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 mini