci) were found to exceed California recreational water quality daily limits at 5 of the 12 sites. However, this excess of bacterial indicators did not correlate with the presence of human adenoviruses in coastal waters. The results of this study call for both a reevaluation of our current recreational water quality standards to reflect the viral quality of recreational waters and monitoring of recreational waters for human viruses on a regular basis. [TOP OF PAGE]

  1270. Mosaic structure of shiga-toxin-2-encoding phages isolated from Escherichia coli O157:H7 indicates frequent gene exchange between lambdoid phage genomes. Johansen,B.K., Wasteson,Y., Granum,P.E., Brynestad,S. (2001). Microbiology 147:1929-1936. Shiga-toxin-2 (stx 2 )-encoding bacteriophages were isolated from Norwegian Escherichia coli O157: H7 isolates of cattle and human origin. The phages were characterized by restriction enzyme analysis, hybridization with probes for toxin genes and selected phage DNA such as the P gene, integrase gene and IS1203, and by PCR studies and partial sequencing of selected DNA regions in the integrase to stx 2 region of the phages. The stx 2 -phage-containing bacteria from which inducible phages were detected produced similar amounts of toxin, as shown by a Vero cell assay. The results indicate that the population of stx 2 -carrying phages is heterogeneous, although the phages from epidemiologically linked E. coli O157: H7 isolates were similar. There appears to have been frequent recombination of stx 2 phages with other lambdoid phages. [TOP OF PAGE]

  1271. Effects of disinfectants on Shiga-like toxin converting phage from enterohemorrhagic Escherichia coli O157 : H7. Kajiura,T., Tanaka,M., Wada,H., Ito,K., Koyama,Y., Kato,F. (2001). J. Health Sci. 47:203-207. Inactivation of free phage carrying slt2 from enterohemorrhagic Escherichia coli (E. coli) O157 : H7 by four kinds of common disinfectants in Japan was examined under conditions with (dirty) and without (clean) interfering substance. Ethanol (EtOH) inactivated the phage within one minute under both conditions. The effect of sodium hypochlorite (NaOCl) on this phage decreased under the dirty condition, but was potentiated by increasing the concentration and contact time to the degree that could be sufficient for practical use. Use of benzalkonium chloride (BAC) at a high concentration: 0.2%, would be effective. Alkyldiaminoethylglycine hydrochloride (DAG) was not effective on this phage. [TOP OF PAGE]

  1272. Phage conversion of staphylococcal bi-component toxin. Kaneko,J. (2001). Nippon Nogeikagaku Kaishi 75:939-947. [TOP OF PAGE]

  1273. Removal of bacterial indicators and pathogens from dairy wastewater by a multi-component treatment system. Karpiscak,M.M., Sanchez,L.R., Freitas,R.J., Gerba,C.P. (2001). Water Sci. Technol. 44:183-190. Microbial removal by a multi-component treatment system for dairy and municipal wastewater is being studied in Arizona, USA. The system consists of paired solids separators, anaerobic lagoons, aerobic ponds and constructed wetlands cells. The organisms under study include: total coliform, fecal coliform, enterovirus, Listeria monocytogenes, Clostridium perfringens, coliphage, Giardia lamblia and Cryptosporidium parvum. Organism removal rates from dairy wastewater varied from 13.2 per cent for fecal coliform to 94.9 per cent for coliphage. It appears that the much higher turbidity of the dairy wastewater, nearly 1,300 NTU, decreased the treatment systems' ability to remove some microbial indicators and pathogens. Information from this study can be used to determine the adequacy of multi-component treatment systems for the control of wastewater-borne pathogens, both in municipal treatment systems as well as in confined animal feeding operations (CAFO). This information also can assist municipalities and the CAFO industry in the implementation of rational and efficient treatment strategies for appropriate reuse of wastewaters. [TOP OF PAGE]

  1274. Mechanism of the photocatalytic inactivation of Lactobacillus casei phage PL-1 by titania thin film. Kashige,N., Kakita,Y., Nakashima,Y., Miake,F., Watanabe,K. (2001). Curr. Microbiol. 42:184-189. The mechanism of the inactivation of Lactobacillus casei phage PL-1 suspended in a phosphate buffer by black-light (BL)-catalytic titanium dioxide (TiO2) thin film was studied. Generation of both superoxide anions (O2-) and hydroxyl radicals (•OH) was confirmed in the aqueous medium in which TiO2 film was settled with BL irradiation under gentle shaking. With BL-irradiation alone without TiO2 film, only O2- was generated to some extent. The genome DNA inside the phage particles was found to be fragmented by the treatment of PL-1 phages with BL-catalytic TiO2 film. The phage inactivation by BL-catalytic TiO2 film was inhibited by the addition of albumin in a concentration-dependent manner. BL-catalytic TiO2 film was considered to cause primarily the damage to the capsid protein through the generation of active oxygen species such as •OH, followed by damage to the genome DNA inside the phage particles. [TOP OF PAGE]

  1275. Elimination of fecal coliforms and F-specific RNA coliphage from oysters (Crassostrea virginica) relaid in floating containers. Kator,H., Rhodes,M. (2001). J. Food Prot. 64:796-801. Declining oyster (Crassostrea virginica) production in the Chesapeake Bay has stimulated aquaculture based on floats for off-bottom culture. While advantages of off-bottom culture are significant, the increased use of floating containers raises public health and microbiological concerns, because oysters in floats may be more susceptible to fecal contamination from storm runoff compared to those cultured on-bottom. We conducted four commercial-scale studies with market-size oysters naturally contaminated with fecal coliforms (FC) and a candidate viral indicator, F-specific RNA (FRNA) coliphage. To facilitate sampling and to test for location effects, 12 replicate subsamples, each consisting of 15 to 20 randomly selected oysters in plastic mesh bags, were placed at four characteristic locations within a 0.6- by 3.0-m "Taylor" float, and the remaining oysters were added to a depth not exceeding 15.2 cm. The float containing approximately 3,000 oysters was relaid in the York River, Virginia, for 14 days. During relay, increases in shellfish FC densities followed rain events such that final mean levels exceeded initial levels or did not meet an arbitrary product end point of 50 FC/100 ml. FRNA coliphage densities decreased to undetectable levels within 14 days (16 to 28 degrees C) in all but the last experiment, when temperatures fell between 12 and 16 degrees C. Friedman (nonparametric analysis of variance) tests performed on FC/Escherichia coli and FRNA densities indicated no differences in counts as a function of location within the float. The public health consequences of these observations are discussed, and future research and educational needs are identified. [TOP OF PAGE]

  1276. A unique group of self-splicing introns in bacteriophage T4. Khan,A.U., Ajamaluddin,M., Ahmad,M. (2001). Indian journal of biochemistry & biophysics 38:289-293. We describe in this review, the salient splicing features of group I introns of bacteriophage T4 and propose, a hypothetical model to fit in the self-splicing of nrdB intron of T4 phage. Occurrence of non-coding sequences in prokaryotic cells is a rare event while it is common in eukaryotic cells, especially the higher eukaryotes. Therefore, T4 bacteriophage can serve as a good model system to study the evolutionary aspects of splicing of introns. Three genes of T4 phage were found to have stretches of non-coding sequences which belonged to the group IA type introns of self-splicing nature. [TOP OF PAGE]

  1277. Comparative study of vaginal Lactobacillus phages isolated from women in the United States and Turkey: prevalence, morphology, host range, and DNA homology. Kilic,A.O., Pavlova,S.I., Alpay,S., Kilic,S.S., Tao,L. (2001). Clin. Diag. Lab. Immunol. 8:31-39. Lactobacilli play an important role in maintaining vaginal health. However, during bacterial vaginosis lactobacilli decrease for unknown reasons. Our preliminary study showed that phages could infect vaginal lactobacilli. Therefore, the aim of this study was to analyze the distribution, virulence, and types of vaginal Lactobacillus phages isolated from women of two countries: the United States and Turkey. A total of 209 vaginal lactobacilli were isolated from reproductive-aged women in the United States (n = 107) and Turkey (n = 102). By analysis of 16S rRNA gene sequence and by comparison of protein profiles, most lactobacilli were identified as L. crispatus, L. gasseri, and L. jensenii. After mitomycin C induction, 28% of American lactobacilli and 36% of Turkish lactobacilli released phages. A total of 67 phages were isolated and further characterized by their host range, electron microscopy, and DNA homology. All 67 phages were infective against lactobacilli from both collections. The host ranges of most phages were broad, including multiple Lactobacillus species. Even though the phages were all temperate, they were able to cause lytic infection in various strains. The electron micrographs of these phages showed a hexagon-shaped head and a long tail with or without a contractile tail sheath. Based on their morphology, these phages belonged to Bradley's phage groups A and B, and could be further classified into four morphotypes. All four types were found among American phages, but only three were found among Turkish isolates. DNA hybridization with labeled probes of the four types of phages revealed that additional genetic types existed within each morphotype among these phages. The phage genomic sizes ranged between 34 and 55 kb. Many of the lysogenic Lactobacillus strains released phages spontaneously at a high frequency of 10-3 to 10-4 PFU/cell. In conclusion, lysogeny in vaginal lactobacilli is widely spread. Some lysogenic lactobacilli spontaneously release phages with a broad host range. [TOP OF PAGE]

  1278. Octamer-based genome scanning distinguishes a unique subpopulation of Escherichia coli O157:H7 strains in cattle. Kim,J., Nietfeldt,J., Benson,A.K. (2001). Proc. Natl. Acad. Sci. USA 96:13288-13293. Multilocus-genotyping methods have shown that Escherichia coli O157:H7 is a geographically disseminated clone. However, high-resolution methods such as pulse-field gel electrophoresis demon-strate significant genomic diversity among different isolates. To assess the genetic relationship of human and bovine isolates of E. coli O157:H7 in detail, we have developed an octamer-based genome-scanning methodology, which compares the distance be-tween over-represented, strand-biased octamers that occur in the genome. Comparison of octamer-based genome-scanning prod-ucts derived from >1 megabase of the genome demonstrated the existence of two distinct lineages of E. coli O157:H7 that are disseminated within the United States. Human and bovine isolates are nonrandomly distributed among the lineages, suggesting that one of these lineages may be less virulent for humans or may not be efficiently transmitted to humans from bovine sources. Restric-tion fragment length polymorphism analysis with lambdoid phage genomes indicates that phage-mediated events are associated with divergence of the lineages, thereby providing one explana-tion for the degree of diversity that is observed among E. coli O157:H7 by other molecular-fingerprinting methods. [TOP OF PAGE]

  1279. Behavior of restriction-modification systems as selfish mobile elements and their impact on genome evolution. Kobayashi,I. (2001). Nucleic Acids Research 29:3742-3756. Restriction-modification (RM) systems are composed of genes that encode a restriction enzyme and a modification methylase. RM systems sometimes behave as discrete units of life, like viruses and transposons. RM complexes attack invading DNA that has not been properly modified and thus may serve as a tool of defense for bacterial cells. However, any threat to their maintenance, such as a challenge by a competing genetic element (an incompatible plasmid or an allelic homologous stretch of DNA, for example) can lead to cell death through restriction breakage in the genome. This post-segregational or post-disturbance cell killing may provide the RM complexes (and any DNA linked with them) with a competitive advantage. There is evidence that they have undergone extensive horizontal transfer between genomes, as inferred from their sequence homology, codon usage bias and GC content difference. They are often linked with mobile genetic elements such as plasmids, viruses, transposons and integrons. The comparison of closely related bacterial genomes also suggests that, at times, RM genes themselves behave as mobile elements and cause genome rearrangements. Indeed some bacterial genomes that survived post-disturbance attack by an RM gene complex in the laboratory have experienced genome rearrangements. The avoidance of some restriction sites by bacterial genomes may result from selection by past restriction attacks. Both bacteriophages and bacteria also appear to use homologous recombination to cope with the selfish behavior of RM systems. RM systems compete with each other in several ways. One is competition for recognition sequences in post-segregational killing. Another is super-infection exclusion, that is, the killing of the cell carrying an RM system when it is infected with another RM system of the same regulatory specificity but of a different sequence specificity. The capacity of RM systems to act as selfish, mobile genetic elements may underlie the structure and function of RM enzymes. [TOP OF PAGE]

  1280. Isolation of a lysogenic bacteriophage carrying the stx(1(OX3)) gene, which is closely associated with Shiga toxin-producing Escherichia coli strains from sheep and humans. Koch,C., Hertwig,S., Lurz,R., Appel,B., Beutin,L. (2001). J. Clin. Microbiol. 39:3992-3998. A specific PCR for the detection of a variant of the gene encoding Shiga toxin 1 (stx(1)) called stx(1(OX3)) (GenBank accession no. Z36901) was developed. The PCR was used to investigate 148 Stx(1)-producing Escherichia coli strains from human patients (n = 72), cattle (n = 27), sheep (n = 48), and a goat (n = 1) for the presence of the stx(1(OX3)) gene. The stx(1(OX3)) gene was present in 38 Shiga toxin-producing E. coli (STEC) strains from sheep belonging to serogroups O5, O125, O128, O146, and OX3 but was absent from Stx(1)-positive ovine STEC O91 strains. The stx(1(OX3)) gene was also detected in 22 STEC strains from humans with nonbloody diarrhea and from asymptomatic excreters. Serotypes O146:H21 and O128:H2 were most frequently associated with stx(1(OX3))-carrying STEC from sheep and humans. In contrast, Stx(1)-producing STEC strains from cattle and goats and 50 STEC strains from humans were all negative for the stx(1(OX3)) gene. The stx(1(OX3))-negative strains belonged to 13 serotypes which were different from those of the stx(1(OX3))-positive STEC strains. Moreover, the stx(1(OX3)) gene was not associated with STEC belonging to enterohemorrhagic E. coli (EHEC) serogroups O26, O103, O111, O118, O145, and O157. A bacteriophage carrying the stx(1(OX3)) gene (phage 6220) was isolated from a human STEC O146:H21 strain. The phage was able to lysogenize laboratory E. coli K-12 strain C600. Phage 6220 shared a similar morphology and a high degree of DNA homology with Stx(2)-encoding phage 933W, which originates from EHEC O157. In contrast, few similarities were found between phage 6220 and Stx(1)-encoding bacteriophage H-19B from EHEC O26. [TOP OF PAGE]

  1281. Phage facts. Konforti,B. (2001). Nature Struct. Biol. 8:19-20. [first paragraph] Often the simplest experiments lead to the most remarkable insights. So it was with the famous fluctuation experiments of Luria and Delbrück and the Waring blender experiments of Hershey and Chase for which they were awarded the Nobel Prize in Physiology or Medicine in 1969. While the results of these experiments are permanently etched into every first year biology student's brain, it is worth recalling what was known at the time these experiments were conducted and the conclusions the authors drew. [TOP OF PAGE]

  1282. Antacid increases survival of Vibrio vulnificus and Vibrio vulnificus phage in a gastrointestinal model. Koo,J., Marshall,D.L., Depaola,A. (2001). Appl. Environ. Microbiol. 67:2895-2902. Viable counts of three strains of Vibrio vulnificus and its phage were determined during exposure to a mechanical gastrointestinal model with or without antacid for 9 h at 37 degrees C. V. vulnificus was eliminated (>4-log reduction) within 30 min in the gastric compartment (pH decline from 5.0 to 3.5). Viable V. vulnificus cells delivered from the gastric compartment during the first 30 min of exposure reached 106 to 108 CFU/ml in the intestinal compartment after 9 h (pH 7.0). Phages were eliminated within 45 min in the gastric compartment (pH decline from 5.1 to 2.5). Less than a 2-log reduction of phage was observed in the intestinal compartment after 9 h (pH 7.0). When the gastric compartment contained antacid V. vulnificus counts decreased slightly (<2 log) during 2 h of exposure (pH decline from 7.7 to 6.0), while counts in the intestinal compartment (pH 7.5) reached 107 to 109 CFU/ml. Phage numbers decreased 1 log after 2 h in the gastric compartment (pH decline from 7.7 to 5.7) containing antacid and decreased 1 log in the intestinal compartment (pH 7.6) after 9 h. Presence of antacid in the gastric compartment of the model greatly increased the ability of both V. vulnificus and its phage to survive simulated gastrointestinal transit and may be a factor involved with oyster-associated illness. [TOP OF PAGE]

  1283. Cloning of genomic DNA of Lactococcus lactis that restores phage sensitivity to an unusual bacteriophage sk1-resistant mutant. Kraus,J., Geller,B.L. (2001). Appl. Environ. Microbiol. 67:791-798. An unusual, spontaneous, phage sk1-resistant mutant (RMSK1/1) of Lactococcus lactis C2 apparently blocks phage DNA entry into the host. Although no visible plaques formed on RMSK1/1, this host propagated phage at a reduced efficiency. This was evident from center-of-infection experiments, which showed that 21% of infected RMSK1/1 formed plaques when plated on its phage-sensitive parental strain, C2. Moreover, viable cell counts 0 and 4 h after infection were not significantly different from those of an uninfected culture. Further characterization showed that phage adsorption was normal, but burst size was reduced fivefold and the latent period was increased from 28.5 to 36 min. RMSK1/1 was resistant to other, but not all, similar phages. Phage sensitivity was restored to RMSK1/1 by transformation with a cloned DNA fragment from a genomic library of a phage-sensitive strain. Characterization of the DNA that restored phage sensitivity revealed an open reading frame with similarity to sequences encoding lysozymes (beta-1,4-N-acetylmuramidase) and lysins from various bacteria, a fungus, and phages of Lactobacillus and Streptococcus and also revealed DNA homologous to noncoding sequences of temperate phage of L. lactis, DNA similar to a region of phage sk1, a gene with similarity to tRNA genes, a prophage attachment site, and open reading frames with similarities to sun and to sequences encoding phosphoprotein phosphatases and protein kinases. Mutational analyses of the cloned DNA showed that the region of homology with lactococcal temperate phage was responsible for restoring the phage-sensitive phenotype. The region of homology with DNA of lactococcal temperate phage was similar to DNA from a previously characterized lactococcal phage that suppresses an abortive infection mechanism of phage resistance. The region of homology with lactococcal temperate phage was deleted from a phage-sensitive strain, but the strain was not phage resistant. The results suggest that the cloned DNA with homology to lactococcal temperate phage was not mutated in the phage-resistant strain. The cloned DNA apparently suppressed the mechanism of resistance, and it may do so by mimicking a region of phage DNA that interacts with components of the resistance mechanism. [TOP OF PAGE]

  1284. Phage therapy in terms of bacteriophage genetics: Hopes, prospects, safety, limitations. Krylov,V.N. (2001). Rus. J. Genet. 37:715-730. The appearance and spreading of multidrug-resistant bacterial pathogens is a consequence of the large-scale use of antibiotics in medicine. In view of this, claims for the phage therapy were renewed: in recent studies, the natural phages and their products neutralizing various proteins, as well as the bacterial products often controlled by defective prophages (bacteriocins) were applied for treatment of bacterial infections. Constructs obtained by gene engineering are increasingly used to change bacteriophage properties to expand the spectrum of their lytic activity and to eliminate therapeutic drawbacks of some natural phages. In this review, the problem of phage therapy is discussed in general with respect to bacteriophage properties, their genetics, structure, evolution, taking into account long-term experience of the author in the field of bacteriophage genetics. Note that the general concept of phage therapy should be developed to ensure long-term, efficient and harm-less phage therapy. [TOP OF PAGE]

  1285. [Phagotherapy in terms of bacteriophage genetics: hopes, perspectives, safety, limitations]. Krylov,V.N. (2001). Genetika 37:869-887. The appearance and spreading of multidrug-resistant bacterial pathogens is a consequence of the large-scale use of antibiotics in medicine. In view of this, claims for the phage therapy were renewed: in recent studies, the natural phages and their products neutralizing various proteins, as well as the bacterial products often controlled by defective prophages (bacteriocins) were applied for treatment of bacterial infections. Constructs obtained by gene engineering are increasingly used to change some bacteriophage: properties to expand the spectrum of their lytic activity and to eliminate therapeutic drawbacks of some natural phages. In this review, the problem of phage therapy is discussed in general with respect to bacteriophage properties, their genetics, structure, evolution, taking into account long-term experience of the author in the field of bacteriophage genetics. Note that the general concept of phage therapy should be developed to ensure long-term, efficient and harmless phage therapy. [TOP OF PAGE]

  1286. The bacteriophage lambda attachment site in wild strains of Escherichia coli. Kuhn,J., Campbell,A. (2001). J. Mol. Evol. 53:607-614. The attachment site (attlambda) of bacteriophage lambda was examined in wild strains of Escherichia coli. Although the att region is non-coding, the DNA sequence was invariant in the 13 strains examined. Two other non-coding regions showed nine changes, all associated with a single strain. In four of 33 strains, sequences were inserted in or near the attlambda site and in two of these the insert was related to lambda. Among strains that can be lysogenized by lambda, integration was via the attlambda site in all cases. Some resistant strains can be lysogenized, and these have been termed "lenient". Most of these fail to give normal phage yield after induction. In some cases rare lysogens have been formed in cells that belong to a mutant sub-population. [TOP OF PAGE]

  1287. Use of bacteriophage therapy in surgical practice. Lakhno,V.M., Bordunovskii,V.N. (2001). Vestnik Khirurgii Imeni I. I. Grekova 160:122-125. [TOP OF PAGE]

  1288. Population dynamics and diversity of phytoplankton, bacteria and viruses in a seawater enclosure. Larsen,A., Castberg,T., Sandaa,R.-A., Brussaard,C.P.D., Egge,J.K., Heldal,M., Paulino,A., Thyrhaug,R., van Hannen,E.J., Bratbak,G. (2001). Mar. Ecol. Prog. Ser. 221:47-57. We now know that the abundance of free viruses in most marine environments is high. There is still, however, a lack of understanding of their occurrence and distribution and of in situ relationships between viral and host communities in natural environments. This may be partly due to methodological limitations. Our main aim was therefore to perform a case study in which a variety of methods were applied in order to give an improved, high-resolution description of the microbial communities in a natural environment, In order to do this we combined light microscopy (LM), transmission electron microscopy (TEM), flow cytometry (FCM), PCR denaturing gradient gel electrophoresis (PCR-DGGE) and pulsed-field gel electrophoresis (PFGE) and studied the diversity and succession of algae, bacteria and viruses in a nutrient enriched seawater enclosure. In the enclosure we experienced a situation where the development of the dominating algal population, which consisted of several flagellate species, was followed by proliferation of several different size-classes of viruses. The total bacterial number decreased markedly during the flagellate bloom but the community composition was maintained and the diversity remained high. Our results indicate a close linkage between various algal, bacterial and viral populations and show that virioplankton do not necessarily terminate algal and bacterial blooms but that they keep the host populations at non-blooming levels. [TOP OF PAGE]

  1289. A novel virus (HaNIV) causes lysis of the toxic bloom-forming alga Heterosigma akashiwo (Raphidophyceae). Lawrence,J.E., Chan,A.M., Suttle,C.A. (2001). J. Phycol. 37:216-222. We describe a previously unknown virus that causes lysis of the toxic blopm-forming alga Heterosigma akashiwo (Hada) Hara et Chihara (Raphidophyceae). Heterosigma akashiwo nuclear inclusion virus (HaNIV) does not resemble other algal viruses described to date. HaNIV is small (ca. 30 nm diameter), is assembled in the nucleus, and forms crystalline arrays. We estimate that approximately 105 HaNIV particles are released during lysis of a cell. During a time-course experiment, TEM revealed the first signs of HaNIV infection 24 h after viral addition, and by 74 h 98% of observed cells were visibly infected. The onset of cell lysis, as indicated by a decrease in the relative fluorescence of the cultures, was apparent by 42 h postinfection. The heterochromatin of infected cells is frequently found at the margin of the nucleoplasm, which is consistent with virus-mediated programmed cell death, or apoptosis. HaNIV is clearly different from other described viruses that infect alg ae, including other viral pathogens of H, akashiwo. These results indicate that viruses other than Phycodnaviridae are pathogens and cause mortality of microalgae in marine systems. It is Likely that HaNIV plays an integral role in the population dynamics of H. akashiwo. [TOP OF PAGE]

  1290. Where are the pseudogenes in bacterial genomes? Lawrence,J.G., Hendrix,R.W., Casjens,S. (2001). Trends Microbiol. 9:535-540. Most bacterial genomes have very few pseudogenes; notable exceptions include the genomes of the intracellular parasites Rickettsia prowazekii and Mycobacterium leprae. As DNA can be introduced into microbial genomes in many ways, the compact nature of these genomes suggests that the rate of DNA influx is balanced by the rate of DNA deletion. We propose that the influx of dangerous genetic elements such as transposons and bacteriophages selects for the maintenance of relatively high deletion rates in most bacteria; the sheltered lifestyle of intracellular parasites removes this threat, leading to reduced deletion rates and larger pseudogene loads. [TOP OF PAGE]

  1291. Viruses in the plankton of freshwater and saline Antarctic lakes. Laybourn-Parry,J., Hofer,J.S., Sommaruga,R. (2001). Freshw. Biol. 46:1279-1287. 1. Virus-like particle (VLP) abundances in nine freshwater to saline lakes in the Vestfold Hills, Eastern Antarctica (68degree S) were determined in December 1999. In the ultra-oligotrophic to oligotrophic freshwater lakes, VLP abundances ranged from 1.01 to 3.28 X 106 mL-1 in the top 6 m of the water column. In the saline lakes the range was between 6.76 and 36.5 X 106 mL-1. The lowest value was found in meromictic Ace Lake and the highest value in hypersaline Lake Williams. Virus to bacteria ratios (VBR) were lowest in the freshwater lakes and highest in the saline lakes, with a maximum of 23.4 in the former and 50.3 in the latter. 2. A range of morphologies among VLP was observed, including phages with short (Podoviridae) and long tails, icosahedric viruses of up to 300 nm and star-like particles of about 80 nm diameter. 3. In these microbially dominated ecosystems there was no correlation between VLP and either bacterial numbers of chlorophyll a. There was a significant correlation between VLP abundances and dissolved organic carbon concentration (r = 0.845, P < 0.01). 4. The data suggested that viruses probably attack a spectrum of bacteria and protozoan species. Virus-like particle numbers in the freshwater lakes were lower than values reported for lower latitude systems. Those in the saline lakes were comparable with abundances reported from other Antarctic lakes, and were higher than most values published for lower latitude lakes and many marine systems. Across the salinity spectrum from freshwater through brackish to hypersaline, VLP concentrations increased roughly in relation to increasing trophy. 5. Given that Antarctic lakes have a plankton almost entirely made up of bacteria and protists, and that VLP abundances are high, it is likely that viruses play a pivotal role in carbon cycling in these extreme ecosystems. [TOP OF PAGE]

  1292. Efficacy of bacteriophage use in complex treatment of the patients with burn wounds. Lazareva,E.B., Smirnov,S.V., Khvatov,V.B., Spiridonova,T.G., Bitkova,E.E., Darbeeva,O.S., Mayskaya,L.M., Parphenyuk,R.L., Menshikov,D.D. (2001). Antibiot. Khimoter. 46:10-14. Results of clinical and laboratory evaluation of the treatment with pyobacteriophage in tablets of the patients with burn wounds are presented. It was shown that phagotherapy provided more rapid cure of pyoseptic complications, temperature normalization, wounds purification and lower lethality Bacteriological analysis of wound secretions revealed that after the treatment staphylococci and streptococci were cultured 2 times rarely, Proteus spp. Were isolated 1.5 times rarely, E. coli was not isolated. The amount of positive haemocultures also diminished. Investigation of immunologic status demonstrated statistically significant normalization of immunity on cell level. Phagocytosis level didn't change while in control group (without bacteriophage use) it became lower. Antibody level enhanced but less extensively than in control group. The results of trial demonstrates positive effect of phagotherapy use at the patients with burns. [TOP OF PAGE]

  1293. The effect of bacteriophage treatment to reduce the rapid dissemination of Salmonella typhimurium in pigs. Lee,N., Harris,D.L. (2001). Proceedings of the American Association of Swine Veterinarians 32:555-557. Bacteriophage treatment significantly reduced the rapid dissemination of Salmonella typhimurium in tonsil and cecum, where the highest number of Salmonella was recovered in pigs experimentally infected with S. typhimurium. The rapid dissemination of Salmonella in market weight pigs prior to slaughter may pose a potential risk in contaminating pork products. Phage treatment should be considered as an intervention strategy to reduce the number of Salmonella in pigs. [TOP OF PAGE]

  1294. Examination of bacteriophage as a biocontrol method for Salmonella on fresh-cut fruit: a model study. Leverentz,B., Conway,W.S., Alavidze,Z., Janisiewicz,W.J., Fuchs,Y., Camp,M.J., Chighladze,E., Sulakvelidze,A. (2001). J. Food Prot. 64:1116-1121. The preparation and distribution of fresh-cut produce is a rapidly developing industry that provides the consumer with convenient and nutritious food. However, fresh-cut fruits and vegetables may represent an increased food safety concern because of the absence or damage of peel and rind, which normally help reduce colonization of uncut produce with pathogenic bacteria. In this study, we found that Salmonella enteritidis populations can (i) survive on fresh-cut melons and apples stored at 5 degrees C, (ii) increase up to 2 log units on fresh-cut fruits stored at 10 degrees C, and (iii) increase up to 5 log units at 20 degrees C during a storage period of 168 h. In addition, we examined the effect of lytic, Salmonella-specific phages on reducing Salmonella numbers in experimentally contaminated fresh-cut melons and apples stored at various temperatures. We found that the phage mixture reduced Salmonella populations by approximately 3.5 logs on honeydew melon slices stored at 5 and 10 degrees C and by approximately 2.5 logs on slices stored at 20 degrees C, which is greater than the maximal amount achieved using chemical sanitizers. However, the phages did not significantly reduce Salmonella populations on the apple slices at any of the three temperatures. The titer of the phage preparation remained relatively stable on melon slices, whereas on apple slices the titer decreased to nondetectable levels in 48 h at all temperatures tested. Inactivation of phages, possibly by the acidic pH of apple slices (pH 4.2 versus pH 5.8 for melon slices), may have contributed to their inability to reduce Salmonella contamination in the apple slices. Higher phage concentrations and/or the use of low-pH-tolerant phage mutants may be required to increase the efficacy of the phage treatment in reducing Salmonella contamination of fresh-cut produce with a low pH. [TOP OF PAGE]

  1295. Colloidal interactions in suspensions of rods. Lin,K., Crocker,J.C., Zeri,A.C., Yodh,A.G. (2001). Phys. Rev. Lett. 87:088301 We report direct measurements of entropic interactions of colloidal spheres in suspensions of rodlike fd bacteriophage. We investigate the influence of sphere size, rod concentration, and ionic strength on these interactions. Although the results compare favorably with a recent calculation, small discrepancies reveal entropic effects due to rod flexibility. At high salt concentrations, the potential turns repulsive as a result of viral adsorption on the spheres and viral bridging between the spheres. [TOP OF PAGE]

  1296. Mechanism of host cell death induced by infection of Escherichia coli with the c2 clear-plaque mutant of phage f1. Lin,S.H., Chen,W.P., Kuo,T.T. (2001). Botanical Bulletin of Academia Sinica (Taipei) 42:45-52. The c2 clear-plaque mutant arose spontaneously from the turbid plaque-inducing wild-type strain of bacteriophage f1. The mechanism of host cell death induced by infection of Escherichia coli with c2 has now been investigated. A marked decrease in cell membrane potential was apparent as early as 30 min after infection with c2, and leakage of cell contents was apparent after 4 h. Transmission electron microscopy also revealed the accumulation of granular membrane-like structures within cells at early stages of c2 infection. Electrophoretic analysis showed that the abundance of several bacterial outer membrane proteins was markedly reduced 2 h after infection with c2. Furthermore, substantial amounts of the phage coat protein (gpVIII) and single-stranded DNA-binding protein (gpV) were apparent in the inner membrane of c2-infected cells 2 h after infection. These data support the hypothesis that the death of c2-infected cells results from phage-induced damage to the bacterial cell membrane. [TOP OF PAGE]

  1297. Vibrio detection by 6 species of bacteriophages of Vibrionaceae. Lin,Y., Chen,K., Ou,J. (2001). Zhonghua Weishengwuxue He Mianyixue Zazhi 21:108-110. Objective: To predigest the detection procedure of 6 species of pathogenic bacteria of Vibrionaceae. Methods: Six species of specific bacteriophages were isolated and filtered to identify and type corresponding pathogenic bacteria. Results: 440 strains of bacteriophages, among which 54 strains were selected to form the typing bacteriophage groups, were isolated from 1400 environmental specimens. The typing bacteriophage groups could identify and type 80%-90% of bacteria in laboratory or those from environment. Conclusion: The application of specific bacteriophages to identify the corresponding pathogenic bacteria is fast, economic and easy to operate, so bacteriophage typing is an effective method in epidemiology investigation. [TOP OF PAGE]

  1298. Depolymerization of the capsular polysaccharide from Vibrio cholerae O139 by a lyase associated with the bacteriophage JA1. Linnerborg,M., Weintraub,A., Albert,M.J., Widmalm,G. (2001). Carbohydrate Research 333:263-269. We have studied the interaction between the Vibrio cholerae O139 specific phage JA1, belonging to the Podoviridae family, and the capsular polysaccharide (CPS) of the parent strain from which the phage was isolated. Upon incubation of the JA1 phage with the CPS, oligosaccharides were isolated and purified. The oligosaccharides derived from one (shown below) and two repeating units of the CPS were characterized using NMR spectroscopy, mass spectrometry and sugar analysis (structure: see text). The cleavage was found to occur by beta-elimination at the 4-substituted alpha-linked galacturonic acid, which results in a 4-deoxy-beta-L-threo-hex-4-enopyranosyl uronic acid group (Sug). The enzyme associated with the JA1 phage responsible for the depolymerization of the V. cholerae O139 CPS is thus a lyase. [TOP OF PAGE]

  1299. The effects of seasonal variability and weather on microbial fecal pollution and enteric pathogens in a subtropical estuary. Lipp,E.K., Kurz,R., Vincent,R., Rodriguez-Palacios,C., Farrah,S.R., Rose,J.B. (2001). Estuaries 24:266-276. The Charlotte Harbor estuary in southwest Florida was sampled monthly for one year at twelve stations, in the lower reaches of the Myakka and Peace Rivers. The objectives of the study were to address the distribution and seasonal changes in microbial indicators and human pathogen levels in Charlotte Harbor shellfish and recreational waters, and to determine those factors that may be important in the transport and survival of pathogens. Monthly water samples and quarterly sediment samples were analyzed for fecal coliform bacteria, enterococci, Clostridium perfringens, and coliphage. Quarterly samples also were analyzed for the enteric human pathogens, Cryptosporidium spp., Giardia spp., and enteroviruses. Fecal indicator organisms were generally concentrated in areas of low salinity and high densities of septic systems; however, pollution became widespread during wet weather in the late fall and winter of 1997-1998, coincident with a strong El Nino event. Between December 1997 and February 1998, enteroviruses were detected at 75% of the sampling stations; none were detected in other months. Enteric protozoa were detected infrequently and were not related to seasonal influences. Fecal indicators and enteroviruses were each significantly associated with rainfall, streamflow, and temperature. Regression models suggest that temperature and rainfall can predict the occurrence of enteroviruses in 93.7% of the cases. Based on findings in this watershed, factors such as variability in precipitation, streamflow, and temperature show promise in modeling and forecasting periods of poor coastal water quality. [TOP OF PAGE]

  1300. Physiological function of exopolysaccharides produced by Lactococcus lactis. Looijesteijn,P.J., Trapet,L., de Vries,E., Abee,T., Hugenholtz,J. (2001). Int. J. Food Microbiol. 64:71-80. The physiological function of EPS produced by Lactococcus lactis was studied by comparing the tolerance of the non-EPS producing strain L. lactis ssp. cremoris MG1614 and an EPS producing isogenic variant of this strain to several anti-microbial factors. There was no difference in the sensitivity of the strains to increased temperatures, freezing or freeze-drying and the antibiotics, penicillin and vancomycin. A model system showed that EPS production did not affect the survival of L. lactis during passage through the gastrointestinal tract although the EPS itself was not degraded during this passage. The presence of cell associated EPS and EPS in suspension resulted in an increased tolerance to copper and nisin. Furthermore, cell associated EPS also protected the bacteria against bacteriophages and the cell wall degrading enzyme lysozyme. However, it has not been possible, so far, to increase EPS production using the presence of copper, nisin, lysozyme or bacteriophages as inducing factors. [TOP OF PAGE]

  1301. Distribution, isolation, host specificity, and diversity of cyanophages infecting marine Synechococcus spp. in river estuaries. Lu,J., Chen,F., Hodson,R.E. (2001). Appl. Environ. Microbiol. 67:3285-3290. The abundance of cyanophages infecting marine Synechococcus spp. increased with increasing salinity in three Georgia coastal rivers. About 80% of the cyanophage isolates were cyanomyoviruses. High cross-infectivity was found among the cyanophages infecting phycoerythrin-containing Synechococcus strains. Cyanophages in the river estuaries were diverse in terms of their morphotypes and genotypes. [TOP OF PAGE]

  1302. Elution, detection, and quantification of polio I, bacteriophages, Salmonella montevideo, and Escherichia coli O157:H7 from seeded strawberries and tomatoes. Lukasik,J., Bradley,M.L., Scott,T.M., Hsu,W.Y., Farrah,S.R., Tamplin,M.L. (2001). J. Food Prot. 64:292-297. This study compared the effect of different physical and chemical treatments of strawberries and tomatoes to determine their ability to recover seeded viral and bacterial pathogens from produce surfaces. Solutions of salts, amino acids, complex media, and detergents were compared as eluants. Phosphate-buffered saline (PBS) containing 0.1% Tween 80 eluted the highest number of seeded microorganisms. Elution with this defined solution was then compared under different conditions of physical agitation. Rotary shaking for 20 min at 36 degrees C eluted higher numbers of viruses and bacteria than did low- or high-speed stomaching. Commercially available and laboratory prepared bacteriological differential media were compared for their ability to recover and distinguish eluted Salmonella montevideo and Escherichia coli O157:H7 strains from seeded produce. The recovery of seeded bacterial pathogens was low when differential media containing selective ingredients were used (MacConkey sorbitol agar, XLD agar, MacConkey agar). Highest recoveries were obtained on a medium consisting of tryptic soy agar supplemented with sodium thiosulfate and ferric ammonium citrate compared with selective media that inhibited up to 50% of the growth of the eluted microorganisms. [TOP OF PAGE]

  1303. The genome of archaeal prophage PsiM100 encodes the lytic enzyme responsible for autolysis of Methanothermobacter wolfeii. Luo,Y., Pfister,P., Leisinger,T., Wasserfallen,A. (2001). J. Bacteriol. 183:5788-5792. Methanothermobacter wolfeii (formerly Methanobacterium wolfei), a thermophilic methanoarchaeon whose cultures lyse upon hydrogen starvation, carries a defective prophage called PsiM100 on its chromosome. The nucleotide sequence of PsiM100 and its flanking regions was established and compared to that of the previously sequenced phage PsiM2 of Methanothermobacter marburgensis (formerly Methanobacterium thermoautotrophicum Marburg). The PsiM100 genome extends over 28,798 bp, and its borders are defined by flanking 21-bp direct repeats of a pure-AT sequence, which very likely forms the core of the putative attachment site where the crossing over occurred during integration. A large fragment of 2,793 bp, IFa, apparently inserted into PsiM100 but is absent in the genome of PsiM2. The remaining part of the PsiM100 genome showed 70.8% nucleotide sequence identity to the whole genome of PsiM2. Thirty-four open reading frames (ORFs) on the forward strand and one ORF on the reverse strand were identified in the PsiM100 genome. Comparison of PsiM100-encoded ORFs to those encoded by phage PsiM2 and to other known protein sequences permitted the assignment of putative functions to some ORFs. The ORF28 protein of PsiM100 was identified as the previously known autolytic enzyme pseudomurein endoisopeptidase PeiW produced by M. wolfeii. [TOP OF PAGE]

  1304. Presence of bacteriophages in animal feed as indicators of fecal contamination. Maciorowski,K.G., Pillai,S.D., Ricke,S.C. (2001). J. Environ. Sci. Health Part B Pestic. 36:699-708. The objectives of this study were to determine if indigenous male specific and somatic bacteriophages could be detected in animal feeds and if isolated phages contained RNA or DNA. Seven fresh feeds, 2 fresh feed ingredients, 7 stored feeds, 2 stored feed ingredients, and 8 samples of poultry diets suspected to contain Salmonella spp. were enriched and spot plated for indigenous phages using Escherichia coli Famp and CN-13 as hosts. Bacteriophage numbers were below detection without enrichment, but both male specific and somatic coliphages were detected in all animal feeds, feed ingredients, and poultry diets after 16 h of enrichment, even after the samples had been stored for 14 months of storage at -20 C. Five out of 9 fresh feeds and 7 out of 8 stored feeds contained RNA somatic phages. [TOP OF PAGE]

  1305. Analysis of the genetic switch and replication region of a P335-type bacteriophage with an obligate lytic lifestyle on Lactococcus lactis. Madsen,S.M., Mills,D., Djordjevic,G., Israelsen,H., Klaenhammer,T.R. (2001). Appl. Environ. Microbiol. 67:1128-1139. The DNA sequence of the replication module, part of the lysis module, and remnants of a lysogenic module from the lytic P335 species lactococcal bacteriophage variant phi31 was determined, and its regulatory elements were investigated. The identification of a characteristic genetic switch including two divergent promoters and two cognate repressor genes strongly indicates that variant phi31 was derived from a temperate bacteriophage. Regulation of the two early promoters was analyzed by primer extension and transcriptional promoter fusions to a lacLM reporter. The regulatory behavior of the promoter region differed significantly from the genetic responses of temperate Lactococcus lactis phages. The cro gene homologue regulates its own production and is an efficient repressor of cI gene expression. No detectable cI gene expression could be measured in the presence of cro. cI gene expression in the absence of cro exerted minor influences on the regulation of the two promoters within the genetic switch. Homology comparisons revealed a replication module which is most likely expressed from the promoter located upstream of the cro gene homologue. The replication module encoded genes with strong homology to helicases and primases found in several Streptococcus thermophilus phages. Downstream of the primase homologue, an AT-rich noncoding origin region was identified. The characteristics and location of this region and its ability to reduce the efficiency of plaquing of variant phi31 106-fold when present at high copy number in trans provide evidence for identification of the phage origin of replication. Phage variant phi31 is an obligately lytic phage that was isolated from commercial dairy fermentation environments. Neither a phage attachment site nor an integrase gene, required to establish lysogeny, was identified, explaining its lytic lifestyle and suggesting its origin from a temperate phage ancestor. Several regions showing extensive DNA and protein homologies to different temperate phages of Lactococcus, Lactobacillus, and Streptococcus were also discovered, indicating the likely exchange of DNA cassettes through horizontal gene transfer in the dynamic ecological environment of dairy fermentations. [TOP OF PAGE]

  1306. Sequence analysis and molecular characterization of the Lactococcus lactis temperate bacteriophage BK5-T. Mahanivong,C., Boyce,J.D., Davidson,B.E., Hillier,A.J. (2001). Appl. Environ. Microbiol. 67:3564-3576. The Lactococcus lactis temperate bacteriophage BK5-T is one of twelve type phages that define L. lactis phage species. This paper describes the nucleotide sequence and analysis of a 21-kbp region of the BK5-T genome and completes the nucleotide sequence of the genome of this phage. The 40,003-nucleotide linear genome encodes 63 open reading frames. Sequence runoff experiments showed that the cohesive ends of the BK5-T genome contained a 12-bp 3' single-stranded overhang with the sequence 5'-CACACACATAGG-3'. Two major BK5-T structural proteins, of approximately 30 and 20 kDa, were identified, and N-terminal sequence analysis determined that they were encoded by orf7 and orf12, respectively. A 169-bp fragment containing a 37-bp direct repeat and several smaller repeat sequences conferred resistance to BK5-T infection when introduced in trans to the host cell and is likely a part of the BK5-T origin of replication (ori). [TOP OF PAGE]

  1307. Growth and survival of clinical vs. environmental species of Aeromonas in tap water. Mary,P., Buchet,G., Defives,C., Hornez,J.P. (2001). Int. J. Food Microbiol. 69:191-198. The ability of four species of Aeromonas (two of clinical and two of environmental origin) to survive and/or grow in tap water microcosms supplemented with sodium thiosulphate was tested. After bottling, the autochthonous microflora reached 6 x 105 cfu ml-1 after a 5-day incubation period in tap water unfiltered and which was non-autoclaved. In filtered tap water, "ultramicrocells" were detected and final populations of ca. 106 cfu ml-1 after 7 days were obtained. Aeromonas was inoculated at an initial cell concentration of ca. 104 cfu ml-1. All strains were able to grow in tap water samples, which were filtered and autoclaved, and a final concentration of 105-106 cfu ml-1 was observed. Any inherent capability of Aeromonas to grow in tap water was eliminated by the presence of autochthonous microflora and "ultramicrocells" bacteria. Survival rates were strain- and microcosm-dependent. In unfiltered-non-autoclaved water, viable counts declined to below the detection limit (i.e. 1 log cfu ml-1) in 1.5 to 20 days. The declines in viable counts were even more pronounced in the filtered microcosm. Although inoculation ratios (100/1 in unfiltered-non-autoclaved and 1,000/1 in filtered microcosms) were favourable for aeromonads, at least for I to 3 days, the organisms disappeared in these microcosms. Thus, competition for nutrients was an unlikely cause of the limitation of aeromonads. The bacteriolytic effect of enzymes released by membrane vesicles from the autochthonous microflora and of "tail phage-like particles" bacteriocins were suggested as an in situ control of aeromonad populations. The present study showed that environmental strains of Aeromonas had no ecological advantage over clinical isolates. Thus, waterborne infections and contaminations of foods by pathogenic Aeromonas species could not be discounted. [TOP OF PAGE]

  1308. Mu-like prophage in serogroup B Neisseria meningitidis coding for surface-exposed antigens. Masignani,V., Giuliani,M.M., Tettelin,H., Comanducci,M., Rappuoli,R., Scarlato,V. (2001). Infect. Immun. 69:2580-2588. Sequence analysis of the genome of Neisseria meningititdis serogroup B revealed the presence of an apprx35-kb region inserted within a putative gene coding for an ABC-type transporter. The region contains 46 open reading frames, 29 of which are colinear and homologous to the genes of Escherichia coli Mu phage. Two prophages with similar organizations were also found in serogroup A meningococcus, and one was found in Haemophilus influenzae. Early and late phage functions are well preserved in this family of Mu-like prophages. Several regions of atypical nucleotide content were identified. These likely represent genes acquired by horizontal transfer. Three of the acquired genes are shown to code for surface-associated antigens, and the encoded proteins are able to induce bactericidal antibodies. [TOP OF PAGE]

  1309. Bacteriophage WO and virus-like particles in Wolbachia, an endosymbiont of arthropods. Masui,S., Kuroiwa,H., Sasaki,T., Inui,M., Kuroiwa,T., Ishikawa,H. (2001). Biochem. Biophys. Res. Com. 283:1099-1104. Wolbachia are intracellular symbionts mainly found in arthropods, causing various sexual alterations on their hosts by unknown mechanisms. Here we report the results that strongly suggest that Wolbachia have virus-like particles of phage WO, which was previously identified as a prophage-like element in the Wolbachia genome. Wolbachia (strain wTai) infection in an insect was detected with the antibody against Wsp, an outer surface protein of Wolbachia, by fluorescence microscopy and immunoelectron-microscopy for the first time. Virus-like particles in Wolbachia were observed by electron-microscopy. The 0.22-&mgr;m filtrate of insect ovary contained DAPI-positive particles, and PCR analysis demonstrated that a phage WO DNA passed through the filter while Wolbachia DNA were eliminated, suggesting that the DAPI-positive particles were phage WO. [TOP OF PAGE]

  1310. Turning the phage on produce pathogens. McBride,J. (2001). Agricultural Research 2001(July), 12. Even bacteria have their nemesis. Tiny viruses, called phages, infect and kill bacteria naturally, including the foodborne pathogens that sometimes make humans so sick, they wish they were dead. ¶ So why not put these phages to work on fresh-cut fruit, thought ARS plant pathologists Britta Leverentz and William S. Conway at the Produce Quality and Safety Laboratory in Beltsville, Maryland. ¶ Since phages home in on a bacterium's surface proteins, they are very selective about their hosts. Phages specific for Salmonella, for instance, would leave beneficial bacteria free to multiply on fresh-cut produce and crowd out potential pathogens, Leverentz explains. ¶ What's more, these tiny viruses are natural, safe, and ubiquitous. A small dropperful of fresh water from a stream or lake, for example, contains an average 250 million phages. Before antibiotics, phages were used to treat human infections in the United States and are still used therapeutically in other parts of the world. ¶ Phages are already under study to control pathogens in poultry, meat, and eggs. Leverentz and Conway are the first to investigate their potential to reduce pathogens on fruits and vegetables— both whole and fresh-cut. They are working under a cooperative research and development agreement with Intralytix in Baltimore, Maryland, which is providing known phages for Salmonella Enteritidis. A patent application has been filed on the use of phages with produce. [TOP OF PAGE]

  1311. Improvement and optimization of two engineered phage resistance mechanisms in Lactococcus lactis. McGrath,S., Fitzgerald,G.F., van Sinderen,D. (2001). Appl. Environ. Microbiol. 67:608-616. Homologous replication module genes were identified for four P335 type phages. DNA sequence analysis revealed that all four phages exhibited more than 90% DNA homology for at least two genes, designated rep2009 and orf17. One of these genes, rep2009, codes for a putative replisome organizer protein and contains an assumed origin of phage DNA replication (ori2009), which was identical for all four phages. DNA fragments representing the ori2009 sequence confer a phage-encoded resistance (Per) phenotype on lactococcal hosts when they are supplied on a high-copy-number vector. Furthermore, cloning multiple copies of the ori2009 sequence was found to increase the effectiveness of the Per phenotype conferred. A number of antisense plasmids targeting specific genes of the replication module were constructed. Two separate plasmids targeting rep2009 and orf17 were found to efficiently inhibit proliferation of all four phages by interfering with intracellular phage DNA replication. These results represent two highly effective strategies for inhibiting bacteriophage proliferation, and they also identify a novel gene, orf17, which appears to be important for phage DNA replication. Furthermore, these results indicate that although the actual mechanisms of DNA replication are very similar, if not identical, for all four phages, expression of the replication genes is significantly different in each case. [TOP OF PAGE]

  1312. Novel in vivo use of a polyvalent Streptomyces phage to disinfest Streptomyces scabies-infected seed potatoes. McKenna,F., El-Tarabily,K.A., Hardy,G.E.S.T., Dell,B. (2001). Plant Pathol. 50:666-675. A highly virulent and polyvalent Streptomyces phage was isolated from a potato field near Albany, Western Australia. The efficacy of the isolated phage to disinfest seed potato tubers artificially inoculated with a common scab-causing streptomycete was evaluated. The phage suspension was prepared in a mini-bioreactor. Diseased potatoes were bathed in a phage suspension (1X109 plaque-forming units per mL) for 24 h. The suspension was constantly circulated within a novel 25 L phage bath by means of an air-sparging pipe driven from an air compressor. Phage-treated scab-affected seed potatoes planted into free-draining polystyrene boxes containing steam-pasteurized field soil produced tuber progeny with significantly (P<0.05) reduced levels of surface lesions of scab (1.2%) compared with tubers harvested from nonphage-treated tubers (23%). The number of scab lesions was also significantly reduced (P<0.05) by phage treatment of mother tubers. No significant differences were recorded in weight, size or number of harvested tubers from phage-treated or nontreated mother tubers. This is the first in vivo study that has used Streptomyces phage to significantly disinfest seed potatoes of Streptomyces scabies and thereby reduce contamination of soil from seed-tuber-borne inoculum and reduce infection of daughter tubers. [TOP OF PAGE]

  1313. Viral and chemical tracer movement through contrasting soils. McLeod,M., Aislabie,J., Smith,J., Fraser,R., Roberts,A., Taylor,M. (2001). J. Environ. Qual. 30:2134-2140. Land treatment of animal or human waste can result in chemical and microbial contamination of shallow ground water and/or water-ways. We investigated the fate of a host-specific Salmonella bacteriophage and a nonreactive chemical (Br-) tracer when applied to large intact lysimeter soil cores (500 mm diam. by 700 mm high). The soils included a poorly drained Gley Soil and well-drained Pumice, Allophanic, and Recent Soils. A depth of 30 mm of water containing the bacteriophage and Br- was applied to the soil at a rate of 5 mm h(-1) followed by up to about 1.8 pore volumes of simulated rainfall. Resulting leachates, collected continuously over at least one pore volume were analyzed for the bacteriophage and bromide (Br-) tracers. Bromide moved uniformly through the Pumice and Allophanic Soils with peak concentrations at about 1 pore volume, while the bacteriophage was detected only at trace levels or not at all. In contrast, both Br- and bacteriophage tracers moved rapidly through Gley and Recent Soils, appearing early in the leachate and then tailing off. Such flow patterns in the Gley and Recent Soils are indicative of bypass flow. Coarse soil structure in the Gley Soil, and finger-flow due to water repellency in the sandy Recent Soil are considered responsible for the observed bypass flow in these two soils. Allophanic and Pumice Soils have finer, more porous soil structure leading to a predominance of matrix flow over bypass flow. This study suggests vertical movement of viruses varies significantly with soil type. [TOP OF PAGE]

  1314. Phi29 family of phages. Meijer,W.J., Horcajadas,J.A., Salas,M. (2001). Microbiol. Mol. Biol. Rev. 65:261-287. Continuous research spanning more than three decades has made the Bacillus bacteriophage phi29 a paradigm for several molecular mechanisms of general biological processes, such as DNA replication, regulation of transcription, phage morphogenesis, and phage DNA packaging. The genome of bacteriophage phi29 consists of a linear double-stranded DNA (dsDNA), which has a terminal protein (TP) covalently linked to its 5' ends. Initiation of DNA replication, carried out by a protein-primed mechanism, has been studied in detail and is considered to be a model system for the protein-primed DNA replication that is also used by most other linear genomes with a TP linked to their DNA ends, such as other phages, linear plasmids, and adenoviruses. In addition to a continuing progress in unraveling the initiation of DNA replication mechanism and the role of various proteins involved in this process, major advances have been made during the last few years, especially in our understanding of transcription regulation, the head-tail connector protein, and DNA packaging. Recent progress in all these topics is reviewed. In addition to phi29, the genomes of several other Bacillus phages consist of a linear dsDNA with a TP molecule attached to their 5' ends. These phi29-like phages can be divided into three groups. The first group includes, in addition to phi29, phages PZA, phi15, and BS32. The second group comprises B103, Nf, and M2Y, and the third group contains GA-1 as its sole member. Whereas the DNA sequences of the complete genomes of phi29 (group I) and B103 (group II) are known, only parts of the genome of GA-1 (group III) were sequenced. We have determined the complete DNA sequence of the GA-1 genome, which allowed analysis of differences and homologies between the three groups of phi29-like phages, which is included in this review. [TOP OF PAGE]

  1315. Characterization of two novel Rhizobium leguminosarum bacteriophages from a field release site of genetically-modified rhizobia. Mendum,T.A., Clark,I.M., Hirsch,P.R. (2001). Antonie van Leeuwenhoek J. Microbiol. 79:189-197. Two Rhizobium leguminosarum biovar viceae bacteriophages with contrasting properties were isolated from a field site in which the survival of genetically modified R. leguminosarum inoculants had been monitored for several years. Inoculant strain RSM2004 was used as the indicator for phage isolation and propagation. One phage, RL1RES, was temperate and could not replicate in any of the 42 indigenous R. leguminosarum field isolates tested although nested PCR indicated that phage sequences were present in six of the isolates. The second phage, RL2RES, was virulent, capable of generalised transduction, contained DNA with modified cytosine residues, and was capable of infecting all field isolates tested although the GM inoculant strain CT0370 was resistant. Sequence with homology to RL2RES was detected by nested PCR in six of the 42 field-isolates. These were not the same isolates that showed homology to RL1RES. The implication of these findings for the survival of rhizobial inoculants, and the ecology of phages and their host bacteria, are discussed. [TOP OF PAGE]

  1316. Evidence for bacteriophages within gram-negative cocci: Obligate endoparasitic bacteria of Naegleria sp. Michel,R., Schmid,E.N., Gmeiner,G., Mueller,K.D., Hauroeder,B. (2001). Acta Protozoologica 40:229-232. Gram-negative cocci observed as endocytobionts within the cytoplasm of a Naegleria strain isolated from a garden pond harboured small hexagonal particles of about 70 nm identified as bacteriophages called "Neo-Ph/2". These phages resembled the recently described phages; strain "Neo-Ph/1" observed for the first time within the Chlamydia-like endocytobiont Neochlamydia hartmannellae (Parachlamydiaceae) multiplying within Hartmannella vermiformis (Schmid et al. 2001). The possible reasons for this obvious similarity are object for discussion in this article. [TOP OF PAGE]

  1317. Effects of bacteriophages on the population dynamics of four strains of pelagic marine bacteria. Middelboe,M., Hagstrom,A., Blackburn,N., Sinn,B., Fischer,U., Borch,N.H., Pinhassi,J., Simu,K., Lorenz,M.G. (2001). Microb. Ecol. 42:395-406. Viral lysis of specific bacterial populations has been suggested to be an important factor for structuring marine bacterioplankton communities. In the present study, the influence of bacteriophages on the diversity and population dynamics of four marine bacterial phage-host systems was studied experimentally in continuous cultures and theoretically by a mathematical model. By use of whole genome DNA hybridization toward community DNA, we analyzed the dynamics of individual bacterial host populations in response to the addition of their specific phage in continuous cultures of mixed bacterial assemblages. In these experiments, viral lysis had only temporary effects on the dynamics and diversity of the individual bacterial host species. Following the initial lysis of sensitive host cells, growth of phage-resistant clones of the added bacteria resulted in a distribution of bacterial strains in the phage-enriched culture that was similar to that in the control culture without phages after about 50-60 h incubation. Consequently, after a time frame of 5-10 generations after lysis, it was the interspecies competition rather than viral lysis of specific bacterial strains that was the driving force in the regulation of bacterial species composition in these experiments. The clonal diversity, on the other hand, was strongly influenced by viral activity, since the clonal composition of the four species in the phage-enriched culture changed completely from phage-sensitive to phage-resistant clones. The model simulation predicted that viral lysis had a strong impact on the population dynamics, the species composition, and the clonal composition of the bacterial community over longer time scales (weeks). However, according to the model, the overall density of bacteria in the system was not affected by phages, since resistant clones complemented the fluctuations caused by viral lysis. Based on the model analysis, we therefore suggest that viral lysis can have a strong influence on the dynamics of bacterial populations in planktonic marine systems. [TOP OF PAGE]

  1318. Environmental bacteriophage-host interactions: Factors contribution to natural transduction. Miller,R.V. (2001). Antonie van Leeuwenhoek J. Microbiol. 79:141-147. Over the past two decades the potential for the exchange of bacterial genes in natural environments through transduction (bacteriophage-mediated gene transfer) has been well established. Studies carried out by various laboratories throughout the world have demonstrated that both chromosomal and plasmid DNA can be successfully transduced in natural environments ranging from sewer plants to rivers and lakes. Transduction has been shown to take place in the gills of oysters and the kidneys of mice. Model studies have demonstrated the ability of transduction to maintain genetic material in bacterial gene pools that would otherwise be lost because of negative fitness. Thus, transduction may affect the course of bacterial evolution. Identification of natural transduction has led to the investigation of the dynamics of bacteriophage host interactions in natural aquatic environments and to the exploration of various environmental factors that affect virus-host interactions. Two important environmental factors which affect virus-host interactions are the metabolic state of the host and the exposure of the host to DNA-damaging stresses such as solar UV light. Recent researches on these two areas of virus-host relationships are reviewed. [TOP OF PAGE]

  1319. Use of Lacticin 481 to facilitate delivery of the bacteriophage resistance plasmid, pCBG104 to cheese starters. Mills,S., Ross,R.P., O'Sullivan,L., Stokes,D., Hill,C., Fitzgeral,G.F., Coffey,A. (2001). J. Appl. Microbiol. 92:238-246. [TOP OF PAGE]

  1320. Validity of Escherichia coli, enterovirus, and F-specific RNA bacteriophages as indicators of viral shellfish contamination. Miossec,L., Le Guyader,F., Pelletier,D., Haugarreau,L., Caprais,M.P., Pommepuy,M. (2001). J. Shelfish Res. 20:1223-1227. The sanitary classification of harvesting areas for bivalve mollusks in France is based on the level of Escherichia coli contamination detected in shellfish meat, as defined in EC Directive 91/492 EEC. However, outbreaks of gastroenteritis or hepatitis after consumption of shellfish meeting current bacteriological standards suggest that E. coli is a poor indicator of viral contamination. The purpose of this study was to assess the adequacy of enterovirus and F-specific RNA bacteriophages as new indicators of human enteric viruses. Shellfish were sampled over a 37-mo period to characterize microbial contamination in two coastal areas subjected to different sewage contamination inputs. Contamination by E. coli, F-specific RNA bacteriophages (F+ RNA) and human enteric viruses (enterovirus, EV; hepatitis A virus, HAV; Norwalk-like virus, NLV; astrovirus, AV; and rotavirus, RV) was measured in the same samples. E. coli analysis was performed by conductance measurement, enteric viruses were detected by reverse-transcription polymerase chain reaction (RT-PCR) and hybridization, and F+ RNA was evaluated by culture according to the ISO 10705-1 method. Statistical analysis based on bootstrap methods was performed on 95 series of paired observations. The validity of E. coli, enterovirus, and F-specific RNA bacteriophages as viral indicators was evaluated by measuring their sensitivity and specificity in the presence of enteric viruses. None of the tested indicators proved adequate to protect the public from viral shellfish contamination. The sensitivity of all indicators was better in the highly contaminated zone, and enteroviruses showed the highest specificity for both sites. [TOP OF PAGE]

  1321. Multiple infection dynamics has pronounced effects on the fitness of RNA viruses. Miralles,R., Ferrer,R., Sole,R.V., Moya,A., Elena,S.F. (2001). J. Evol. Biol. 14:654-662. Several factors play a role during the replication and transmission of RNA viruses. First, as a consequence of their enormous mutation rate, complex mixtures of genomes are generated immediately after infection of a new host. Secondly, differences in growth and competition rates drive the selection of certain genetic variants within an infected host. Thirdly, but not less important, a random sampling occurs at the moment of viral infectious passage from an infected to a healthy host. In addition, the availability of hosts also influences the fate of a given viral genotype. When new hosts are scarce, different viral genotypes might infect the same host, adding an extra complexity to the competition among genetic variants. We have employed a two-fold approach to analyse the role played by each of these factors in the evolution of RNA viruses. First, we have derived a model that takes into account all the preceding factors. This model employs the classic Lotka-Volterra competition equations but it also incorporates the effect of mutation during RNA replication, the effect of the stochastic sampling at the moment of infectious passage among hosts and, the effect of the type of infection (single, coinfection or superinfection). Secondly, the predictions of the model have been tested in an in vitro evolution experiment. Both theoretical and experimental results show that in infection passages with coinfection viral fitness increased more than in single infections. In contrast, infection passages with superinfection did not differ from the single infection. The coinfection frequency also affected the outcome: the larger the proportion of viruses coinfecting a host, the larger increase in fitness observed. [TOP OF PAGE]

  1322. Transfer of the Salmonella type III effector sopE between unrelated phage families. Mirold,S., Rabsch,W., Tschaepe,H., Hardt,W.-D. (2001). J. Mol. Biol. 312:7-16. Salmonella spp. are pathogenic enterobacteria that employ type III secretion systems to translocate effector proteins and modulate responses of host cells. The repertoire of translocated effector proteins is thought to define host specificity and epidemic virulence, and varies even between closely related Salmonella strains. Therefore, horizontal transfer of effector protein genes between Salmonella strains plays a key role in shaping the Salmonella-host interaction. Several effector protein genes are located in temperate phages. The P2-like phage SopEF encodes SopE and the ?-like GIFSY phages encode several effector proteins of the YopM/IpaH-family. Lysogenic conversion with these phages is responsible for much of the diversity of the effector protein repertoires observed among Salmonella spp. However, free exchange of effector proteins by lysogenic conversion can be restricted by superinfection immunity. To identify genetic mechanisms that may further enhance horizontal transfer of effector genes, we have analyzed sopE loci from Salmonella spp. that do not harbor P2-like sequences of SopEF. In two novel sopE loci that were identified, the 723 nt sopE gene is located in a conserved 1.2 kb cassette present also in SopEF. Most strikingly, in Salmonella enterica subspecies I serovars Gallinarum, Enteritidis, Hadar and Dublin, the sopE-cassette is located in a cryptic l-like prophage with similarity to the GIFSY phages. This provides the first evidence for transfer of virulence genes between different phage families. We show that such a mechanism can circumvent restrictions to phage-mediated gene transfer and thereby enhances reassortment of the effector protein repertoires in Salmonella spp. [TOP OF PAGE]

  1323. Effect of phage on survival of Salmonella enteritidis during manufacture and storage of cheddar cheese made from raw and pasteurized milk. Modi,R., Hirvi,Y., Hill,A., Griffiths,M.W. (2001). J. Food Prot. 64:927-933. The ability of Salmonella enteritidis to survive in the presence of phage, SJ2, during manufacture, ripening, and storage of Cheddar cheese produced from raw and pasteurized milk was investigated. Raw milk and pasteurized milk were inoculated to contain 104 CFU/ml of a luminescent strain of Salmonella enteritidis (lux) and 108 PFU/ml SJ2 phage. The milks were processed into Cheddar cheese following standard procedures. Cheese samples were examined for Salmonella enteritidis (lux), lactic acid bacteria, molds and yeasts, coliforms, and total counts, while moisture, fat, salt, and pH values were also measured. Salmonella enteritidis (lux) was enumerated in duplicate samples by surface plating on MacConkey novobiocin agar. Bioluminescent colonies of Salmonella enteritidis were identified in the NightOwl molecular imager. Samples were taken over a period of 99 days. Counts of Salmonella enteritidis (lux) decreased by 1 to 2 log cycles in raw and pasteurized milk cheeses made from milk containing phage. In cheeses made from milks to which phage was not added, there was an increase in Salmonella counts of about 1 log cycle. Lower counts of Salmonella enteritidis (lux) were observed after 24 h in pasteurized milk cheese containing phage compared to Salmonella counts in raw milk cheese with phage. Salmonella enteritidis (lux) survived in raw milk and pasteurized milk cheese without phage, reaching a final concentration of 103 CFU/g after 99 days of storage at 8 degrees C. Salmonella did not survive in pasteurized milk cheese after 89 days in the presence of phage. However, Salmonella counts of approximately 50 CFU/g were observed in raw milk cheese containing phage even after 99 days of storage. In conclusion, this study demonstrates that the addition of phage may be a useful adjunct to reduce the ability of Salmonella to survive in Cheddar cheese made from both raw and pasteurized milk. [TOP OF PAGE]

  1324. Bacteriophage biology and Kenneth Schaffner's rendition of developmentalism. Morgan,G.J. (2001). Biology & Philosophy 16:85-92. In this paper I consider Kenneth Schaffner's (1998) rendition of "developmentalism" from the point of view of bacteriophage biology. I argue that the fact that a viable phage can be produced from purified DNA and host cellular components lends some support to the anti-developmentalist, if they first show that one can draw a principled distinction between genetic and environmental effects. The existence of host-controlled phage host range restriction supports the developmentalist's insistence on the parity of DNA and environment. However, in the case of bacteriophage, the developmentalist stands on less firm ground than when organisms with nervous systems, such as Schaffner's C. elegans, are considered. [TOP OF PAGE]

  1325. Characterization of VPI pathogenicity island and CTXphi prophage in environmental strains of Vibrio cholerae. Mukhopadhyay,A.K., Chakraborty,S., Takeda,Y., Nair,G.B., Berg,D.E. (2001). J. Bacteriol. 183:4737-4746. Environmental isolates of Vibrio cholerae of eight randomly amplified polymorphic DNA (RAPD) fingerprint types from Calcutta, India, that were unusual in containing toxin-coregulated pilus or cholera toxin genes but not O1 or O139 antigens of epidemic strains were studied by PCR and sequencing to gain insights into V. cholerae evolution. We found that each isolate contained a variant form of the VPI pathogenicity island. Distinguishing features included (i) four new alleles of tcpF (which encodes secreted virulence protein; its exact function is unknown), 20 to 70% divergent (at the protein level) from each other and canonical tcpF; (ii) a new allele of toxT (virulence regulatory gene), 36% divergent (at the protein level) in its 5' half and nearly identical in its 3' half to canonical toxT; (iii) a new tcpA (pilin) gene; and (iv) four variant forms of a regulatory sequence upstream of toxT. Also found were transpositions of an IS903-related element and function-unknown genes to sites in VPI. Cholera toxin (ctx) genes were found in isolates of two RAPD types, in each case embedded in CTXphi-like prophages. Fragments that are inferred to contain only putative repressor, replication, and integration genes were present in two other RAPD types. New possible prophage repressor and replication genes were also identified. Our results show marked genetic diversity in the virulence-associated gene clusters found in some nonepidemic V. cholerae strains, suggest that some of these genes contribute to fitness in nature, and emphasize the potential importance of interstrain gene exchange in the evolution of this species. [TOP OF PAGE]

  1326. Role of biofilms in the survival of Legionella pneumophila in a model potable-water system. Murga,R., Forster,T.S., Brown,E., Pruckler,J.M., Fields,B.S., Donlan,R.M. (2001). Microbiology (Reading) 147:3121-3126. Legionellae can infect and multiply intracellularly in both human phagocytic cells and protozoa. Growth of legionellae in the absence of protozoa has been documented only on complex laboratory media. The hypothesis upon which this study was based was that biofilm matrices, known to provide a habitat and a gradient of nutrients, might allow the survival and multiplication of legionellae outside a host cell. This study determined whether Legionella pneumophila can colonize and grow in biofilms with and without an association with Hartmannella vermiformis. The laboratory model used a rotating disc reactor at a retention time of 6.7 h to grow biofilms on stainless steel coupons. The biofilm was composed of Pseudomonas aeruginosa, Klebsiella pneumoniae and a Flavobacterium sp. The levels of L. pneumophila cells present in the biofilm were monitored for 15 d, with and without the presence of H. vermiformis, and it was found that, although unable to replicate in the absence of H. vermiformis, L. pneumophila was able to persist. [TOP OF PAGE]

  1327. A visualization method of filamentous phage infection and phage-derived proteins in Escherichia coli using biotinylated phages. Nakamura,M., Tsumoto,K., Ishimura,K., Kumagai,I. (2001). Biochem. Biophys. Res. Com. 289:252-256. Direct visualization of filamentous phage infection in Escherichia coli (E. coli) was attempted using biotinylated phages (BIO-phages). The biotinylation of the phages did not influence their infectivity into E. coli. E. coli infected with BIO-phages could be detected by using fluorescein-conjugated avidin with confocal laser scanning microscopy, and BIO-phages and BIO-phage-derived proteins in E. coli could be directly observed by using the avidin-biotin-peroxidase complex method with electron microscopy. This is the first report of direct visualization of phage infection and phage-derived proteins in the host cell using a biotin-avidin interaction. This simple and powerful method is applicable to the study of infection by various viruses. [TOP OF PAGE]

  1328. Phage conversion of Panton-Valentine leukocidin in Staphylococcus aureus: molecular analysis of a PVL-converting phage, phiSLT. Narita,S., Kaneko,J., Chiba,J., Piemont,Y., Jarraud,S., Etienne,J., Kamio,Y. (2001). Gene 268:195-206. Staphylococcal Panton-Valentine leukocidin (PVL) is an important virulence factor, which causes leukocytolysis and tissue necrosis. Our previous report on the existence of the PVL genes (lukS-PV and lukF-PV) on the genome of prophage phiPVL in the Staphylococcus aureus strain V8 suggested the horizontal transmission of PVL genes by temperate bacteriophage among S. aureus (Kaneko, et al., 1998. Gene 215, 57-67). Here, we demonstrated the phage conversion of S. aureus leading to the production of PVL by discovery of a novel PVL-carrying phage, phiSLT (Staphylococcal Leukocytolytic Toxin) from a clinical isolate of S. aureus. phiSLT was able to lysogenize several clinical isolates of PVL-negative S. aureus strains as well as strain RN4220 at the conserved 29-bp sequence (attB) and all the lysogenized S. aureus strains had the ability to produce PVL. phiSLT had an elongated head of about 100x50 nm and a flexible tail of 400 nm long, that was quite different from phiPVL which had an isometric hexagonal head of about 60 nm diameter. The linear double-stranded phiSLT genome comprised 42,942 bp with 29-bp attachment core sequences and contained 62 open reading frames. Only 6.4 kbp region containing lysis cassette, PVL genes, attP, integrase, and orf204 of phiSLT was identical to that of phiPVL, while other regions were different from those of phiPVL. Thus, it can be concluded that PVL genes are carried by different temperate phages, which have the same attachment site. [TOP OF PAGE]

  1329. The UL6 gene product forms the portal for entry of DNA into the herpes simplex virus capsid. Newcomb,W.W., Juhas,R.M., Thomsen,D.R., Homa,F.L., Burch,A.D., Weller,S.K., Brown,J.C. (2001). J. Virol. 75:10923-10932. During replication of herpes simplex virus type 1 (HSV-1), viral DNA is synthesized in the infected cell nucleus, where DNA-free capsids are also assembled. Genome-length DNA molecules are then cut out of a larger, multigenome concatemer and packaged into capsids. Here we report the results of experiments carried out to test the idea that the HSV-1 UL6 gene product (pUL6) forms the portal through which viral DNA passes as it enters the capsid. Since DNA must enter at a unique site, immunoelectron microscopy experiments were undertaken to determine the location of pUL6. After specific immunogold staining of HSV-1 B capsids, pUL6 was found, by its attached gold label, at one of the 12 capsid vertices. Label was not observed at multiple vertices, at nonvertex sites, or in capsids lacking pUL6. In immunoblot experiments, the pUL6 copy number in purified B capsids was found to be 14.8 ± 2.6. Biochemical experiments to isolate pUL6 were carried out, beginning with insect cells infected with a recombinant baculovirus expressing the UL6 gene. After purification, pUL6 was found in the form of rings, which were observed in electron micrographs to have outside and inside diameters of 16.4 ± 1.1 and 5.0 ± 0.7 nm, respectively, and a height of 19.5 ± 1.9 nm. The particle weights of individual rings as determined by scanning transmission electron microscopy showed a majority population with a mass corresponding to an oligomeric state of 12. The results are interpreted to support the view that pUL6 forms the DNA entry portal, since it exists at a unique site in the capsid and forms a channel through which DNA can pass. The HSV-1 portal is the first identified in a virus infecting a eukaryote. In its dimensions and oligomeric state, the pUL6 portal resembles the connector or portal complexes employed for DNA encapsidation in double-stranded DNA bacteriophages such as 29, T4, and P22. This similarity supports the proposed evolutionary relationship between herpesviruses and double-stranded DNA phages and suggests the basic mechanism of DNA packaging is conserved. [TOP OF PAGE]

  1330. Three-component-mediated serotype conversion in Pseudomonas aeruginosa by bacteriophage D3. Newton,G.J., Daniels,C., Burrows,L.L., Kropinski,A.M., Clarke,A.J., Lam,J.S. (2001). Mol. Microbiol. 39:1237-1247. Bacteriophage D3 is capable of lysogenizing Pseudomonas aeruginosa PAO1 (serotype O5), converting the O-antigen from O5 to O16 and O-acetylating the N-acetylfucosamine moiety. To investigate the mechanism of lysogenic conversion, a 3.6 kb fragment from the D3 genome was isolated capable of mediating serotypic conversion identical to the D3 lysogen strain (AK1380). The PAO1 transformants containing this 3.6 kb of D3 DNA exhibited identical lipopolysaccharide (LPS) banding patterns to serotype O16 in silver-stained SDS-PAGE gels and displayed reactivity to an antibody specific for O-acetyl groups. Further analysis led to the identification of three open reading frames (ORFs) required for serotype conversion: an alpha-polymerase inhibitor (iap); an O-acetylase (oac); and a beta-polymerase (wzybeta). The alpha-polymerase inhibitor (lap) is capable of inhibiting the assembly of the serotype-specific O5 B-band LPS and allows the phage-encoded beta-polymerase (Wzybeta) to form new beta-linked B-band LPS. The D3 phage also alters the LPS by the addition of O-acetyl groups to the FucNAc residue in the O-antigen repeat unit by the action of the D3 O-acetylase (Oac). These three components form a simple yet elegant system by which bacteriophage D3 is capable of altering the surface of P. aeruginosa PAO1. [TOP OF PAGE]

  1331. DNA inversion in the tail fiber gene alters the host range specificity of carotovoricin Er, a phage-tail-like bacteriocin of phytopathogenic Erwinia carotovora subsp. carotovora Er. Nguyen,H.A., Tomita,T., Hirota,M., Kaneko,J., Hayashi,T., Kamio,Y. (2001). J. Bacteriol. 183:6274-6281. Carotovoricin Er is a phage-tail-like bacteriocin produced by Erwinia carotovora subsp. carotovora strain Er, a causative agent for soft rot disease in plants. Here we studied binding and killing spectra of carotovoricin Er preparations for various strains of the bacterium (strains 645Ar, EC-2, N786, and P7) and found that the preparations contain two types of carotovoricin Er with different host specificities; carotovoricin Era possessing a tail fiber protein of 68 kDa killed strains 645Ar and EC-2, while carotovoricin Erb with a tail fiber protein of 76 kDa killed strains N786 and P7. The tail fiber proteins of 68 and 76 kDa had identical N-terminal amino acid sequences for at least 11 residues. A search of the carotovoricin Er region in the chromosome of strain Er indicated the occurrence of a DNA inversion system for the tail fiber protein consisting of (i) two 26-bp inverted repeats inside and downstream of the tail fiber gene that flank a 790-bp fragment and (ii) a putative DNA invertase gene with a 90-bp recombinational enhancer sequence. In fact, when a 1,400-bp region containing the 790-bp fragment was amplified by a PCR using the chromosomal DNA of strain Er as the template, both the forward and the reverse nucleotide sequences of the 790-bp fragment were detected. DNA inversion of the 790-bp fragment also occurred in Escherichia coli DH5alpha when two compatible plasmids carrying either the 790-bp fragment or the invertase gene were contrasformed into the bacterium. Furthermore, hybrid carotovoricin CGE possessing the tail fiber protein of 68 or 76 kDa exhibited a host range specificity corresponding to that of carotovoricin Era or Erb, respectively. Thus, a DNA inversion altered the C-terminal part of the tail fiber protein of carotovoricin Er, altering the host range specificity of the bacteriocin. [TOP OF PAGE]

  1332. A field study of virus removal in septic tank drainfields. Nicosia,L.A., Rose,J.B., Stark,L., Stewart,M.T. (2001). J. Environ. Qual. 30:1933-1939. Two field studies were conducted at a research station in Tampa, Florida to assess the removal of bacteriophage PRD1 from wastewater in septic tank drainfields. Infiltration cells were seeded with PRD1 and bromide and the effects of effluent hydraulic loading rate and rainfall on virus removal were monitored. Septic tank effluent samples were collected after passage through 0.6 m of unsaturated fine sand and PRD1 was detected over an average of 67 d. Bacteriophage PRD1 breakthrough was detected at approximately the same time as bromide in all three cells except for the low-load cell (Study 1), where bromide was never detected. Log10 removals of PRD1 were 1.43 and 1.91 for the high-load cells (hydraulic loading rate = 0.063 m/d) and 2.21 for the low-load cell (hydraulic loading rate = 0.032 m/d). Virus attenuation is attributed to dispersion, dilution, and inactivation. Significant increases in PRD1 elution with rainfall were observed in the first 10 d of the study. Approximately 125 mm of rainfall caused a 1.2 log10 increase of PRD1 detected at the 0.6-m depth. Current Florida onsite wastewater disposal standards, which specify a 0.6-m distance from the drainfield to the water table, may not provide sufficient removal of viruses, particularly during the wet season. [TOP OF PAGE]

  1333. Detection of homologous recombination among bacteriophage P2 relatives. Nilsson,A.S., Haggard-Ljungquist,E. (2001). Mol. Phyl. Evol. 21:259-269. Sequencing of five late genes from 18 isolates of P2-like bacteriophages showed that these are at least 96% identical to the genes of phage P2. A maximum-parsimony phylogenetic analysis of these genes showed excess homoplasy of a magnitude three to six times higher than that expected. Examination of the distribution of the number of homoplasies at parsimoniously informative sites and incompatibility matrices of such sites revealed a pattern typical for extensive recombination. It has been shown that phage P2 probably incorporated some functionally complete genes or gene modules by recombination with other phages or with different hosts, but homologous recombination within genes has previously not been shown. In this paper we demonstrate that homologous recombination between P2-like bacteriophages occurs randomly at multiple breakpoints in five late genes. The rate of recombination is high but, since some phages were sampled decades apart and in different parts of the world, this has to be viewed on an evolutionary time scale. The applicability of different methods used for detection of recombination breakpoints and estimation of rates of recombination in bacteriophages is discussed. [TOP OF PAGE]

  1334. Estimating viral proliferation in aquatic samples. Noble,R., Steward,G. (2001). Meth. Microbiol. 30:67-84. [first paragraph] It is only within the last decade that marine viruses were determined to be consistently the most abundant biological entities in the sea (Fuhrman, 1999). Since then, many advances have been made in understanding viral ecology (Fuhrman, 1999, Wilhelm and Suttle, 1999). Initial discoveries showed that viruses are both abundant in the ocean and that many bacteria are infected with viruses (Bergh et al., 1989; Proctor and Fuhrman, 1990). These data led researchers to believe that viruses are an important source of mortality in marine microbial food webs, but only provided a static picture. Subsequent studies have shown that virus populations are extremely dynamic, and can change quickly over short time scales (Bratbak et al 1990, 1996). Estimates of viral production and decay rates provided the valuable confirmation that viruses are active members of the marine community (Heldal and Bratbak, 1991; Steward et al., 1992b). The production of viruses implies the lysis of host cells and the release of cellular material as dissolved and colloidal organic carbon. Therefore, measurements of viral replication rates are also useful for assessing the contribution of viruses to bacterial mortality and organic matter cycling in the ocean. By assuming a burst size, viral productivity can be used to estimate rates of bacterial lysis. This approach provides an additional means to assess bacterial mortality along with the original electron microscopy-based method of Proctor and Fuhrman (1990). Accurate measurements of viral productivity and turnover are required if we are to properly model their dynamics and impact within the microbial food web. So far, however, there is no standard method for measuring viral productivity. A wide variety of different approaches have been used each with associated advantages and disadvantages. These methods include:
    1. Quantifying net increases in viral abundance over time (Bratbak et al., 1990)
    2. Measuring rates of viral decay (Heldal and Bratbak, 1991)
    3. Estimating viral DNA synthesis rates by radiolabeling (Steward et al., 1992a,b)
    4. Calculating expected viral release rates from estimated rates of bacterial lysis and an assumed burst size (Weinbauer et al., 1993)
    5. Measuring tracer dilution rates using fluorescently labeled viruses (FLV) as tracers (Noble and Fuhrman, submitted). [TOP OF PAGE]

  1335. Enumeration of viruses. Noble,R.T. (2001). Meth. Microbiol. 30:43-51. [first paragraph] Viruses are now known to be the most numerically abundant component of marine plankton (Bergh et al., 1989; Bratbak et al., 1990; Fulirman and Suttle, 1993; Hennes and Suttle, 1995; Noble and Fuhrman, 1998; Fuhrman, 1999). In the late 1980s, some of the first reports were published documenting the high abundances of marine viruses at 10^? particles per liter of seawater, exceeding the typical abundance of bacteria (Proctor et al., 1988; Bergh et al., 1989). Since then, studies have demonstrated high numbers of viruses in all types of marine environments, from eutrophic coastal waters to deep blue open-ocean waters, from the sea surface to the depths of the sea, and from the polar to the tropical regions (Bratbak et al., 1990; Cochlan et al., 1993; Guixa-Boixareu et al., 1996; Stelvard rt nl., 1996). Multiple groups of researchers have identified important roles of Viruses in the mortality of heterotrophic bacterioplankton, cyanobacteria, and phytoplankton. They also play a role in biogeochemical cycling and control of species diversity (Fuhrman, 1999; \8t7illielm and Suttle, 1999). Specifically, it has been shown by a number of researchers that viruses are capable of causing a significant portion of the heterotrophic bacterial mortality in certain marine environments (Fuhrman and Noble, 1993; Guixa-Boixareu et al., 1996; Steward et al., 1996; Weinbauer and Hofle, 1998). [TOP OF PAGE]

  1336. Increased mutation rate of E. coli K12 lambda cultures maintained in continuous logarithmic growth. Northrop,J.H. (2001). J. Gen. Physiol. 50:369-377. Continuous logarithmic growth of E. coli K12 lambda in an automatic culture cell resulted in marked increases in the proportion of several mutants. The P1 phage-resistant cells increased 10 to 3000 times, the T2 phage-resistant cells 1 to 1000 times, the neomycin-resistant cells 1 to 10 times, and the virus-producing cells 30 to 70 times. No change occurred in the penicillin-resistant cells. Calculation of the growth curves and direct determination of the mutation rates by the null fraction method showed that the increases in the proportion of mutants were due to increases in the mutation rates. [TOP OF PAGE]

  1337. Pathogenicity and resistance islands of staphylococci. Novick,R.P., Schlievert,P., Ruzin,A. (2001). Microbes Infect. 3:585-594. Variable genetic elements including plasmids, transposons and prophages are involved in pathogenesis and antibiotic resistance, and are an important component of the staphylococcal genome. This review covers a set of newly described variable chromosomal elements, pathogenicity and resistance islands, carrying superantigen and resistance genes, especially toxic shock and methicillin resistance, respectively. [TOP OF PAGE]

  1338. Naturally occurring lactococcal plasmid pAH90 links bacteriophage resistance and mobility functions to a food-grade selectable marker. O'Sullivan,D., Ross,R.P., Twomey,D.P., Fitzgerald,G.F., Hill,C., Coffey,A. (2001). Appl. Environ. Microbiol. 67:929-937. The bacteriophage resistance plasmid pAH90 (26,490 bp) is a natural cointegrate plasmid formed via homologous recombination between the type I restriction-modification specificity determinants (hsdS) of two smaller lactococcal plasmids, pAH33 (6,159 bp) and pAH82 (20,331 bp), giving rise to a bacteriophage-insensitive mutant following phage challenge (D. O'Sullivan, D. P. Twomey, A. Coffey, C. Hill, G. F. Fitzgerald, and R. P. Ross, Mol. Microbiol. 36:866-876; 2000). In this communication we provide evidence that the recombination event is favored by phage infection. The entire nucleotide sequence of plasmid pAH90 was determined and found to contain 24 open reading frames (ORFs) responsible for phenotypes which include restriction-modification, phage adsorption inhibition, plasmid replication, cadmium resistance, cobalt transport, and conjugative mobilization. The cadmium resistance property, encoded by the cadA gene, which has an associated regulatory gene (cadC), is of particular interest, as it facilitated the selection of pAH90 in other phage-sensitive lactococci after electroporation. In addition, we report the identification of a group II self-splicing intron bounded by two exons which have the capacity to encode a relaxase implicated in conjugation in gram-positive bacteria. The functionality of this intron was evident by demonstrating splicing in vivo. Given that pAH90 encodes potent phage defense systems which act at different stages in the phage lytic cycle, the linkage of these with a food-grade selectable marker on a replicon that can be mobilized among lactococci has significant potential for natural strain improvement for industrial dairy fermentations which are susceptible to phage inhibition. [TOP OF PAGE]

  1339. Diversification of Escherichia coli genomes: are bacteriophages the major contributors? Ohnishi,M., Kurokawa,K., Hayashi,T. (2001). Trends Microbiol. 9:481-485. Determination of the genome sequence of enterohemorrhagic Escherichia coli O157 Sakai and genomic comparison with the laboratory strain K-12 has revealed that the two strains share a highly conserved 4.1-Mb sequence and that each also contains a larger amount of strain-specific sequence. The analysis also revealed the presence of a surprisingly larger number of prophages in O157, most of which are lambda-like phages that resemble each other. Based on these results, we discuss how bacteriophages contributed to this process. We also describe possible mechanisms by which O157 acquired many closely related phages, and raise the possibility that such bacteria might function as 'phage factories', releasing a variety of chemeric or mosaic prophages into the environment. [TOP OF PAGE]

  1340. Filamentous bacteriophage stability in non-aqueous media. Olofsson,L., Ankarloo,J., Andersson,P.O., Nicholls,I.A. (2001). Chemistry and Biology 8:661-671. Background: Filamentous bacteriophage are used as general cloning vectors as well as phage display vectors in order to study ligand-receptor interactions. Exposure to biphasic chloroform-water interface leads to specific contraction of phage, to non-infective I- or S-forms. Results: Upon exposure, phage were inactivated (non-infective) at methanol, ethanol and l-propanol concentrations inversely dependent upon alcohol hydrophobicity. Infectivity loss of phage at certain concentrations of l-propanol or ethanol coincided with changes in the spectral properties of the fl virion in ultraviolet fluorescence and circular dichroism studies. Conclusions: The alcohols inactivate filamentous phage by a general mechanism-solvation of coat protein-thereby disrupting the capsid in a manner quite different from the previously reported I- and S-forms. The infectivity retention of phagemid pG8H6 in 99% acetonitrile and the relatively high general solvent resistance of the phage strains studied here open up the possibility of employing phage display in non-aqueous media. [TOP OF PAGE]

  1341. [Bacteria-killing viruses, Stalinists and "superbugs"]. Olsen,I., Handal,T., Lokken,P. (2001). Tidsskr Nor Laegeforen 121:3197-3200. In June 2000, the WHO warned that the level of resistance to drugs used to treat common infectious diseases is now reaching a crisis point. If world governments do not control infections better in order to slow down the development of drug resistance, entire populations could be wiped out by superbugs against which there is no efficient treatment. Development of resistance is due to both underuse and overuse of drugs, and strategies have been worked out, to slow down the development of resistance for instance by the Norwegian Ministry of Health and Social Affairs. The present article deals with an old principle, mainly developed behind the Iron Curtain, which is now attracting renewed attention in the west: the application of bacterial viruses (bacteriophages) in the fight against bacteria. According to clinical trials in Eastern Europe, mostly uncontrolled, phages have been used successfully in treatments against antibiotic-resistant bacteria, for instance in suppurative wound infections, gastroenteritis, sepsis, osteomyelitis and pneumonia. These encouraging data are supported by recent findings in well-controlled animal models demonstrating that phages can rescue animals from a variety of fatal infections. The present review discusses possible advantages and limitations of phage treatment in humans. [TOP OF PAGE]

  1342. Bacteriophage P4282, a parasite of Ralstonia solanacearum, encodes a bacteriolytic protein important for lytic infection of its host. Ozawa,H., Tanaka,H., Ichinose,Y., Shiraishi,T., Yamada,T. (2001). Mol. Gen. Genom. 265:95-101. To enhance bacterial wilt resistance in tobacco expressing a foreign protein, we isolated the bacteriolytic gene from a bacteriophage that infects Ralstonia solanacearum. The bacteriolytic protein of phage P4282 isolated in Tochigi Prefecture was purified from a lysate of R. solanacearum M4S cells infected with the phage, and its bacteriolytic activity was assayed by following the decrease in the turbidity of suspensions of R. solanacearum M4S cells. The molecular weight of the bacteriolytic protein was approximately 71 kDa, and the sequence of the N-terminal 13 amino acids was determined. We used oligonucleotide probes based on this amino acid sequence to isolate the bacteriolytic gene from phage P4282 DNA. This gene of 2061 bp encodes a product of 687 amino acids, whose calaculated molecular weight was 70.12 kDa. The bacteriolytic gene was placed under the control of an inducible promoter, and the plasmid was transformed into Escherichia coli NM522. The soluble proteins extracted from E. coli NM522 cells harboring the plasmid with the bacteriolytic gene showed obvious bacteriolytic activities against several strains of R. solanacearum isolated in various districts in Japan. DNA fragments from five phages, isolated in Niigata, Aomori, Okinawa, Fukushima and Yamaguchi Prefectures, hybridized to the bacteriolytic gene of phage P4282. These observations indicate that the bacteriolytic protein shows nonspecific activity against R. solanacearum strains, and a sequence similar to that of the bacteriolytic gene is conserved in the DNA of other bacteriophages. These results indicate that the generation of transgenic (tobacco) plants expressing the bacteriolytic gene of phage P4282 might result in enhanced resistance to bacterial wilt in tobacco. [TOP OF PAGE]

  1343. Biotech firm tries a novel tactic in fighting bacteria. Ozretich,J. (2001). Puget Sound Business Journal 21(51), 6. A small Bothell-based biotechnology company is working on the medical equivalent of fighting fire with fire. To combat infections caused by bacteria, it is using a class of viruses that infect the bacteria themselves. ¶ Phage Therapeutics Inc. hopes to use genetically engineered versions of these naturally occurring viruses, called bacteriophages, to treat diseases where antibiotic-resistant bacteria are becoming an increasing problem, such as staph infections and tuberculosis. [TOP OF PAGE]

  1344. Complete genomic sequence of the lytic bacteriophage fYeO3-12 of Yersinia enterocolitica serotype O:3. Pajunen,M.I., Kiljunen,S.J., Soderholm,M.E.L., Skurnik,M. (2001). J. Bacteriol. 183:1928-1937. fYeO3-12 is a T3-related lytic bacteriophage of Yersinia enterocolitica serotype O:3. The nucleotide sequence of the 39,600-bp linear double-stranded DNA (dsDNA) genome was determined. The phage genome has direct terminal repeats of 232 bp, a GC content of 50.6%, and 54 putative genes, which are all transcribed from the same DNA strand. Functions were assigned to 30 genes based on the similarity of the predicted products to known proteins. A striking feature of the phiYeO3-12 genome is its extensive similarity to the coliphage T3 and T7 genomes; most of the predicted fYeO3-12 gene products were >70% identical to those of T3, and the overall organizations of the genomes were similar. In addition to an identical promoter specificity, fYeO3-12 shares several common features with T3, nonsubjectibility to F exclusion and growth on Shigella sonnei D2371-48 (M. Pajunen, S. Kiljunen, and M. Skurnik, J. Bacteriol. 182:5114-5120, 2000). These findings indicate that phiYeO3-12 is a T3-like phage that has adapted to Y. enterocolitica O:3 or vice versa. This is the first dsDNA yersiniophage genome sequence to be reported. [TOP OF PAGE]

  1345. [Stability of Lactococcus lactis phages treated with sodium hypochlorite and during storage]. Parada,J.L., de Fabrizio,S.V. (2001). Revista Argentina de Microbiologia 33:89-95. Survival of lytic bacteriophages active against Lactococcus lactis ssp. lactis and ssp. cremoris was determined after treatment with sodium hypochlorite and during at 4degreeC. Three phages were isolated from dairy plants in Argentina (ARG) and the other phages were isolated in the United States of America (US). All of them represent phages that infected cheese manufacture industries and belong to different morphological or serological groups. These phages showed higher survival in M17 broth, buffered with sodium glycerophosphate, than in trypteine soy broth (TSB). Phage populations did not decrease significantly during 14 weeks in M17 broth, whereas in TSB the titers of phage suspensions began to decline around 9 days. In addition, the effect of sodium hypochlorite was more marked in broth than in milk. A higher surviving fraction was obtained in milk, even when tenfold higher concentrations of chloride were used. The effect of hypochlorite on phages of the same serological group was quite similar and independent of phage morphology. However, phage 137-1, which belongs to other serological group, showed lower resistance to sodium hypochlorite. Comparing the hypochlorite inactivation for ARG and US phages, it was observed that they have their own inactivation values, independently of their origin and morphological group. Long periods of time and high concentrations of chlorine were necessary to reduce the surviving fraction in milk. This indicates that hypochlorite concentrations and times of contact can be critical for the efficiency of the operative sanitization processes. [TOP OF PAGE]

  1346. Estabilidad de fagos de Lactococcus lactis frente al hipoclorito de sodio y durante el almacenamiento [Stability of Lactococcus lactis phages treated with sodium hypochlorite and during storage]. Parada,J.L., de Fabrizio,S.V. (2001). Revista Argentina de Microbiologia 33:89-95. Survival of lytic bacteriophages active against Lactococcus lactis ssp. lactis and ssp. cremoris was determined after treatment with sodium hypochlorite and during storage at 4 degrees C. Three phages were isolated from dairy plants in Argentina (ARG) and the other phages were isolated in the United States of America (US). All of them represent phages that infected cheese manufacture industries and belong to different morphological or serological groups. These phages showed higher survival in M17 broth, buffered with sodium glycerophosphate, than in trypteine soy broth (TSB). Phage populations did not decrease significantly during 14 weeks in M17 broth, whereas in TSB the titers of phage suspensions began to decline around 9 days. In addition, the effect of sodium hypochlorite was more marked in broth than in milk. A higher surviving fraction was obtained in milk, even when tenfold higher concentrations of chlorine were used. The effect of hypochlorite on phages of the same serological group was quite similar and independent of phage morphology. However, phage 137-1, which belongs to other serological group, showed lower resistance to sodium hypochlorite. Comparing the hypochlorite inactivation for ARG and US phages, it was observed that they have their own inactivation values, independently of their origin and morphological group. Long periods of time and high concentrations of chlorine were necessary to reduce the surviving fraction in milk. This indicates that hypochlorite concentrations and times of contact can be critical for the efficiency of the operative sanitization processes. [TOP OF PAGE]

  1347. Lysogeny and transduction. Paul,J.H., Jiang,S.C. (2001). Meth. Microbiol. 30:105-125. [first paragraph] Lysogeny and transduction describe a type of phage/host interaction and a method of bacterial gene transfer (procaryotic sex), respectively. Although they are often reviewed together, these topics are linked only in that one type of transduction (specialized) has an obligate requirement for a lysogenic interaction. In this chapter we describe the background for understanding both of these processes, and give methods that we have found useful in studying lysogeny and transduction in the marine environment. [TOP OF PAGE]

  1348. Understanding bacteriophage therapy as a density-dependent kinetic process. Payne,R.J.H., Jansen,V.A.A. (2001). J. Theor. Biol. 208:37-48. Studies of bacteriophage as therapeutic agents have had mixed and unpredictable outcomes. We argue that interpretation of these apparently paradoxical results requires appreciation of various density-dependent threshold effects. We use a mathematical model to delineate different categories of outcome, including therapy by simple inundation, by active biocontrol, and by delayed active biocontrol. Counter-intuitively, there are situations in which earlier inoculation can be less efficacious, and simultaneous inoculation with antibiotics can be detrimental. Predictions of therapeutic responses are made using formulae dependent on biologically meaningful parameters; experimental measurement of the parameters will be a prerequisite of application of the model to particular study systems. Such modelling can point to which aspects of phage biology might most fruitfully be engineered so as to enhance the viability of bacteriophage therapy. [TOP OF PAGE]

  1349. Sequences and replication of genomes of the archaeal rudiviruses SIRV1 and SIRV2: Relationships to the archaeal lipothrixvirus SIFV and some eukaryal viruses. Peng,X., Blum,H., She,Q., Mallok,S., Brügger,K., Garrett,R.A., Prangishvili,D. (2001). Virology 291:226-234. The double-stranded DNA genomes of the viruses SIRV1 and SIRV2, which infect the extremely thermophilic archaeon Sulfolobus and belong to the family Rudiviridae, were sequenced. They are linear, covalently closed at the ends, and 32,312 and 35,502 bp long, respectively, with an A1T content of 75%. The genomes of SIRV1 and SIRV2 carry inverted terminal repeats of 2029 and 1628 bp, respectively, which contain multiple direct repeats. SIRV1 and SIRV2 genomes contain 45 and 54 ORFs, respectively, of which 44 are homologous to one another. Their predicted functions include a DNA polymerase, a Holliday junction resolvase, and a dUTPase. The genomes consist of blocks with well-conserved sequences separated by nonconserved sequences. Recombination, gene duplication, horizontal gene transfer, and substitution of viral genes by homologous host genes have contributed to their evolution. The finding of head-to-head and tail-to-tail linked replicative intermediates suggests that the linear genomes replicate by the same mechanism as the similarly organized linear genomes of the eukaryal poxviruses, African swine fever virus and Chlorella viruses. SIRV1 and SIRV2 both contain motifs that resemble the binding sites for Holliday junction resolvases of eukaryal viruses and may use common mechanisms for resolution of replicative intermediates. The results suggest a common origin of the replication machineries of the archaeal rudiviruses and the above-mentioned eukaryal viruses. About 1/3 of the ORFs of each rudivirus have homologs in the Sulfolobus virus SIFV of the family Lipothrixviridae, indicating that the two viral families form a superfamily. The finding of inverted repeats of at least 0.8 kb at the termini of the linear genome of SIFV supports this inference. [TOP OF PAGE]

  1350. Modeling virus inactivation on salad crops using microbial count data. Petterson,S.R., Teunis,P.F., Ashbolt,N.J. (2001). Risk Anal 21:1097-1108. Microbial counts of the persistent Bacteroides fragilis bacteriophage B40-8 from a virus decay experiment conducted under glasshouse conditions were used to model the decay of viruses on wastewater-irrigated lettuce and carrot crops. The modeling approach applied gave specific consideration to the discrete nature of microbial count data. The experimental counts were best fit by a negative binomial distribution indicating highly dispersed distribution of viruses on lettuce and carrot crops following irrigation with wastewater. In addition, there was evidence for biphasic inactivation of viruses, signifying the presence of a persistent subpopulation of viruses that decayed slowly, resulting in virus accumulation on the crop surface over subsequent irrigations. Maximum likelihood estimates of initial and persistent subpopulation inactivation rates were 2.48 day(-1) and 0.51 day(-1) for lettuces and 0.84 day(-1) and 0.046 day(-1) for carrots. Maximum likelihood estimates of the persistent virus subpopulation size were 0.12% and 2% for lettuce and carrots, respectively. [TOP OF PAGE]

  1351. Viruses of the extremely therophilic archaeon Sulfolobus. Prangishvili,D., Stedman,K., Zillig,W. (2001). Trends Microbiol. 9:39-42. Viruses of Sulfolobus are highly unusual in their morphology, and genome structure and sequence. Certain characteristics of the replication strategies of these viruses and the virus-host interactions suggest relationships with eukaryal and bacterial viruses. Moreover, studying these viruses led to the discovery of archaeal promoters and has provided tools for the development of the molecular genetics of these organisms. The Sulfolobus viruses contain unique regulatory features and structures that undoubtedly hold surprises for researchers in the future. [TOP OF PAGE]

  1352. Identification of cell wall proteins of Bacteroides fragilis to which bacteriophages B40-8 binds specifically. Puig,A., Arujo,R., Jofre,J., Frias-Lopez,J. (2001). Microbiology (Reading) 147:281-288. Bacteriophage infecting Bacteroides fragilis, one of the most abundant bacteria in the human colon, have been proposed as indicators of virological faecal pollution. The first identification of a receptor for a bacteriophage in B.fragilis is reported here. First, resistant mutants were characterized following phage inactivation, and it was shown that cell wall proteins are involved in phage binding. Then the proteins involved were identified by various approaches: (i) comparison of the protein profiles of wild-type B.fragilis HSP40 and phage-resistant mutants; (ii) application of a modification of the virus overlay protein blot assay (VOPBA). At least two proteins of B.fragilis, with apparent molecular masses of 35 ± 5kDa and 65 ± 5kDa, bind to B40-8. This result was later confirmed by running a complex consisting of this phage bound to radiolabelled proteins of B. fragilis on an immunoaffinity column loaded with a specific antibody against the phage. Cell proteins retained in the column also coincided with the proteins that differed in the profiles of resistant mutants. Finally, to identify the potential function of these two proteins, their N-terminal sequences were determined and compared to published sequences, but no homologies were found. [TOP OF PAGE]

  1353. Contingent neutrality in competing viral populations. Quer,J., Hershey,C.L., Domingo,E., Holland,J.J., Novella,I.S. (2001). J. Virol. 75:7315-7320. The replicative fitness of a genetically marked (MARM-C) population of vesicular stomatitis virus was examined in competition assays in BHK-21 cells. In standard fitness assays involving up to eight competition passages of the mixed populations, MARM-C competes equally with the wild type (wt), but very prolonged competitions always led to the wt gaining dominance over MARM-C in a very slowed, nonlinear manner (J. Quer et al., J. Mol. Biol. 264:465-471, 1996). In the present study we show that a number of quite unrelated environmental perturbations, which decreased virus replication during competitions, all led to an accelerated dominance of the wt over MARM-C. These perturbations were (i) the presence of added (or endogenously generated) defective interfering particles, (ii) the presence of the chemical mutagen 5-fluorouracil (5-FU), or (iii) an increase in temperature to 40.5 degreesC. Thus, the "neutral fitness" of the MARM-C population is contingent. We have determined the entire genomic consensus sequence of MARM-C and have identified only six mutations. Clearly, some or all of these mutations allowed the MARM-C quasispecies population to compete equally with wt in a defined constant host environment, but the period of neutrality was shortened when the environment was perturbed during competitions. Interestingly, when four passages of each population were carried out independently in the presence of 5-FU (but in the absence of competition), no significant differences were detected in the fitness changes of wt and MARM-C, nor was there a difference in their subsequent abilities to compete with each other in a standard fitness assay. We propose a model for this contingent neutrality. The conditions employed to generate the MARM-C quasispecies population selected a small number of mutations in the consensus sequence. It appears that the MARM-C quasispecies population has moved into a segment of sequence space in which the average fitness value is neutral but, under environmental stress, beneficial mutations cannot be generated rapidly enough to compete with those being generated concurrently by competing wt virus quasispecies populations. [TOP OF PAGE]

  1354. Removal of pathogenic and indicator microorganisms by a constructed wetland receiving untreated domestic wastewater. Quinonez-Diaz,M.d., Karpiscak,M.M., Ellman,E.D., Gerba,C.P. (2001). J. Environ. Sci. Health Part A A36:1311-1320. Wetlands containing floating, emergent and submergent aquatic plants, and other water-tolerant species have been found to economically provide a mechanism of enhancing the quality of domestic wastewater. The use of constructed wetlands for the removal of indicator bacteria (total and fecal coliforms), coliphages, protozoan parasites (Giardia and Cryptosporidium) and enteric viruses was investigated. A pilot scale constructed wetland consisting of two cells, one planted with bulrush and the other unplanted bare sand, were used to compare their efficiency in removing pathogens from raw sewage. Overall more than 90 percent of all microorganisms studied were removed by either of the two systems with a 1 to 2 day retention time. Removal of all mentioned microorganisms was greater from the surface flow in the unplanted cell than in the planted cell, except for Giardia and Cryptosporidium, although the differences were not statistically significant. Enteric viruses, coliphages and indicator bacteria were found to penetrate 2 m below the surface, although concentrations were reduced by greater than 99 percent in both cells. Less virus penetration into the sand occurred in the planted wetland versus the unplanted wetland. Water temperature was found to be the most important factor in the removal of enteric bacteria and viruses, while turbidity reduction was related to Giardia removal. These results demonstrate that significant reductions of pathogenic microorganisms can occur in constructed wetlands receiving untreated domestic wastewater with only a 1-2 day retention time. [TOP OF PAGE]

  1355. Phage spread dynamics in clonal bacterial populations is depending on features of the founder cell. Ramirez,E., Carbonell,X., Villaverde,A. (2001). Microbiological Research 156:35-40. Plate-cultured bacterial colonies are intriguing models to study host-parasite interactions in senescent populations. During the growth of bacteriophage-infected colonies there is a synchronous prophage induction episode among lysogenic cells that allows a dramatic but time-restricted amplification of viral particles. We report here that the dynamics of phage spread depends on the history of the lysogenic cell that establishes the clonal population, the duration of the pre-burst period being shorter when the founder, infected cell derives from older colonies. These results offer a physiologic explanation for the self-contained progression of the viral spread in closed environments, that ensures both viral dissemination but also survival of most of the host cells. [TOP OF PAGE]

  1356. Gene transfer in bacterial biofilms. Roberts,AP., Mullany,P., Wilson,M., Doyle,R.J. (2001). Meth. Enzymol. 336:60-65. [TOP OF PAGE]

  1357. Evolutionary role of restriction/modification systems as revealed by comparative genome analysis. Rocha,E.P.C., Danchin,A., Viari,A. (2001). Genome Res. 11:946-958. Type II restriction modification systems (RMSs) have been regarded either as defense tools or as molecular parasites of bacteria. We extensively analyzed their evolutionary role from the study of their impact in the complete genomes of 26 bacteria and 35 phages in terms of palindrome avoidance. This analysis reveals that palindrome avoidance is not universally spread among bacterial species and that it does not correlate with taxonomic proximity. Palindrome avoidance is also not universal among bacteriophage, even when their hosts code for RMSs, and depends strongly on the genetic material of the phage. Interestingly, palindrome avoidance is intimately correlated with the infective behavior of the phage. We observe that the degree of palindrome and restriction site avoidance is significantly and consistently less important in phages than in their bacterial hosts. This result brings to the fore a larger selective load for palindrome and restriction site avoidance on the bacterial hosts than on their infecting phages. It is then consistent with a view where type II RMSs are considered as parasites possibly at the verge of mutualism. As a consequence, RMSs constitute a nontrivial third player in the host-parasite relationship between bacteria and phages. [TOP OF PAGE]

  1358. Reduction of enteric microorganisms at the Upper Occoquan Sewage Authority Water Reclamation Plant. Rose,J.B., Huffman,D.E., Riley,K., Farrah,S.R., Lukasik,J.O., Hamann,C.L. (2001). Water Environ. Res. 73:711-720. The Upper Occoquan Sewage Authority (UOSA) Water Reclamation Plant, Centreville, Virginia, is a state-of-the-art wastewater treatment plant that was created to treat area wastewater and provide protection for the Occoquan Reservoir. This study investigated UOSA's unit processes as barriers to pathogenic as well as altemative and traditional-indicator microorganisms. Samples were collected once a month for 1 year from eight sites within UOSA's advanced wastewater reclamation plant. The eight sites were monitored for indicator bacteria total and fecal coliforms, enterococci, Clostridium, coliphage (the virus that infects Escherichia coli), human enteroviruses, and enteric protozoa. Overall, the plant was able to achieve a 5- to 7-log10 reduction of bacteria, 5-log10 reduction of enteroviruses, 4-log10 reduction for Clostridium, and 4.6-log10 reduction of protozoa. Total coliforms, enterococci, Clostridium, coliphage, Cryptosporidium, and Giardia were all detected in four or fewer samples of the final effluent. No enteroviruses or fecal coliforms were detected in the final effluent. The microbiological quality of reclaimed water and the reservoir water were compared. In every case, the treated wastewater was of a better quality than the ambient water in the reservoir, thus indicating that the reclaimed water will not adversely affect the water quality for downstream users. [TOP OF PAGE]

  1359. Low-molecular-weight plasmid of Salmonella enterica serovar Enteritidis codes for retron reverse transcriptase and influences phage resistance. Rychlik,I., Sebkova,A., Gregorova,D., Karpiskova,R. (2001). J. Bacteriol. 183:2852-2858. Retron reverse transcriptases are unusual procaryotic enzymes capable of synthesis of low-molecular-weight DNA by reverse transcription. All of the so-far-described DNA species synthesized by retron reverse transcriptases have been identified as multicopy single-stranded DNA. We have shown that Salmonella enterica serovar Enteritidis is also capable of synthesis of the low-molecular-weight DNA by retron reverse transcriptase. Surprisingly, Salmonella serovar Enteritidis-produced low-molecular-weight DNA was shown to be a double-stranded DNA with single-stranded overhangs (sdsDNA). The sdsDNA was 72 nucleotides (nt) long, of which a 38-nt sequence was formed by double-stranded DNA with 19- and 15-nt single-stranded overhangs, respectively. Three open reading frames (ORFs), encoded by the 4,053-bp plasmid, were essential for the production of sdsDNA. These included an ORF with an unknown function, the retron reverse transcriptase, and an ORF encoding the cold shock protein homologue. This plasmid was also able to confer phage resistance onto the host cell by a mechanism which was independent of sdsDNA synthesis. [TOP OF PAGE]

  1360. Seasonal variations in the microbial population density present in biological sludge. Saleem,M., Al-Malack,M.H., Bukhari,A.A. (2001). Environ. Technol. 22:255-259. Sludge produced during the treatment of wastewater is being used as fertilizer in several Gulf countries. The Water and Sewage Authority of Saudi Arabia has targeted the reuse of the total amount of sludge in the future. However, these sludges should be properly treated before reuse as they contain a large number of pathogens and parasites. Little information is available on the microbial characteristics of sludge produced in wastewater treatment plants operating in this region. Variations in the population densities measured by Standard Plate Count, total coliform, fecal coliform, coliphage, and Clostridium perfringens present in the sludge, were monitored during a one year study at Al-Khobar wastewater treatment plant so that the effect of seasonal variations on the fate of these five indicator microorganisms could be investigated. This paper covers an evaluation of the fate of indicator microorganisms in the drying sludge. Insight gained in this study will be helpful in establishing guidelines for the use of sludge as fertilizer for agriculture purposes. [TOP OF PAGE]

  1361. Isolation and characterization of two viruses with large genome size infecting Chrysochromulina ericina (Prymnesiophyceae) and Pyramimonas orientalis (Prasinophyceae). Sandaa,R.-A., Heldal,M., Castberg,T., Thyrhaug,R., Bratbak,G. (2001). Virology 290:272-280. Two lytic viruses specific for Chrysochromulina ericina (Prymnesiophyceae) and for Pyramimonas orientalis (Prasinophyceae) were isolated from Norwegian coastal waters in June 1998. The lytic cycle was 14-19 h for both viruses; the burst size was estimated at 1800-4100 viruses per host cell for the Chrysochromulina virus and 800-1000 for the Pyramimonas virus. Thin sections of infected cells show that both viruses replicate in the cytoplasm and that they have a hexagonal cross section, indicating i