Bacteriophage Ecology Group Reference Abstracts (1999)
	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 Wednesday, December 26, 2001

1. Viruses help fight bacteria that resist antibiotics. ??? (1999). The Patriot Ledger Quincy, MA 13(183?), 183? (News Section)-same? Scientists have harnessed nature's way of tackling antibiotic- resistant bacteria. An injection of a virus that attacks bacteria only has saved the life of a patient after all other drugs proved useless. The technique -- the use of bacteriophages, or bacteria-eaters -- was pioneered in the former Soviet Union at around the time of the discovery of much more swiftly effective antibiotics. Although penicillin and other such drugs changed medicine, one team in Tbilisi, Georgia, kept research in phages going to the present day. [TOP OF PAGE]

2. Genetic exchange between microorganisms. ??? (1999). p. ???-??? In Lengeler, J.W., Drews, G., and Shlegel, H.G. (eds.), Biology of Prokaryotes. Blackwell Science, Inc., [TOP OF PAGE]

3. Bacteriophage T4 resistance to lysis-inhibition collapse. Abedon, S.T. (1999). Genet. Res. 74:1-11. Lysis-inhibition is a mechanism of latent-period extension and burst-size increase that is induced by the T4 bacteriophage adsorption of T4-infected cells. Mutants of T4 genes imm, sp, and 5 (specifically the ts1 mutant of the latter) display some lysis inhibition. However, these mutants experience lysis-inhibition collapse, the lysis of lysis-inhibited cells, earlier than wild type-infected cells (i.e., their collapse occurs prematurely). Lysis from without is a lysis induced by excessive T4 adsorption. Gp5 is an inducer of lysis from without while gpimm and gpsp effect resistance to lysis from without. This paper shows that interfering with the adsorption of phages to imm-, sp-, or 5ts1-mutant-infected cells, in a variety of contexts, inhibits premature lysis-inhibition collapse. From these data, it is inferred that wild-type T4-infected cells display resistance to lysis-inhibition collapse by a mechanism resembling resistance to lysis from without. [TOP OF PAGE]

4. Longitudinal study on the susceptibility to bacteriophages of Staphylococcus aureus strains isolated from dairy farms in Trinidad. Adesiyun, A.A., Romain, H.T. (1999). Zentralblatt Fur Veterinarmedizin - Reihe B 46:567-581. A 6-month longitudinal study was conducted on 30 dairy cows in early lactation and their human handlers on six farms across Trinidad. Weekly samples of bulk milk, composite milk and anterior nares and hand swabs from human handlers were collected and cultured for Staphylococcus aureus on Baird-Parker agar (BPA). The susceptibility of S. aureus strains to bacteriophages and the relatedness of strains isolated over the study period were determined. Sixty-three (51.2%) of 123 strains of S. aureus from bulk milk were typable compared with 111 (57.3%) of 194 and 82 (61.7%) of 133 strains isolated from composite milk and human handlers, respectively. The differences were not statistically significant (P > 0.05; chi 2). Bovine phage 42D lysed 3.3% (4 of 123), 16.5% (32 of 194) and 12.0% (16 of 133) of S. aureus strains isolated from bulk milk, composite milk and human handlers, respectively. The differences were statistically significant (P < 0.001; chi 2). Amongst bulk milk isolates of S. aureus, 35 (31.8%) of 110 exhibited relatedness in 11 groups based on their phage patterns and groups. The mean maximum interval between the first and last detection of related S. aureus strains in a group was 11.5 +/- 7.3 weeks. Amongst composite milk strains of S. aureus, 23 (46.0%) of 50, 25 (62.5%) of 40 and 22 (53.7%) of 41 exhibited relatedness on farms IB 2, IB 27 and IC 23, respectively, but the differences were not statistically significant (P > 0.05; chi 2). On farm IB 2, five groups of related strains of S. aureus were detected with a mean maximum interval of detection of 18.2 +/- 8.5 weeks compared to farm IB 27 where five groups of related strains were also observed but with an interval of 13.8 +/- 8.2 weeks. On farm IC 23, a total of seven groups of related S. aureus strains were detected with a mean interval of 8.0 +/- 5.5 weeks. For human strains of S. aureus from farm IB 2, nine (56.3%) of 16 strains isolated from anterior nares exhibited relatedness in three groups with a mean maximum interval of 13.3 +/- 4.7 weeks compared to four (25.0%) of 16 hand swab isolates which exhibited relatedness in two groups with mean interval of detection of 11.0 +/- 1.4 weeks. The differences were not statistically significant (P > 0.05; chi 2). On farm IB 27, for anterior nares isolates, eight (72.7%) of 11 exhibited relatedness in two groups with a mean maximum interval of detection of 20.5 +/- 2.1 weeks compared to hand swab isolates, with six (50.0%) of 12 showing relatedness in two groups and a mean interval of 10.5 +/- 2.1 weeks. It was concluded that dairy cows and their human handlers carried particular strains of S. aureus at various sites for extended periods, which served as continuous sources of contamination of milk and may play a significant role in the occurrence of subclinical mastitis, with an obvious economic impact. [TOP OF PAGE]

5. Iron modulates phenotypic variation and phosphorylation of P270 in double-stranded RNA virus-infected Trichomonas vaginalis. Alderete, J.F. (1999). Infect. Immun. 67:4298-4302. Trichomonas vaginalis infected with a double-stranded RNA virus undergoes phenotypic variation on the basis of surface versus cytoplasmic expression of the immunogenic protein P270. Examination of batch cultures by flow cytofluorometry with monoclonal antibody (MAb) to P270 yields both fluorescent and nonfluorescent trichomonads. Greater numbers and intensity of fluorescent organisms with surface P270 reactive with MAb were evident in parasites grown in medium depleted of iron. Placement of iron-limited organisms in medium supplemented with iron gave increased numbers of nonfluorescent trichomonads. Purified subpopulations of trichomonads with and without surface P270 obtained by fluorescence-activated cell sorting reverted to nonfluorescent and fluorescent phenotypes when placed in high- and low-iron media, respectively. No similar regulation by iron of P270 was evident among virus-negative T. vaginalis isolates or virus-negative progeny trichomonads derived from virus-infected isolates. Equal amounts of P270 were detectable by MAb on immunoblots of total proteins from identical numbers of parasites grown in low- and high-iron media. Finally, P270 was found to be highly phosphorylated in high-iron parasites. Iron, therefore, plays a role in modulating surface localization of P270 in virus-harboring parasites. [TOP OF PAGE]

6. Primary structure and features of the genome of the Lactobacillus gasseri temperate bacteriophage phiadh. Altermann, E., Klein, J.R., Henrich, B. (1999). Gene (Amsterdam) 236:333-346. The complete DNA sequence of the Lactobacillus (Lb.) gasseri temperate phage phiadh was determined. The linear and double-stranded genome consists of 43.785 bp with a G+C content of 35.3% and 3' protruding cohesive ends of 12 nt. Sixty-two possible ORFs were identified. On the basis of homology comparisons, some of them could be assigned to possible functions, such as a helicase, a nucleic acid polymerase and a protease. In a non-coding area of the phiadh genome, structural features of a potential replication origin were detected. After subcloning, this region was functional as a replicon in Lb. gasseri and Lactococcus lactis. N-terminal aa sequencing and electron microscopic analysis of intact and defective phage particles enabled the identification of two capsid protein genes. One of their products, the major head protein, seems to be processed on the posttranslational level. [TOP OF PAGE]

7. Stable expression of the Lactobacillus casei bacteriophage A2 repressor blocks phage propagation during milk fermentation. Alverez, M.A., Rodriguez, A., Suarez, J.E. (1999). Journal of Applied Microbiology 86:812-816. A general strategy was applied to implement resistance against temperate bacteriophages that infect food fermentation starters through cloning and expression of the phage repressor. Lactobacillus casei ATCC 393 and phage A2 were used to demonstrate its feasibility as milk fermentation is drastically inhibited when the strain is infected by this phage. The engineered strain Lact. casei EM40::cI, which has the A2 repressor gene (cI) integrated into the genome, was completely resistant and able to ferment milk whether phage was present or not. In addition, viable phages were eliminated from the milk, probably through adsorption to the cell wall. Finally, the integration of cI in the genome resulted in a stable resistance phenotype, being unnecessary selective pressure during milk fermentation. [TOP OF PAGE]

8. Test tube evolution catches time in a bottle. Appenzeller, T. (1999). Science 284(June 25, 1999), 2108-2110. By running experiments on microbes for thousands of generations, researchers are exploring the roles of chance and history in evolution. [TOP OF PAGE]

9. Application of bacteriophages as surrogates for mammalian viruses: a case for use in filter validation based on precedents and current practices in medical and environmental virology. Aranha-Creado, H., Brandwein, H. (1999). PDA Journal of Pharamaceutical Science and Technology 53:75-82. Infectivity-based assays are the assays of choice for the detection of pathogenic mammalian viruses. While it is intuitively appropriate to conduct testing and validation studies with the known viral burden or a closely related mammalian species, logistic considerations often dictate otherwise. Consequently, bacteriophages have served as suitable surrogates for mammalian viruses in both medical and environmental virology applications. The wide range of bacteriophages available offers a powerful analytical tool amenable to several different applications: filter validation studies (where removal is based on size exclusion), investigations into virus contamination control issues, evaluation of barrier materials, etc. There is a considerable body of evidence to suggest and support the use of bacteriophages as surrogates for mammalian viruses. Use of appropriately sized bacteriophages provides an innocuous, efficacious and expeditious method for economical testing and validation of viral clearance capabilities of virus removal filters, thus facilitating performance of filter validation studies in biopharmaceuticals under product- and process-specific conditions in an overall effort towards ensuring the virological safety of biologicals. This paper discusses the limitations associated with mammalian virus assays and provides a rationale for the use of bacteriophages as surrogates for mammalian viruses. Data from published literature documenting applicability of bacteriophages in filter validation studies, especially when removal is based on size exclusion, is reviewed along with examples of studies from the fields of medical and environmental virology. [TOP OF PAGE]

10. The genetic element pSSVx of the extremely thermophilic crenarchaeon Sulfolobus is a hybrid between a plasmid and a virus. Arnold, H.P., She, Q., Phan, H., Stedman, K., Prangishvili, D., Holz, I., Kristjansson, J.K., Garrett, R., Zillig, W. (1999). Molecular Microbiology 34:217-226. A new Sulfolobus islandicus strain, REY15/4, harboured both a novel fusellovirus, SSV2, and a small plasmid, pSSVx. The plasmid spread in S. solfataricus P1 together with the virus after infection with either the supernatant of a culture of REY15/4 or purified virus. Spreading of the plasmid required co-transfection with either SSV2 or the related SSV1 as helpers. Virus purified from REY15/4 constituted a mixture of two sizes of particles, one with the dimensions of a normal fusellovirus and the other smaller. Cloned SSV2 produced only the larger particles and only SSV2 DNA, indicating that the smaller particles contained pSSVx packaged into capsids made up of SSV2 components. The 5.7 kb genome of pSSVx revealed regions of high sequence similarity to the cryptic Sulfolobales plasmids pRN1, pRN2 and pDL10. Thus, pSSVx belongs to the family of pRN plasmids that share a highly conserved region, which probably constitutes the minimal replicon. They also contain a variable region showing no sequence similarity. In pSSVx, this region contains three open reading frames (ORFs), two of which are juxtapositioned and show high sequence similarity to a tandem of ORFs in fusellovirus genomes. Neither pRN1 nor pRN2, which lack this tandem, spread in the presence of the fuselloviruses, which implies that the sequences of these ORFs enable pSSVx to use the packaging system of the viral helpers for spreading. [TOP OF PAGE]

11. Characterization of six bacteriophages of Serratia liquefaciens CP6 isolated from the sugar beet phytosphere. Ashelford, K.E., Fry, J.C., Bailey, M.J., Jeffries, A.R., Day, M.J. (1999). Appl. Environ. Microbiol. 65:1959-1965. Six phages (Phi CP6-1 to (Phi CP6-6) that are commonly found in the phytosphere of sugar beet (Beta vulgaris var. Amethyst) were investigated, and their relative impacts on their host (Serratia liquefaciens CP6) were compared. There were fundamental differences between the two most abundant predators of CP6 Phi CP6-1 and Phi CP6-4), Like Phi CP6-2 and Phi CP6-5, Phi CP6-1 belonged to the family Siphoviridae, while Phi CP6-4 exhibited the morphology of the family Podoviridae, The other phages were members of the family Myoviridae, DNA-DNA crosshybridization revealed that Phi CP6-1 and Phi CP6-4 had little common DNA, although all of the other phages exhibited some genetic similarity. Like Phi CP6-2, Phi CP6-3, and Phi CP6-5, Phi CP6-1 was capable of forming a lysogenic association with its host, while Phi CP6-4 and Phi CP6-6 appeared to be entirely virulent. Single-step growth curve experiments revealed that Phi CP6-4 had a much shorter latent period and a smaller burst size than Phi CPC-1. Also, Phi CP6-1 could transduce a number of host chromosomal markers with transfer frequencies of 2.9 x 10(-9) to 3.9 x 10(-7), whereas Phi CP6-4 could not transduce S. liquefaciens CP6 genes. When viewed in the context of the strikingly different temporal niches of these phages, our data provide an insight into how bacteriophage interactions with their hosts might reflect the natural ecology of bacteriophages, Our data also illustrate how the potential for gene transfer changes over time in an environment that supports several different phages. [TOP OF PAGE]

12. In situ population dynamics of bacterial viruses in a terrestrial environment. Ashelford, K.E., Day, M.J., Bailey, M.J., Lilley, A.K., Fry, J.C. (1999). Appl. Environ. Microbiol. 65:169-174. Predation by bacteriophages is thought to control bacterial numbers and facilitate gene transfer among bacteria in the biosphere. A thorough understanding of phage population dynamics is therefore necessary if their significance in natural environments is to be fully appreciated. Here we describe the in situ population dynamics of three separate phage populations predating on separate bacterial species, living on the surface of field-grown sugar beet (Beta vulgaris var. Amethyst), as recorded over a 9-month period. The distributions of the three phage populations were different and fluctuated temporally in 1996 (peak density, approximately 10(3) PFU g-1). One of these populations, predating on the indigenous phytosphere bacterium Serratia liquefaciens CP6, consisted of six genetically distinct DNA phages that varied in relative abundance to the extent that an apparent temporal succession was observed between the two most abundant phages, phi CP6-1 and phi CP6-4. [TOP OF PAGE]

13. Toward selection of internalizing antibodies from phage libraries. Becerril, B., Poul, M.A., Marks, J.D. (1999). Biochemical & Biophysical Research Communications 255:386-393. Antibodies which bind cell surface receptors in a manner whereby they are endocytosed are useful molecules for the delivery of drugs, toxins, or DNA into the cytosol of mammalian cells for therapeutic applications. Traditionally, internalizing antibodies have been identified by screening hybridomas. For this work, we studied a human scFv (C6.5) which binds ErbB2 to determine the feasibility of directly selecting internalizing antibodies from phage libraries and to identify the most efficient display format. Using wild-type C6.5 scFv displayed monovalently on a phagemid, we demonstrate that anti-ErbB2 phage antibodies can undergo receptor-mediated endocytosis. Using affinity mutants and dimeric diabodies of C6.5 displayed as either single copies on a phagemid or multiple copies on phage, we define the role of affinity, valency, and display format on phage endocytosis and identify the factors that lead to the greatest enrichment for internalization. Phage displaying bivalent diabodies or multiple copies of scFv were more efficiently endocytosed than phage displaying monomeric scFv and recovery of infectious phage was increased by preincubation of cells with chloroquine. Measurement of phage recovery from within the cytosol as a function of applied phage titer indicates that it is possible to select for endocytosable antibodies, even at the low concentrations that would exist for a single phage antibody member in a library of 10(9). [TOP OF PAGE]

14. Viral evolution revealed by bacteriophage PRD1 and human adenovirus coat protein structures. Benson, S.D., Bamford, J.K., Bamford, D.H., Burnett, R.M. (1999). Cell 98:825-833. The unusual bacteriophage PRD1 features a membrane beneath its icosahedral protein coat. The crystal structure of the major coat protein, P3, at 1.85 A resolution reveals a molecule with three interlocking subunits, each with two eight-stranded viral jelly rolls normal to the viral capsid, and putative membrane-interacting regions. Surprisingly, the P3 molecule closely resembles hexon, the equivalent protein in human adenovirus. Both viruses also have similar overall architecture, with identical capsid lattices and attachment proteins at their vertices. Although these two dsDNA viruses infect hosts from very different kingdoms, their striking similarities, from major coat protein through capsid architecture, strongly suggest their evolutionary relationship. [TOP OF PAGE]

15. Biological UV dosimeters in the assessment of the biological hazard from environmental radiation. Berces, A., Fekete, A., Gaspar, S., Grof, P., Rettberg, P., Horneck, G., Ronto, G. (1999). Journal of Photochemistry and Photobiology B, Biology 53:36-43. To determine the impact of environmental UV radiation, biological dosimeters that weight directly the incident UV components of sunlight have been developed, improved and evaluated in the frame of the BIODOS project. Four DNA-based biological dosimeters ((i) phage T7, (ii) uracil thin layer, (iii) spore dosimeter and (iv) DLR-biofilm) have been assessed from the viewpoint of their biological relevance, spectral response and quantification of their biological effectiveness. The biological dosimeters have been validated by comparing their readings with weighted spectroradiometer data, by comparison with other biological doses, as well as with the determined amounts of DNA UV photoproducts. The data presented here demonstrate that the biological dosimeters are potentially reliable field dosimeters for measuring the integrated biologically effective irradiance for DNA damage. [TOP OF PAGE]

16. Isolation of Shigella sonnei lysogenic for a bacteriophage encoding gene for production of Shiga toxin. Beutin, L., Strauch, E., Fischer, I. (1999). Lancet 353:1498-1498. [TOP OF PAGE]

17. Reconsidering the relationship between virally induced bacterial mortality and frequency of infected cells. Binder, B. (1999). Aquat. Microb. Ecol. 18:207-215. The relative contribution of viral lysis to overall mortality in aquatic bacterial populations is often estimated as twice the frequency of infected cells (FIC). The `factor-of-two rule' upon which this estimate is based assumes (1) steady-state conditions, (2) that latent period is equivalent to generation time, and (3) that infected cells are not grazed. FIC values for this calculation are themselves derived from measurements of the frequency of visibly infected cells (FVIC) by the use of a simple conversion factor. A steady-state model was developed to more rigorously define the relationships between FIC, FVIC, and the fraction of mortality from viral lysis (FMVL). This model shows that even under the restrictive assumptions listed above, the factor-of-two rule systematically overestimates FMVL for typically reported values of FVIC. The model also shows that although grazing on infected cells further reduces FMVL for a given estimate of FIC, at the same time such grazing increases FIC for a given measurement of FVIC. In combination, these 2 effects minimize the influence of grazing on the calculation of FMVL from FVIC. Overall, the relationship between FMVL and FVIC is well approximated as follows: FMVL epsilon FVIC/[ gamma ln(2) (1 - epsilon - FVIC)], where gamma = the ratio between the latent period and generation time, and \g?\ = the fraction of the latent period during which viral particles are not yet visible. Using typically observed values of FVIC, and assuming that gamma = 1 (per assumption 2, above) and \g?\ = 0.186 (per literature estimates), the model suggests that, on average, viral lysis accounts for approximately 22% (range: 4.5 to 45%) of total bacterial mortality in a range of aquatic environments, corresponding to a mean overestimate of 24% (range: 4 to 44%) by the factor-of-two rule. Perhaps most importantly, the model shows that calculations of FMVL from FIC or FVIC are very sensitive to changes in the relative length of the latent period ( gamma ) and in the assumed proportion of the latent period during which viral particles are not recognizable ( epsilon ). Constraining these 2 factors would greatly improve the reliability of FMVL calculations. [TOP OF PAGE]

18. Vaginal bacterial phaginosis? Blackwell, A.L. (1999). Sexually Transmitted Infections 75:352-353. The hypothesis that a sexually transmitted lactobacillus phage may specifically destroy the endogenous healthy lactobacillus vaginal flora and secondarily permit overgrowth of endogenous aerobic bacteria and G. vaginalis may explain why anaerobic vaginosis behaves epidemiologically as a sexually transmitted agent but recurrence rate is unaffected by antibacterial treatment of male partners. Unfortunately this hypothesis raises as many questions as answers. Are phages capable of destroying lactobacilli carried on the penis or are the lactobacillus phages derived from the woman's own gut flora and merely transferred into the vagina by sexually activity. Are there any phage resistant strains of lactobacilli which could be of therapeutic use? Is t here any possibility that a vaccine could be developed that could be given to women with recurrent anaerobic vaginosis, particularly perhaps, those who smoke or who have cervical intraepithelial neoplasia. Until the pathogenesis of anaerobic vaginosis is more fully understood, argument will undoubtedly remain concerning the best name for the conditions (?vaginal bacterial phaginosis) and treatment will be unsatisfactory. As a result some women will be burdened not only by the social consequences of recurrent genital malodour but may also be at risk of a plethora of complications. [see: http://sti.bmjjournals.com/cgi/reprint/75/5/352.pdf for full-text pdf]. [TOP OF PAGE]

19. Rezervoary, interakcie a stabilita genov rezistencie na antibiotika. Syndrom "easy to get--hard to lose". [Reservoirs, interactions and stability of genetic resistance to antibiotics. The "easy to get--hard to lose" syndrome]. Blahova, J., Kralikova, K., Krcmery, V. (1999). CASOPIS LEKARU CESKYCH 138:424-428. Enthusiasm after discovery of antibiotics and their use in clinical practice led to presumption that problems of bacterial infections will be soon resolved and forgotten and attention will be turned to other serious problems, such as viral infections or neoplastic diseases. However, instead of disappearance of bacterial infections, bacterial pathogens become more resistant to many antibiotics. The ability of bacterial strains to acquire resistance genes from other bacteria, even of different species, causes increasing stability of resistance of bacteria. Transferable elements--resistance genes--often interact and create changed structures; this enables to preserve, stabilize, or under special conditions, transfer resistance genes. Transferable elements include plasmids, transposons, integrins and gene cassettes. Conjugation of bacteria, transduction by bacteriophages and transformation are the mechanisms by which these elements are transferred. A very significant property of transferable, mobilizable and transposable genetic systems of resistance is their stability and ability to adapt to new hosts. They do not lose it in the absence of antibiotics. The generally pessimistic view on future antibacterial chemotherapy should be a challenge to prevent the existence and spread of resistant strains of bacteria. It is much simpler and more convenient than "quench the fire" later. Best scheme is to stop resistance before it starts. [TOP OF PAGE]

20. Transduction of antibiotic resistance in Pseudomomas aeruginosa: relationship between lytic and transducing activity of phage isolate AP-423. Blahova, J., Kralikova, K., Krcmery, V.S., Jezek, P. (1999). Acta Virol. 43:395-398. Isolation and propagation of a wild type phage, isolate AP-423, from an apparently lysogenic strain of Pseudomonas aeruginosa, resistant to a series of anti-pseudomonadal antibiotics, and its use for transduction of resistance determinants is described. The phage isolate AP-423 showed a phenomenon of host restriction, i.e. it was lysogenic only for some of the recipient strains tested. Its transduction capacity, both in sets of genes transduced and frequency of transduction, was different in two recipient strains of P. aeruginosa. This phage showed also some restriction in titers, to which it could be propagated, only in certain recipient strains. [TOP OF PAGE]

21. High-frequency transduction (HFT) of resistance to ceftazidime and other antibiotics by a wild-type Pseudomonas aeruginosa phage. Blahová, J., Králiková, K., Krcméry, V., Sr., Bartoníková, N., Mikovicova, A. (1999). Zentralblatt Fuer Bakteriologie 289:179-183. [TOP OF PAGE]

22. High-frequency transduction of antibiotic resistance in Pseudomonas aeruginosa by a wild-type bacteriophage with restricted specificity for recipient strains. Blahová, J., Králiková, K., Krcméry, V., Sr., Bartoníková, N. (1999). European Journal of Clinical Microbiology & Infectious Diseases 18:152-154. [TOP OF PAGE]

23. Epistatic interactions can lower the cost of resistance to multiple consumers. Bohannan, B.J.M., Travisano, M., Lenski, R.E. (1999). Evolution 53:292-295. It is widely assumed that resistance to consumers (e.g., predators or pathogens) comes at a "cost"; i.e., that when the consumer is absent the resident organisms are less fit than their susceptible counterparts. It is unclear what factors determine this cost. We demonstrate that epistasis between genes that confer resistance to two different consumers can alter the cost of resistance. We used as a model system the bacterium Escherichia coli and two different viruses (bacteriophage), T4 and l, that prey upon E. coli. Epistasis tended to reduce the costs of multiple resistance in this system. However, the extent of cost savings and its statistical significance depended on the environment in which fitness was measured, whether the null hypothesis for gene interaction was additive or multiplicative, and subtle differences among mutations that conferred the same resistance phenotype. [TOP OF PAGE]

24. Effect of prey heterogeneity on the response of a food chain to resource enrichment. Bohannan, B.J.M., Lenski, R.E. (1999). Am. Nat. 153:73-82. We demonstrated that the presence of invulnerable prey can result in a shift in the balance between top-down and bottom-up control of a model food chain. Our model food chain consisted of the bacterium Escherichia coli and the bacteriophage T4 (a virus that feeds on E. coli) in chemostats supplied with different concentrations of glucose. The E. coli population consisted of individuals that were susceptible to predation by T4 ("edible" E. coli) and individuals that were resistant to predation by T4 ("inedible" E. coli). The equilibrium density of a hetergeneous prey population (consisting of edible and inedible E. coli) increased strongly in response to an enrichment of its resources. This response consisted of an increase in the inedible fraction of the prey population but no change in the edible fraction. In contrast, a homogeneous prey population (edible E. coli only) increased only marginally. The equilibrium density of the predator population (bacteriophage T4) did not significantly increase in response to enrichment when its prey were heterogeneous, but it increased when its prey were homogeneous. [TOP OF PAGE]

25. Effect of resource supply rate on host-pathogen dynamics. Bohannan, B.J.M. (1999). AnonymousProceedings of the 8th International Symposium on Microbial Ecology. The dynamics of model host cell (E. coli) and model pathogen (bacteriophage) populations were studied in chemostats with different resource supply rates. Resource supply rate was manipulated by altering the concentration of the limiting resource (glucose) in the incoming media. Population responses to increased resourse supply rate were influenced by the vulnerability of the host cells to infection. When the host cell population consisted entirely of cells equally vulnerable to infection, both pathogen and host cells responded to increased resource supply rate with an increase in their average densities. In contrast, when the host cell contained some cells that were less vulnerable to infection (i.e., partially phage-resistant E. coli), only the pathogen population responded to increased supply rate with a signficant increase in average density. Furthermore, when the host cell population contained some cells completely invulnerable to infection (i.e., phage-resistant E. coli) only the host cell population responded to increased supply rate with an increase in average density. These responses were in general agreement with the predictions of mechanistic models of resource-consumer interactions. [TOP OF PAGE]

26. Antimicrobial properties and morphological characteristics of two Photorhabdus luminescens strains. Bondi, M., Messi, P., Sabia, C., Baccarani Contri, M., Manicardi, G. (1999). New Microbiologica 22:117-127. The biological properties of two Photorhabdus luminescens isolates (MU1 and MU2) of environmental source and the activity of antimicrobial agar diffusible agents (AADA) produced by the same are reported. With regard to cultural features, two variant forms for P. luminescens MU1 and three for P. luminescens MU2 (including an intermediate phase I-like form) have been found. These three forms differ in biological and biochemical properties: beta-lactamase, urease, bioluminescence and antimicrobial agar diffusible substance production associated with the phase I form, were less evident in the intermediate phase I-like MU2 and were absent in phase II form. Antimicrobial activity was present in both strains, with the production of a large amount of a diffusible compound with a wide spectrum of action against bacteria of other genera; a reduced activity against correlated species was also observed. Examination by electron microscopy of MU1 and MU2 purified broth cultures revealed the presence of particles belonging to the class of the phage tail-like bacteriocins, described in recent studies as responsible for antibacterial activity against correlated bacteria, a result never confirmed "in vitro". A plasmid of 21 Mdal was observed in all the form variants of P. luminescens MU2, suggesting that plasmids are not involved in the transition from primary to secondary phase; no plasmid was detected in P. luminescens MU1. [TOP OF PAGE]

27. Alternative mechanism of cholera toxin acquisition by Vibrio cholerae: Generalized transduction of CTXPHI by bacteriophage CP-T1. Boyd, E.F., Waldor, M.K. (1999). Infect. Immun. 67:5898-5905. Horizontal transfer of genes encoding virulence factors has played a central role in the evolution of many pathogenic bacteria. The unexpected discovery that the genes encoding cholera toxin (ctxAB), the main cause of the profuse secretory diarrhea characteristic of cholera, are encoded on a novel filamentous phage named CTXPHI, has resulted in a renewed interest in the potential mechanisms of transfer of virulence genes among Vibrio cholerae. We describe here an alternative mechanism of cholera toxin gene transfer into nontoxigenic V. cholerae isolates, including strains that lack both the CTXPHI receptor, the toxin coregulated pilus (TCP), and attRS, the chromosomal attachment site for CTXPHI integration. A temperature-sensitive mutant of the V. cholerae generalized transducing bacteriophage CP-T1 (CP-T1ts) was used to transfer a genetically marked derivative of the CTX prophage into four nontoxigenic V. cholerae strains, including two V. cholerae vaccine strains. We demonstrate that CTXPHI transduced by CP-T1ts can replicate and integrate into these nontoxigenic V. cholerae strains with high efficiency. In fact, CP-T1ts transduces the CTX prophage preferentially when compared with other chromosomal markers. These results reveal a potential mechanism by which CTXPHI+ V. cholerae strains that lack the TCP receptor may have arisen. Finally, these findings indicate an additional pathway for reversion of live-attenuated V. cholerae vaccine strains. [TOP OF PAGE]

28. Use of plaque assay to detect enteric viruses in a rural watershed. Brenner, F.J., Brenner, E.K., Schwartz, T.E. (1999). Journal of Environmental Quality 28:845-849. Water samples were collected from four locations within the Munnell Run Watershed in Mercer County, Pennsylvania, and analyzed for fecal coliforms by MPN and enteric phages by plaque assay using Salmonella typhimurium WG 49 and Bacteroides fragilis HSP 40 as hosts. Fecal coliform concentrations and the number of phages varied seasonally (P < 0.001), as well as among the different sampling stations (P < 0.001). At all sampling stations positive for phages, S. typhimurium WG 49 PFUs outnumbered B. fragilis HSP 40 PFUs. Phages also were isolated from a septic discharge pipe and a wetland receiving septic drainage, but not from three avian species, 12 mammalian species, two streams without human wastes or from natural wetland, indicating that these viruses were of human origin. MPNs of fecal coliforms and S. typhimurium and B. fragilis PFUs were correlated with stream temperature (P < 0.001) and rainfall (P < 0.01). Only fecal coliforms and S. typhimurium WG 49 phages were correlated with suspended solids concentrations (P < 0.01). Likewise, there was a significant interaction among these parameters and MPNs of fecal coliform and HSP 40 and WG 49 PFUs (P < 0.001). The presence of host specific phages indicate the existence of septic discharges in the watershed, but both fecal coliforms and enteric viruses persist in stream systems, especially during the summer months. [TOP OF PAGE]

29. Iodine disinfection of a model bacteriophage, MS2, demonstrating apparent rebound. Brion, G.M., Silverstein, J. (1999). Water Res. 33:169-179. MS2 coliphage viruses suspended in buffered distilled water were rapidly inactivated by < 5 mg/L iodine doses, losing 6 logs (99.9999%) of infectivity within less than 3 min contact time. The effect of pH on MS2 inactivation within the range of 6 to 8 was not statistically significant. However, in the presence of dissolved organic substances, such as detergents and proteins, the inactivation of MS2 viruses decreased significantly to less than 4 logs (99.99%). Of special interest was that in the presence of beef extract proteins, an apparent reversal of MS2 inactivation, dubbed rebound, was observed. It was observed that after an initial 5 to 6 log reduction in infectivity, a consistent and statistically significant increase in the number of plaque forming units (PFU), as much as 2 logs, was measured. MS2 rebound occurred only when the oxidized iodine residual had been quickly consumed by beef extract proteins in solution. Neither virus particle aggregation nor water salinity were found to account for the increase in PFU values. Based on other investigators' suggestions that iodine disinfection caused changes to viral protein coats, it was hypothesized that conformational changes in MS2's protein coat caused by iodine would result in a change in the isoelectric focusing point of whole MS2 virions. A shift in isoelectric focusing point from an acidic pH value of 3.9 to more basic values, and a dispersion of the virus band after exposure to high levels of iodine was observed, supporting the hypothesis that iodine caused changes in the charge distribution characteristics of the protein coat. [TOP OF PAGE]

30. Unexplored reservoirs of pathogenic bacteria: protozoa and biofilms. Brown, M., Barker, J. (1999). Trends in Microbiology 7:???-??? [TOP OF PAGE]

31. Flow cytometric analyses of virus infection in two marine phytoplankton species, Micromonas pusilla (Prasinophyceae) and Phaeocystis pouchetii (Prymnesiophyceae). Brussaard, C.P.D., Thyrhaug, R., Marie, D., Bratbak, G. (1999). Journal of Phycology 35:941-948. Cell characteristics of two axenic marine phytoplankton species, Micromonas pusilla (Butscher) Manton et Parke and Phaeocystis pouchetii (Hariot) Lagerheim, were followed during viral infection using flow cytometry, Distinct differences between noninfected and infected cultures were detected in the forward scatter intensities for both algal species. Changes in side scatter signals on viral infection were found only for P, pouchetii. Chlorophyll red fluorescence intensity per cell decreased gradually over time in the infected cultures, DNA analyses were performed using the nucleic acid-specific fluorescent dye SYBR Green I. Shortly after infection the fraction of algal cells with more than one genome equivalent increased for both species because of the replication of viral DNA in the infected cells. Over time, a population of algal cells with low red autofluorescence and low DNA fluorescence developed, likely representing algal cells just prior to viral lysis, The present study provides insight into basic virus-algal host cell interactions. It shows that flow cytometry can be a useful tool to discriminate between virus infected and noninfected phytoplankton cells. [TOP OF PAGE]

32. Evolution by small steps and rugged landscapes in the RNA virus phi6. Burch, C.L., Chao, L. (1999). Genetics 151:921-927. Fisher's geometric model of adaptive evolution argues that adaptive evolution should generally result from the substitution of many mutations of small effect because advantageous mutations of small effect should be more common than those of large effect. However, evidence for both evolution by small steps and for Fisher's model has been mixed. Here we report supporting results from a new experimental test of the model. We subjected the bacteriophage phi6 to intensified genetic drift in small populations and caused viral fitness to decline through the accumulation of a deleterious mutation. We then propagated the mutated virus at a range of larger population sizes and allowed fitness to recover by natural selection. Although fitness declined in one large step, it was usually recovered in smaller steps. More importantly, step size during recovery was smaller with decreasing size of the recovery population. These results confirm Fisher's main prediction that advantageous mutations of small effect should be more common. We also show that the advantageous mutations of small effect are compensatory mutations whose advantage is conditional (epistatic) on the presence of the deleterious mutation, in which case the adaptive landscape of phi6 is likely to be very rugged. [TOP OF PAGE]

33. Induction and characterization of Pediococcus acidilactici temperate bacteriophage. Caldwell, S.L., McMahon, D.J., Oberg, C.J., Broadbent, J.R. (1999). Systematic and Applied Microbiology. 22:514-519. Mitomycin C was used to induce temperate bacteriophage from three strains of Pediococcus acidilactici. The new bacteriophage, designated pa97, pa40, and pa42, were characterized based on morphology, DNA homology, and major protein profiles. Morphological attributes (small isometric heads with non-contractile tails) place these bacteriophages within the B1 group of the family Siphovirdae. Restriction endonuclease digests suggested that the bacteriophage genomes were linear molecules without cohesive ends, and between 33 and 37 kilobases in length. All three bacteriophages possessed one major protein with an estimated mass of 30 to 35 kilodaltons. Bacteriophage pa42 also contained a second major protein of approximately 47 kilodaltons. DNA-DNA hybridization showed bacteriophages pa40 and pa42 were homologous to each other, but not to pa97, suggesting that Pediococcus acidilactici bacteriophage fall into at least two different species. [TOP OF PAGE]

34. Phage therapy: past history and future prospects. Carlton, R.M. (1999). Archivum Immunologii et Therapiae Experimentalis 47:267-274. Bacterial viruses (bacteriophages, also called "phages") can be robust antibacterial agents in vitro. However, their use as therapeutic agents, during a number of trials from the 1920s to the 1950s, was greatly handicapped by a number of factors. In part, there were certain limitations inherent in phage physiology (e. g. narrow host range, and rapid clearance from the body); in part there were technological limitations in the era (e.g. lysogeny not yet discovered); but the greatest limitation was the highly inadequate scientific methodologies used by practitioners at the time (e.g., their failure to conduct placebo-controlled studies, to remove endotoxins from the preparations, and to re-confirm phage viability after adding sterilizing agents to the preparations). In recent years, well-controlled animal models have demonstrated that phages can rescue animals from a variety of fatal infections, while non-controlled clinical reports published in Eastern Europe have shown that phages can be effective in treating drug-resistant infections in humans. This encouraging data, combined with the fact that drug-resistant bacteria have become a global crisis, have created a window of opportunity for phage therapy to be tested anew, this time using modem technologies and placebo-controlled designs. If successful, it can be used as a stand-alone therapy when bacteria are fully resistant to antibiotics, and as a valuable adjunct to antibiotics when the bacteria are still susceptible. [TOP OF PAGE]

35. Adsorption of bacteriophages on clay minerals. Chattopadhyay, S., Puls, R.W. (1999). Environmental Science & Technology 33:3609-3614. The ability to predict the fate of microorganisms in soil is dependent on an understanding of the process of their sorption on soil and subsurface materials. Presently, we have focused on studying the thermodynamics of sorption of bacteriophages (T-2, MS-2, and variant phiX-174) on clays (hectorite, saponite, kaolinite, and clay fraction of samples collected from a landfill site). The thermodynamic study not only determines the feasibility of the process but also provides information on the relative magnitudes of the different forces under a particular set of conditions. The total free energy of interaction during sorption of bacteriophages on clays (DELTAG) has been assumed to be the summation of DELTAGH (DELTAG due to hydrophobic interactions) and DELTAGEL (DELTAG due to electrostatic interactions). The magnitude of DELTAGH was determined from the different interfacial tensions (gamma) present in the system, while DELTAGEL was calculated from zeta-potentials of the colloidal particles. Calculated results show that surface hydrophobicities of the selected sorbents and sorbates dictate sorption. Among the selected bacteriophages, maximum sorption was observed with T-2, while hectorite has the maximum sorption capacity. Experimental results obtained from the batch adsorption studies also corroborated those obtained from the theoretical study. [TOP OF PAGE]

36. The primary immunity determinant in modulating the lysogenic immunity of the filamentous bacteriophage cf [published erratum appears in J Mol Biol 1999 Nov 5;293(4):987]. Cheng, C.M., Wang, H.J., Bau, H.J., Kuo, T.T. (1999). J. Mol. Biol. 287:867-876. Bacteriophage cf is the first single-stranded DNA phage that has been shown to set up a stable lysogenic state with its genome integrated into the host chromosome. From the isolation and characterization of a virulent mutant, cf-tv2, we report the first investigation into the mechanisms of the immunity established by the filamentous bacteriophage. The mutation in cf-tv2 enables the phage to produce plaques on lawns of cf lysogenic cells. The mutation was defined as a 49-nucleotide deletion located in a 0.59 kb NcoI/KpnI fragment of cf replicative form DNA. Two messages, cM1 and cM2, transcribed from the immunity region of wild-type cf but in opposite directions, were detected. In cf-tv2, the 49-nucleotide deletion abolishes cM2 transcription. The primer extension assay suggests a possible RNA-RNA interaction directed by base-pairing of the cM1 and cM2 RNAs. A frameshift mutation of the open reading frame ORF 165, encoded by cM2, resulted in a 10(5) plating efficiency on the cf lysogen. These observations suggest that both RNA-RNA interaction and repressor protein inhibition are involved in the mechanism of cf immunity. A model is proposed for the regulation of cf immunity. [TOP OF PAGE]

37. Procaryotic infections in the mussel Mytilus galloprovinciallis and in its parasite the turbellarian Urastoma cyprinae. Comps, M., Tige, G. (1999). Diseases of Aquatic Organisms 38:211-217. Mussels Mytilus galloprovincialis from the Thau lagoon (Mediterranean coast of France) were regularly sampled to determine the prevalence and intensity of parasitic infections. Microscopically, hepatopancreatic tubules of the mussel appeared infected by a rickettsia-like organism (RLO). Each RLO were surrounded by 2 unit membranes, and colonies composed of several bacteria were enclosed within a vacuolar membrane of the host cell. In addition, examination by transmission electron microscopy revealed that the RLO was infected by phage particles. Histological investigations of the turbellarian Urastoma cyprinae parasitizing the mussels have shown that this ectoparasite was also infected by 2 types of procaryotes, a chlamydia-like organism (CLO) and a mollicute-like organism (MLO). The CLO displayed characteristic developmental stages of the Chlamydiales and was secondarily infected by electron-dense particles presumed to be phage particles. The MLO exhibited some morphological characteristics of the mollicutes, in that the microorganisms were bounded by a single membrane sharing a trilaminar structure. Neither of these microorganisms have previously been reported in Platyhelminthes. [TOP OF PAGE]

38. Inactivation of faecal indicator microorganisms in waste stabilisation ponds: Interactions of environmental factors with sunlight. Davies-Colley, R.J., Donnison, A.M., Speed, D.J., Ross, C.M., Nagels, J.W. (1999). Water Res. 33:1220-1230. Sunlight exposure is considered to be the most important cause of "natural" disinfection in waste stabilisation ponds (WSPs). We examined the influence of dissolved oxygen (DO), pH, and particulate and dissolved constituents in WSP effluent, on sunlight inactivation of faecal micro-organisms, using small reactors operated under controlled physico-chemical conditions. Inactivation of both enterococci and F-RNA phages increased strongly as DO was increased, and also depended on light-absorbing pondwater constituents, but pH was not influential over the range investigated (7.5 to 10). Inactivation of E. coli increased strongly when pH increased above 8.5, as well as being strongly dependent on DO. Inactivation of F-DNA phage was independent of the factors investigated. These results are consistent with the F-DNA phages being inactivated as a result of direct DNA damage by UVB in sunlight, whereas the other three microbiological indicators are inactivated as a result of photo-oxidative damage, although the target of damage is apparently different. Our findings of diverse influences of physico-chemical conditions suggest difficulties in interpreting data for a single micro-organism to indicate WSP effluent quality. However, sunlight remains the factor of over-riding importance, and disinfection in WSPs may be enhanced by increasing sunlight exposure. [TOP OF PAGE]

39. The Vibrio cholerae O139 Calcutta bacteriophage CTXphi is infectious and encodes a novel repressor. Davis, B.M., Kimsey, H.H., Chang, W., Waldor, M.K. (1999). J. Bacteriol. 181:6779-6787. CTXphi is a lysogenic, filamentous bacteriophage. Its genome includes the genes encoding cholera toxin (ctxAB), one of the principal virulence factors of Vibrio cholerae; consequently, nonpathogenic strains of V. cholerae can be converted into toxigenic strains by CTXphi infection. O139 Calcutta strains of V. cholerae, which were linked to cholera outbreaks in Calcutta, India, in 1996, are novel pathogenic strains that carry two distinct CTX prophages integrated in tandem: CTX(ET), the prophage previously characterized within El Tor strains, and a new CTX Calcutta prophage (CTX(calc)). We found that the CTX(calc) prophage gives rise to infectious virions; thus, CTX(ET)phi is no longer the only known vector for transmission of ctxAB. The most functionally significant differences between the nucleotide sequences of CTX(calc)phi and CTX(ET)phi are located within the phages' repressor genes (rstR(calc) and rstR(ET), respectively) and their RstR operators. RstR(calc) is a novel, allele-specific repressor that regulates replication of CTX(calc)phi by inhibiting the activity of the rstA(calc) promoter. RstR(calc) has no inhibitory effect upon the classical and El Tor rstA promoters, which are instead regulated by their cognate RstRs. Consequently, production of RstR(calc) renders a CTX(calc) lysogen immune to superinfection by CTX(calc)phi but susceptible (heteroimmune) to infection by CTX(ET)phi. Analysis of the prophage arrays generated by sequentially integrated CTX phages revealed that pathogenic V. cholerae O139 Calcutta probably arose via infection of an O139 CTX(ET)phi lysogen by CTX(calc)phi. [TOP OF PAGE]

40. Rapid transport of viruses in a floodplain aquifer. DeBorde, D.C., Woessner, W.W., Kiley, Q.T., Ball, P. (1999). Water Res. 33:2229-2238. An unconfined floodplain aquifer near Missoula, MT, was instrumented with 89 monitoring wells and 20 four-port multilevel samplers. Bromide, bacteriophages MS2, PRD1 and phiX174 and the attenuated enterovirus, polio virus (type-1 CHAT strain), were seeded into the aquifer as slug injections. Bromide transport rates ranged between 22-29 m/d. Input concentrations of the tracers and the placement of monitoring wells limited detection of bromide and polio virus to 19.4 m and the detection of three bacteriophage to 40.5 m downgradient from the injection point. After 7.5 m of transport, the calculated relative attenuations (Harvey R. W and Garabedian S. P. (1991) Env. Sci. Tech. 25, 178-185) for MS2, PRD-1, phiX174 and attenuated polio virus were 49, 71, 65 and 99%, respectively. During the 72-h experiment, die-off was negligible (less than 1%) and attachment of virus to sediment surfaces resulted in the overall differences in bromide and virus behavior. Although relative attenuations at downgradient monitoring wells indicated that the virus tracers were attaching to aquifer material along the flowpath, virus peaks arrived at observation wells at rates similar to the bromide peak. The high collision efficiency of the attenuated polio virus resulted in breakthrough curve truncation. Natural attenuation of slug input virus over a "typical" source-supply set-back distance of 30.5 m would most likely not reduce virus concentrations to proposed acceptable risk levels in this or a similar cold-water high-velocity groundwater system. [TOP OF PAGE]

41. An estimator of the mutant frequency in assays using transgenic animals. Delongchamp, R.R., Malling, H.V., Chen, J.B., Heflich, R.H. (1999). Mutation Research 440:101-108. The Poisson distribution is a fundamental probability model for count data, and is a natural model for the observed plaque counts in mutation assays using animals with lambda or PhiX174 transgenes. The Poisson likelihood for observed counts is a function of the mutant fraction, and it is straightforward to derive the associated maximum likelihood estimate of the mutant fraction and its variance. The estimate is easy to calculate, and if not the same, very similar to ad hoc estimates in current use. The model indicates the proper way to combine data from a number of plates, possibly prepared with different sample dilutions. The estimator of the mutant fraction is biased as a consequence of dividing by a random variable, the plaque count used to calculate the total recovered plaque-forming units. Fortunately, the bias becomes negligible as this count becomes large. On the other hand, increasing this count can increase the variance by decreasing the amount of sample assayed for mutant phages. Concurrent heed to the bias and the variance provides some guidance as to the optimum allocation of a sample into portions assayed for mutant phages and total recovered phages. The distribution of the estimate of the mutant fraction is related to the binomial distribution. This relationship implies a binomial distribution for the mutant count conditional on an overall count (either the sum of mutant and counted total plaques or the sum of counted mutant and non-mutant plaques). A special but important case occurs when each plate can be evaluated for mutant plaques and non-mutant plaques. Then, the observed proportion of mutants estimates the mutant fraction. More generally, the relationship to a binomial distribution provides a procedure for calculating a confidence interval. [TOP OF PAGE]

42. Genetic organization and functional analysis of a novel phage abortive infection system, AbiL, from Lactococcus lactis. Deng, Y.M., Liu, C.Q., Dunn, N.W. (1999). Journal of Biotechnology 67:135-149. A plasmid-encoded phage abortive infection mechanism (AbiL) was identified from Lactococcus lactis biovar. diacetylactis LD10-1. AbiL conferred complete resistance to the small isometric-headed phage phi 712 (936 species) and partial resistance to the prolate-headed phage phi c2 (c2 species) when introduced into L. lactis LM0230. However, AbiL was not effective against the small isometric-headed phage ul36 (P335 species). The AbiL determinant was sequenced and it consists of two open reading frames, abiLi and abiLii. Their encoded proteins did not share significant homology with any known proteins in the protein databases. Transcriptional analysis indicated that abiLi and abiLii are organized as a single operon. Deletion within abiLii abolished the phage resistance. The levels of four phi c2-specific transcripts, three within the early transcribed region and one within the late transcribed region, were examined by RT-PCR, no effect of AbiL on synthesis of these transcripts was detected, suggesting that AbiL may act at a point after the transcription of phi c2 in L. lactis. [TOP OF PAGE]

43. Comparative sequence analysis of the DNA packaging, head, and tail morphogenesis modules in the temperate cos-site Streptococcus thermophilus bacteriophage Sfi21. Desiere, F., Lucchini S, Brussow, H. (1999). Virology 260:244-253. The temperate Streptococcus thermophilus bacteriophage Sfi21 possesses 15-nucleotide-long cohesive ends with a 3' overhang that reconstitutes a cos-site with twofold hyphenated rotational symmetry, Over the DNA packaging, head and tail morphogenesis modules, the Sfi21 sequence predicts a gene map that is strikingly similar to that of lambdoid coliphages in the absence of any sequence similarity. A nearly one to one gene correlation was found with the phage lambda genes Nul to H, except for gene B-to-E complex, where the Sfi21 map resembled that of coliphage HK97. The similarity between Sfi21 and HK97 was striking: both major head proteins showed an N-terminal coiled-coil structure, the mature major head proteins started at amino acid positions 105 and 104, respectively, and both major head genes were preceded by genes encoding a possible protease and portal protein. The purported Sfi21 protease is the first viral member of the CipP protease family The prediction of Sfi21 gene functions by reference to the gene map of intensively investigated coliphages was experimentally confirmed for the major head and tail gene. Phage Sfi21 shows nucleotide sequence similarity with Lactococcus phage BK5-T and a lactococcal prophage and amino acid sequence similarity with the Lactobacillus phage A2 and the Staphylococcus phage PVL. PVL is a missing link that connects the portal proteins from Sfi21 and HK97 with respect to sequence similarity. These observations and database searches, which demonstrate sequence similarity between proteins of phage from gram-positive bacteria, proteobacteria, and Archaea, constrain models of phage evolution. [TOP OF PAGE]

44. [An accelerated method for detecting coliphages in the drinking water]. Dmitrieva, R.A., Doskina, T.V., Nedachin, A.E., Sidorenko, S.G. (1999). Gigiena i Sanitariia 71-72. [TOP OF PAGE]

45. Presence of bacteriophages in different stages of wastewater treatment. Donia, D., Divizia, M., Pana, A., Gabrieli, R., Gasbarro, M., Capuani, L., Morelli, A.L. (1999). Igiene Moderna 111:239-251. The presence and correlation among somatic coliphages, F-specific phages and the phage of Bacteroides fragilis in a wastewater treatment plant was evaluated. The efficiency of the treatment for bacteriological parameters was confirmed with a reduction of total coliforms of 99,9%, fecal coliforms of 96,0% and fecal streptococci of 97,9%. A similar abatement was present for F-specific phages and the phage of Bacteroides fragilis (2.0 log) whereas the somatic coliphages appeared stable. Cytopathogenic enteric viruses were also recovered in the different withdrawal points with different efficiency according to the methods used. A positive correlation was found in the inlet samples among coliphages, phage of Bacteroides fragilis and enteric viruses. [TOP OF PAGE]

46. Horizontal gene transfer among bacteria in terrestrial and aquatic habitats as assessed by microcosm and field studies. Droege, M., Puehler, A., Selbitschka, W. (1999). Biology and Fertility of Soils 29:221-245. Genetic interactions among bacteria are mediated by one of the three distinct gene-exchange mechanisms: conjugation, transformation or transduction. Conjugative gene exchange relies on mobile elements, such as plasmids, which transfer between donor and recipient cells. In natural transformation, competent cells take up DNA and incorporate it into their genome. Gene transfer via transduction is mediated by bacteriophages which accidentally package donor DNA in their phage head and transfer it to recipient cells. Driven mainly by biosafety research and research into the rapid dissemination of antibiotic resistance, the evaluation of gene flux among bacteria in their natural habitats has become a focus of scientific interest in recent years. Accordingly, gene transfer has been assessed in laboratory-based studies employing model ecosystems, as well as in field experiments. Conjugative gene exchange has been shown to occur under a wide range of environmental conditions. Factors identified as conducive for conjugation include the presence of nutrients provided by the rhizosphere of plants. Studies addressing gene transfer via transformation have demonstrated that naturally transformable bacteria develop competence and take up DNA under in situ conditions. Moreover, DNA has been shown to persist to some extent in the environment, and thus be available for uptake by naturally competent cells. Gene exchange via transduction has been demonstrated under conditions of nutrient depletion and low densities of host cells. Whereas gene transfer is readily observed in the laboratory, more importantly, field studies have provided direct evidence that all three gene transfer mechanisms also occur in nature. DNA transfer frequencies observed in the environment in some cases differed considerably from those obtained under laboratory conditions. Transfers of low frequency observed in laboratory-based experiments have been readily detected in the environment in the presence of selective forces. [TOP OF PAGE]

47. Survival, physiology, and lysis of Lactococcus lactis in the digestive tract. Drouault, S. (1999). Appl. Environ. Microbiol. 65:4881-4886. The survival and the physiology of lactococcal cells in the different compartments of the digestive tracts of rats were studied in order to know better the fate of ingested lactic acid bacteria after oral administration. For this purpose, we used strains marked with reporter genes, the luxA-luxB gene of Vibrio harveyi and the gfp gene of Aequora victoria, that allowed us to differentiate the inoculated bacteria from food and the other intestinal bacteria. Luciferase was chosen to measure the metabolic activity of Lactococcus lactis in the digestive tract because it requires NADH, which is available only in metabolically active cells. The green fluorescent protein was used to assess the bacterial lysis independently of death. We report not only that specific factors affect the cell viability and integrity in some digestive tract compartments but also that the way bacteria are administrated has a dramatic impact. Lactococci which transit with the diet are quite resistant to gastric acidity (90 to 98% survival). In contrast, only 10 to 30% of bacteria survive in the duodenum. Viable cells are metabolically active in each compartment of the digestive tract, whereas most dead cells appear to be subject to rapid lysis. This property suggests that lactococci could be used as a vector to deliver specifically into the duodenum the proteins produced in the cytoplasm. This type of delivery vector would be particularly appropriate for targeting digestive enzymes such as lipase to treat pancreatic deficiencies. [TOP OF PAGE]

48. Integrity of powder-free examination gloves to bacteriophage penetration. Edlich, R.F. (1999). J. Biomed. Mater. Res. 48:755-758. The purpose of this study was to compare the resistance to viral penetration of powder-free synthetic examination gloves with powder-free latex examination gloves commonly used in hospitals. Because these gloves had no holes, this study examined viral penetration through a membrane. Using a standard bacteriophage penetration model, no bacteriophage penetration was detected through the membrane for any of the gloves tested. The new powder-free nitrile and polyvinyl chloride synthetic examination gloves provided comparable resistance to viral penetration as did the powder-free latex examination gloves. [TOP OF PAGE]

49. Molecular evidence for a new bacteriophage of Borrelia burgdorferi. Eggers, C.H., Samuels, D.S. (1999). J. Bacteriol. 181:7308-7313. We have recovered a DNase-protected, chloroform-resistant molecule of DNA from the cell-free supernatant of a Borrelia burgdorferi culture. The DNA is a 32-kb double-stranded linear molecule that is derived from the 32-kb circular plasmids (cp32s) of the B. burgdorferi genome. Electron microscopy of samples from which the 32-kb DNA molecule was purified revealed bacteriophage particles. The bacteriophage has a polyhedral head with a diameter of 55 nm and appears to have a simple 100-nm-long tail. The phage is produced constitutively at low levels from growing cultures of some B. burgdorferi strains and is inducible to higher levels with 10 microg of 1-methyl-3-nitroso-nitroguanidine (MNNG) ml(-1). In addition, the prophage can be induced with MNNG from some Borrelia isolates that do not naturally produce phage. We have isolated and partially characterized the phage associated with B. burgdorferi CA-11.2A. To our knowledge, this is the first molecular characterization of a bacteriophage of B. burgdorferi. [TOP OF PAGE]

50. Bacteriophage-like particles associated with the gene transfer agent of Methanococcus voltae PS. Eiserling, F., Pushkin, A., Gingery, M., Bertani, G. (1999). J. Gen. Virol. 80:3305-3308. The methanogenic archaeobacterium Methanococcus voltae (strain PS) is known to produce a filterable, DNase-resistant agent (called VTA, for voltae transfer agent), which carries very small fragments (4400 bp) of bacterial DNA and is able to transduce bacterial genes between derivatives of the strain. Examination by electron microscopy of two preparations of VTA that were concentrated and partially purified by different methods showed virus-like particles with isometric heads, about 40 nm in diameter, and with 61 nm long tails. These particles co-sedimented with the minute bacteriophage fX174 in a sucrose density gradient. [TOP OF PAGE]

51. Development of reduced acridines as antiprophage agents. El-Bermawy, M.A., Kadry, A., El-Didamony, G., Amin, M. (1999). Chinese Pharmaceutical Journal (Taipei) 51:191-200. A set of 9,10-diphenyldecahydroacridine-1, 8-diones 3-7 with different electronic characteristics were synthesized and tested as antiprophages, curing and as antimicrobial agents. All compounds except the 4-nitrophenyl 7 showed inhibition of prophage lambda (lambda) induction. These compounds appeared to have different levels of antiprophage activity. The 4-methyl derivative 5 has the highest inhibition of the prophage lambda induction. The prepared acridines were tested against standard strains of microorganisms. The minimum inhibitory concentrations (MICs) were 0.5, 0.6, 3.0, 3.5 and 4.0 mg/mL for compounds 7, 5, 6, 3 and 4 respectively. The mutagenic activity of the prophage inducing agent 7 was also investigated. Compound 7 has a curing activity on the plasmids of clinical isolates of E. coli. [TOP OF PAGE]

52. Little evidence for synergism among deleterious mutations in a nonsegmented RNA virus. Elena, S.F. (1999). J. Mol. Evol. 49:703-707. Several models have been proposed to account for the segmentation of RNA viruses. One of the best known models suggests that segmentation, and mixing of segments during coinfections, is a way to eliminate deleterious mutations from the genome. However, for validity, this model requires that deleterious mutations interact in a synergistic way. That is, two mutations together should have a more deleterious effect than the result of adding their individual effects. Here I present evidence that deleterious mutations in foot-and-mouth disease virus produce a decline in fitness but that the relationship between the number of mutations fixed and the magnitude of fitness decline is compatible mainly with a nonsynergistic model. However, the statistical uncertainties associated with the data still give some room for the existence of very weak synergistic epistasis. [TOP OF PAGE]

53. Rate of deleterious mutation and the distribution of its effects on fitness in vesicular stomatitis virus. Elena, S.F., Moya, A. (1999). Journal of Evolutionary Biology 12:1078-1088. Despite their importance, the parameters describing the spontaneous deleterious mutation process have not been well described in many organisms. If mutations are important for the evolution of every living organism, their importance becomes critical in the case of RNA-based viruses, in which the frequency of mutation is orders of magnitude larger than in DNA-based organisms. The present work reports minimum estimates of the deleterious mutation rate, as well as the characterization of the distribution of deleterious mutational effects on the total fitness of the vesicular stomatitis virus (VSV). The estimates are based on mutation-accumulation experiments in which selection against deleterious mutations was minimized by recurrently imposing genetic bottlenecks of size one. The estimated deleterious mutation rate was 1.2 mutations per genome and generation, with a mean fitness effect of -0.39% per generation. At the end of the mutation-accumulation experiment, the average reduction in fitness was 38% and the distribution of accumulated deleterious effects was, on average, left-skewed. The magnitude of the skewness depends on the initial fitness of the clone analysed. The implications of our findings for the evolutionary biology of RNA viruses are discussed. [TOP OF PAGE]

54. Evaluation of a bacteriophage-based assay (Phage amplified biologically assay) as a rapid screen for resistance to isoniazid, ethambutol, streptomycin, pyrazinamide, and ciprofloxacin among clinical isolates of Mycobacterium tuberculosis. Eltringham, I.J. (1999). J. Clin. Microbiol. 37:3528-3532. Rapid molecular assays for the detection of mutations associated with rifampin resistance in Mycobacterium tuberculosis are commercially available. However, they are complex and expensive and have predictive values of 90 to 95%. Molecular assays for other drugs are less predictive of resistance. Ideally, assays based on phenotypic markers should be used for susceptibility testing, but these can take weeks to complete. We previously described a rapid phenotypic assay, the phage amplified biologically (PhaB) assay, for the rapid determination of rifampin and isoniazid susceptibility in clinical isolates of M. tuberculosis. In this study, we extended the assay to the study of ethambutol, pyrazinamide, streptomycin, and ciprofloxacin. After the optimization of antibiotic concentrations and incubation conditions, the assay was applied to each drug for a total of 157 isolates. The correlations between the results of the PhaB assay and the resistance ratio method were 94% for isoniazid, 96% for streptomycin, 100% for ciprofloxacin, 88% for ethambutol, and 87% for pyrazinamide. For ciprofloxacin, ethambutol, and pyrazinamide, significantly better correlations were found when a 90% reduction in plaque count was used as the cutoff. Turnaround times for the PhaB assay were 2 to 3 days, compared with 10 days for the resistance ratio method. We believe that this low-cost assay may have widespread applicability for the rapid screening of drug resistance in M. tuberculosis isolates, especially in developing countries. [TOP OF PAGE]

55. Evaluation of reverse transcription-PCR and a bacteriophage-based assay for rapid phenotypic detection of rifampin resistance in clinical isolates of Mycobacterium tuberculosis. Eltringham, I.J. (1999). J. Clin. Microbiol. 37:3524-3527. New rapid phenotypic assays for the detection of rifampin resistance in Mycobacterium tuberculosis have recently been described, but most of these require liquid cultures, which reduces the utility of many tests in terms of turnaround times. In the United Kingdom, over 90% of rifampin-resistant isolates are also resistant to isoniazid, so rifampin resistance can be used as a sensitive marker for multidrug-resistant tuberculosis. In this study, two new rapid phenotypic assays were compared to the standard resistance ratio method on 91 clinical isolates of M. tuberculosis. One, the phage amplified biologically (PhaB) assay, has been described previously and is based on the inability of susceptible isolates of M. tuberculosis to support the replication of bacteriophage D29 in the presence of inhibitory doses of rifampin. The other employed reverse transcription (RT)-PCR to demonstrate a reduction in inducible dnaK mRNA levels in susceptible isolates treated with rifampin. After incubation for 18 h with 4 g of rifampin per ml, the PhaB assay showed concordance with the resistance ratio method for 46 of 46 (100%) susceptible and 31 of 31 (100%) resistant isolates, while RT-PCR showed concordance for 46 of 48 (96%) susceptible and 35 of 36 (97%) resistant isolates. We believe these assays provide a reliable rapid means of susceptibility testing with a total turnaround time of only 48 h, although the PhaB assay is better in terms of its lower technical demand and cost and its applicability to tuberculosis susceptibility testing in developing countries. [TOP OF PAGE]

56. Lysogenic Conversion of Environmental Vibrio mimicus Strains by CTXF. Faruque, S.M., Rahman, M.M., Asadulghani, K.M., Islam, N., Mekalanos, J.J. (1999). Infect. Immun. 67:5723-5729. The filamentous bacteriophage CTXF, which encodes cholera toxin (CT) in toxigenic Vibrio cholerae, is known to propagate by infecting susceptible strains of V. cholerae by using the toxin coregulated pilus (TCP) as its receptor and thereby causing the origination of new strains of toxigenic V. cholerae from nontoxigenic progenitors. Besides V. cholerae, Vibrio mimicus strains which are normally TCP negative have also been shown to occasionally produce CT and cause diarrhea in humans. We analyzed nontoxigenic V. mimicus strains isolated from surface waters in Bangladesh for susceptibility and lysogenic conversion by CTXF and studied the expression of CT in the lysogens by using genetically marked derivatives of the phage. Of 27 V. mimicus strains analyzed, which were all negative for genes encoding TCP but positive for the regulatory gene toxR, 2 strains (7.4%) were infected by CTX-KmF, derived from strain SM44(P27459 ctx::km), and the phage genome integrated into the host chromosome, forming stable lysogens. The lysogens spontaneously produced infectious phage particles in the supernatant fluids of the culture, and high titers of the phage could be achieved when the lysogens were induced with mitomycin C. This is the first demonstration of lysogenic conversion of V. mimicus strains by CTXF. When a genetically marked derivative of the replicative form of the CTXF genome carrying a functional ctxAB operon, pMSF9.2, was introduced into nontoxigenic V. mimicus strains, the plasmid integrated into the host genome and the strains produced CT both in vitro and inside the intestines of adult rabbits and caused mild-to-severe diarrhea in rabbits. This suggested that in the natural habitat infection of nontoxigenic V. mimicus strains by wild-type CTXF may lead to the origination of toxigenic V. mimicus strains which are capable of producing biologically active CT. The results of this study also supported the existence of a TCP-independent mechanism for infection by CTXF and showed that at least one species of Vibrio other than V. cholerae may contribute to the propagation of the phage. [TOP OF PAGE]

57. Inducible prophages contribute to Salmonella virulence in mice. Figueroa-Bossi, N., Bossi, L. (1999). Molecular Microbiology 33:167-176. We show that Salmonella typhimurium harbours two fully functional prophages, Gifsy-1 and Gifsy-2, that can be induced by standard treatments or, more effectively, by exposing bacteria to hydrogen peroxide. Curing bacteria for the Gifsy-2 prophage significantly reduces Salmonella's ability to establish a systemic infection in mice. Cured strains recover their virulence properties upon relysogenization. Phage Gifsy-2 carries the sodC gene for a periplasmic [Cu,Zn]-superoxide dismutase previously implicated in the bacterial defences against killing by macrophages. The contribution of the Gifsy-1 prophage to virulence - undetectable in the presence of Gifsy-2 as prophage - becomes significant in cells that lack Gifsy-2 but carry the sodC gene integrated in the chromosome. This confirms the involvement of Gifsy-2-encoded SodC protein in Salmonella pathogenicity and suggests that the Gifsy-1 prophage carries one or more additional virulence genes that have a functional equivalent on the Gifsy-2 genome. [TOP OF PAGE]

58. Identification of four phage resistance plasmids from Lactococcus lactis subsp. cremoris HO2. Forde, A., Daly, C., Fitzgerald, G.F. (1999). Appl. Environ. Microbiol. 65:1540-1547. The bacteriophage-host sensitivity patterns of 16 strains of Lactococcus lactis originally isolated from a mixed strain Cheddar cheese starter culture were determined. Using phages obtained from cheese factory whey, four of the strains were found to be highly phage resistant. One of these isolates, Lactococcus lactis subsp. cremoris HO2, was studied in detail to determine the mechanisms responsible for the phage insensitivity phenotypes. Conjugal transfer of plasmid DNA from strain HO2 allowed a function to be assigned to four of its six plasmids. A 46-kb molecule, designated pCI646, was found to harbor the lactose utilization genes, while this and plasmids of 58 kb (pCI658), 42 kb (pCI642), and 4.5 kb (pCI605) were shown to be responsible for the phage resistance phenotypes observed against the small isometric-headed phage phi712 (936 phage species) and the prolate-headed phage phic2 (c2 species). pCI658 was found to mediate an adsorption-blocking mechanism and was also responsible for the fluffy pellet phenotype of cells containing the molecule. pCI642 and pCI605 were both shown to be required for the operation of a restriction-modification system. [TOP OF PAGE]

59. Bacteriophage defence systems in lactic acid bacteria. Forde, A. (1999). Antonie van Leeuwenhoek 76:89-113. The study of the interactions between lactic acid bacteria and their bacteriophages has been a vibrant and rewarding research activity for a considerable number of years. In the more recent past, the application of molecular genetics for the analysis of phage-host relationships has contributed enormously to the unravelling of specific events which dictate insensitivity to bacteriophage infection and has revealed that while they are complex and intricate in nature, they are also extremely effective. In addition, the strategy has laid solid foundations for the construction of phage resistant strains for use in commercial applications and has provided a sound basis for continued investigations into existing, naturally-derived and novel, genetically-engineered defence systems. Of course, it has also become clear that phage particles are highly dynamic in their response to those defence systems which they do encounter and that they can readily adapt to them as a consequence of their genetic flexibility and plasticity. This paper reviews the exciting developments that have been described in the literature regarding the study of phage-host interactions in lactic acid bacteria and the innovative approaches that can be taken to exploit this basic information for curtailing phage infection. [TOP OF PAGE]

60. Phage typing of Campylobacter jejuni and Campylobacter coli and its use as an adjunct to serotyping. Frost, J.A., Kramer, J.M., Gillanders, S.A. (1999). Epidemiology and Infection 123:47-55. Campylobacter is the most commonly reported cause of gastro-intestinal infection in England and Wales, with over 50000 reported cases in 1997. The majority of human campylobacter isolates in England and Wales are C. jejuni (c. 90%) with most of the remainder being C. coli. We describe the use of phage typing as an extension to serotyping for more detailed characterization within these two species. The scheme was piloted during a study of 2407 C. jejuni and 182 C. coli strains isolated in Wales between April 1996 and March 1997. Fifty-seven C. jejuni phage types were identified, with the ten most prevalent phage types accounting for 60% of isolates tested; 16% of isolates were untypable. The most common phage type was PT 1 which represented c. 20% of isolates. A further 7% of isolates reacted with the phages but did not conform to a designated type (RDNC). Only 12 phage types were identified among C. coli, with the two most common types, PT 2 and PT 7 accounting for 75.2% ofisolates. When used in conjunction with serotyping, the ability of phage typing to identify between 6 and 29 subtypes within each of the predominant HS types has enabled a further level of discrimination to be achieved that enhances the epidemiological typing of C. jejuni and C. coli. [TOP OF PAGE]

61. Causative agents of bacterial mortality and the consequences to marine food webs. Fuhrman, J.A., Noble, R.T. (1999). p. ???-??? In Bell, C.R., Brylinsky, M., and Johnson-Green, P. (eds.), Microbial Biosystems: New Frontiers. Proc 8th Int Symp Microb. Ecol. Atlantic Canada Society for Microbial Ecology, Halifax, Canada. [TOP OF PAGE]

62. Marine viruses and their biogeochemical and ecological effects. Fuhrman, J.A. (1999). Nature 399:541-548. Viruses are the most common biological agents in the sea, typically numbering ten billion per litre. They probably infect all organisms, can undergo rapid decay and replenishment, and influence many biogeochemical and ecological processes, including nutrient cycling, system respiration, particle size-distributions and sinking rates, bacterial and algal biodiversity and species distributions, algal bloom control, dimethyl sulphide formation and genetic transfer. Newly developed fluorescence and molecular techniques leave the field poised to make significant advances towards evaluating and quantifying such efforts. [TOP OF PAGE]

63. Impact of viruses on planktonic bacteria. Fuhrman, J.A. (1999). p. ???-??? In Kirchman, D.L. (ed.), Microbial Ecology of the Oceans. Wiley & Sons, ??? [TOP OF PAGE]

64. The microbial quality of a Wetland Reclamation Facility used to produce an effluent for unrestricted non-potable reuse. Fujioka, R.S., Bonilla, A.J., Rijal, G.K. (1999). Water Science and Technology 40:369-374. An auxiliary Wetland Reclamation Facility (WRF) was constructed to receive stabilization pond treated sewage and further treat it with water hyacinth ponds, chemical flocculation, filtration and ultraviolet light disinfection. This was the first facility in Hawaii which was approved to produce the highest quality reclaimed water using alternative treatment schemes. We assessed the effectiveness of the WRF by monitoring water samples after each of the WRF treatment schemes for five genetically different groups of sewage borne microorganisms (fecal coliform, enterococci, C. perfringens, FRNA phage, total heterotrophic bacteria). The concentrations of all fecal indicator microorganisms, especially FRNA phage were low in the influent water to the WRF indicating that extended pond treatment may be especially effective in removing human viruses from sewage. The WRF treatment scheme was calculated to be able to reduce >99.99% of fecal coliform and therefore was able to produce an effluent meeting the non-potable, unrestricted reuse standard of a geometric mean of <1 fecal coliform/100 ml. [TOP OF PAGE]

65. Horizontal gene transfer in glycosyl hydrolases inferred from codon usage in Escherichia coli and Bacillus subtilis. Garcia-Vallve, S., Palau, J., Romeu, A. (1999). Molecular Biology and Evolution 16:1125-1134. Glycosyl hydrolase (GH) genes from Escherichia coli and Bacillus subtilis were used to search for cases of horizontal gene transfer. Such an event was inferred by G + C content, codon usage analysis, and a phylogenetic congruency test. The codon usage analysis used is a procedure based on a distance derived from a Pearson linear correlation coefficient determined from a pairwise codon usage comparison. The distances are then used to generate a distance-based tree with which we can define clusters and rapidly compare codon usage. Three genes (yagH from E. coli and xynA and xynB from B. subtilis) were determined to have arrived by horizontal gene transfer and were located in E. coli CP4-6 prophage, and B. subtilis prophages 6 and 5, respectively. In this study, we demonstrate that with codon usage analysis, the proposed horizontally transferred genes can be distinguished from highly expressed genes. [TOP OF PAGE]

66. Bacteriophages of dairy propionibacteria. Gautier, M., Rouault, A., Herve, C., Sommer, P., eret, V., Jan, G., Fraslin, J.M., Prevot, F., Coste, A. (1999). Lait- 79:93-104. Characteristics of 2 types of phages infecting propionic acid bacteria that were isolated from Swiss-type cheeses were examined. One type belonged to group B1 of Bradley's classification and the other was a filamentous phage, the 1st of its type identified in Gram positive bacteria. Diversity, host spectrum and origin of the phages were studied. The phages were detected in raw milk and cheeses, but not in curd or cooked curd. Group 1 phages showed high homology and were probably derived from a common ancestor. All phages showed a very narrow host spectrum. Information on phages infecting Propionibacterium freudenreichii was used in the development of a cloning system for this bacterium. [This paper was presented at the 2nd Symposium on Propionibacteria, which was held in Cork, Republic of Ireland on 25-27 June 1998.]. [TOP OF PAGE]

67. Optimization of artificial wetland design for removal of indicator microorganisms and pathogenic protozoa. Gerba, C.P., Thurston, J.A., Falabi, J.A., Watt, P.M., Karpiscak, M.M. (1999). Water Science and Technology 40:363-368. The enhancement of water quality by artificial wetland systems is increasingly being employed throughout the world. Three wetlands were studied in Tucson, AZ to evaluate their individual performance in the removal of indicator bacteria (coliforms), coliphage, and enteric pathogens (Giardia and Cryptosporidium). A duckweed-covered pond, a multi-species subsurface flow (SSF) and a multi-species surface flow (SF) wetland were studied. Removal of the larger microorganisms, Giardia and Cryptosporidium, was the greatest in the duckweed pond at 98 and 89 percent, respectively. The lowest removal occurred in the SF wetland, 73 percent for Giardia and 58 percent removal for Cryptosporidium. In contrast, the greatest removal of coliphage, total and fecal coliforms occurred in the SSF wetland, 95, 99, and 98 percent respectively, whereas the pond had the lowest removals (40, 62, and 61 percent, respectively). Sedimentation may be the primary removal mechanism within the duckweed pond since the removal was related to size, removal of the largest organisms being the greatest. However, the smaller microorganisms were removed more efficiently in the SSF wetland, which may be related to the large surface area available for adsorption and filtration. This study suggests that in order to achieve the highest treatment level of secondary unchlorinated wastewater, a combination of aquatic ponds and subsurface flow wetlands may be necessary. [TOP OF PAGE]

68. Biocontrol of Erwinia amylovora using bacteriophage. Gill, J.J., Svircev, A.M., Myers, A.L., Castle, A.J. (1999). Phytopathology 89:S27 [TOP OF PAGE]

69. Roles of Pseudomonas aeruginosa las and rhl quorum-sensing systems in control of twitching motility. Glessner, A., Smith, R.S., Iglewski, B.H., Robinson, J.B. (1999). J. Bacteriol. 181:1623-1629. Pseudomonas aeruginosa is a ubiquitous environmental bacterium and an important human pathogen. The production of several virulence factors by P. aeruginosa is controlled through two quorum-sensing systems, las and rhl. We have obtained evidence that both the las and rhl quorum-sensing systems are also required for type 4 pilus-dependent twitching motility and infection by the pilus-specific phage D3112cts. Mutants which lack the ability to synthesize PAI-1, PAI-2, or both autoinducers were significantly or greatly impaired in twitching motility and in susceptibility to D3112cts. Twitching motility and phage susceptibility in the autoinducer-deficient mutants were partially restored by exposure to exogenous PAI-1 and PAI-2. Both twitching motility and infection by pilus-specific phage are believed to be dependent on the extension and retraction of polar type 4 pili. Western blot analysis of whole-cell lysates and enzyme-linked immunosorbent assays of intact cells were used to measure the amounts of pilin on the cell surfaces of las and rhl mutants relative to that of the wild type. It appears that PAI-2 plays a crucial role in twitching motility and phage infection by affecting the export and assembly of surface type 4 pili. The ability of P. aeruginosa cells to adhere to human bronchial epithelial cells was also found to be dependent on the rhl quorum-sensing system. Microscopic analysis of twitching motility indicated that mutants which were unable to synthesize PAI-1 were defective in the maintenance of cellular monolayers and migrating packs of cells. Thus, PAI-1 appears to have an essential role in maintaining cell-cell spacing and associations required for effective twitching motility. [TOP OF PAGE]

70. Development and characterization of a fluorescent-bacteriophage assay for detection of Escherichia coli O157:H7. Goodridge, L., Chen, J., Griffiths, M. (1999). Appl. Environ. Microbiol. 65:1397-1404. In this paper we describe evaluation and characterization of a novel assay that combines immunomagnetic separation and a fluorescently stained bacteriophage for detection of Escherichia coli O157:H7 in broth. When it was combined with flow cytometry, the fluorescent-bacteriophage assay (FBA) was capable of detecting 10(4) cells/ml. A modified direct epifluorescent-filter technique (DEFT) was employed in an attempt to estimate bacterial concentrations. Using regression analysis, we calculated that the lower detection limit was between 10(2) and 10(3) cells/ml; however, the modified DEFT was found to be an unreliable method for determining bacterial concentrations. The results of this study show that the FBA, when combined with flow cytometry, is a sensitive technique for presumptive detection of E. coli O157:H7 in broth cultures. [TOP OF PAGE]

71. The use of a fluorescent bacteriophage assay for detection of Escherichia coli O157:H7 in inoculated ground beef and raw milk. Goodridge, L., Chen, J., Griffiths, M. (1999). Int. J. Food Microbiol. 47:43-50. The objective of this study was to develop a fluorescent bacteriophage assay (FBA) for the detection of E. coli O157:H7 in ground beef and raw milk. The FBA is a two step assay that combines immunomagnetic separation, to separate the target organism from mixed culture, with a highly specific fluorescently stained bacteriophage to label the E. coli O157:H7 cells. When used in conjunction with flow cytometry, the FBA was able to detect 2.2 CFU/g of artificially contaminated ground beef following a 6 h enrichment. Between 10(1) and 10(2) CFU/ml of artificially contaminated raw milk were detectable after a 10 h enrichment step. The results show that the FBA is potentially useful as a rapid technique for the preliminary detection of E. coli O157:H7 in food. [TOP OF PAGE]

72. Bacteriophage lambda: The untold story. Gottesman, M. (1999). J. Mol. Biol. 293:177-180. The study of bacteriophage lambda has provided key insights into fundamental biological processes. This review recalls some highlights in the history of lambda research, and relates how simple (but elegant) experiments yielded major scientific breakthroughs. What we know about recombination, gene regulation, and protein folding, for example, derives in large part from bacteriophage lambda genetics. Lambda not only represents a model system of scientific logic in a technology-driven age, but continues to reveal new principles of molecular biology. [TOP OF PAGE]

73. Removal of MS-2 and PRD-1 bacteriophages from an ultrapure water system. Governal, R.A., Gerba, C.P. (1999). Journal of Industrial Microbiology & Biotechnology 23:166-172. Viruses must be removed from the ultrapure water environment, as they have the potential to deposit on microelectronic devices and generate killer defects. Controlled and well-defined challenges by MS-2 and PRD-1 bacteriophages were treated in a pilot-scale ultrapure water system using ultraviolet radiation (UV), ozone, mixed bed ion exchange adsorption, and reverse osmosis filtration technologies typical of those used in industrial systems. Applying a first order kinetic model to the data generated rate constants for MS-2 removal by UV-185, 50 mg L^-1 ozone, mixed bed ion exchange or reverse osmosis filtration of 15.5, 12.9, 3.9, and 10.4 min^-1, respectively, and PRD-1 removal of 13.8, 15.5, 8.2, and 11.9 min^-1, respectively. In all cases, removal of viruses by oxidative mechanisms such as ozone and UV were far superior to adsorption and filtration mechanisms. A theoretical viral population balance was generated to model the removal of the bacteriophages by these unit operations.This model relates the inlet time-dependent profile of viruses to the output, destruction, and accumulation profiles; it also relates these profiles to the unit operation's treatment mechanisms including oxidation, adsorption, and filtration. This model is the first step in generating a site-independent theoretical model to project the persistence of viruses in ultrapure water systems. [TOP OF PAGE]

74. Elimination of viruses, phages, bacteria and Cryptosporidium by a new generation Aquaguard point-of-use water treatment unit. Grabow, W.O., Clay, C.G., Dhaliwal, W., Vrey, M.A., Muller, E.E. (1999). Zentralblatt fur Hygiene und Unweltmedizin 202:399-410. The elimination of human viruses, phages, bacteria and Cryptosporidium oocysts by a new generation commercial Aquaguard purifier for the domestic treatment of drinking water, has been evaluated. The unit basically consists of a candle prefilter, activated carbon filter and ultraviolet irradiation compartment. Drinking water seeded with selected laboratory test strains of resistant micro-organisms was passed through the unit. Similar tests were carried out with sewage-contaminated river water and secondary treated waste water containing naturally occurring organisms. Test procedures were based on internationally accepted principles for the evaluation of point-of-use water treatment units, including a standard test protocol of the United States Environmental Protection Agency. Reduction in numbers of seeded test organisms at several log levels higher than those expected in water for which the unit is intended, was determined by the cultivation of viable organisms. In the case of seeded viruses and Cryptosporidium parvum oocysts the qualitative absence of nucleic acid was determined by the reverse transcriptase polymerase chain reaction (RT-PCR). At the design flow rate of one litre per minute, numbers of polio, hepatitis A, adeno types 2 and 41, rota SA11, human rota and astro viruses, as well as somatic and MS2 coliphages, and Escherichia coli, Streptococcus faecalis, Clostridium perfringens, total coliform bacteria, enterococci, heterotrophic bacteria and C. parvum oocysts, were reduced by more than 99.999% in all waters tested. This efficiency conforms to specifications for such units. The quality of the treated water was well within microbiological limits of international specifications for drinking water. [TOP OF PAGE]

75. Virus removal by filtration. Graf, E.G., Jander, E., West, A., Pora, H., Aranha-Creado, H. (1999). Dev. Biol. Standard. 99:89-94. Advances in membrane technology have allowed the expansion of the size-exclusion removal principle to viruses of concern in the processing of pharmaceutical drug products derived from biological fluids and cell-culture techniques. Direct flow- and cross-flow filters are complementary techniques for virus removal and may be used either independently or as an adjunct to other virus clearance methods. Representative virus titre reduction data for microfiltration and ultrafiltration membranes are presented along with a validation model using bacteriophages as challenge viruses. Non-destructive filter integrity tests before and after filtration and a stringent process validation regime are applied to enhance product safety. [TOP OF PAGE]

76. Viral lysis and bacterivory during a phytoplankton bloom in a coastal water microcosm. Guixa-Boixareu, N., Lysnes, K., Pedros-Alio, C. (1999). Appl. Environ. Microbiol. 65:1949-1958. The relative importance of viral lysis and bacterivory as causes of bacterial mortality were estimated. A laboratory experiment was carried out to check the kind of control that viruses could exert over the bacterial assemblage in a non-steady-state situation. Virus-like particles (VLP) were determined by using three methods of counting (DAPI [4',6-diamidino-2-phenylindole] staining, YOPRO staining, and transmission electron microscopy). Virus counts increased from the beginning until the end of the experiment. However, different methods produced significantly different results. DAPI-stained VLP yielded the lowest numbers, while YOPRO-stained VLP yielded the highest numbers. Bacteria reached the maximal abundance at 122 h (3 x 10 super(7) bacteria ml super(-1)), after the peak of chlorophyll a (80 mu g liter super(-1)). Phototrophic nanoflagellates followed the same pattern as for chlorophyll a. Heterotrophic nanoflagellates showed oscillations in abundance throughout the experiment. The specific bacterial growth rate increased until 168 h (2.6 day super(-1)). The bacterivory rate reached the maximal value at 96 hours (0.9 day super(-1)). Bacterial mortality due to viral infection was measured by using two approaches: measuring the percentage of visibly infected bacteria (%VIB) and measuring the viral decay rates (VDR), which were estimated with cyanide. The %VIB was always lower than 1% during the experiment. VDR were used to estimate viral production. Viral production increased 1 order of magnitude during the experiment (from 10 super(6) to 10 super(7) VLP ml super(-1) h super(-1)). The percentage of heterotrophic bacterial production consumed by bacterivores was higher than 60% during the first 4 days of the experiment; afterwards, this percentage was lower than 10%. The percentage of heterotrophic bacterial production lysed by viruses as assessed by the VDR reached the highest values at the beginning (100%) and at the end (50%) of the experiment. Comparing both sources of mortality at each stage of the bloom, bacterivory was found to be higher than viral lysis at days 2 and 4, and viral lysis was higher than bacterivory at days 7 and 9. A balance between bacterial losses and bacterial production was calculated for each sampling interval. At intervals of 0 to 2 and 2 to 4 days, viral lysis and bacterivory accounted for all the bacterial losses. At intervals of 4 to 7 and 7 to 9 days, bacterial losses were not balanced by the sources of mortality measured. At these time points, bacterial abundance was about 20 times higher than the expected value if viral lysis and bacterivory had been the only factors causing bacterial mortality. In conclusion, mortality caused by viruses can be more important than bacterivory under non-steady-state conditions. [TOP OF PAGE]

77. Evolutionary relationships among diverse bacteriophages and prophages: All the world's a phage. Hendrix, R.W., Smith, M.C.M., Burns, R.N., Ford, M.E., Hatfull, G.F. (1999). Proc. Natl. Acad. Sci. USA 96:2192-2197. We report DNA and predicted protein sequence similarities, implying homology, among genes of dsDNA bacteriophages and prophages spanning broad phylogenetic range of host bacteria. The sequence matches reported here establish genetic connections not always direct, among the lambdoid phages of Escherichia coli, phage fC31 of Streptomyces, phages of Mycobacterium, a previously unrecognized cryptic prophage, fFlu, in the Haemophilus influenzae genome, and two small prophage-like elements fRv1 and fRV2, in the genome of M. tuberculosis. The results imply that these phage genomes, and very possibly all of the dsDNA tailed phages, share common ancestry. We propose a model for the genetic structure and dynamics of the global phage population in which all dsDNA 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 phage. [TOP OF PAGE]

78. Evolution: the long evolutionary reach of viruses. Hendrix, R.W. (1999). Current Biology 9:R914-R917 The structure of a phage capsid protein provides good evidence this phage shares ancestry with an animal virus. In this and similar cases, either the viral lineages predate the emergence of the three contemporary domains of life, or viruses have been leaping the phylogenetic chasms that separate the domains. [TOP OF PAGE]

79. Removal of viruses by microfiltration membranes at different solution environments. Herath, G., Yamamoto, K., Urase, T. (1999). Water Science and Technology 40:331-338. Rejection change by nuclepore microfiltration membranes with solution environment was investigated by using the RNA coliphages Qbeta, MS2, fr and DNA coliphage T4. The obtained rejection results showed a higher rejection at lower pH than at higher pH for all viruses. The highest rejection for all viruses were obtained at pH closer to their isoelectric points and also the rejection variation indicates a similar pattern of behavior. This phenomenon of higher virus rejection at lower pH is explained with a possible viruses aggregation with each other due to their own electrostatic charge and isoelectric points. Also it was observed that the virus rejection was enhanced when they are in mixed environment. Finally the effect of protein was studied where, the virus rejection below pH 5.0 showed protein influence. [TOP OF PAGE]

80. Preparation of phage-insensitive strains of Tetragenococcus halophila and its application for soy sauce fermentation. Higuchi, T., Uchida, K., Abe, K. (1999). Biosci. Biotech. Biochem. 63:415-417. Production of _D-10 phage-insensitive mutants of Tetragenococcus halophila D-10, used in industrial fermentation of soy sauce, is reported. Phage contact during selection initially resulted in lysogeny. Subsequently, phage-insensitive mutants were screened by replica plating so that mutant cells did not touch the phage during selection. 2 strains were selected from approx. 150 000. They grew normally in soy sauce mash (moromi) in the presence of phage _D-10, although had a similar extent of adsorption of _D-10, as did the parent strain. Industrial application of these strains in moromi fermentation is discussed. [TOP OF PAGE]

81. The catalytic group-I introns of the psbA gene of Chlamydomonas reinhardtii : core structures, ORFs and evolutionary implications. Holloway, S.P., Deshpande, N.N., Herrin, D.L. (1999). Current Genetics 36:69-78. The sequences and predicted secondary structures of the four catalytic group-I introns in the psbA gene of Chlamydomonas reinhardtii, Cr.psbA-1-Cr.psbA-4, have been determined. Cr.psbA-1 and Cr.psbA-4 are subgroup-IA1 introns and have similar secondary structures, except at the 3 end where Cr.psbA-1 contains a large inverted-repeat domain. Cr.psbA-4 is closely related to intron 1 of the Chlamydomonas moewusii psbA gene, with which it shares the same location, high nucleotide identity in the core, and an identically placed ORF that shows 58% amino-acid identity. Cr.psbA-2 is a subgroup-IA3 intron, and shows similarities to the Chlamydomonas eugametos rRNA intron, Ce.LSU-1. Cr.psbA-3 is a subgroup-IA2 intron, and is remarkably similar to the T4 phage intron, sunY. Interestingly, a degenerate version of Cr.psbA-3 is located in the intergenic region between the chloroplast petA and petD genes. All four introns contain ORFs, which potentially code for basic proteins of 11-38 kDa. The ORFs in introns 2 and 3 contain variants of the GIY-YIG motif; however, the Cr.psbA-2 ORF is free-standing, whereas the Cr.psbA-3 ORF is contiguous and in-frame with the upstream exon. The Cr.psbA-4 ORF contains an H-N-H motif, and possibly a GIY-YIG motif. These data indicate that the C. reinhardtii psbA introns have multiple origins, and illustrate some of the evolutionary DNA dynamics associated with group-I introns in Chlamydomonas. [TOP OF PAGE]

82. Uptake and intracellular fate of phage display vectors in mammalian cells. Ivanenkov, V.V., Felici, F., Menon, A.G. (1999). Biochim. Biophys. Acta 1448:450-462. Receptor-mediated endocytosis is exploited in experimental systems for selective delivery of genes and drugs into specific cells. To improve targeting efficiency of delivery vectors, we have used phage display technology to isolate novel ligands for endocytosed receptors. We show here that phage vectors internalized by mammalian cells via integrin-mediated endocytosis can be rescued by cell lysis and quantitated by infection of bacteria. Immediately following uptake, phage enter an intracellular compartment where they remain intact, with phage titer unaffected by the addition of chloroquine. Phage are then translocated to a second intracellular compartment in which they are inactivated and their titer affected by chloroquine. Immunofluorescence microscopy showed an association of the second compartment with supranuclear organelles. The ability to recover internalized phage in an infectious form from two distinctive intracellular compartments provides a means to select novel ligands from phage libraries for targeted delivery of macromolecules into mammalian cells. [TOP OF PAGE]

83. Changing consumer water-use patterns and their effect on microbiological water quality as a result of an engineering intervention. Jagals, P., Bokako, T.C., Grabow, W.O.K. (1999). Water S A (Pretoria) 25:297-326. A previous study done during 1994-1995 in a section of a large, low socio-economic urban development with limited sanitary facilities and drinking-water provision indicated that the community was exposed to water-related health risks when consuming the water supplied. The study indicated that, although the public supplied water was of a good quality, the stored water, once fetched from the standpipes, deteriorated to a quality often not safe for human consumption. Based on the findings of this previous study, the local authority decided to install standpipes for each individual family in the area concerned and these were placed in the house yards. The closer proximity of the standpipes immediately altered the water-fetching and storing patterns of the community. The consequent study, on which this abstract is based, assessed the potential risk of infection posed to health by the altered water-use pattern. Weekly water samples were collected from standpipes outside as well as from containers kept inside houses of selected families. Total coliforms, faecal coliforms, heterotrophic plate counts, Clostridium perfringens and somatic coliphages were used as microbiological indicators. Although the improvement of water accessibility enhanced the microbiological quality of stored water, the results indicated that hygienic quality still deteriorated. This situation indicated that a suitable education and information programme to enhance the quality gains of such engineering interventions should accompany engineering improvement of water accessibility. [TOP OF PAGE]

84. Development of lytic Lactococcus lactis bacteriophages in a Cheddar cheese plant. Josephsen, J., Petersen, A., Neve, H., Nielsen, E.W. (1999). Int. J. Food Microbiol. 50:163-171. The mixed TK5 starter culture was used in a Danish factory as the only starter for production of Cheddar cheese for more than 11 years before the factory experienced serious bacteriophage attacks with inhibition of the acid production in the curd. The cheese whey contained some phages from the beginning, and gradually new phages appeared able to infect an increasing number of isolates. Three bacteriophages jw30, jw31 and jw32 were isolated from the factory whey collected in 1989-94 and compared with lytic bacteriophages isolated in the period 1982-86 (Josephsen et al., 1994) DNA hybridisation showed that the type phage P008 had high homology to the new phages jw30, jw31 and jw32 as had the phages isolated in 1982-84. The new phages had broader host ranges and higher burst sizes than the previously isolated phages, showing that the lytic phages had become more virulent with time. [TOP OF PAGE]

85. [Cryostabilization of biological properties of plague phages]. Kadetov, V.V., Kudriakova, T.A., Terent'ev, A.N., Kachkina, G.V., Borodina, T.N., Saiamov, S.R. (1999). Vopr. Virusol. 44:136-139. Conditions of cryostabilization of Yersinia pestis phages preserving their biological properties at very low temperature are studied. [TOP OF PAGE]

86. Bacteriocin-like inhibitory activities among various species of Listeria. Kalmokoff, M.L., Daley, E., Austin, J.W., Farber, J.M. (1999). Int. J. Food Microbiol. 50:191-201. Three hundred Listeria isolates were examined for inhibitory activities using a deferred antagonism plating assay. Approximately 75% of the surveyed isolates produced inhibitory activity, the majority of which (71%) resulted from the production of bacteriophage or defective bacteriophage particles. Twenty-three isolates (8%) produced inhibitory activities distinct from those resulting from bacteriophage. Four of these isolates (Listeria innocua 743, L. innocua 755, L. innocua 228, L. monocytogenes 538) produced heat-stable, protease sensitive peptides, which demonstrated broad-spectrum inhibitory activities against all L. monocytogenes serotypes tested. [TOP OF PAGE]

87. Characterization of wild lambdoid bacteriophages: detection of a wide distribution of phage immunity groups and identification of a nus-dependent, nonlambdoid phage group. Kameyama, L., Fernandez, L., Calderon, J., Ortiz-Rojas, A., Patterson, T.A. (1999). Virology 263:100-111. Temperate phages were isolated from fresh human fecal samples. Lambdoid phages were screened for growth on Nus+ but not Nus- bacteria. Approximately 100 independent lysogens of Nus-dependent phages were constructed and tested for immunity to superinfection by the same Nus-dependent phages. This identified 20 different phage immunity groups, 18 of which belonged to the lambdoid phage family. The DNA from the majority of these phages hybridized with a lambda DNA probe, and approximately 50% were recognized by anti-lambda antibodies. Furthermore most were inducible by UV light. Eleven phage recombinants with different immunity were obtained when a phage from each group was coinfected with lambda or its derivative lambdaBLK20. We also identified another immunity group with 48 members. None of these hybridized with either lambda or phi80 DNA probes nor were they recognized by anti-lambda serum. Most were not induced by UV light treatment, and no recombinants were obtained when crossed with either lambda or lambdaBLK20. Consequently, this group of Nus-dependent phages represent a new nonlambdoid phage family. [TOP OF PAGE]

88. An extrachromosomal prophage naturally associated with Bacillus thuringiensis serovar israelensis. Kanda, K., Ohderaotoshi, T., Shimojyo, A., Kato, F., Murata, A. (1999). Letters in Applied Microbiology 28:305-308. Bacillus thuringiensis serovar israelensis, an entomopathogen for mosquito larvae, was demonstrated to be lysogenized by temperate phage SU-11 whose genome was located extrachromosomally in the cell. The prophage SU-11 was cured at high frequency from the parental strain by continuous sub-culture at high temperature, but the ability to produce delta-endotoxin remained in the prophage cured strain. Moreover, phage induction was found to occur after mating of serovar israelensis with its prophage cured strain, as well as with B. thuringiensis serovar thuringiensis, B. cereus and B. subtilis. [TOP OF PAGE]

89. A bacteriophage encoding a pathogenicity island, a type-IV pilus and a phage receptor in cholera bacteria [see comments]. Karaolis, D.K., Somara, S., Maneval, D.R.Jr., Johnson, J.A., Kaper, J.B. (1999). Nature 399:375-379. The virulence properties of many pathogenic bacteria are due to proteins encoded by large gene clusters called pathogenicity islands, which are found in a variety of human pathogens including Escherichia coli, Salmonella, Shigella, Yersinia, Helicobacter pylori, Vibrio cholerae, and animal and plant pathogens such as Dichelobacter nodosus and Pseudomonas syringae. Although the presence of pathogenicity islands is a prerequisite for many bacterial diseases, little is known about their origins or mechanism of transfer into the bacterium. The bacterial agent of epidemic cholera, Vibrio cholerae, contains a bacteriophage known as cholera-toxin phage (CTXphi), which encodes the cholera toxin, and a large pathogenicity island called the VPI (for V. cholerae pathogenicity island) which itself encodes a toxin-coregulated pilus that functions as a colonization factor and as a CTXphi receptor. We have now identified the VPI pathogenicity island as the genome of another filamentous bacteriophage, VPIphi. We show that VPIphi is transferred between V. cholerae strains and provide evidence that the TcpA subunit of the toxin-coregulated type IV pilus is in fact a coat protein of VPIphi. Our results are the first description of a phage that encodes a receptor for another phage and of a virus-virus interaction that is necessary for bacterial pathogenicity. [TOP OF PAGE]

90. Shiga toxins even when different are encoded at identical positions in the genomes of related temperate bacteriophages. Karch, H., Schmidt, H., Janetzki-Mittmann, C., Scheef, J., Kroger, M. (1999). Molecular and General Genetics 262:600-607. The nucleotide sequence of an 11,142-bp region including the stx2 operon in the genome of the temperate bacteriophage 933W in the EDL933 strain of Escherichia coli 0157 was determined and compared to the respective regions derived from other lambdoid bacteriophages. In phage 933W, a region of ORFs interlinked by overlapping start-stop codons (ATGA) was detected preceding the toxin gene. These ORFs show a high degree of sequence identity to genes of the nin region of phage lambda. Immediately downstream of these nin genes we identified an ORF that may code for an anti-terminator similar to the lambda Q protein. It is concluded that toxin expression is directly associated with the initiation of cell lysis. Downstream of the stx2 operon we identified an ORF that is homologous to the holin gene S of bacteriophage PA-2. PCR primers were designed, which, based on a comparison of the phage sequences, appeared to be common to both stx1- and stx2-harbouring phages. However, only seven of the 22 STEC strains investigated from serogroups 0157, 026, 0103 and 0111 yielded the expected PCR amplification product. The data reported here may be useful in developing new strategies for inhibiting the expression of Stx and for developing universal diagnostic primers for use in tracking the origin and evolution of Shiga toxins and the phages that carry them. [TOP OF PAGE]

91. Genetic selection of phage engineered for receptor-mediated gene transfer to mammalian cells. Kassner, P.D., Burg, M.A., Baird, A., Larocca, D. (1999). Biochemical & Biophysical Research Communications 264:921-928. Although phage display is a powerful way of selecting ligands against purified target proteins, it is less effective for selecting functional ligands for complex targets like living cells. Accordingly, phage display has had limited utility in the development of targeting agents for gene therapy vectors. By adapting a filamentous bacteriophage for gene delivery to mammalian cells, however, we show here that it is possible to screen phage libraries for functional ligands capable of delivering DNA to cells. For example, when targeted with epidermal growth factor (EGF), M13 bacteriophage were capable of delivering a green fluorescent protein (GFP) gene to EGF receptor bearing cells in a ligand-, time-, and phage concentration-dependent manner. The EGF-targeted phage transduced COS-1 cells in a highly specific manner as demonstrated by competition with excess free EGF or alternatively with anti-EGF receptor antibodies. We further demonstrate that EGF-phage can be selected, by their ability to transduce EGF receptor bearing cells from libraries of peptide display phage. When phage were incubated with COS-1 cells, EGF ligand-encoding sequences were recovered by PCR from FACsorted, GFP-positive cells and the EGF-displaying phage were enriched 1 million-fold by four rounds of selection. These data suggest the feasibility of applying molecular evolution to phage gene delivery to select novel cell-specific DNA-targeting ligands. The same approach could be used to select genetically altered phage that are specifically designed and evolved as gene therapy vectors. [TOP OF PAGE]

92. Anticodon nuclease: a bacterial RNA restriction enzyme. Kaufmann, G. (1999). TIBS ???:???-??? The tRNA^Lys-specific anticodon nuclease exists in an E. coli isolate in latent form, complexed with a DNA restriction enzyme. A phage T4-encoded alleviator of DNA restriction disrupts this masking interaction but other phage proteins repair the damaged tRNA^Lys. Detection of a homologous system in Neisseria and a different anticodon nuclease in Colicin E5 suggest widespread occurrence of versatile tRNA restriction endonucleases. Studying them may provide new insights into the evolution of RNA recognition and cleavage mechanisms. [TOP OF PAGE]

93. Biological projectiles (phage, yeast, bacteria) for genetic transformation of plants. Kikkert, J.R., Humiston, G.A., Roy, M.K., Sanford, J.C. (1999). In Vitro Cellular & Developmental Biology Plant 35:43-50. Bacteriophage lambda particles, yeast cells, and bacterial cells were tested as projectiles to deliver marker/reporter genes into plant cells via the biolistic process. When phage particles were complexed to tungsten or gold particles and used to bombard tobacco cells, fewer than 15 cell clusters per plate transiently expressed beta-glucuronidase (GUS). Cells of wild-type Saccharomyces cerevisiae were too large to be effective projectiles, but use of a reduced-size mutant resulted in a small number of transformants. Escherichia coli cells complexed with tungsten were the most effective projectile for plant transformation. Various methods to prepare E. coli were tested to reduce particle size, improve binding of bacteria to metal particles, and/or minimize particle clumping. In maize, the number of transformants was highest when bacteria/tungsten particles were air-dried onto macrocarriers from an aqueous solution. When maize cells were bombarded with bacteria/tungsten projectiles, rates of transient gene expression (2000 per plate) and stable transformation (50 per plate) were only two- to threefold lower than when purified DNA was used. Transformation of tobacco with E. coli projectiles was improved when the bacteria were treated with a series of ethanol and ether washes, then dried into a powder. Nevertheless, tobacco transformation was still 24- (transient) and 200-fold (stable) less than when purified DNA was used. Biological projectiles can be effective for plant transformation and are advantageous because once a DNA construct is made and put into the appropriate microorganism, the need to isolate and purify DNA for the biolistic process is eliminated, which saves time and lessens DNA shear. Such projectiles may be especially well suited where high molecular weight DNA constructs are needed. [TOP OF PAGE]

94. Alternative origins for nannobacteria-like objects in calcite. Kirkland, B.L., Folk, R.L., Lynch, F.L., McLean, R.J.C., Molineux, I.J., Rahnis, M.A. (1999). Geology (Boulder) 27:347-350. More than 40 calcite-precipitation experiments were performed under sterile conditions in order to investigate the origins of 25-300 nm spherical-, rod-, and ovoid-shaped objects that have been widely interpreted as evidence of nanometer-scale life (i.e., nannobacteria). Individual experiments included the addition of soluble organic compounds, common species of eubacteria, or phage-induced eubacterial lysates. These experiments indicate that many of the nanometer-scale objects have inorganic or nonnannobacterial origins. In the precipitation experiments, calcite formed euhedral crystals 50-800 nm in diameter and smaller (<50 nm) anhedral or rounded particles or protocrystals. The small anhedral or rounded solids resembled nannobacteria. The relative amount of anhedral or rounded calcite was greatest in experiments with a dissolved organic component. These controlled experiments are in accord with observations that rounded nanometer-scale objects are more common in minerals formed in organic-rich environments. Bacterial fragments occur as rounded to irregularly shaped particles that included cell-wall fragments, expulsed cytoplasm, and relict capsules that also closely resembled nannobacteria. Acid etching of the large euhedral crystals produced in the precipitation experiments also resulted in the formation of nanometer-scale features that resembled nannobacteria in natural carbonates. The shapes of the etching artifacts vary as a function of the strength of the acid and the duration of etching. Much caution is advisable in interpreting the origin of rounded features <50 nm. [TOP OF PAGE]

95. Genetic homogeneity and phage susceptibility of ruminal strains of Streptococcus bovis isolated in Australia. Klieve, A.V., Heck, G.L., Prance, M.A., Shu, Q. (1999). Letters in Applied Microbiology 29:108-112. The genetic homogeneity of 37 strains of ruminal streptococci was investigated by comparing DNA fragment profiles on agarose gels following restriction endonuclease digestion with Hae III, Cfo I and Msp I. Thirty strains were indistinguishable from Streptococcus bovis strains, 2B, H24 and AR3. The remaining three strains were similar but not identical to a ruminal strain of Strep. intermedius (AR36). In addition, the susceptibility of these strains to infection by five bacteriophages was examined. Three of the phages (phi Sb02, phi Sb03 and phi Sb04) were specific to the strain of Strep. bovis from which they were isolated, while phages 2BV and phi Sb01 infected one and two strains, respectively, in addition to their primary host. It was concluded that although Strep. bovis is relatively homogeneous genetically, broad host range phages appear to be uncommon with this bacterial species. [TOP OF PAGE]

96. Biocontrol of Escherichia coli O157 with O157-specific bacteriophages. Kudva, I.T., Jelacic, S., Tarr, P.I., Youderian, P., Hovde, C.J. (1999). Appl. Environ. Microbiol. 65:3767-3773. Escherichia coli O157 antigen-specific bacteriophages were isolated and tested to determine their ability to lyse laboratory cultures of Escherichia coli O157:H7. A total of 53 bovine or ovine fecal samples were enriched for phage, and 5 of these samples were found to contain lytic phages that grow on E. coli O157:H7. Three bacteriophages, designated KH1, KH4, and KH5, were evaluated. At 37 or 4 degrees C, a mixture of these three O157-specific phages lysed all of the E. coli O157 cultures tested and none of the non-O157 E. coli or non-E. coli cultures tested. These results required culture aeration and a high multiplicity of infection. Without aeration, complete lysis of the bacterial cells occurred only after 5 days of incubation and only at 4 degrees C. Phage infection and plaque formation were influenced by the nature of the host cell O157 lipopolysaccharide (LPS). Strains that did not express the O157 antigen or expressed a truncated LPS were not susceptible to plaque formation or lysis by phage. In addition, strains that expressed abundant mid-range-molecular-weight LPS did not support plaque formation but were lysed in liquid culture. Virulent O157 antigen-specific phages could play a role in biocontrol of E. coli O157:H7 in animals and fresh foods without compromising the viability of other normal flora or food quality. [TOP OF PAGE]

97. Gene transfer to mammalian cells using genetically targeted filamentous bacteriophage. Larocca, D., Kassner, P.D., Witte, A., Ladner, R.C., Pierce, G.F., Baird, A. (1999). FASEB Journal 13:727-734. We have genetically modified filamentous bacteriophage to deliver genes to mammalian cells. In previous studies we showed that noncovalently attached fibroblast growth factor (FGF2) can target bacteriophage to COS-1 cells, resulting in receptor-mediated transduction with a reporter gene. Thus, bacteriophage, which normally lack tropism for mammalian cells, can be adapted for mammalian cell gene transfer. To determine the potential of using phage-mediated gene transfer as a novel display phage screening strategy, we transfected COS-1 cells with phage that were engineered to display FGF2 on their surface coat as a fusion to the minor coat protein, pIII. Immunoblot and ELISA analysis confirmed the presence of FGF2 on the phage coat. Significant transduction was obtained in COS-1 cells with the targeted FGF2-phage compared with the nontargeted parent phage. Specificity was demonstrated by successful inhibition of transduction in the presence of excess free FGF2. Having demonstrated mammalian cell transduction by phage displaying a known gene targeting ligand, it is now feasible to apply phage-mediated transduction as a screen for discovering novel ligands. [TOP OF PAGE]

98. Occurrence and levels of phages proposed as surrogate indicators of enteric viruses in different types of sludges. Lasobras, J., Dellunde, J., Jofre, J., Lucena, F. (1999). Journal of Applied Microbiology 86:723-729. A method based on the treatment of sludge with beef extract recovered, with similar efficiency, the three groups of bacteriophages studied from different kinds of sludges. The three groups of bacteriophages were found in high numbers in the different sludge types, the highest value being that of somatic coliphages in primary sludge of a biological treatment plant (1.1 x 10(5) pfu g-1) and the lowest being that of Bacteroides fragilis phages (110 pfu g-1) in de-watered, anaerobically, mesophilically-digested sludge. All phages studied accumulated in the sludges. In primary and activated sludges, all three types accumulated similarly but in lime-treated sludge and de-watered, anaerobically, mesophilically-digested sludge, the relative proportion of F-specific bacteriophages decreased significantly with respect to somatic coliphages and bacteriophages infecting B. fragilis. All phages survived successfully in stored sludge, depending on the temperature, and again, F-specific bacteriophages survived less successfully than the others. [TOP OF PAGE]

99. The R-type pyocin of Pseudomonas aeruginosa C is a bacteriophage tail-like particle that contains single-stranded DNA. Lee, F.K.N., Dudas, K.C., Hanson, J.A., Nelson, M.B., Loverde, P.T., Apicella, M.A. (1999). Infect. Immun. 67:717-725. Pseudomonas aeruginosa R-type pyocin particles have been described as bacteriocins that resemble bacteriophage tail-like structures. Because of their unusual structure, we reexamined whether they contained nucleic acids. Our data indicated that pyocin particles isolated from P. aeruginosa C (pyocin C) contain DNA. Probes generated from this DNA by the random-primer extension method hybridized to distinct bands in restriction endonuclease-digested P. aeruginosa C genomic DNA. These probes also hybridized to genomic DNA from 6 of 18 P. aeruginosa strains that produced R-type pyocins. Asymmetric PCR, complementary oligonucleotide hybridization, and electron microscopy indicated that pyocin C particles contained closed circular single-stranded DNA, approximately 4.0 kb in length. Examination of total intracellular DNA from mitomycin C-induced cultures revealed the presence of two extrachromosomal DNA molecules, a double-stranded molecule and a single-stranded molecule, which hybridized to pyocin DNA. Sequence analysis of 7,480 nucleotides of P. aeruginosa C chromosomal DNA containing the pyocin DNA indicated the presence of pyocin open reading frames with similarities to open reading frames from filamentous phages and cryptic phage elements. We did not observe any similarities to known phage structural proteins or previously characterized pseudomonal prt genes expressing R-type pyocin structural proteins. These studies demonstrate that pyocin particles from P. aeruginosa C are defective phages that contain a novel closed circular single-stranded DNA and that this DNA was derived from the chromosome of P. aeruginosa C. [TOP OF PAGE]

100. [Reconstruction of possible paths of the origin and morphological evolution of bacteriophages]. Letarov, A.V. (1999). Genetika 34:1461-1469. The problem of the origin and evolution of viruses and, in particular, the origin and evolution of bacteriophages is of considerable interest. However, so far, this problem has not been solved with quantitative methods of molecular systematics. In the present study, an attempt to reconstruct the possible paths of appearance and evolution of bacteriophages based on their structural features and morphogenesis, as well as general characteristics of their life cycles and genome organization, was carried out. A scheme describing phylogeny of the main bacteriophage groups and evolution of their life cycles is suggested. Existence of two independently evaluating types of morphogenesis ("budding outward" and "budding inward") is postulated. [TOP OF PAGE]

101. Rekonstruktsiia vozmozhnykh putei proiskhozhdeniia i morfologicheskoi evoliutsii bakteriofagov [Reconstruction of possible paths of the origin and morphological evolution of bacteriophages]. Letarov, A.V. (1999). Genetika 34:1461-1469. The problem of the origin and evolution of viruses and, in particular, the origin and evolution of bacteriophages is of considerable interest. However, so far, this problem has not been solved with quantitative methods of molecular systematics. In the present study, an attempt to reconstruct the possible paths of appearance and evolution of bacteriophages based on their structural features and morphogenesis, as well as general characteristics of their life cycles and genome organization, was carried out. A scheme describing phylogeny of the main bacteriophage groups and evolution of their life cycles is suggested. Existence of two independently evaluating types of morphogenesis ("budding outward" and "budding inward") is postulated. [TOP OF PAGE]

102. [The history of the discovery and study of Brucella bacteriophages]. Liapustina, L.V., Liamkin, L.I., Taran, I.F. (1999). Zhurnal Mikrobiologii, Epidemiologii i Immunobiologii 123-124. [TOP OF PAGE]

103. Identification of a Vibrio cholerae RTX toxin gene cluster that is tightly linked to the cholera toxin prophage. Lin, W., Fullner, K.J., Clayton, R., Sexton, J.A., Rogers, M.B., Calia, K.E., Calderwood, S.B., Fraser, C., Mekalanos, J.J. (1999). Proc. Natl. Acad. Sci. USA 96:1071-1076. We identify and characterize a gene cluster in El Tor Vibrio cholerae that encodes a cytotoxic activity for HEp-2 cells in vitro. This gene cluster contains four genes and is physically linked to the cholera toxin (CTX) element in the V. cholerae genome. We demonstrate by using insertional mutagenesis that this gene cluster is required for the cytotoxic activity. The toxin, RtxA, resembles members of the RTX (repeats in toxin) toxin family in that it contains a GD-rich repeated motif. Like other RTX toxins, its activity depends on an activator, RtxC, and an associated ABC transporter system, RtxB and RtxD. In V. cholerae strains of the classical biotype, a deletion within the gene cluster removes rtxC and eliminates cytotoxic activity. Other strains, including those of the current cholera pandemic, contain a functional gene cluster and display cytotoxic activity. Thus, the RTX gene cluster in El Tor O1 and O139 strains might have contributed significantly to their emergence. Furthermore, the RTX toxin of V. cholerae may be associated with residual adverse properties displayed by certain live, attenuated cholera vaccines. [TOP OF PAGE]

104. Bacteriophages: An altertnative to antibiotics? Lorch, A. (1999). Biotechnology and Development Monitor 39:14-17. Bacterial resistance to antibiotics has become a serious medical problem. Treatment with bacteriophages might pose an effective alternative that has long been known but has been ignored outside the former Soviet Union. The development of phage therapies exemplifies positive as well as negative implications for scientific development that is restricted in its access to the mainstream, English-language dominated scientific community. [TOP OF PAGE]

105. Defektnye fagi kak faktor antagonizma u blizkorodstvennykh batsill [Defective pages as an antagonistic factor in closely-related bacilli]. Lotareva, O.V., Prozorov, A.A. (1999). Mikrobiologica 67:788-791. The antagonistic effect produced by the detective phage PBSX during cocultivation of the mutant strain B. subtilis 168, in which this phage is heat-inducible, and strain B. subtilis NRS231, which also bears a defective phage, was investigated. As soon as in the first hours of cocultivation under conditions of PBSX induction, the number of viable cells of strain NRS231 decreased by two orders of magnitude. However, the effect was not observed if the temperature of cocultivation was noninducing. The results confirm the supposition that defective phages may play a role in the competition between closely related bacilli. [TOP OF PAGE]

106. Comparative genomics of Streptococcus thermophilus phage species supports a modular evolution theory. Lucchini S., Desiere, F., Brussow, H. (1999). J. Virol. 73:8647-8656. The comparative analysis of five completely sequenced Streptococcus thermophilus bacteriophage genomes demonstrated that their diversification was achieved by a combination of DNA recombination events and an accumulation of point mutations. The five phages included lytic and temperate phages, both pac site and cos site, from three distinct geographical areas. The units of genetic exchange were either large, comprising the entire morphogenesis gene cluster, excluding the putative tail fiber genes, or small, consisting of one or maximally two genes or even segments of a gene. Many indels were flanked by DNA repeats. Differences in a single putative tail fiber gene correlated with the host ranges of the phages. The predicted tail fiber protein consisted of highly conserved domains containing conspicuous glycine repeats interspersed with highly variable domains. As in the T-even coliphage adhesins, the glycine-containing domains were recombinational hot spots. Downstream of a highly conserved DNA replication region, all lytic phages showed a short duplication; in three isolates the origin of replication was repeated. The lytic phages could conceivably be derived from the temperate phages by deletion and multiple rearrangement events in the lysogeny module, giving rise to occasional selfish phages that defy the superinfection control systems of the corresponding temperate phages. [TOP OF PAGE]

107. Similarly organized lysogeny modules in temperate Siphoviridae from low GC content gram-positive bacteria. Lucchini, S., Desiere, F., Brussow, H. (1999). Virology 263:427-435. Temperate Siphoviridae from an evolutionarily related branch of low GC content gram-positive bacteria share a common genetic organization of lysogeny-related genes and the predicted proteins are linked by many sequence similarities. Their compact lysogeny modules [integrase/1-2 orfs (phage exclusion? and metalloproteinase motif proteins)/cl-like repressor/cro-like repressor/antirepressor (optional)] differ clearly from that of h-like and L5-like viruses, the two currently established genera of temperate Siphoviridae, while they resemble those of the P2-like genus of Myoviridae. In all known temperate Siphoviridae from low GC content gram-positive bacteria the lysogeny module is flanked by the lysis module and the DNA replication module. This modular organization is again distinct from that of the known genera of temperate Siphoviridae. On the basis of comparative sequence analysis we propose a new genus of Siphoviridae: "Sfi21-like" phages. With a larger database of phage sequences it might be possible to establish a genomics-based phage taxonomy and to retrace the evolutionary history of selected phage modules or individual phage genes. The antirepressor of Sfi21-like phages has an unusual widespread distribution since proteins with high aa similarity (40%) were found not only in phages from gramnegative bacteria, but also in insect viruses. [TOP OF PAGE]

108. The genetic relationship between virulent and temperate Streptococcus thermophilus bacteriophages: Whole genome comparison of cos-site phages Sfi19 and Sfi21. Lucchini, S., Desiere, F., Brussow, H. (1999). Virology 260:232-243. The virulent cos-site Streptococcus thermophilus bacteriophage Sfi19 has a 37,392-bp-long genome consisting of 44 open reading frames all encoded on the same DNA strand. The genome of the temperate cos-site S. thermophilus phage Sfi21 is 3.3 kb longer (40,740 bp, 53 orfs). Both genomes are very similarly organized and differed mainly by gene deletion and DNA rearrangement events in the lysogeny module; gene replacement, duplication, and deletion events in the DNA replication module, and numerous point mutations. The level of point mutations varied from 15% (DNA packaging and head morphogenesis modules). A dotplot analysis showed nearly a straight line over the left 25 kb of their genomes. Over the right genome half, a more variable dotplot pattern was observed. The entire lysogeny module from Sfi21 comprising 12 genes was replaced by 7 orfs in Sfi19, six showed similarity with genes from temperate pac-site S. thermophilus phages. Noneof the genes implicated in the establishment of the lysogenic state (integrase, superinfection immunity, repressor) or remnants of it were conserved in Sfi19, while a Cro-like repressor was detected. Downstream of the highly conserved DNA replication module 11 and 13 orfs were found in Sfi19 and phiSfi21, respectively: Two orfs from Sfi21 were replaced by a different gene and a duplication of the phage origin of replication in Sfi19; a further orf was only found in Sfi21. All other orfs from this region, which included a second putative phage repressor, were closely related between both phages. Two noncoding regions of Sfi19 showed sequence similarity to pST1, a small cryptic plasmid of S. thermophilus. [TOP OF PAGE]

109. Removal of microorganisms from water by columns containing sand coated with ferric and aluminum hydroxides. Lukasik, J., Cheng, Y.F., Lu, F., Tamplin, M., Farrah, S.R. (1999). Water Res. 33:769-777. Tap water seeded with different microorganisms or untreated waste water was passed through columns containing sand modified by the in situ precipitation of metallic hydroxides or unmodified sand. Columns (35.5 X 5.0 cm) packed with 1 kg of sand modified with a combination of ferric and aluminum hydroxide removed greater than 99% of Escherichia coli, Vibrio cholerae, poliovirus 1 and coliphage MS-2 from dechlorinated tap water. This removal efficiency was consistent throughout the passage of 1201 of water during a 30 day test. After the passage of 1921 of tap water, these columns were still able to remove 99.9% MS-2, 80% E. coli and 90% of poliovirus 1. Columns containing modified sand efficiently removed microorganism from water samples at the various pH and temperature values tested while columns containing unmodified sand did not. In addition, these modified sand filters were able to remove coliform bacteria and coliphage from raw sewage. More than a 4 log10 reduction in the numbers of these microorganisms was achieved at room temperature. The modified sand seems to better remove microorganisms due to increased electrostatic interactions. Neither E. coli nor MS-2 were inactivated by the modified sand column effluents. The metal used for coating the sand could not be detected in the column effluents, indicating that the coatings were stable. [TOP OF PAGE]

110. Establishment of bacteriophages in an immobilized cells system used for continuous inoculation of lactococci. Macedo, M.G., Champagne, C.P., Vuillemard, J.C., Lacroix, C. (1999). International Dairy Journal 9:437-445. Effects of high dilution rates (10-30 h---1) on development of a low phage population inoculated in an immobilized cell technology continuous milk inoculation system were evaluated. Effects of medium on phage and bacterial kinetics of the bioreactor were also examined. Lactococcus lactis subsp. lactis CRA-1 and the appropriate bacteriophage _CRA-1 (homologue to L. lactis CRA-1) were used. Lactococci were immobilized in a 2-phase dispersion procedure, in 2.75% kappa-carrageenan/0/25% locust bean gum gel beads which were then used to continuously inoculate rehydrated low heat skim milk powder (9% solids); a synthetic medium (containing lactose, yeast extract, tryptone, MgSO4, KCl, CaCl2 and sodium citrate) was used for some fermentations at a dilution rate of 30 h---1. Acidifying activity tests were performed with fermented medium samples collected from the bioreactor. Even by reduction of phage contamination level from 10-5 to 10-2 pfu/ml and increasing dilution rate to 30 h---1, bacteriophages were not washed out and developed in the continuous immobilized bioreactor system with milk and synthetic media. High dilution rates effective against psychrotrophs and yeasts were not, therefore, effective against phages, so strategies are required to prevent problems associated with phage contamination. In terms of use of immobilized cells for production of lactic starters, it may be that phage contamination could be controlled by use of phage-resistant media and/or conditions which prevent phage contamination. For continuous inoculation, it may be possible to immobilize phage-resistant or multiple phage-unrelated strains, to alleviate effects of contamination in pasteurized milk. Population dynamics of mixed cultures need to be examined. [TOP OF PAGE]

111. Enteroviruses in the recreational waters of Lake Orta. Maiello, A., Guidetti, A., Poncetta, D., Ossola, O., Guidetti, L., Buttinelli, G., Fiore, L., Ruggenini, A.M. (1999). Lakes Reservoirs Research and Management 4:93-99. The water quality of Lake Orta was evaluated for recreational use. This lake was the only Italian lake to undergo a 'liming treatment' which neutralizes water acidity by adding carbonates. Chemical, bacteriological and virological parameters were monitored for 3 years after the treatment ended. Chemical and bacteriological studies were performed according to national standard methods (DPR 470/82) whereas F' specific bacteriophage were enumerated according to an international standard (ISO 10705-1). Enteroviruses were detected by the observation of a cytophatic effect on buffalo green monkey cultures. Also, the possibility of applying molecular biological techniques for enterovirus detection directly to waters concentrated without previous isolation of viruses in cell culture was verified. The sensitivity, specificity and feasibility of both methods were evaluated. The association between the direct detection of enteroviruses and the indirect indication of their presence by the presenceof bacteriophages, and the relationship between bacteriophage presence and bacteriological contamination were also evaluated. In the course of surveillance, none of the samples infringed the law with respect to the physical-chemical parameter (pH < 6). As far as bacteriological characteristics were concerned, 16.6% of the samples taken in 1993 infringed the law, as well as 19.4% of samples in 1994 and 19.4% in 1995. The level of faecal coliforms most frequently exceeded the given limits. The detection test for enteroviruses was positive in 5.1% of the samples using the traditional method with cell culture, and it was positive in 7.6% using the antigen capture polymerase chain reaction (AC-PCR) method directly applied to concentrated waters, indicating the feasibility and a higher sensitivity of the AC-PCR method compared to cell culture. Bacteriophages were present in all the samples that were positive in the virological analysis, as well as in 46.7% of negative samples. [TOP OF PAGE]

112. Complete nucleotide sequence of the prophage VT2-Sakai carrying the verotoxin 2 genes of the enterohemorrhagic Escherichia coli O157:H7 derived from the Sakai outbreak. Makino, K., Yokoama, K., Kubota, Y., Yutsudo, C.H., Kimura, S., Kurokawa, K., Ishii, K., Hattori, M., Tatsuno, I., Abe, H., Lida, T., Yamamoto, K., Onishi, M., Hayashi, T., Yasunaga, T., Honda, T., Sasakawa, C., Shinagawa, H. (1999). Genes and Genetic Systems 74:227-239. The enterohemorrhagic Escherichia coli (EHEC) O157:H7 strain RIMD 0509952, derived from an outbreak in Sakai city, Japan, in 1996, produces two kinds of verotoxins, VT1 and VT2, encoded by the stx1 and stx2 genes. In the EHEC strains, as well as in other VT-producing E. coli strains, the toxins are encoded by lysogenic bacteriophages. The EHEC O157:H7 strain RIMD 0509952 did not produce plaque-forming phage particles upon inducing treatments. We have determined the complete nucleotide sequence of a prophage, VT2-Sakai, carrying the stx2A and stx2B genes on the chromosome, and presumed the putative functions of the encoded proteins and the cis-acting DNA elements based on sequence homology data. To our surprise, the sequences in the regions of VT2-Sakai corresponding to the early gene regulators and replication proteins, and the DNA sequences recognized by the regulators share very limited homology to those of the VT2-encoding 933W phage carried by the EHEC O157:H7 strain EDL933 reported by Plunkett et al. (J. Bacteriol., p1767-1778, 181, 1999), although the sequences corresponding to the structural components are almost identical. These data suggest that these two phages were derived from a common ancestral phage and that either or both of them underwent multiple genetic rearrangements. An IS629 insertion was found downstream of the stx2B gene and upstream of the lysis gene S, and this might be responsible for the absence of plaque-forming activity in the lysate obtained after inducing treatments. [TOP OF PAGE]

113. Prophages inserted in archaebacterial genomes. Makino, S.I., Amano, N., Koike, H., Suzuki, M. (1999). Proceedings of the Japan Academy Series B Physical and Biological Sciences 75:166-171. By analyzing archaebacterial genomic DNA sequences, three new prophages have been found inserted in tRNA genes of the host genomes: proPOF1 and proPOF2 in the genome of Pyrococcus sp. OT3, and proMJF1 in that of Methanococcus jannaschii. The three prophages possess a gene coding for site-specific tyrosine integrase, and are characterized with pairs of elements of the same 46-65 base sequences, that are positioned on the borders to the host genomes (i.e. the attachments). If the two ends of proPOF1 are connected to form a circle by overlapping the two attachments, proPOF1 possesses 33 ORFs in 9 putative transcription units (i.e. 5 operons and 4 independently transcribed ORFs) in 4 clusters inside each of which the direction of transcription is kept the same. This prophage is more likely to inherit a potential to be activated to a phage than is proPOF2, which has only 3 ORFs. In proPOF1 and proPOF2 the attachments are designed, so that their nucleotide sequences are complementary to the 3'-terminal halves of the host tRNA genes. These attachments are part of the integrase genes in the prophages. Upon the integration the integrase genes were divided at the attachments into two segments and are positioned at the two ends of the prophages. In proMJF1 the integrase gene is undivided, and is positioned between the two attachments whose nucleotide sequence is the same as that of the 3'-terminal half of the host tRNA gene. Thus, the type of phage-integration that created proMJF1 is different from that created proPOF1 and proPOF2. [TOP OF PAGE]

114. Seasonal changes in densities of cyanophage infectious to Microcystis aeruginosa in a hypereutrophic pond. Manage, P., Kawabata, Z., Nakano, S. (1999). Hydrobiologia. 211-216. Seasonal changes in densities of cyanophages infectious to Microcystis aeruginosa were studied in a hypereutrophic pond from March 1997 to January 1998 to elucidate the potential impact of the cyanophage on M. aeruginosa mortality. Densities of M. aeruginosa ranged between 1.8 X 10⁴ and 9.4 X 10⁵ cells ml-1, while those of the cyanophages were between 2.0 X 10² and 4.2 X 10⁴ PFU ml^-1. Sharp decreases in densities of M. aeruginosa were detected on 10 June and 24 September, as densities of the cyanophages increased, suggesting release of the cyanophages due to the lysis of infected M. aeruginosa. Thus, infection by cyanophages may have a substantial effect on cyanobacterial succession in the pond. Densities of cyanophages became undetectable when those of M. aeruginosa were at low levels during winter. We suggest that there is a tight host-pathogen relationship between M. aeruginosa and the cyanophage in the pond. [TOP OF PAGE]

115. Enumeration of marine viruses in culturea nd natural samples by flow cytometry. Marie, D., Brussaard, C.P.D., Thyrhaug, G., Bratbak, G., Vaulot, D. (1999). Appl. Environ. Microbiol. 65:45-52. Flow cytometry (FCM) was successfully used to enumerate viruses in seawater after staining with the nucleic acid-specific dye SYBR Green-I. The technique was first optimized by using the Phaeocystis lytic virus PpV-01. Then it was used to analyze natural samples from different oceanic locations. Virus samples were fixed with 0.5% glutaraldehyde and deep frozen for delayed analysis. The samples were then diluted in Tris-EDTA buffer and analyzed in the presence of SYBR Green-I. A duplicate sample was heated at 80 degree C in the presence of detergent before analysis. Virus counts obtained by FCM were highly correlated to, although slightly higher than, those obtained by epifluorescence microscopy or by transmission electron microscopy (r = 0.937, n = 14, and r = 0.96, n = 8, respectively). Analysis of a depth profile from the Mediterranean Sea revealed that the abundance of viruses displayed the same vertical trend as that of planktonic cells. FCM permits us to distinguish between at least two and sometimes three virus populations in natural samples. Because of its speed and accuracy, FCM should prove very useful for studies of virus infection in cultures and should allow us to better understand the structure and dynamics of virus populations in natural waters. [TOP OF PAGE]

116. Cyanophages. Martin, E.L., Kokjohn, T.A. (1999). pp. 324-332. In In Granoff, A. and Webster, R.G. (eds.), Encyclopedia of Virology second edition. Academic Press, San Diego. [no abstract?]. [TOP OF PAGE]

117. Induction of prophages of enterohemorrhagic Escherichia coli O157:H7 with norfloxacin. Matsushiro, A., Sato, K., Miyamoto, H., Yamamura, T., Honda, T. (1999). J. Bacteriol. 181:2257-2260. Norfloxacin (NFLX) caused induction of prophages VT1 and VT2 of enterohemorrhagic Escherichia coli O157 at subinhibitory concentrations. In time course experiments, we observed the following sequential events: upon induction, the phage genomes underwent multiplication; the amount of stx genes increased; and subsequently, large quantities of toxins VT1 and VT2 were produced. Further studies showed that the molecular mechanism of prophage induction is closely related to the RecA system since the prophage VT2 was not induced with NFLX in a recA mutant strain. [TOP OF PAGE]

118. Vibriophage KVP40 and coliphage T4 genomes share a homologous 7-kbp region immediately upstream of the gene encoding the major capsid protein. Matsuzaki, S., Kuroda, M., Kimura, S., Tanaka, S. (1999). Archives of Virology 144:2007-2012. Vibriophage KVP40, a large tailed DNA phage morphologically similar to T-even coliphages, has a major capsid protein (Mcp) homologous to the equivalent protein, gp23(*), of coliphage T4. The sequence analysis was extended to a 7-kbp region immediately upstream of the mcp gene encoding the precursor of Mcp. The region as a whole was fairly homologous to the corresponding region of the T4 genome and contained 8 ORFs homologous to T4 genes 17, 18, 19, 20, 67, 68, 21, and 22 in the same order as in T4. These findings thus strongly suggest that these two phages are phylogenetically related. [TOP OF PAGE]

119. Major capsid proteins of certain Vibrio and Aeromonas phages are homologous to the equivalent protein, gp23(*), of coliphage T4. Matsuzaki, S., Kuroda, K., Kimura, S., Tanaka, S. (1999). Archives of Virology 144:1647-1651. N-terminal amino acid sequences of major capsid proteins (Mcps) of three vibriophages (KVP20, KVP40 and nt-1), two aeromonad phages (Aeh 1 and 65) and coliphage T4 were compared. All these phages are morphologically similar, belonging to family Myoviridae and the vernacular genus name "T4-like phages". A dendrogram constructed from homology data indicated that (i) the three vibriophages were closely related, (ii) the two aeromonad phages were also fairly related and (iii) these five phages were all distantly, but definitely, related to coliphage T4. These results suggest that Mcps of morphologically similar phages are highly conserved and may serve as a measure to assess the phylogenetic relationships among different phages of similar morphology. [TOP OF PAGE]

120. Bacterial and viral indicators of fecal pollution in Mexico City's southern aquifer. Mazari-Hiriart, M., Torres-Beristain, B., Velazquez, E., Calva, J.J., Pillai, S.D. (1999). Journal of Environmental Science and Health Part A Toxic-Hazardous Substances & Environmental Engineering 34:1715-1735. Mexico City with a population of about 18 million people relies on groundwater to supply about 70% of its water needs. In order to understand the extent of microbial pathogen contamination of these reserves, a 10 month long monitoring study of the southern aquifer was undertaken. Groundwater samples were collected from five different locations and analyzed (100 mL) for total coliforms, fecal coliforms, and fecal streptococci. Larger volume samples (5 L) were collected and concentrated for quantitative and qualitative (presence/absence) determination of microorganisms including bacteriophages. Gene amplification (PCR) approaches were employed to screen for Escherichia coli/Shigella specific (uid) sequences. Laboratory microcosms were conducted to evaluate the potential survival of pathogenic viruses in the groundwater using MS-2 and PRD-1 as model viruses. Coliphage as a single indicator, or in conjunction with fecal coliforms and fecal streptococci were found to have value as an indicator of fecal pollution in this geographical region. The results indicate that the southern aquifer underlying metropolitan Mexico City can pose a significant risk to public health when water is distributed and used without adequate disinfection. The pumping wells located in the transition and mountain areas indicated the presence of extensive microbial pathogen contamination. There was surprisingly, no difference between the dry and rainy seasons in terms of the presence of fecal pollution microbial indicators. [TOP OF PAGE]

121. TB: the return of the phage. A review of fifty years of mycobacteriophage research. McNerney, R. (1999). Int. J. Tuberc. Lung Dis. 3:179-184. The first mycobacteriophage was isolated in 1947, and since that time over 250 of these viruses have been identified. Phages have made a significant contribution to our knowledge of mycobacteria over the past 50 years, and following the development of typing techniques in the 1960s and 1970s they were widely used in epidemiological studies of tuberculosis. Unfortunately, attempts to use lytic phages therapeutically during tuberculosis infection have so far failed to elicit cure in experimentally infected animals. During the past decade phages have become important in molecular studies of mycobacteria, both in terms of studying phage biology and as tools in recombinant DNA technology, thus facilitating the investigation of mycobacterial pathogenesis. Today their potential as diagnostics reagents is also being realized with the development of exciting new techniques for rapid bacterial detection and drug susceptibility testing. This review outlines the history of these remarkable organisms, from their discovery fifty years ago to the current developments in rapid diagnostic techniques. [TOP OF PAGE]

122. Virulence evolution in a virus obeys a trade-off. Messenger, S.L., Molineux, I.J., Bull, J.J. (1999). Proc. R. Soc. Lond. B 266:297-404. The evolution of virulence was studied in a virus subjected to alternating episodes of vertical and horizontal transmission. Bacteriophage f1 was used as the parasite because it establishes a debilitating but non-fatal infection that can be transmitted vertically (from a host to its progeny) as well as horizontally (infection of new hosts). Horizontal transmission was required of all phage at specific intervals, but was prevented otherwise. Each episode of horizontal transmission was followed by an interval of obligate vertical transmission, followed by an interval of obligate horizontal transmission etc. The duration of vertical transmission was eight times longer per episode in one treatment than in the other, thus varying the relative intensity of selection against virulence while maintaining selection for some level of virus production. Viral lines with the higher enforced rate of infectious transmission evolved higher virulence and higher rates of virus production. These results support the trade-off model for the evolution of virulence. [TOP OF PAGE]

123. Bacteriophages in the evolution of pathogen-host interactions. Miao, E.A., Miller, S.I. (1999). Proc. Natl. Acad. Sci. USA 96:9452-9454. The term "emerging infectious diseases" has recently been popularized as a way to describe the introduction of new infectious agents to human populations (1). Bacterial infectious diseases can "emerge" by different methods. Emergence may involve the discovery that a disease of unknown etiology has a microbial etiology, such as peptic ulcers caused by infection with Helicobacter pylori bacteria (2). Infectious diseases can also emerge as a result of exposure of specific human populations to microorganisms that are new to those populations. Such strains could emerge as new epidemics or pandemics, as was the case with the spread of the bacterial cause of cholera, Vibrio cholerae, from Asia to South America (3). In addition to these well appreciated mechanisms, advances in the understanding of the molecular basis of microbial pathogenesis have led to the hypothesis that more virulent bacterial strains could emerge through recent acquisition of virulence factors. The current worldwide epidemic of the Gram-negative enterobacteriaciae Salmonella has significant public health implications (4) and may provide an important example to illustrate this principle. [see http://www.pnas.org/cgi/reprint/96/17/9452.pdf for full-text pdf]. [TOP OF PAGE]

124. Comparative study of techniques used to recover viruses from residual urban sludge. Mignotte, B., Maul, A., Schwartzbrod, L. (1999). Journal of Virological Methods 78:71-80. Eight virus extraction techniques were compared on three types of residual urban sludge for simultaneous detection of infectious enteroviruses, somatic coliphages, F-specific RNA bacteriophages and Bacteroides fragilis bacteriophages. The highest virus counts were found in extracts obtained using three extraction techniques described by respectively using a 10% beef extract solution at pH 9 and sonication, using a 0.3 M NaCl/7% beef extract solution at pH 7.5 and freon, and finally using a 0.1 M borate buffer/3% beef extract solution at pH 9 and sonication. [TOP OF PAGE]

125. Isolation of additional bacteriophages with genomes of segmented double-stranded RNA. Mindich, L., Qiao, X., Qiao, J., Onodera, S., Romantschuk, M., Hoogstraten, D. (1999). J. Bacteriol. 181:4505-4508. Eight different bacteriophages were isolated from leaves of Pisum sativum, Phaseolus vulgaris, Lycopersicon esculentum, Daucus carota sativum, Raphanus sativum, and Ocimum basilicum. All contain three segments of double-stranded RNA and have genomic-segment sizes that are similar but not identical to those of previously described bacteriophage phi6. All appear to have lipid-containing membranes. The base sequences of some of the viruses are very similar but not identical to those of phi6. Three of the viruses have little or no base sequence identity to phi6. Two of the viruses, phi8 and phi12, contain proteins with a size distribution very different from that of phi6 and do not package genomic segments of phi6. Whereas phi6 attaches to host cells by means of a pilus, several of the new isolates attach directly to the outer membrane. Although the normal hosts of these viruses seem to be pseudomonads, those viruses that attach directly to the outer membrane can establish carrier states in Escherichia coli or Salmonella typhimurium. One of the isolates, phi8, can form plaques on heptoseless strains of S. typhimurium. [TOP OF PAGE]

126. Effect of population patchiness and migration rates on the adaptation and divergence of vesicular stomatitis virus quasispecies populations. Miralles, R., Moya, A., Elena, S.F. (1999). J. Gen. Virol. 80:2051-2059. The effect of migration among different isolated virus quasispecies populations on their adaptation and diversity was analysed through experimental evolution. An in vitro cell system was employed to simulate migration of vesicular stomatitis virus between isolated homogeneous host cell populations. The results clearly demonstrated a positive correlation between the migration rate and the magnitude of the mean fitness reached by the virus quasispecies populations. The results also showed, although less clearly, that fitness differences among quasispecies decreased with the magnitude of migration. These results are in close agreement with predictions of standard population genetics theory. These results can be explained in terms of the spread of beneficial mutations, originating in a single isolated quasispecies, through the entire system formed by the different quasispecies populations contained in different host cell populations. [TOP OF PAGE]

127. Clonal interference and the evolution of RNA viruses. Miralles, R., Gerrish, P.J., Moya, A., Elena, S.F. (1999). Science 285:1745-1747. In asexual populations, beneficial mutations that occur in different lineages compete with one another. This phenomenon, known as clonal interference, ensures that those beneficial mutations that do achieve fixation are of Large effect. Clonal interference also increases the time between fixations, thereby stowing the adaptation of asexual populations. The effects of clonal interference were measured in the asexual RNA virus vesicular stomatitis virus; rates and average effects of beneficial mutations were quantified. [TOP OF PAGE]

128. Isolation of a temperate bacteriophage encoding the type III effector protein SopE from an epidemic Salmonella typhimurium strain. Mirold, S., Rabsch, W., Rohde, M., Stender, S., Tschaepe, H., Ruessmann, H., Igwe, E., Hardt, W.D. (1999). Proc. Natl. Acad. Sci. USA 96:9845-9850. Salmonella typhimurium employs the specialized type III secretion system encoded in pathogenicity island 1 (SPI1) to translocate effector proteins into host cells and to modulate host cell signal transduction. The SPI1 type III system and the effector proteins are conserved among all salmonellae and are thought to be acquired by horizontal gene transfer. The genetic mechanisms mediating this horizontal transfer are unknown. Here, we describe that SopE, a SPI1-dependent translocated effector protein, is present in relatively few S. typhimurium isolates. We have isolated a temperate phage that encodes SopE. Phage morphology and DNA hybridization, as well as partial sequence information, suggest that this phage (SopEPHI) is a new member of the P2 family of bacteriophages. By lysogenic conversion this phage can horizontally transfer genes between different S. typhimurium strains. Strikingly, most of the isolates harboring SopEPHI belong to the small group of epidemic strains of S. typhimurium that have been responsible for a large percentage of human and animal salmonellosis and have persisted for a long period of time. Our data suggest that horizontal transfer of type III dependent effector proteins by lysogenic infection with bacteriophages (lysogenic conversion) may provide an efficient mechanism for fine-tuning the interaction of Salmonella spp. with their hosts. [TOP OF PAGE]

129. [Natural interspecific hybrids of transposable phages of Pseudomonas aeruginosa]. Mit'kina, L.N., Krylov, V.N. (1999). Genetika 35:1182-1190. Bacterial viruses of Pseudomonas aeruginosa assigned to two groups, D3112 and B3, recombine with very low frequencies. Previous study of the genome structure of intergroup hybrids suggested the incompatibility of some genetic modules of these bacteriophages. In this work, several natural hybrid transposable phages that had the genomes largely consisting of modules of phages from group D3112 and B3, were described. The discovery of these phages suggests the continuous genetic exchange in nature of these viruses belonging to different species. This model is considered as promising from the viewpoint of monitoring virus evolution. [TOP OF PAGE]

130. Escherichia coli O157 strains which caused Japanese outbreaks have residues of bacteriophage sequences. Miyahara, M., Konuma, H. (1999). Biological and Pharmaceutical Bulletin 22:1372-1375. Twelve strains of Escherichia coli O157 which caused outbreaks in Japan were used as DNA sources. The sequences of the gene encoding the Shiga toxin 2 in all 12 strains were almost identical and the sequences downstream of this gene were similar to that of bacteriophage 933W. [TOP OF PAGE]

131. The so-called chromosomal verotoxin genes are actually carried by defective prophages. Mizutani, S., Nakazono, N., Sugino, Y. (1999). DNA Research 6:141-143. [TOP OF PAGE]

132. Transmission of the methicillin resistance from Staphylococcus epidermidis to Staphylococcus aureus in mixed cultures. Mlynarczyk, A., Mlynarczyk, G., Jeljaszewicz, J. (1999). Medycyna Doswiadczalna i Mikrobiologia 51:199-205. In mixed cultures of staphylococci a transfer of the resistance to methicillin and penicillinase plasmids as well as tetracycline and chloramphenicol plasmids was investigated. It was shown that the resistance to methicillin was transferred in mixed cultures from one strain of S. aureus to another and from S. epidermidis to S. aureus. In both cases transfer of methicillin resistance required, the presence of penicillinase plasmid in recipient or donor strain. In the case of other markers transmission was independent. Moreover it was shown that the transfer of resistance genes in mixed cultures was mediated by bacteriophage of the serologic group A. [TOP OF PAGE]

133. The role of the spectral sensitivity curve in the selection of relevant biological dosimeters for solar UV monitoring. Modos, K., Gaspar, S., Kerekgyarto, T., Vink, A.A., Roza, L., Fekete, A. (1999). Journal of Photochemistry and Photobiology B Biology 53:20-25. To estimate the risk of enhanced UV-B radiation due to stratospheric ozone depletion, phage T7 and uracil thin-layer biological dosimeters have been developed, which weight the UV irradiance according to induced DNA damage. To study the molecular basis of the biological effects observed after UV irradiation, the spectral sensitivity curves of the two dosimeters and induction of the two major DNA photoproducts, cyclobutane pyrimidine dimers (CPDs) and 6-4 photoproducts ((6-4)PDs), in phage T7 have been determined for polychromatic UV sources. CPDs and (6-4)PDs are determined by lesion-specific monoclonal antibodies in an immunodotblot assay. Phage T7 and uracil biological dosimeters together with a Robertson-Berger (RB) meter have been used for monitoring environmental radiation from the polar region to the equator. The biologically effective dose (BED) established with the three different dosimeters increases according to the changes in the solar angle and ozone column, but the degree of the change differs significantly. The results can be explained based on the different spectral sensitivities of the dosimeters. A possible method for determining the trend of the increase in the biological risk due to ozone depletion is suggested. [TOP OF PAGE]

134. Applications of phage resistance in lactic acid bacteria. Moineau, S. (1999). Antonie van Leeuwenhoek 76:377-382. [TOP OF PAGE]

135. Codon usage and lateral gene transfer in Bacillus subtilis. Moszer, I., Rocha, E.P., Danchin, A. (1999). Curr Opin Microbiol 2:524-528. Bacillus subtilis possesses three classes of genes, differing by their codon preference. One class corresponds to prophages or prophage-like elements, indicative of the existence of systematic lateral gene transfer in this organism. The nature of the selection pressure that operates on codon bias is beginning to be understood. [TOP OF PAGE]

136. Study of the potential relationship between the morphology of infectious somatic coliphages and their persistence in the environment. Muniesa, M., Lucena, F., Jofre, J. (1999). Journal of Applied Microbiology 87:402-409. The proportions of different morphological types of infectious somatic coliphages were determined in faecally polluted freshwaters. Myoviridae, followed by Siphoviridae, were the most frequently isolated morphological types in raw sewage, treated sewage and river water collected a few metres downstreams from a sewage outfall. However, in river water collected further downstream from the pollution point, in river water after 'in situ' inactivation experiments and in chlorinated raw and treated sewage significant changes in the proportions of the different somatic coliphage morphological types occurred. In all cases, Siphoviridae, especially those with flexible and curled tails, became more abundant to the detriment of Myoviridae. [TOP OF PAGE]

137. Comparative survival of free shiga toxin 2-encoding phages and Escherichia coli strains outside the gut. Muniesa, M., Lucena, F., Jofre, J. (1999). Appl. Environ. Microbiol. 65:5615-5618. The behavior outside the gut of seeded Escherichia coli O157:H7, naturally occurring E. coli, somatic coliphages, bacteriophages infecting O157:H7, and Shiga toxin 2 (Stx2)-encoding bacteriophages was studied to determine whether the last persist in the environment more successfully than their host bacteria. The ratios between the numbers of E. coli and those of the different bacteriophages were clearly lower in river water than in sewage of the area, whereas the ratios between the numbers of the different phages were similar. In addition, the numbers of bacteria decreased between 2 and 3 log units in in situ survival experiments performed in river water, whereas the numbers of phages decreased between 1 and 2 log units. Chlorination and pasteurization treatments that reduced by approximately 4 log units the numbers of bacteria reduced by less than 1 log unit the numbers of bacteriophages. Thus, it can be concluded that Stx2-encoding phages persist longer than their host bacteria in the water environment and are more resistant than their host bacteria to chlorination and heat treatment. [TOP OF PAGE]

138. Occurrence and numbers of bacteriophages and bacterial indicators in faeces of yellow-legged seagull (Larus cachinnans). Muniesa, M., Jofre, J., Lucena, F. (1999). Letters in Applied Microbiology 29:421-423. Faeces from feral populations of yellow-legged seagulls from the northern coastal area of Catalonia (North-eastern Spain) contained variable amounts of faecal coliforms, faecal streptococci, somatic coliphages, F-specific bacteriophages and Bacteroides fragilis bacteriophages. Occurrence and numbers of bacterial indicators and bacteriophages in the faeces of yellow-legged seagulls are in the ranges described in the faeces of different animals. The ratios between numbers of bacterial indicators and numbers of bacteriophages are much higher in faeces of seagulls than in treated or raw sewage contributed by out-falls of the same area. [TOP OF PAGE]

139. Phage-inactivating effect of riboflavin phosphate and flavin-adenine dinucleotide. Murata, A., Oda, K., Sakai, S. (1999). Agric. Biol. Chem. 49:1881-1883. [TOP OF PAGE]

140. Model systems to study the parameters determining the success of phage antibody selections on complex antigens. Mutuberria, R., Hoogenboom, H.R., van, d.L., de, B.A., Roovers, R.C. (1999). Journal of Immunological Methods 231:65-81. Phage antibody display technology offers a powerful tool for the isolation of specific antibodies to defined target antigens. Most selection strategies described to date have relied on the availability of purified and often recombinant antigen, providing the possibility to perform selections on a well-defined antigen source. However, when the target antigen cannot be purified (e.g., an integral membrane protein), or if the antigen is unknown (e.g., when searching for novel markers on cells or tissues), panning of phage antibody libraries has to be performed on complex antigen sources such as cell surfaces or tissue sections, or even by in vivo selection methods. This provides a series of technical and experimental challenges. One focus of our research is to select antibodies directed to novel cancer-induced antigens expressed by tumours and by the tumour vasculature. To understand the parameters governing selection on complex antigen sources and to assess the efficiency of these phage library selections, we have set up two model selection systems in which both tumour cells and vascular endothelial cells serve as target "antigen". We describe a model based on phage antibodies directed to the tumour antigen epithelial glycoprotein-2, to compare phage antibody selections on a range of different antigen sources including purified and recombinant antigen, whole live cells, tissue cryosections and in vivo grown solid tumours. Secondly, we describe a model based on a phage antibody directed against the endothelial cell inducible adhesion molecule E-selectin. We compare selections on cultured cell monolayers with selections on cell suspensions immobilised on columns, to determine which selection approach is most suitable for the identification of novel tumour endothelial cell markers. Our data provide insight into the efficiency and thus potency of different selection strategies and show that there are very large differences in the recovery and enrichment of binding phage between the different methods tested. Our results further demonstrate the feasibility of phage antibody selections on whole, intact cells and show that these may sometimes compare favourably to selections on purified antigen. Selections on endothelial cells immobilised on columns compare favourably with selections on cell-monolayers; the most favourable conditions for both selection procedures are described. The implications of our data for phage antibody selections on these different complex antigen sources using either non-immune or immune phage antibody repertoires are discussed. The use of model systems such as the ones described here will help to determine optimal experimental conditions for phage library selections on complex antigens and aid in developing more powerful selection procedures for target discovery. [TOP OF PAGE]

141. Protective effects of bacteriophage on experimental Lactococcus garvieae infection in yellowtail. Nakai, T., Sugimoto, R., Park, K.-H., Matsuoka, S., Mori, K., Nishioka, T., Maruyama, K. (1999). Diseases of Aquatic Organisms 37:33-41. The present study describes the in vitro and in vivo survival of Lactococcus garvieae bacteriophages and the potential of the phage for controlling experimental L. garvieae infection in yellowtail. Anti-L. garvieae phages persisted well in various physicochemical (water temperature, salinity, pH) and biological (feed, serum and alimentary tract extracts of yellowtail) conditions, except for low acidity. In the in vivo, the phage PLgY-16 was detected in the spleens of yellowtail until 24 h after intraperitoneal (i.p.) injection, or the phage was recovered from the intestine of yellowtail 3 h after the oral administration of phage-impregnated feed but undetectable 10 h later. Simultaneous administration of live L. garvieae and phage enhanced recovery of the phage from the spleen or intestine. The survival rate was much higher in yellowtail that received i.p. injection of the phage after i.p. challenge with L. garvieae, compared with that of control fish without phage injection. When fish were i.p.-injected with phage at different hours after L. garvieae challenge, higher protective effects were demonstrated in fish that received phage treatment at the earlier time. Protection was also obtained in yellowtail receiving phage-impregnated feed, in which fish were challenged by an anal intubation with L. garvieae. Anal-intubated L. garvieae were detected constantly in the spleens of the control fish, while they were detected sporadically and disappeared from the phage-treated fish 48 h later. On the other hand, orally administered phage was detected at high plaque-forming units from the intestines and spleens of the phage-treated fish until 48 h later. These results indicate that intraperitoneally or orally administered anti-L. garvieae phage prevented fish from experimental L. garvieae infection, suggesting potential use of the phage for controlling the disease. [TOP OF PAGE]

142. A filamentous phage of Vibrio parahaemolyticus O3:K6 isolated in Laos. Nakasone, N., Ikema, M., Higa, N., Yamashiro, T., Iwanaga, M. (1999). Microbiology and Immunology 43:385-388. A filamentous phage, 'lvpf5,' of Vibrio parahaemolyticus O3:K6 strain LVP5 was isolated and characterized. The host range was not restricted to serotype O3:K6, but 7 of 99 V. parahaemolyticus strains with a variety of serotypes were susceptible to the phage. The phage was inactivated by heating at 80 C for 10 min and by treating with chloroform. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the phage exhibited a 3.8 kDa protein. The amino-terminal amino acid sequence of the coat protein was determined as AEGGAADPFEAIDLLGVATL. The phage genome consisted of a single-stranded DNA molecule. The activity of the phages was inhibited by anti-Na2 pili antibody. [TOP OF PAGE]

143. The complete nucleotide sequence of phi CTX, a cytotoxin-converting phage of Pseudomonas aeruginosa: implications for phage evolution and horizontal gene transfer via bacteriophages. Nakayama, K., Kanaya, S., Ohnishi, M., Terawaki, Y., Hayashi, T. (1999). Molecular Microbiology 31:399-419. phi CTX is a cytotoxin-converting phage isolated from Pseudomonas aeruginosa. In this study, we determined the complete nucleotide sequence of the phi CTX phage genome. The precise genome size was 35,538 bp with 21 base 5'-extruding cohesive ends. Forty-seven open reading frames (ORFs) were identified on the phi CTX genome, including two previously identified genes, ctx and int. Among them, 15 gene products were identified in the phage particle by protein microsequencing. The most striking feature of the phi CTX genome was an extensive homology with the coliphage P2 and P2-related phages; more than half of the ORFs (25 ORFs) had marked homology to P2 genes with 28.9-65.8% identity. The gene arrangement on the genome was also highly conserved for the two phages, although the G + C content and codon usage of most phi CTX genes were similar to those of the host P. aeruginosa chromosome. In addition, phi CTX was found to share several common features with P2, including the morphology, non-inducibility, use of lipopolysaccharide core oligosaccharide as receptor and Ca(2+)-dependent receptor binding. These findings indicate that phi CTX is a P2-like phage well adapted to P. aeruginosa, and provide clear evidence of the intergeneric spread and evolution of bacteriophages. Furthermore, comparative analysis of genome structures of phi CTX, P2 and other P2 relatives revealed the presence of several hot-spots where foreign DNAs, including the cytotoxin gene, were inserted. They appear to be deeply concerned in the acquisition of various genes that are horizontally transferred by bacteriophage infection. [TOP OF PAGE]

144. Quantitative assessment of the inactivation of pathogenic and indicator viruses in natural water sources. Nasser, A.M., Oman, S.D. (1999). Water Res. 33:1748-1752. Suitable monitoring of drinking water sources may prevent the occurrence of waterborne disease. Furthermore, determining the environmental factors which affect virus inactivation may help in predicting the fate of pathogenic microorganisms in water sources. This study was conducted to evaluate the suitability of F+ bacteriophages as indicators for microbial contamination of water sources and to determine the effects of water quality, temperature and virus type on virus inactivation. The inactivation of hepatitis A virus (HAV), poliovirus 1, F+ bacteriophages and E. coli was compared in groundwater and wastewater effluents at various temperatures. Similar inactivation patterns were observed for HAV and poliovirus 1 under various experimental conditions. Male-specific bacteriophages persisted for the longest time in the various water types, whereas E. coli inactivation was the fastest in groundwater at 4 and 37degreeC. F+ bacteriophages have been found more suitable than E. coli to predict the inactivation of pathogenic viruses in natural water sources. The effects of temperature and microorganism type have been found significant in all studied water types. The interaction between temperature and microorganism type was highly significant in groundwater and phosphate buffered saline and less significant in raw wastewater, indicating that wastewater may enhance virus persistence. The difference between the inactivation of F+ bacteriophages and E. coli was significant under all experimental conditions, indicating their suitability to signal the persistence of pathogenic viruses in natural water sources. [TOP OF PAGE]

145. A whole-glove method for the evaluation of surgical gloves as barriers to viruses. Nelson, J.R., Roming, T.A., Bennet, J.K. (1999). American Journal of Contact Dermatitis 10:183-189. BACKGROUND: Today, because of the wide variety of infectious agents encountered in the health care environment, clinicians must be particularly concerned about the potential for small-sized virus penetration through glove defects. OBJECTIVE: To describe a method for testing gloves that evaluates the entire glove and allows for detection of low levels of virus penetration. Ten sets of 10 different gloves from 4 manufacturers were evaluated using this method. METHODS: Barrier properties were evaluated using the bacteriophage, phiX174. Gloves were filled with surfactant solution placed in flasks containing 10(6) viruses per mL. Flasks were agitated at 37 degrees C +/- 2 degrees C and assayed for 180 minutes. RESULTS: Virus penetration was detected in 8% of the 100 gloves tested using the quantitative assay. The qualitative assay determined that 14% of the gloves tested allowed penetration. [TOP OF PAGE]

146. Bacterial and chemical quality of water supply in the Dertig village settlement. Nevondo, T.S., Cloete, T.E. (1999). Water S A (Pretoria) 25:215-220. Water contaminated with microbiological and chemical constituents can cause a variety of diseases. Water intended for human consumption should be safe, palatable and aesthetically pleasing. Water sources have different qualities influenced by natural or anthropological pollution. In South Africa, the availability of safe and clean water is a serious problem, especially in rural areas. Most people in such areas use water directly from available sources without any treatment and therefore are exposed to a variety of water-related diseases. The objective of this study was to determine the chemical and microbiological quality of drinking water supply to a rural community in order to estimate the health implications thereof. Water samples were collected weekly from five water sources, that is, Lefatlheng Well, Tlhaloganyo groundwater, Tlhaloganyo rain water, Matlaisane groundwater and Tshwane River in the Dertig/Lefatlheng village settlement which is in Hammanskraal, about 55 km north of Pretoria. To provide an indication of the microbiological quality of the water resources, indicator organisms including heterotrophic bacteria, faecal coliform, total coliform, Salmonella and coliphages were used. In order to support the results, bacterial isolates were identified using both the 20E and 20NE API systems to confirm their isolation. For the chemical quality analyses, different chemical quality variables including temperature, pH, dissolved oxygen (DO), aluminium (A1), iron (Fe), manganese (Mn), fluoride (F), nitrate (NO3), nitrite (NO2) and colour were determined. The chemical quality of all the water sources analysed was acceptable. In contrast, however, the microbiological quality of all the water sources exceeded the standard for potable water and the sources pose a serious health risk to consumers. [TOP OF PAGE]

147. Genetic variation of chlorella viruses: Variable regions localized on the CVK2 genomic DNA. Nishida, K., Kimura, Y., Kawasaki, T., Fujie, M., Yamada, T. (1999). Virology 255:376-384. [TOP OF PAGE]

148. Breakdown and microbial uptake of marine viruses and other lysis products. Noble, R.T., Fuhrman, J.A. (1999). Aquat. Microb. Ecol. 20:1-11. To understand the roles of marine viruses in marine microbial food webs, it is important to determine rates and mechanisms of virus degradation and subsequent uptake of degraded virus material and other cell lysis products by heterotrophic marine bacteria. We radiolabeled and concentrated viruses and viral lysis products from either pure cultures (³H) or natural communities (³H and ³³P) and added them to seawater samples of differing trophic status from coastal (mesotrophic) and offshore (oligotrophic) California waters and French Mediterranean waters (oligotrophic). Rates of degradation were determined by the loss of high molecular weight radiolabel over time and the fate of the degraded material (microbial uptake or accumulation in low molecular weight pools) was followed by size fractionation and/or acid extraction. Preliminary experiments with ³H-labeled, single-stranded RNA phage MS2 and marine phage H11/1 demonstrated that MS2 degraded significantly faster in coastal Santa Monica Bay seawater (2.5 ± 0.6% h^-1), than the marine phage, H11/1 (0.99 ± 0.1% h^-1). For labeled virus material from natural populations, rates of degradation were slower in oligotrophic waters (ranges from 1.0 to 3.3% h^-1) than in mesotrophic waters (ranges from 4.9 to 6.0% h^-1), corresponding to turnover rates of 1 to 4 d for this material. Degradation rates of labeled virus material are likely underestimates, because during preparation, degradation and uptake are continually occurring, resulting in accumulation of the less reactive products. The proportion of radiolabeled material taken up by microbes was greatest in oligotrophic waters, especially in the phosphate-limited Villefranche Bay, France, where most of the ³³PO₄-labeled material was taken up in less than 7 h. In contrast, the majority of degraded ³H-labeled material was not accumulated into biomass, and in 3 of 4 samples, accumulation was hardly detectable. The results suggest that viruses and lysis products are labile and turn over relatively rapidly, but often may not be efficiently incorporated into bacterial biomass. [TOP OF PAGE]

149. The effects of viral enrichment on the mortality and growth of heterotrophic bacterioplankton. Noble, R.T., Middelboe, M., Fuhrman, J.A. (1999). Aquat. Microb. Ecol. 18:1-13. [TOP OF PAGE]

150. The fates of viruses in the marine environment. Noble, R.T. (1999). University of Southern California. Viruses are an important component of the marine microbial food web, as they are capable of contributing to a significant fraction of the mortality of heterotrophic bacterioplankton. To better understand the ecological roles of viruses in the ocean and their possible influences upon biogeochemical cycles, I studied the fates of viruses in relation to other components of the microbial food web. the fates of viruses were studied by examining loss of infectivity, biochemical degradation, the effects of viral enrichment on bacterial mortality, and virus production. Spatio-temporal analysis of surface seawater of Santa Monica Bay over five years demonstrated significantly higher viral and bacterial abundances during the rainy season, with nearly constant virus to bacterial ratios of about 10. Loss of infectivity was studied with the use of eight laboratory cultured host/virus systems. The decay of infectivity of these viruses was assessed in seawater, and was partitioned according to singular causative agents of decay, such as ultraviolet light, heat labile material such as extracellular enzymes, and/or particles for adsorption. Virus isolates native to Santa Monica Bay consistently degraded more slowly in full sunlight than bacteriophages isolated from the North Sea, and although sunlight was an important contributing factor to virus decay, decay due to particles and dissolved substances in seawater was also significant. [TOP OF PAGE]

151. Exponential fitness gains of RNA virus populations are limited by bottleneck effects. Novella, I.S., Quer, J., Domingo, E., Holland, J.J. (1999). J. Virol. 73:1668-1671. Fitness is a parameter that quantitatively measures adaptation of a virus to a given environment. We have previously reported exponential fitness gains of large populations of vesicular stomatitis virus replicating in a constant environment (I. S. Novella et al., Proc. Natl. Acad. Sci. USA 92:5841-5844, 1995). In this paper, we report that during long-term passage of such large viral populations, fitness values reached a high-fitness plateau during which stochastic fitness variations were observed. This effect appears likely to be due to bottleneck effects on very high fitness populations. [TOP OF PAGE]

152. Lack of evolutionary stasis during alternating replication of an arbovirus in insect and mammalian cells. Novella, I.S., Hershey, C.L., Escarmis, C., Domingo, E., Holland, J.J. (1999). J. Mol. Biol. 287:459-465. The evolution of vesicular stomatitis virus (VSV) in a constant environment, consisting of either mammalian or insect cells, has been compared to the evolution of the same viral population in changing environments consisting in alternating passages in mammalian and insect cells. Fitness increases were observed in all cases. An initial fitness loss of VSV passaged in insect cells was noted when fitness was measured in BHK-21 cells, but this effect could be attributed to a difference of temperature during VSV replication at 37 degrees C in BHK-21 cells. Sequencing of nucleotides 1-4717 at the 3' end of the VSV genome (N, P, M and G genes) showed that at passage 80 the number of mutations accumulated during alternated passages (seven mutations) is similar or larger than that observed in populations evolving in a constant environment (two to four mutations). Our results indicate that insect and mammalian cells can constitute similar environments for viral replication. Thus, the slow rates of evolution observed in natural populations of arboviruses are not necessarily due to the need for the virus to compromise between adaptation to both arthropod and vertebrate cell types. [TOP OF PAGE]

153. Phage-lift for game theory. Nowak, M.A., Sigmund, K. (1999). Nature 398:367-368. The prisoner's dilemma is a classic of game theory in which acting for individual advantage is pitted against acting for collective benefit. An example has been identified among clones of a virus that infects bacteria. [TOP OF PAGE]

154. A possible role of temperate phage in the regulation of Trichodesmium biomass. Ohki, K. (1999). Bulletin de l'Institut Oceanographique (Monaco) 0:287-291. [TOP OF PAGE]

155. Microbiological studies in the Dead Sea: Future challenges toward the understanding of life at the limit of salt concentrations. Oren, A. (1999). Hydrobiologia 1-9. no abstract. [TOP OF PAGE]

156. Bacteriophage: tools toward a cell-targeted delivery [comment]. Paillard, F. (1999). Human Gene Therapy 9:2307-2308. [TOP OF PAGE]

157. Microbial gene transfer: an ecological perspective. Paul, J.H. (1999). J. Mol. Microbiol. Biotechnol. 1:45-50. Microbial gene transfer or microbial sex is a means of exchanging loci amongst prokaryotes and certain eukaryotes. Historically viewed as a laboratory artifact, recent evidence from natural populations as well as genome research has indicated that this process may be a major driving force in microbial evolution. Studies with natural populations have taken two approaches-either adding a defined donor with a traceable gene to an indigenous community, and detecting the target gene in the indigenous bacteria, or by adding a model recipient to capture genes being transferred from the ambient microbial flora. However, both approaches usually require some cultivation of the recipient, which may result in a dramatic underestimation of the ambient transfer frequency. Novel methods are just evolving to study in situ gene transfer processes, including the use of green fluorescent protein (GFP)-marked plasmids, which enable detection of transferrants by epifluorescence microscopy. A transduction-like mechanism of transfer from viral-like particles produced by marine bacteria and thermal spring bacteria to Escherichia coli has been documented recently, indicating that broad host range transduction may be occurring in aquatic environments. The sequencing of complete microbial genomes has shown that they are a mosaic of ancestral chromosomal genes interspersed with recently transferred operons that encode peripheral functions. Archaeal genomes indicate that the genes for replication, transcription, and translation are all eukaryotic in complexity, while the genes for intermediary metabolism are purely bacterial. And in eukaryotes, many ancestral eukaryotic genes have been replaced by bacterial genes believed derived from food sources. Collectively these results indicate that microbial sex can result in the dispersal of loci in contemporary microbial populations as well as having shaped the phylogenies of microbes from multiple, very early gene transfer events. [see http://www.jmmb.net/v1n1/07/07.html for full-text entry]. [TOP OF PAGE]

158. Poor efficacy of residual chlorine disinfectant in drinking water to inactivate waterborne pathogens in distribution systems. Payment, P. (1999). Can. J. Microbiol. 45:709-715. To evaluate the inactivating power of residual chlorine in a distribution system, test microorganisms (Escherichia coli, Clostridium perfringens, bacteriophage phi-X 170, and poliovirus type 1) were added to drinking water samples obtained from two water treatment plants and their distribution system. Except for Escherichia coli, microorganisms remained relatively unaffected in water from the distribution systems tested. When sewage was added to the water samples, indigenous thermotolerant coliforms were inactivated only when water was obtained from sites very close to the treatment plant and containing a high residual chlorine concentration. Clostridium perfringens was barely inactivated, suggesting that the most resistant pathogens such as Giardia lamblia, Cryptosporidium parvum, and human enteric viruses would not be inactivated. Our results suggest that the maintenance of a free residual concentration in a distribution system does not provide a significant inactivation of pathogens, could even mask events of contamination of the distribution, and thus would provide only a false sense
     of safety with little active protection of public health. Recent epidemiological studies that have suggested a significant waterborne level of endemic gastrointestinal illness could then be explained by undetected intrusions in the distribution system, intrusions resulting in the infection of a small number of individuals without eliciting an outbreak situation. [TOP OF PAGE]

159. Effect of biocides commonly used in the hospital environment on the transfer of antibiotic-resistance genes in Staphylococcus aureus. Pearce, H., Messager, S., Maillard, J.Y. (1999). Journal of Hospital Infection 43:101-107. The effect of sub-minimal inhibitory concentrations of biocides, commonly used in the hospital environment, on the conjugation and transduction of plasmid pWG613 was investigated in three strains of Staphylococcus aureus. The highest transfer frequency was obtained in the conjugation experiments. A low concentration of povidone-iodine was found to significantly reduce transfer frequency by 10-fold in S. aureus SAU3/13136 mating, while other biocides had no effect at low concentrations. Cetrimide (0.0001%) was found to increase significantly transduction efficiency in S. aureus RF2 when the biocide was included in the recovery media. A low concentration of chlorhexidine or povidone-iodine reduced transduction efficiency in the same recipient. This study showed that reduction in transduction efficiency was caused by the direct effect of biocides on the recipient strains rather than on the phage 80 alpha particles. [TOP OF PAGE]

160. Induction of the phage resistance in the progeny of an infected bacterial cell. Pererva, T.P. (1999). Biopolimery i Kletka 15:63-66. Using bacteriophages MS2, P17 and lambda-vir infecting Escherichia coli AB 259 (Hfr 3000), E. coli M17 and E. coli C600 cells appropriately the author demonstration that both RNA- and DNA-containing phages induce the development of phage-resistant forms appearing with high frequency in the progeny of infected cells. [TOP OF PAGE]

161. [Evaluation of the usefulness of new international experimental phages for typing methicillin resistant Staphylococcus aureus (MRSA)]. Piechowicz, L., Wisniewska, K., Galinski, J. (1999). Medycyna Doswiadczalna i Mikrobiologia 51:31-36. The aim of the study was to determine the usefulness of the set of experimental phages obtained from the Central Public Health Laboratory in London for typing of MRSA strains in Poland. The study was performed on 150 MRSA strains isolated from various clinical materials in various regions of the country. The set of 10 experimental phages and the international basic set of 23 phages were used for typing. The results of the study showed that 76.8% of MRSA strains were typing with the experimental set of phages. The frequency of inhibition reactions was 19.9%. Only 3.3% of the strains were nontypable with the new phages while nearly half of the studied strains were nontypable with the basic set of phages. The studied strains were divided into 19 phagotypes. There was a high frequency of typable strains among MRSA typable and nontypable strains and those inhibited by the basic set of phages (71.4%-85.7%). These data indicate that the set of 10 experimental phages is useful for typing of MRSA strains isolated in Poland except for phage M3 which failed to react with almost all the strains and should be excluded from the proposed set. [TOP OF PAGE]

162. Dependence of phage infection efficiency on the Staphylococcus cells energetic status. Polishko, T.N., Vinnikov, A.I. (1999). Ukrainskii Biokhimicheskii Zhurnal 71:28-32. Studies of phage infection efficiency in Staphylococcus aureus showed its dependence on the energy processes. Addition of KCN, dycyclohexylcarbodiimide and chlorocarbonylcyanide-phenylhydrazone at the moment of cells contact with bacteriophages lowered phagolysis efficiency to 49,5-68,0%. These inhibitors influenced on cell specifically leading to suppressing generators of protonmotive force and to dissipating membrane potential. Adding phages to the suspension of bacterial led to the dissipation of membrane potential. [TOP OF PAGE]

163. An evaluation of the efficacy of a wastewater treatment plant. Poncetta, D., Maiello, A., Guidetti, L., Moiraghi, R.A. (1999). Igiene Moderna 112:1-15. Effectiveness of wastewater treatment systems for microbiological pollution removal is a significant hygienic-sanitary problem, principally for the evaluation of effects on basins where the depuration final effluents flow. This study aimed to verify, from a microbiological viewpoint, the effectiveness of the activated sludge depuration process of Verbania and the environmental impact that its effluent has in comparison with the Lago Maggiore waters it flows into. The bacteric pollution extent has been evaluated quantifying the presence of fecal pollution indicators (total and fecal coliforms, fecal streptococci), sulfite-reducing clostridia spores and the presence of pathogens bacteria (salmonella) through classic colony techniques (membrane filter, MPN and pour plate method). The presence of enterovirus and bacteriophages have also been detected through isolation on cell cultures and lysis plaques. The results indicate a substantial reduction of all detected microorganisms of at least 2 log after the activated sludge treatment, and at least 3 log after chloration, with the disappearance of bacteriophages and salmonella in many samples. Enterovirus detection, based on qualitative methods, gave positive results in 95% of samples collected at the entry to the plant, whereas at the exit 63,2% of the tested samples resulted positive for ECP and this percentage fell to 15,8% after disinfection treatment. The positive samples were confirmed by biological molecular techniques. The comparison of the microbiological features surveyed in the coastal waters where the treatment plant effluent flows before and after its coming into operation shows an improvement of hygienic conditions. This situation is probably was due to the discharged effluent quality and to the convey to the plant of built-up area wastewater, which, in the past, directly reached the lake without passing through any depuration treatment. [TOP OF PAGE]

164. Targeted gene delivery to mammalian cells by filamentous bacteriophage. Poul, M.A., Marks, J.D. (1999). J. Mol. Biol. 288:203-211. We report that prokaryotic viruses can be re-engineered to infect eukaryotic cells resulting in expression of a reporter gene inserted into the bacteriophage genome. Phage capable of binding mammalian cells expressing the growth factor receptor ErbB2 and undergoing receptor-mediated endocytosis were isolated by selection of a phage antibody library on breast tumor cells and recovery of infectious phage from within the cell. As determined by immunofluorescence, F5 phage were efficiently endocytosed into 100 % of ErbB2 expressing SKBR3 cells. To achieve reporter gene expression, F5 phage were engineered to package the green fluorescent protein (GFP) reporter gene driven by the CMV promoter. These phage when applied to cells underwent ErbB2-mediated endocytosis leading to GFP expression. GFP expression occurred only in cells overexpressing ErbB2, was dose-dependent reaching, 4 % of cells after 60 hours and was detected with phage titers as low as 2.0 x 10(7) cfu/ml (500 phage/cell). The results demonstrate that bacterial viruses displaying the appropriate antibody can bind to mammalian receptors and utilize the endocytic pathway to infect eukaryotic cells, resulting in expression of a reporter gene inserted into the viral genome. This represents a novel method to discover targeting molecules capable of delivering a gene intracellularly into the correct trafficking pathway for gene expression by directly screening phage antibodies. This should significantly facilitate the identification of appropriate targets and targeting molecules for gene therapy or other applications where delivery into the cytosol is required. This approach can be adapted to directly select, rather than screen, phage antibodies for targeted gene expression. The results also demonstrate the potential of phage antibodies as an in vitro or in vivo targeted gene delivery vehicle. [TOP OF PAGE]

165. Virus removal from sewage effluents during saturated and unsaturated flow through soil columns. Powelson, D.K., Gerba, C.P. (1999). Water Res. 28:2175-2181. Recharge of sewage effluents may lead to contamination of groundwater with viruses. The goal of this research was to quantify virus removal in representative subsurface transport conditions. Soil column and batch studies were conducted to evaluate how virus type, effluent type and water saturation affect virus adsorption and removal. Three viruses were used: MS2 and PRD1 bacteriophages and poliovirus type 1. In the first column study, secondary- or tertiary-treated sewage containing the viruses percolated through coarse-sand columns under unsaturated conditions. In the second column study, the viruses suspended in secondary-treated sewage percolated through the columns under saturated or unsaturated conditions. A batch adsorption study was conducted to determine equilibrium adsorption of these viruses to the sand. Effluent type had no significant effect on first-order virus removal coefficients or retardation of virus transport. Virus "removal" was considered to be inactivation or irreversible adsorption. Unsaturated conditions resulted in an average removal coefficient (mu^-s = 0.31 h^-1) more than three times greater than saturated conditions (mu^-s= 0.095 h^-1), a significant difference at the 0.01 level. Poliovirus had a greater retardation coefficient (R = 5.2) than the bacteriophages (MS2, R = 1.4; and PRD1, R = 2.2), a significant difference at the 0.001 level. Column retardations of virus transport were only 0.8-8.0% of that predicted by adsorption coefficients determined from the batch studies. Equations developed in this paper may aid in estimating virus removal during recharge of effluents if the water residence times in ponds, the vadose zone and the aquifer are known. [TOP OF PAGE]

166. A novel virus family, the Rudiviridae: Structure, virus-host interactions and genome variability of the sulfolobus viruses SIRV1 and SIRV2. Prangishvili, D., Arnold, H.P., Gotz, D., Ziese, U., Holz, I., Kristjansson, J.K., Zillig, W. (1999). Genetics 152:1387-1396. The unenveloped, stiff-rod-shaped, linear double-stranded DNA viruses SIRV1 and SIRV2 from Icelandic Sulfolobus isolates form a novel virus family, the Rudiviridae. The sizes of the genomes are 32. 3 kbp for SIRV1 and 35.8 kbp for SIRV2. The virions consist of a tube-like superhelix formed by the DNA and a single basic 15.8-kD DNA-binding protein. The tube carries a plug and three tail fibers at each end. One turn of the DNA-protein superhelix measures 4.3 nm and comprises 16.5 turns of B DNA. The linear DNA molecules appear to have covalently closed hairpin ends. The viruses are not lytic and are present in their original hosts in carrier states. Both viruses are quite stable in these carrier states. In several laboratory hosts SIRV2 was invariant, but SIRV1 formed many different variants that completely replaced the wild-type virus. Some of these variants were still variable, whereas others were stable. Up to 10% nucleotide substitution was found between corresponding genome fragments of three variants. Some variants showed deletions. Wild-type SIRV1, but not SIRV2, induces an SOS-like response in Sulfolobus. We propose that wild-type SIRV1 is unable to propagate in some hosts but surmounts this host range barrier by inducing a host response effecting extensive variation of the viral genome. [TOP OF PAGE]

167. Diversity of Bacteroides fragilis strains in their capacity to recover phages from human and animal wastes and from fecally polluted wastewater. Puig, A., Queralt, N., Jofre, J., Araujo, R. (1999). Appl. Environ. Microbiol. 65:1772-1776. Great differences in capability to detect bacteriophages from urban sewage of the area of Barcelona existed among 115 strains of Bacteroides fragilis. The capability of six of the strains to detect phages in a variety of feces and wastewater was studied. Strains HSP40 and RYC4023 detected similar numbers of phages in urban sewage and did not detect phages in animal feces. The other four strains detected phages in the feces of different animal species and in wastewater of both human and animal origin. Strain RYC2056 recovered consistently higher counts than the other strains and also detected counts ranging from 10¹ to approximately 10³ phages per ml in urban sewage from different geographical areas. This strain detected bacteriophages in animal feces even though their relative concentration with respect to the other fecal indicators was significantly lower in wastewater polluted with animal feces than in urban sewage. [TOP OF PAGE]

168. Inactivation of Lactobacillus helveticus bacteriophages by thermal and chemical treatments. Quiberoni, A. (1999). J. Food. Prot. 62:894-898. The effect of several biocides and thermal treatments on the viability of four Lactobacillus helveticus phages was investigated. Times to achieve 99% inactivation of phages at 63 degrees C and 72 degrees C in three suspension media were calculated. The three suspension media were tris magnesium gelatin buffer (10 mM Tris-HCl, 10 mM MgSO4, and 0.1% wt/vol gelatin), reconstituted skim milk sterile reconstituted commercial nonfat dry skim milk, and Man Rogosa Sharpe broth. The thermal resistance depended on the phage considered, but a treatment of 5 min at 90 degrees C produced a total inactivation of high titer suspensions of all phages studied. The results obtained for the three tested media did not allow us to establish a clear difference among them, since some phages were more heat resistant in Man Rogosa Sharpe broth and others in tris magnesium gelatin buffer. From the investigation on biocides, we established that sodium hypochlorite at a concentration of 100 ppm was very effective in inactivating phages. The suitability of ethanol 75%, commonly used to disinfect utensils and laboratory equipment, was confirmed. Isopropanol turned out to be, in general, less effective than ethanol at the assayed concentrations. In contrast, peracetic acid (0.15%) was found to be an effective biocide for the complete inactivation of all phages studied after 5 min of exposure. The results allowed us to establish a basis for adopting the most effective thermal and chemical treatments for inactivating phages in dairy plant and laboratory environments. [TOP OF PAGE]

169. Bacterial lysis by phage--a theoretical model. Rabinovitch, A., Zaritsky, A., Fishov, I., Einav, M., Hadas, H. (1999). J. Theor. Biol. 201:209-213. The similarity to materials corrosion is invoked to develop a model for phage-infected bacterial lysis based on the statistics of extremes. The importance of cell size, envelope thickness and lysozyme eclipse time on the final probability distribution of lysis is considered. Experiments are suggested to test the model. [TOP OF PAGE]

170. Model for bacteriophage T4 development in Escherichia coli. Rabinovitch, A., Hadas, H., Einav, M., Melamed, Z., Zaritsky, A. (1999). J. Bacteriol. 181:1677-1683. Mathematical relations for the number of mature T4 bacteriophages, both inside and after lysis of an Escherichia coli cell, as a function of time after infection by a single phage were obtained, with the following five parameters: delay time until the first T4 is completed inside the bacterium (eclipse period, nu) and its standard deviation (sigma), the rate at which the number of ripe T4 increases inside the bacterium during the rise period (alpha), and the time when the bacterium bursts (mu) and its standard deviation (beta). Burst size [B = alpha(mu - nu)], the number of phages released from an infected bacterium, is thus a dependent parameter. A least-squares program was used to derive the values of the parameters for a variety of experimental results obtained with wild-type T4 in E. coli B/r under different growth conditions and manipulations (H. Hadas, M. Einav, I. Fishov, and A. Zaritsky, Microbiology 143:179-185, 1997). A "destruction parameter" (zeta) was added to take care of the adverse effect of chloroform on phage survival. The overall agreement between the model and the experiment is quite good. The dependence of the derived parameters on growth conditions can be used to predict phage development under other
     experimental manipulations. [TOP OF PAGE]

171. Prevention of Clostridium difficile-induced ileocecitis with bacteriophage. Ramesh, V., Fralick, J.A., Rolfe, R.D. (1999). Anaerobe 5:69-78. A bacteriophage specific for Clostridium difficile was examined for its ability to prevent ileocecitis in a hamster model. This species- and strain-specific bacteriophage was isolated from a lysogenic strain of C. difficile. Hamsters were maintained in sterile isolation cages to prevent the acquisition of C. difficile from the environment. Bicarbonate neutralization of gastric acidity was necessary for bacteriophage survival in the hamster's gastrointestinal tract. Bacteriophage recovery from the hamster cecum was 2 X 10⁴ plaque forming units/mL of cecal contents 24 h after orogastric challenge with 10⁸ plaque forming units/mL of bacteriophage. However, there was no bacteriophage recovery 48 h post challenge, indicating dissipation of bacteriophage from the hamster intestinal tract within this time frame. Twenty-four hours after being challenged with clindamycin, one group of hamsters was challenged with C. difficile followed by a single dose of bacteriophage (108 plaque forming units/mL). Two additional groups of hamsters received phage doses immediately after C. difficile challenge and subsequently thereafter every 8 h up to 48 and 72 h, respectively. The gastric acidity was neutralized with bicarbonate buffer preceding every bacteriophage treatment. Control animals that received only clindamycin and C. difficile died within 96 h after challenge while the majority of bacteriophage treated hamsters survived. Two weeks after stopping bacteriophage treatment, the surviving hamsters were rechallenged with clindamycin and C. diifficile. All the hamsters died within 96 h indicating susceptibility of the surviving hamsters to C. difficile disease in the absence of bacteriophage treatment. [TOP OF PAGE]

172. RecA-dependent viral burst in bacterial colonies during the entry into stationary phase. Ramirez, E., Schmidt, M., Rinas, U., Villaverde, A. (1999). FEMS Microbiol. Let. 170:313-317. We have explored the nature of the sudden viral amplification observed during the ageing of P22-infected lysogenic colonies of Salmonella typhimurium [Ramirez, E, and Villaverde, A. (1997) Gene 202, 147-149]. By a comparative analysis of the wild-type P22 and a P22 integration mutant, it has been shown that the conditions promoting prophage induction occur in only a small portion of the bacterial population and briefly during the transition between the exponential growth and the stationary phase. The viral burst is RecA-dependent and cannot be reproduced in continuous culture by a mere decrease of the growth rate. This suggests that the limited viral propagation in colonies is probably linked to heterogeneous physiological conditions within colonial populations, distinct from those of the homogeneous liquid cultures. [TOP OF PAGE]

173. A high incidence of prophage carriage among natural isolates of Streptococcus pneumoniae. Ramirez, M., Severina, E., Tomasz, A. (1999). J. Bacteriol. 181:3618-3625. The majority (591 of 791, or 76%) of Streptococcus pneumoniae clinical isolates examined showed the presence of two or more chromosomal SmaI fragments that hybridized with the lytA-specific DNA probe. Only one of these fragments, frequently having an approximate molecular size of 90 kb, was shown to carry the genetic determinant of the pneumococcal autolysin (N-acetylmuramic acid-L-alanine amidase). Strains carrying multiple copies of lytA homologues included both antibiotic-susceptible and -resistant isolates as well as a number of different serotypes and strains recovered from geographic sites on three continents. Mitomycin C treatment of strains carrying several lytA-hybridizing fragments caused the appearance of extrachromosomal DNA hybridizing to the lytA gene, followed by lysis of the bacteria. Such lysates contained phage particles detectable by electron microscopy. The findings suggest that the lytA-hybridizing fragments in excess of the host lytA represent components of pneumococcal bacteriophages. The high proportion of clinical isolates carrying multiple copies of lytA indicates the widespread occurrence of lysogeny, which may contribute to genetic variation in natural populations of pneumococci. [TOP OF PAGE]

174. High bacterial diversity in permanently cold marine sediments. Ravenschlag, K., Sahm, K., Pernthaler, J., Amann, R. (1999). Appl. Environ. Microbiol. 65:3982-3989. A 16S ribosomal DNA (rDNA) clone library from permanently cold marine sediments was established. Screening 353 clones by dot blot hybridization with group-specific oligonucleotide probes suggested a predominance of sequences related to bacteria of the sulfur cycle (43.4% potential sulfate reducers). Within this fraction, the major cluster (19.0%) was affiliated with Desulfotalea sp. and other closely related psychrophilic sulfate reducers isolated from the same habitat. The cloned sequences showed between 93 and 100% similarity to these bacteria. Two additional groups were frequently encountered: 13% of the clones were related to Desulfuromonas palmitatis, and a second group was affiliated with Myxobacteria spp. and Bdellovibrio spp. Many clones (18.1%) belonged to the gamma subclass of the class Proteobacteria and were closest to symbiotic or free-living sulfur oxidizers. Probe target groups were further characterized by amplified rDNA restriction analysis to determine diversity within the groups and within the clone library. Rarefaction analysis suggested that the total diversity assessed by 16S rDNA analysis was very high in these permanently cold sediments and was only partially revealed by screening of 353 clones. [TOP OF PAGE]

175. Bacteriophage therapy of Clostridium difficile-associated intestinal disease in a hamster model. Rdamesh, V., Fralick, J.A., Rolfe, R.D. (1999). Miroecol. Anarobes[sic?] 5:69-??? [TOP OF PAGE]

176. Physicochemical mechanisms responsible for the filtration and mobilization of a filamentous bacteriophage in quartz sand. Redman, J.A., Grant, S.B., Olson, T.M., Adkins, J.M., Jackson, J.L., Castillo, M.S., Yanko, W.A. (1999). Water Res. 33:43-52. This study examines the influence of pore water chemistry on the filtration and physicochemical properties of a mate-specific filamentous bacteriophage isolated from chlorinated effluent of the San Jose Creek Water Reclamation Plant in Los Angeles County, California. The isolate belongs to a class of bacteriophage that are naturally present in sources of sewage, and hence may be an indicator of fecal contamination in groundwater. Furthermore, there is some evidence that this class of bacteriophage are mobilized in the subsurface following rainfall events, although the mechanism responsible for this process is not yet clear. Using a model filtration system consisting of packed columns of quartz sand, we found that the filtration of this isolate was strongly dependent on the concentration and valence of the dominant cation in the pore fluid. In one set of experiments involving columns 19 cm in length, virus retention in the column increased from 0% to 99.999% when the electrolyte composition of the pore fluid was changed from 10 mM NaCl to 10 mM CaCl2. With one exception, filtration efficiencies calculated from the column experiments were inversely proportional to the electrophoretic mobility of the virus, implying that electrostatic interactions between the virus and the quartz surface dominate the filtration dynamics of this particular bacteriophage. From a practical perspective, these results indicate that small changes in the hardness and total dissolved solids of pore fluids - as might occur following a rainfall event - can dramatically affect both the filtration and mobilization of filamentous bacteriophage in subsurface systems. [TOP OF PAGE]

177. Coliphages and indicator bacteria in Boston Harbor, Massachusetts. Ricca, D.M., Cooney, J.J. (1999). Environmental Toxicology 14:404-408. Surface water was tested for two bacterial and two coliphage indicators of fecal pollution at four sites in Boston Harbor, MA over 1 year. Of 108 samples tested, somatic coliphages, fecal coliforms, enterococci, and F-specific phages were present in 107, 105, 73, and 58 water samples, respectively. The means of all samples, per 100 mL, were 195.6 pfu (range: 0-1833) for somatic coliphages, 101.3 cfu (0-2670) for fecal coliforms, 20.0 pfu (0-261) for F-specific phages, and 3.1 cfu (0-32) for enterococci. Somatic coliphages and fecal coliforms were more prevalent indicators of fecal pollution than enterococci and F-specific phages. No indicator correlated well with any of the others. No statistically significant difference in phage numbers was found between samples taken in summer and winter. No correlation was found between salinity and any indicator. There was no increase in counts of any indicator due to localized input from two marinas. [TOP OF PAGE]

178. Phages for methicillin-resistant Staphylococcus aureus: An international trial. Richardson, J.F., Rosdahl, V.T., Van Leeuwen, W.J., Vickery, A.M., Vindel, A., Witte, W. (1999). Epidemiology and Infection 122:227-233. An internationally agreed and validated set of phages is used worldwide for the typing of strains of Staphylococcus aureus of human origin. However, because of the sometimes reduced susceptibility of methicillin-resistant strains (MRSA) to these phages, some of the national typing centres use locally isolated and characterized sets of experimental phages. In this trial, 42 such phages were distributed to 6 centres and tested against 744 isolates of MRSA with the intention of defining a phage set to augment the international set. The use of these experimental phages increased the percentage typability from 75% with the international set to 93% and the number of identifiable lytic patterns from 192 to 424. A subset of 10 experimental phages was selected. When this subset was compared with the experimental panel, the typability rate was 91% and 370 distinct patterns were obtained. This subset of phages has been distributed for international trial. [TOP OF PAGE]

179. Rapid film-based determination of antibiotic susceptibilities of Mycobacterium tuberculosis strains by using a luciferase reporter phage and the Bronx Box. Riska, P.F., Su, Y., Bardarov, S., Freundlich, L., Sarkis, G., Hatfull, G., Carriere, C., Kumar, V., Chan, J., Jacobs, W.J. (1999). Journal of Clinical Microbiology 37:1144-1149. Detecting antibiotic resistance in Mycobacterium tuberculosis is becoming increasingly important with the global recognition of drug-resistant strains and their adverse impact on clinical outcomes. Current methods of susceptibility testing are either time-consuming or costly; rapid, reliable, simple, and inexpensive methods would be highly desirable, especially in the developing world where most tuberculosis is found. The luciferase reporter phage is a unique reagent well-suited for this purpose: upon infection with viable mycobacteria, it produces quantifiable light which is not observed in mycobacterial cells treated with active antimicrobials. In this report, we describe a modification of our original assay, which allows detection of the emitted light with a Polaroid film box designated the Bronx Box. The technique has been applied to 25 M. tuberculosis reference and clinical strains, and criteria are presented which allow rapid and simple discrimination among strains susceptible or resistant to isoniazid and rifampin, the major antituberculosis agents. [TOP OF PAGE]

180. First report of a putative cyanophage, MC-1 of Microcoleus sp. Rosowski, J.R., Shaffer, J.J., Martin, E.L. (1999). Microsc. Microanalysis 5:1142-1143. [TOP OF PAGE]

181. Development of phage resistant starter strains. Ross, R.P. (1999). 5. DPRC. Bacteriophage infection of starter cultures causes significant losses in the manufacture of cheese and other fermented dairy products. In this study, a number of bacteriophage resistant strains were developed using molecular genetic techniques. 3 new natural plasmid (DNA)-associated bacteriophage resistance systems were identified. The detailed genetic make-up of the phage resistance plasmid (pMRC01) was elucidated. Bacteriophages currently evolving in the industrial cheesemaking environment were monitored to facilitate the choice of phage resistance systems for use in commercial starter cultures which can more effectively target the problematic phage types. 2 highly virulent phages targeting important cheese starters were identified in the industrial cheesemaking environment. A reliable food-grade method to facilitate transfer of phage resistance systems to cheesemaking starter strains was developed, based on bacteriocin immunity-linked phage resistance. Phage resistant cheese starter cultures were developed through natural selection and by molecular manipulation using phage genetic resistance plasmids. The phage resistance plasmid pMRC01 was introduced into 31 cheese starter strains. All phage resistant starter strains resulting from the strain improvement research were evaluated under stringent laboratory conditions. Selected strains were then evaluated in pilot-scale Cheddar cheese trials and were subsequently validated in commercial cheese plants. [TOP OF PAGE]

182. Analysis of bacteriophage inactivation and its attenuation by adsorption onto colloidal particles by batch agitation techniques. Rossi, P., Aragno, M. (1999). Canadian Journal of Microbiology 45:9-17. A batch agitation technique was designed to specify the different parameters that influence the inactivation and adsorption mechanisms of viruses in water. The advantage of this method over the classical procedures is that the kinetic reactions of the different subfractions of the virus population can be described simultaneously. A first set of experiments with phage T7 showed that this phage is rapidly inactivated in a constantly agitated liquid medium. This inactivation rate is highly influenced by temperature, but variation of the pH (from 5 to 9) and increase in salt concentration have no effect on it. The addition of colloidal clay particles (CCPs) of montmorillonite and attapulgite into the liquid medium considerably modifies this behavior, even at very low concentrations (0.025 mg/mL). The experiments show that the viruses react quickly with the particles and that bonding is not permanent. Viruses establish a dynamic equilibrium, which is strongly dependent on physicochemical parameters such as pH, ionic concentrations, and the presence of proteins or protein hydrolysates. A major environmental consequence is that the presence of CCPs seems to effectively protect the coliphage T7 from rapid inactivation. [TOP OF PAGE]

183. Lethal toxicity of Vibrio harveyi to cultivated Penaeus monodon induced by a bacteriophage. Ruangpan, L., Danayadol, Y., Direkbusarakom, S., Siurairatana, S., Flegel, T.W. (1999). Diseases of Aquatic Organisms 35:195-201. In Southern Thailand in 1996, intense luminescence in many shrimp rearing ponds was accompanied by massive mortality resulting in total crop loss within 3 or 4 d. Mortality was correlated with gross signs which shrimp farmers called (English translation) tea-brown gill syndrome (TBGS). Histological examination of moribund shrimp revealed massive lesions of the hepatopancreas characterized by hemocytic infiltration and the presence of bacterial cells. Bacterial isolation yielded several strains tentatively identified as Vibrio harveyi on the basis of luminescence and growth on BTB Teepol agar. Representative isolate VH1039 was selected and identified by biochemical tests as V. harveyi. When strain VH1039 and the other luminescent isolates were injected into normal shrimp (1 X 10⁷ cells shrimp-1), no significant mortality was observed in comparison with control shrimp injected without bacteria. Nor was any significant mortality observed after injection of supernatant fluids fromnormal or sonicated bacterial cultures of VH1039 (1 X 10⁸ cells ml^-1). Transmission electron microscopy (TEM) of hepatopancreatic tissue from farmed TBGS shrimp revealed bacterial cells of Vibrio morphology together with large numbers of bacteriophage particles that had round to hexagonal heads of approximately 60 nm diameter and tails of approximately 100 nm length. These were either free, attached to cell walls of intact bacteria or in various stages of replication within bacterial cells. Gills of farmed TBGS shrimp were subsequently homogenized in lobster haemolymph buffer (LHB) and membrane filtered (0.22 mum). Compared to control shrimp injected with LHB, shrimp injected with the 1000X diluted gill filtrate (DGF) showed no significant mortality. However, when DGF was injected together with 1 X 10⁷ cells of strain VH1039, there was total mortality within 48 h. High and rapid mortality concurrent with brown gills was seen only in the mixed injection group. TEM of the artificially infected shrimp tissues did show the presence of bacterial cells, but no mature bacteriophage particles or lysed bacterial cells were found similar to those seen in farmed TBGS shrimp. In further tests, addition of DGF to cultures of VH1039 induced extreme but transitory toxicity of culture filtrates from 24 to 36 h. Treatment of DGF with a germicidal lamp (UV) for 30 min or with heat at 100degreeC for 15 min failed to stop shrimp mortality from mixed DGF/VH1039 injections. However, mortality was stopped if the heated DGF was treated with DNase. The results suggested that a bacteriophage may sometimes mediate the toxicity of V. harveyi in Penaeus monodon by the transfer of a toxin gene(s) or a gene(s) controlling toxin production. [TOP OF PAGE]

184. Hospital Horror. Sardar, Z. (1999). New Statesman , ???-??? Our hospitals are becoming hazardous places. One can go in with a curable illness and come out with an incurable one. The risk of being infected by a "superbug", bacterial infection that is resistant to antibiotic, is very real. It has always been possible to die from surgical infection, but the arrival of superbugs has increased this risk enormously. Within ten years most of these infections will not be treatable with antibiotics. ¶
     This crisis is solely due to overuse of antibiotics. We use antibiotics as a panacea for all illnesses, and doctors have become accustomed to prescribing them as blanket coverage for all complaints. Patients, too, think antibiotics are magic bullets and demand them for every flu of every season. Worse, we use antibacterial agents in household products such as washing-up liquid, bin liners and kitchen utensils. A recent essay in Nature shows how this domestic overuse is leading to resistant bacteria. For example, E coli, one of the most common causes of food poisoning, is developing resistance to triclosan, a common antibacterial agent. ¶ That is the bad news. The good news is that there is a relatively safe and easy cure for drug-resistant strains of infectious bacteria. It's called phage therapy. Bacteriophage, or "bacteria eaters", are viruses extracted from , raw sewage. They thrive wherever bacteria thrive -- in our bodies, waste products, rivers. Phage therapy has been freely available in the former communist world for decades. Even now, a dilapidated factory in Tblisi, Georgia, is producing supplies of bacteriophage under the most difficult conditions. And we in the west, having spent astronomical sums in a vain attempt to contain killer bugs, are beginning to think about learning from them. [TOP OF PAGE]

185. Mycobacteriophages. Sarkis, G.J., Hatfull, G.F. (1999). Methods in Molecular Biology 101:145-173. [TOP OF PAGE]

186. Transduction of enteric Escherichia coli isolates with a derivative of Shiga toxin 2-encoding bacteriophage _3538 isolated from Escherichia coli O157:H7. Schmidt, H., Bielaszewska, M., Karch, H. (1999). Appl. Environ. Microbiol. 65:3855-3861. [Shiga toxin (Stx)-producing Escherichia coli (STEC) strains have emerged as serious foodborne pathogens.] In this study, the ability of a detoxified derivative of a Stx2-encoding bacteriophage to infect and lysogenize enteric E. coli strains and to develop infectious progeny from these lysogenized strains was investigated. The stx2 gene of E. coli O157:H7 strain 3538/95 (a human isolate) was replaced by the chloramphenicol acetyltransferase (cat) gene from plasmid pACYC184. Phage _3538(Deltastx2::cat) was isolated after induction of E. coli O157:H7 strain 3538/95 with mitomycin. A variety of strains of enteropathogenic E. coli (EPEC), enteroinvasive E. coli (EIEC), STEC, enterotoxigenic E. coli (ETEC), enteroaggregative E. coli (EAEC) and E. coli from stool microflora of healthy individuals were infected with _3538(Deltastx2::cat) and plaque formation and lysogenic conversion of wild-type E. coli strains were investigated. With the exception of 1 EIEC strain, none of the E. coli strains supported formation of plaques when used as indicators for _3538(Deltastx2::cat). However, 2 of 11 EPEC, 11 of 25 STEC, 2 of 7 EAEC, 1 of 3 EIEC and 1 of 6 E. coli isolates from stool microflora integrated phage [DNA] in their chromosomes and expressed resistance to chloramphenicol. Following induction with mitomycin, these lysogenic strains released infectious particles of _3538(Deltastx2::cat) that formed plaques on a lawn of E. coli laboratory strain C600. Results demonstrate that _3538(Deltastx2::cat) was able to infect and lysogenize particular enteric strains of pathogenic and nonpathogenic E. coli and that the lysogens produced infectious phage progeny. [It is concluded that] Stx-encoding bacteriophages are able to spread stx genes among enteric E. coli strains. [TOP OF PAGE]

187. Molecular survey of the Salmonella phage typing system of Anderson. Schmieger, H. (1999). J. Bacteriol. 181:1630-1635. Typing phages for Salmonella and the prophages of their typical propagation strains were analyzed at the DNA level. Most of them belong to the P22 branch of the lambdoid phages. Acquisition of new plating properties of the typing phages by propagation in particular strains can be due to different host specific modifications of the DNA or to recombination events with residing prophages which are reflected by changes in the respective DNA restriction patterns. It is concluded that the actually available set of typing phages is a historically unique combination of strains. [TOP OF PAGE]

188. Transduction of multiple drug resistance of Salmonella enterica serovar typhimurium DT104. Schmieger, H., Schicklmaier, P. (1999). FEMS Microbiol. Ecol. 170:251-256. Salmonella enterica serovar typhimurium, definite type (DT)104, has emerged as a new epidemic strain since 1990 and is characterized by multiple drug resistance of various combinations. In order to understand how resistance genes have spread, the possibility of transferring the resistance genes of DT104 strains to various recipients was examined. Transduction occured with the generalized transducing phage ES18 and with phage PDT104 (this phage resides as a prophage in all of the DT104 strains analysed so far). This implies that all DT104 strains carry in their genome a potent vehicle for horizontal transfer and spread of resistance genes. Resistance genes were found to be tightly clustered in the DT104 chromosome. [TOP OF PAGE]

189. Pseudomonas aeruginosa phage lysate as an immunobiological agent. 1. Selection of Pseudomonas aeruginosa clinical strains for phage lysate preparation. Sekaninova, G., Kolarova, M., Pillich, J., Semenka, J., Slavikova, H., Kubickova, D., Zajicova, V. (1999). Folia Microbiologica 44:93-97. A total of 2087 Pseudomonas aeruginosa isolates collected during the period 1994-1997 were used as starting material. Out of 1704 in-patient isolates, 299 strains were selected for the preparation of phage lysates but only five strains provided stable lysates, i.e., maintained the ability to be repeatedly and completely lysed by the appropriate phage in the course of several years. A set of 193 out-patients (189) and water sources (4) isolates failed to yield strains suitable for phage lysate preparation; 190 strains isolated abroad from patients with cystic fibrosis or respiratory infections included three isolates which, despite having a high degree of mucus production, were suitable for lysate preparation. The antigenic pattern of the phage lysates was ascertained by SDS-polyacrylamide gel electrophoresis. [TOP OF PAGE]

190. Advances in the separation of bacteriophages and related particles. Serwer, P., Griess, G.A. (1999). Journal of Chromotography. B, Biomedical Sciences and Applications 722:179-190. Nondenaturing gel electrophoresis is used to both characterize multimolecular particles and determine the assembly pathways of these particles. Characterization of bacteriophage-related particles has yielded strategies for characterizing multimolecular particles in general. Previous studies have revealed means for using nondenaturing gel electrophoresis to determine both the effective radius and the average electrical surface charge density of any particle. The response of electrophoretic mobility to increasing the magnitude of the electrical field is used to detect rod-shaped particles. To increase the capacity of nondenaturing gel electrophoresis to characterize comparatively large particles, some current research is directed towards either determining the structure of gels used for electrophoresis or inducing steric trapping of particles in dead-end regions within the fibrous network that forms a gel. A trapping-dependent technique of pulsed-field gel electrophoresis is presented with which a DNA-protein complex can be made to electrophoretically migrate in a direction opposite to the direction of migration of protein-free DNA. [TOP OF PAGE]

191. Prophage carriage as a molecular epidemiological marker in Streptococcus pneumoniae. Severina, E., Ramirez, M., Tomasz, A. (1999). Journal of Clinical Microbiology 37:3308-3315. The great majority of clinical isolates of Streptococcus pneumoniae carry prophages that may be identified through their hybridization with a DNA probe specific for the pneumococcal lytA gene (M. Ramirez, E. Severina, and A. Tomasz, J. Bacteriol. 181:3618-3625, 1999). We now show that the lytA hybridization pattern of chromosomal SmaI digests is stable for a given strain during extensive serial culturing in the laboratory; the pattern is specific for the strain's clonal type, as defined by pulsed-field gel electrophoretic (PFGE) pattern, and variations in PFGE subtypes may be explained by changes in the number and chromosomal localization of this prophage(s). These observations indicate that the lytA hybridization pattern may be used as a molecular epidemiological marker that offers additional resolution of the genetic background of S. pneumoniae strains. [TOP OF PAGE]

192. Bacteriological evaluation of composting systems in sludge treatment. Shaban, A.M. (1999). Water Science and Technology 40:165-170. Sludge disposal was considered as a serious problem to the authorities. Thus, the treatment of sludge resulting from sewage treatment plants to remove pathogenic microorganisms and to improve its impact on the environment was considered as the main objective of several investigators. Composting is one of the methods of sludge treatment. Different systems of composting (static pile, windrow and natural draft) were applied and evaluated bacteriologically. Faecal coliform and salmonellae were removed completely during the first two weeks in case of forced aeration, but the former are still present till near the end of experiment with natural aeration. For the natural draft system with sawdust base, faecal coliform reduction increased up to 100% after 7 weeks, while faecal streptococci and coliphage decreased gradually and were removed completely at the end of treatment. Salmonellae disappeared after a few days from starting treatment. In case of alkaloids addition (cement and lime), the tested organisms reached acceptable levels with any concentration of alkaloids. Coliphage and faecal streptococci survived till the end of treatment. So, from the previous results it is clear to say, coliphage and faecal streptococci were more resistant to the composting processes than other organisms. [TOP OF PAGE]

193. Use of the polymerase chain reaction and denaturing gradient gel electrophoresis to study diversity in natural virus communities. Short, S.M., Suttle, C.A. (1999). Hydrobiologia 401:19-32. Viruses are abundant members of marine and freshwater microbial communities, and are important players in aquatic ecology and geochemical cycles. Recent methodological developments have allowed the use of the polymerase chain reaction (PCR) to examine the diversity of natural communities of viruses without the need for culture. DNA polymerase genes are highly conserved and are, therefore, suitable targets for PCR analysis of microbes that do not encode rRNA. As natural virus communities are largely made up of dsDNA viruses, and as many dsDNA algal viruses encode their own DNA polymerase, PCR primers can be designed to amplify fragments of these genes. This approach has been used to examine the genetic diversity in natural communities of viruses that infect phytoplankton. Algal-virus-specific primers were used to amplify polymerase fragments from natural virus samples, demonstrating the presence of a diverse community of viruses closely related to those that are known to infect phytoplankton. We have modified this approach by using denaturing gradient gel electrophoresis (DGGE) to rapidly analyze PCR products. DGGE will permit rapid and efficient fingerprinting of natural marine viral communities, and allow spatial and temporal differences in viral community structure to be examined. This paper provides a brief overview of how PCR and DGGE can be used to examine diversity in natural viral communities drawing on viruses that infect phytoplankton as an example. [TOP OF PAGE]

194. Sunlight inactivation of fecal bacteriophages and bacteria in sewage-polluted seawater. Sinton, L.W., Finlay, R.K., Lynch, P.A. (1999). Appl. Environ. Microbiol. 65:3605-3613. Sunlight inactivation rates of somatic coliphages, F-specific RNA bacteriophages (F-RNA phages), and fecal coliforms were compared in seven summer and three winter survival experiments. Experiments were conducted outdoors, using 300-liter 2% (vol/vol) sewage-seawater mixtures held in open-top chambers. Dark inactivation rates (k(D)s), measured from exponential survival curves in enclosed (control) chambers, were higher in summer (temperature range: 14 to 20 degrees C) than in winter (temperature range: 8 to 10 degrees C). Winter k(D)s were highest for fecal coliforms and lowest for F-RNA phages but were the same or similar for all three indicators in summer. Sunlight inactivation rates (k(S)), as a function of cumulative global solar radiation (insolation), were all higher than the k(D)s with a consistent k(S) ranking (from greatest to least) as follows: fecal coliforms, F-RNA phages, and somatic coliphages. Phage inactivation was exponential, but bacterial curves typically exhibited a shoulder. Phages from raw sewage exhibited k(S)s similar to those from waste stabilization pond effluent, but raw sewage fecal coliforms were inactivated faster than pond effluent fecal coliforms. In an experiment which included F-DNA phages and Bacteroides fragilis phages, the k(S) ranking (from greatest to least) was as follows: fecal coliforms, F-RNA phages, B. fragilis phages, F-DNA phages, and somatic coliphages. In a 2-day experiment which included enterococci, the initial concentration ranking (from greatest to least: fecal coliforms, enterococci, F-RNA phages, and somatic coliphages) was reversed during sunlight exposure, with only the phages remaining detectable by the end of day 2. Inactivation rates under different optical filters decreased with the increase in spectral cutoff wavelength (50% light transmission) and indicated that F-RNA phages and fecal coliforms are more susceptible than somatic coliphages to longer solar wavelengths, which predominate in seawater. The consistently superior survival of somatic coliphages in our experiments suggests that they warrant further consideration as fecal, and possibly viral, indicators in marine waters. [TOP OF PAGE]

195. Phenoptosis: programmed death of an organism. Skulachev, V.P. (1999). Biochemistry 64:1418-1426. Programmed cell death (apoptosis) is well-established in many multicellular organisms. Apoptosis purifies a tissue from cells that became useless or even harmful for the organism. Similar phenomena are already described also at subcellular level (suicide of mitochondria, i.e., mitoptosis) as well as at supracellular level (degradation of some organs temporarily appearing in the course of ontogenesis and then disappearing by means of apoptosis of the organ-composing cells). Following the same logic, one may put a question about programmed death of an organism as a mechanism of purification of a kin, community of organisms, or population from individuals who became unwanted for this kin, etc. A putative mechanism of such kind is proposed to be coined "phenoptosis" by analogy with apoptosis and mitoptosis. In a unicellular organism (the bacterium Escherichia coli), three different biochemical mechanisms of programmed death are identified. All of them are actuated by the appearance of phages inside the bacterial cell. This may be regarded as a precedent of phenoptosis which prevents expansion of the phage infection among E. coli cells. Purification of a population from infected individuals looks like an evolutionary invention useful for a species. Such an invention has high chances to be also employed by multicellular organisms. Most probably, septic shock in animals and humans serves as an analog of the phage-induced bacterial phenoptosis. It is hypothesized that the stress-induced ischemic diseases of brain and heart as well as carcinogenesis if they are induced by repeated stresses also represent phenoptoses that, in contrast to sepsis, are age-dependent. There are interrelations of programmed death phenomena at various levels of complexity of the living systems. Thus, extensive mitoptosis in a cell leads to apoptotic death of this cell and extensive apoptosis in an organ of vital importance results in phenoptotic death of an individual. In line with this logic, some cases are already described when inhibition of apoptosis strongly improves the postischemic state of the organism. [TOP OF PAGE]

196. Genetic control of Vibrio cholerae pathogenicity: Temperate filamentous CTX bacteriophage coding for cholera toxin and the "pathogenicity island". Smirnova, N.I. (1999). Molekulyarnaya Genetika Mikrobiologiya i Virusologiya 3-11. Reviews modern data on the genetic control of the key factors of Vibrio cholerae pathogenicity: cholera toxin and toxin-coregulated adhesion phili. Pays special attention to the temperate filamentous CTX bacteriophage, whose genome contains structural genes of cholera toxin, and the "pathogenicity island" carrying tcp genes responsible for the most important factor of the human small intestine colonization with V. cholerae. Discusses the mechanism of coordinated regulation of the activity of the main genes of V. cholerae pathogenicity genes. [TOP OF PAGE]

197. Vibrio cholerae 0139 temperate phage: Characterization and role in modifying the expression of virulence chromosome genes. Smirnova, N.I., Yeroshenko, G.A., Schelkanova, Y., Livanova, L.F., Konnov, N.P. (1999). Molekulyarnaya Genetika Mikrobiologiya i Virusologiya 3-9. Restriction analysis of temperate cholera phage 139 isolated from Vibrio cholerae P16064, serogroup 0139, showed its DNA to be double-stranded linear with cohesive terminals. DNA-DNA hybridization on nylon membranes revealed that many V. cholerae strains of serogroup 0139 isolated in different regions contained a temperate cholera phage 139 in their genomes. Southern blot hybridization of chromosomal DNA PST-fragments with phage probe showed that the temperate phage 139 was identical to the temperate phage of serogroup II V. eltor. The phage integrated in tile chromosome near genes encoding motility (mot) and production of the capsule (cap) and purine (pur). Phage genome is apparently responsible for instability of cap, pur, and mot genes whose products are important for the development of an infectious process in cholera. [TOP OF PAGE]

198. [Vibrio cholerae temperate phage O139: characteristics and role in changing expression of chromosomal virulence genes]. Smirnova, N.I., Eroshenko, G.A., Shchelkanova, E., Livanova, L.F., Konnov, N.P. (1999). Zhurnal Mikrobiologii, Epidemiologii i Immunobiologii ???:3-9. Restriction analysis of temperate cholera phage 139 isolated from Vibrio cholerae P16064, serogroup 0139, showed its DNA to be double-stranded linear with cohesive terminals. DNA-DNA hybridization on nylon membranes revealed that many V. cholerae strains of serogroup 0139 isolated in different regions contained a temperate cholera phage 139 in their genomes. Southern blot hybridization of chromosomal DNA PST-fragments with phage probe showed that the temperate phage 139 was identical to the temperate phage of serogroup II V. eltor. The phage integrated in the chromosome near genes encoding motility (mot) and production of the capsule (cap) and purine (pur). Phage genome is apparently responsible for instability of cap, pur, and mot genes whose products are important for the development of an infectious process in cholera. [TOP OF PAGE]

199. The complete genome sequence of the Streptomyces temperate phage straight phiC31: evolutionary relationships to other viruses. Smith, M.C., Burns, R.N., Wilson, S.E., Gregory, M.A. (1999). Nucleic Acids Research 27:2145-2155. The completed genome sequence of the temperate Streptomyces phage straight phiC31 is reported. straight phiC31 contains genes that are related by sequence similarities to several other dsDNA phages infecting many diverse bacterial hosts, including Escherichia, Arthrobacter, Mycobacterium, Rhodobacter, Staphylococcus, Bacillus, Streptococcus, Lactobacillus and Lactococcus. These observations provide further evidence that dsDNA phages from diverse bacterial hosts are related and have had access to a common genetic pool. Analysis of the late genes was particularly informative. The sequences of the head assembly proteins (portal, head protease and major capsid) were conserved between straight phiC31, coliphage HK97, staphylococcal phage straight phiPVL, two Rhodobacter capsulatus prophages and two Mycobacterium tuberculosis prophages. These phages and prophages (where non-defective) from evolutionarily diverse hosts are, therefore, likely to share a common head assembly mechanism i.e. that of HK97. The organisation of the tail genes in straight phiC31 is highly reminiscent of tail regions from other phage genomes. The unusual organisation of the putative lysis genes in straight phiC31 is discussed, and speculations are made as to the roles of some inessential early gene products. Similarities between certain phage gene products and eukaryotic dsDNA virus proteins were noted, in particular, the primase/helicases and the terminases (large subunits). Furthermore, the complete sequence clarifies the overall transcription map of the phage during lytic growth and the positions of elements involved in the maintenance of lysogeny. [TOP OF PAGE]

200. An in situ enclosure experiment to test the solar UVB impact on plankton in a high altitude mountain lake: II) effects on the microbial food web. Sommaruga, R., Sattller, B., Oberleiter, A., Wille, A., Sommaruga-Wögrath, S., Psenner, R., Felip, M., Camarero, L., Pina, S., Gironés, R., Catalán, J. (1999). Journal of Plankton Research 21:859-879. We studied the impact of ambient levels of solar UVB radiation on the planktonic microbial food web (viruses, heterotrophic bacteria, heterotrophic flagellates and ciliates) of a high-mountain lake (2417 m above sea level) under in situ conditions for 16 days. Enclosures of 1 m3 receiving either the full sunlight spectrum or sunlight without UVB radiation were suspended at the lake surface. We found that the abundance of heterotrophic flagellates was always lower in the +UVB treatment than in the -UVB one. In addition, bacterial consumption, measured by the disappearance of fluorescently labelled bacteria, was significantly (p<0.05) reduced in the +UVB treatment. The abundance of non-filamentous bacteria (<10 µm long) was also lower in the +UVB treatment, suggesting a direct effect of UVB on their growth. This was supported by the significantly (p<0.05) lower cell-specific activity ([3H]-thymidine incorporation) found on the fifth day of the experiment. In contrast, UVB radiation had no effect on filamentous bacteria (>10 µm long) that represented only a small fraction of the total abundance (<4%) but up to ~70 % of the total bacterial biovolume. Ciliates, mainly Urotricha pelagica and U. furcata, were less impacted by UVB radiation although the net growth rate during the first week of the experiment was lower in the +UVB treatment than in the -UVB one (0.22 and 0.39 d-1, respectively). The abundance of virus-like particles during the first week of the experiment was higher in the -UVB treatment. After reaching the maximum value for the interaction viruses x bacteria, their number decreased dramatically (by ~85%) in both treatments with a decay rate of ~0.017 h-1. This study illustrates the complexity in assessing the impact of UVB radiation when more than one trophic level is considered and indicates the existence of different sensitivity to UVB radiation among components of the microbial food web. [TOP OF PAGE]

201. PPL1c, a virulent mutant bacteriophage useful for identification of Paenibacillus larvae subspecies larvae. Stahly, D.P., Alippi, A.M., Bakhiet, N., Campana, C.F., Novak, C.C., Cox, R. (1999). Journal of Invertebrate Pathology 74:295-296. Paenibacillus larvae subspecies larvae is a pathogen of honey bee (Apis mellifera) larvae, causing American foulbrood. We isolated a virulent mutant bacteriophage, PPL1c, from P. larvae subsp. larvae NE that should be of value for rapid identification of P. larvae subsp. larvae. [TOP OF PAGE]

202. (Methodic approaches to studing marine bacteria and viruses interaction) Metodicheskie podkhody k izucheniyu protsessa vzaimodejstviya morskikh bakterij i virusov. Stepanova, O.A., Shaida, V.G. (1999). Ehkologiya morya. Kiev [Ehkol. Morya] 48:96-99. Comparative study of interaction between marine bacteria and viruses with the employment of microcalorimetry and Bioscreen-C automatic analyzer has demonstrated higher sensitivity of microcalorimetric method. Thus method permits studying the dynamics of virus -- bacterium interaction at the level of both lytic phage infection and a variety of enzymatic and other functions induced by lysogeny as the result of viral deoxyribonucleic acid integration into bacterial. [TOP OF PAGE]

203. Skuteczne zastosowanie bakteriofagoterapii w ropnym zapaleniu opon mozgowo-rdzeniowych u noworodka. [Successful treatment with bacteriophage in purulent cerebrospinal meningitis in a newborn]. Stroj, L., Weber-Dabrowska, B., Partyka, K., Mulczyk, M., Wojcik, M. (1999). Neurologia I Neurochirurgia Polska 33:693-698. The subject of this report is the case of purulent meningitis in new-born caused by Klebsiella pneumoniae. As the intensive antibiotic therapy turned out to be ineffective phage therapy was applied. Oral administration of specific phage preparate for the period of 5 weeks resulted in complete sterilization of cerebrospinal fluid and unquestionable improvement of child's health. However, after several ventriculopunctures some complications appeared (haemorrhage into central nervous system, extra infection). They were treated in standard way. Because of increasing internal hydrocephalus and necessity of operation, the child was sent to suitable hospital for further treatment. [TOP OF PAGE]

204. [Successful treatment with bacteriophage in purulent cerebrospinal meningitis in a newborn]. Stroj, L., Weber-Dabrowska, B., Partyka, K., Mulczyk, M., Wojcik, M. (1999). Neurologia I Neurochirurgia Polska 33:693-698. The subject of this report is the case of purulent meningitis in new-born caused by Klebsiella pneumoniae. As the intensive antibiotic therapy turned out to be ineffective phage therapy was applied. Oral administration of specific phage preparate for the period of 5 weeks resulted in complete sterilization of cerebrospinal fluid and unquestionable improvement of child's health. However, after several ventriculopunctures some complications appeared (haemorrhage into central nervous system, extra infection). They were treated in standard way. Because of increasing internal hydrocephalus and necessity of operation, the child was sent to suitable hospital for further treatment. [TOP OF PAGE]

205. Felix d'Herelle and the Origins of Molecular Biology. Summers, W.C. (1999). Yale University Press, New Haven, Connecticut.[TOP OF PAGE]

206. Revenge of the bug zappers. Electrocuted flies spew viruses for feet — and into your food. Susman, E. (1999). MSNBC Chicago (June 3). http://www.msnbc.com/news/276262.asp#BODY ¶ Revenge of the bug zappers. Electrocuted flies spew viruses for feet — and into your food ¶ By Ed Susman MSNBC CHICAGO, June 3 ¶ CHICAGO, June 3 — "Zzzzzap." Aah, the satisfying sound of a barbecue-crashing fly being electronically fried in a bug zapper. But, as it turns out, the insect may get revenge for its electrocution, spewing viruses and bacteria as far as 8 feet from the devices — and into your food. ¶ 'If you must use the units to control flies, at least use them far as possible from the food table inside the house, and away from the grill and condiment table outside.' — JOHN URBAN Kansas State University ¶ RESEARCHERS at Kansas State University in Manhattan said Wednesday that particles of electrocuted insects are spewed all over a room when the critters explode in a blaze of blue light. ¶ "Most people probably think that using electrocuting traps to control insects in one's backyard or around food-handling areas would improve sanitation, but the results of this study suggests their use actually spreads microorganisms," said John Urban, associate professor of microbiology. He spoke here at the general meeting of the American Society for Microbiology. ¶ But you don't need to abandon the devices altogether, he said. "If you must use the units to control flies, at least use them far as possible from the food table inside the house, and away from the grill and condiment table outside." ¶ Even hanging the bug zappers near a home pool would probably be safe, he said, because any viruses or bacteria blasted into the water would succumb to chlorine. ¶ His team had already shown that flies electrocuted in a common bug zapper shower bacteria for about 6 feet. The new study, he said, shows almost identical patterns of virus spread during electrocution. ¶ The zappers use light to attract pests to an electrified metal grid and then electrocute them — producing a sizzling snap, crackle and pop. ¶ For the experiments, they used a virus called FX174, which is similar in size and shape similar to the human poliovirus but does not pose a risk to human health. The experiments were performed in an experimental chamber at the university. ¶ Team member Alberto Broce, a professor of entomology, chose houseflies as the insect model "because they are filth flies and are known to ingest microorganisms in the food they eat," Urban said. "Also, their surfaces are easily contaminated by the filth they walk upon." ¶ SURFACE MICROBES REAL DANGER ¶ When the houseflies hit the traps, their bodies literally exploded, Urban said. However, the electronic zap did not destroy all the viruses that had attached to the insect's body. ¶ As it turns out, microbes on the fly's surface were far more dangerous than any they had ingested. Overall, approximately one virus out of every 4,000 on a fly's surface was spread by electrocution, and virus was spread an average of 6 feet. Only about 1 in 1,000,000 of the viruses inside the fly were released upon electrocution, although they also spewed out as far as 6 feet, the study showed. ¶ "This is potentially significant since flies moving about on filth such as feces are most likely to become surface contaminated," he said. Feces can carry a vast number of organisms that are harmful to human health, causing disorders ranging from the severe diarrhea of roatvirus infection to the cripling cramps of shigella infection. [TOP OF PAGE]

207. Cyanophage. Suttle, C.A. (1999). p. ???-??? In Whitton, B. and Potts, M. (eds.), Ecology of Cyanobacteria: Their Diversity in Time and Spac. ???, ??? [TOP OF PAGE]

208. Do viruses control the oceans? Suttle, C.A. (1999). Natural History 108:48-51. [TOP OF PAGE]

209. Images in Infectious Diseases in Obstetrics and Gynecology. Vaginal Lactobacillus phage plaques and electron micrograph. Tao, L., Pavlova, S.I. (1999). Infectious Diseases in Obstetrics and Gynecology 6:236-236. Normally, lactobacilli are a dominant species in the vagina and play an important role in maintaining vaginal health by producing hydrogen peroxide and lactic acid to inhibit other bacteria, such as pathogenic anaerobes. When bacterial vaginosis occurs, however, vaginal lactobacilli decrease or disappear for unknown reasons. Recently, we have observed that bacteriophages or viruses can infect vaginal lactobacilli. Here we present two figures to show these phages: (A) Photograph of Lactobacillus phage drop assay. Phages were induced from lactobacilli by mitomycin C and dropped onto a Lactobacillus indicator strain on soft agar plates. Note: Each of the small clear plaques in or near a lysis zone is caused by single phage. (B) Electron micrograph of vaginal Lactobacillus phage (bar = 100 nm). [this is the entire study save figures and figure legend]. [TOP OF PAGE]

210. The future of bacterial vaginosis-related research. Taylor-Robinson, D. (1999). International Journal of Gynaecology and Ostetrics 67 Suppl 1:S35-S38 Various ways and criteria are used to diagnose BV. Guidelines should be redrawn and they should embody greater uniformity. The etiology of BV remains enigmatic. However, various observations suggest that host factors, possibly hormonal, cause an imbalance in the vaginal microflora. Exogenous factors, such as semen and antibiotics, may then help to bring about a more prolonged change. This forms a working hypothesis for further exploration. The role of the lactobacillus phage in the development of BV also needs to be determined. Various conditions may occur as a consequence of BV in non-pregnant and pregnant women and BV may also affect men. A subjective assessment of the extent to which these associations occur or are likely to be shown to occur by further investigations is presented in Table 1. The ability to cure acute BV needs to be improved as does the treatment of chronic BV, for which vaginal recolonization with exogenous lactobacilli is an approach to be evaluated further. [TOP OF PAGE]

211. Bacteriophage inactivation at the air-water-solid interface in dynamic batch systems. Thompson, S.S., Yates, M.V. (1999). Appl. Environ. Microbiol. 65:1186-1190. Bacteriophages have been widely used as surrogates for human enteric viruses in many studies on virus transport and fate. In this investigation, the fates of three bacteriophages, MS2, R17, and phiX174, were studied in a series of dynamic batch experiments. Both MS2 and R17 readily underwent inactivation in batch experiments where solutions of each phage were percolated through tubes packed with varying ratios of glass and Teflon beads. MS2 and R17 inactivation was the result of exposure to destructive forces at the dynamic air-water-solid interface. phiX174, however, did not undergo inactivation in similar studies, suggesting that this phage does not accumulate at air-water interfaces or is not affected by interfacial forces in the same manner. Other batch experiments showed that MS2 and R17 were increasingly inactivated during mixing in polypropylene tubes as the ionic strength of the solution was raised (phiX174 was not affected). By the addition of Tween 80 to suspensions of MS2 and R17, phage inactivation was prevented. Our data suggest that viral inactivation in simple dynamic batch experiments is dependent upon (i) the presence of a dynamic air-water-solid interface (where the solid is a hydrophobic surface), (ii) the ionic strength of the solution, (iii) the concentration of surface active compounds in the solution, and (iv) the type of virus used. [TOP OF PAGE]

212. Phage abortive infection of Bacillus licheniformis ATCC 9800; identification of the abiBL11 gene and localisation and sequencing of its promoter region. Tran, L.S.P., Szabo, L., Ponyi, T., Orosz, L., Sik, T., Holczinger, A. (1999). Applied Microbiology and Biotechnology 52:845-852. The virulent bacteriophage BL11 infects almost all Bacillus licheniformis strains tested, including the industrial bacitracin-producing B. licheniformis 19. B. licheniformis ATCC 9800, however, was virtually insensitive to phage BL11 infection, and all of the few surviving progeny phages proved to be mutants. The phage-resistance mechanism was neither inhibition of adsorption, nor restriction or exclusion provided by a resident prophage, but was, instead, of another type. Phage BL11 adsorbed well on to ATCC 9800 cells, its DNA was injected, but replication of phage DNA was inhibited and the infected cells died. Thus, the mechanism of phage resistance was identified as abortive infection (AbiBL11). The so-called abiBL11 gene was identified on the chromosome of strain ATCC 9800 by Tn917PF1 transposon mutagenesis. Part of the abiBL11 gene from the phage-sensitive ATCC 9800::Tn917PFI was cloned. Gene-disruption analysis, based on Campbell-type integration, showed that a 0.3-kb EcoRI fragment contained the 5' end of abiBL11. The promoter region of abiBL11 was identified using promoter- and terminator-probe plasmids. The deduced sequence (206 amino acids) of the N-terminal part of abiBL11 showed no significant homology to known abortive-infection genes, but did show homology to a Saccharomyces cerevisiae gene coding for a serine/threonine protein kinase (RCK1). [TOP OF PAGE]

213. Prisoner's dilemma in an RNA virus. Turner, P.E., Chao, L. (1999). Nature 398:441-443. The evolution of competitive interactions among viruses was studied in the RNA phage phi6 at high and low multiplicities of infection (that is, at high and low ratios of infecting phage to host cells). At high multiplicities, many phage infect and reproduce in the same host cell, whereas at low multiplicities the viruses reproduce mainly as clones. An unexpected result of this study was that phage grown at high rates of co-infection increased in fitness initially, but then evolved lowered fitness. Here we show that the fitness of the high-multiplicity phage relative to their ancestors generates a pay-off matrix conforming to the prisoner's dilemma strategy of game theory. In this strategy, defection (selfishness) evolves, despite the greater fitness pay-off that would result if all players were to cooperate. Viral cooperation and defection can be defined as, respectably, the manufacturing and sequestering of diffusible (shared) intracellular products. Because the low-multiplicity phage did not evolve lowered fitness, we attribute the evolution of selfishness to the lack of clonal structure and the mixing of unrelated genotypes at high multiplicity. [TOP OF PAGE]

214. Hybrid frequencies confirm limit to coinfection in the RNA bacteriophage phi-6. Turner, P.E., Burch, C., Hanley, K., Chao, L. (1999). J. Virol. 73:2420-2424. Coinfection of the same host cell by multiple viruses may lead to increased competition for limited cellular resources, thus reducing the fitness of an individual virus. Selection should favor viruses that can limit or prevent coinfection, and it is not surprising that many viruses have evolved mechanisms to do so. Here we explore whether coinfection is limited in the RNA bacteriophage phi6 that infects Pseudomonas phaseolicola. We estimated the limit to coinfection in phi6 by comparing the frequency of hybrids produced by two marked phage strains to that predicted by a mathematical model based on differing limits to coinfection. Our results provide an alternative method for estimating the limit to coinfection and confirm a previous estimate between two to three phages per host cell. In addition, our data reveal that the rate of coinfection at low phage densities may exceed that expected through random Poisson sampling. We discuss whether phage phi6 has evolved an optimal limit that balances the costly and beneficial fitness effects associated with multiple infections. [TOP OF PAGE]

215. Evaluation of virus removal in membrane separation processes using coliphage Q-beta. Urase, T., Yamamoto, K., Ohgaki, S. (1999). Water Science and Technology 28:9-15. [TOP OF PAGE]

216. The clinically isolated FIZ15 bacteriophage causes lysogenic conversion in Pseudomonas aeruginosa PAO1. Vaca-Pacheco, S., Paniagua-Contreras, G.L., Garcia-Gonzalez, O., de la Garza, M. (1999). Current Microbiology 38:239-243. FIZ15 bacteriophage, from a human clinical isolate of Pseudomonas aeruginosa, causes lysogenic conversion in the P. aeruginosa strain PAO1. The prophage-conferred phenotypes are: (1) increased resistance to phagocytosis by mouse peritoneal macrophages; (2) increased resistance to killing by normal human serum, and (3) increased adhesion to human buccal epithelial cells. These phenotypes are related to the prophage-induced change at the level of its own bacterial receptor, which appears to be the O-antigen. [TOP OF PAGE]

217. Isolation and characterization of APSE-1, a bacteriophage infecting the secondary endosymbiont of Acyrthosiphon pisum. van der Wilk, F. (1999). Virology 262:104-113. A bacteriophage infecting the secondary endosymbiont of the pea aphid Acyrthosiphon pisum was isolated and characterized. The phage was tentatively named bacteriophage APSE-1, for bacteriophage 1 of the A. pisum secondary endosymbiont. The APSE-1 phage particles morphologically resembled those of species of the Podoviridae. The complete nucleotide sequence of the bacteriophage APSE-1 genome was elucidated, and its genomic organization was deduced. The genome consists of a circularly permuted and terminally redundant double-stranded DNA molecule of 36524 bp. Fifty-four open reading frames, putatively encoding proteins with molecular masses of more than 8 kDa, were distinguished. ORF24 was identified as the gene coding for the major head protein by N-terminal amino acid sequencing of the protein. Comparison of APSE-1 sequences with bacteriophage-derived sequences present in databases revealed the putative function of 24 products, including the lysis proteins, scaffolding protein, transfer proteins, and DNA polymerase. This is the first report of a phage infecting an endosymbiont of an arthropod. [TOP OF PAGE]

218. Giant viruses infecting algae. Van Etten, J., Meints, R.H. (1999). Ann. Rev. Microbiol. 53:447-494. Paramecium bursaria chlorella virus (PBCV-1) is the prototype of a family of large, icosohedral, plaque-forming, double-stranded-DNA-containing viruses that replicate in certain unicellular, eukaryotic chlorella-like green algae. DNA sequence analysis of its 330,742-bp genome leads to the predicition that this phycodnavirus has 376 protein encoding genes and 10 transfer RNA genes. The predicited gene products of ~40% of these genes resemble proteins of known function. the chlorella viruses have other features that distinguish them from most viruses, in addition to thier large genome size. These feature include: (a) The viruses encode multiple DNAmethyltransferases and DNA site-specific endonucleases; (b) PBCV-1 encodes at least part, if not the entire machinery to glycosylate its proteins; (c) PBCV-1 has a tleast two types of introns-a self-splicing intron in a transcription factor-like gene and a splicesomal processed type of intron in its DNA polymerase gene. Unlike the chlorella viruses, large double-stranded-DNA-containing viruses that infect marine, filamentous brown algae have a circular genome and a lysogenic phase in their life cycle. [TOP OF PAGE]

219. Algal viruses (Phycodnaviridae). Van Etten, J. (1999). pp. 44-50. In In Webster, R.G. and Granoff, A. (eds.), Encyclopedia of Virology. Academic Press, London. [TOP OF PAGE]

220. Phycodnaviridae. Van Etten, J.L. (1999). pp. 183-193. In AnonymousVirus Taxonomy - Seventh Report. [TOP OF PAGE]

221. Changes in bacterial and eukaryotic community structure after mass lysis of filamentous cyanobacteria associated with viruses. van Hannen, E.J., Zwart, G., van Agterveld, M.P., Gons, H.J., Ebert, J., Laanbroek, H.J. (1999). Appl. Environ. Microbiol. 65:795-801. During an experiment in two laboratory-scale enclosures filled with lake water (130 liters each) we noticed the almost-complete lysis of the cyanobacterial population. Based on electron microscopic observations of viral particles inside cyanobacterial filaments and counts of virus-like particles, we concluded that a viral lysis of the filamentous cyanobacteria had taken place. Denaturing gradient gel electrophoresis (DGGE) of 16S ribosomal DNA fragments qualitatively monitored the removal of the cyanobacterial species from the community and the appearance of newly emerging bacterial species. The majority of these bacteria were related to the Cytophagales and actinomycetes, bacterial divisions known to contain species capable of degrading complex organic molecules. A few days after the cyanobacteria started to lyse, a rotifer species became dominant in the DGGE profile of the eukaryotic community. Since rotifers play an important role in the carbon transfer between the microbial loop and higher trophic levels, these observations confirm the role of viruses in channeling carbon through food webs. Multidimensional scaling analysis of the DGGE profiles showed large changes in the structures of both the bacterial and eukaryotic communities at the time of lysis. These changes were remarkably similar in the two enclosures, indicating that such community structure changes are not random but occur according to a fixed pattern. Our findings strongly support the idea that viruses can structure microbial communities. [TOP OF PAGE]

222. Surface layer variations affecting phage adsorption on seven Lactobacillus helveticus strains. Ventura, M., Callegari, M.L., Morelli, L. (1999). Annali di Microbiologia ed Enzimologia 49:45-53. Previous studies have demonstrated that Lactobacillus helveticus CNRZ 892 is covered by a protein of the S-layer type. The gene coding for this protein was isolated and sequenced. The central region of S-layer protein of this strain was shown to play a role of receptor for the virulent phage CNRZ 832-B1. Data are presented on S-layer encoding genes isolated from 7 L. helveticus strains showing differing sensitivity to the CNRZ 832-B1 phage. Sequence analysis of genes isolated from the phage sensitive strains ATCC 12046, ATCC 15009 and CNRZ 303 showed, in the central part, an identical nucleotide sequence with that obtained from CNRZ 892. In S-layer gene sequences of phage resistant strains, unable to adsorb the phage CNRZ 832-B1 (CNRZ 35, I160, HLMI and M696), point mutations were localized in the same region as the CNRZ 892 isogenic mutants. Data suggest that a change in _1 amino acid in this area seems to affect phage adsorption. [From En summ.]
     DE. [TOP OF PAGE]

223. DNA virus contribution to host evolution. Villarreal, L.P. (1999). pp. 391-420. In AnonymousOrigin and Evolution of Viruses. Academic Press, London. [TOP OF PAGE]

224. Isogenic lysogens of diverse shiga toxin 2-encoding bacteriophages produce markedly different amounts of shiga toxin. Wagner, P.L., Acheson, D.W., Waldor, M.K. (1999). Infect. Immun. 67:6710-6714. We produced isogenic Escherichia coli K-12 lysogens of seven different Shiga toxin 2 (Stx2)-encoding bacteriophages derived from clinical Shiga toxin-producing E. coli (STEC) isolates of serotypes O157:H7, O145, O111, and O83 to assess the variability among these phages and determine if there were phage-related differences in toxin production. Phage genomic restriction fragment length polymorphisms (RFLP) and superinfection resistance studies revealed significant differences among these phages and allowed the seven phages to be placed into five distinct groups. Experiments revealed striking differences in spontaneous phage and toxin production that were correlated with the groupings derived from the RFLP and resistance studies. These results suggest that the genotype of the Stx2 prophage can influence the level of phage release and toxin expression by host strains and thus may be relevant to STEC pathogenesis. [TOP OF PAGE]

225. Increased synthesis of an Escherichia coli membrane protein suppresses F exclusion of bacteriophage T7. Wang, W.F., Margolin, W., Molineux, I.J. (1999). J. Mol. Biol. 292:501-512. Increased synthesis of the protein FxsA alleviates the exclusion of T7 in cells harboring the F plasmid. In contrast to wild-type or cells defective in fxsA, overexpression of fxsA+ allows T7 to form plaques at normal efficiency even though the burst size is reduced to about half that obtained on the isogenic F- strain. No defect in DNA synthesis was observed but late protein synthesis remains partially inhibited and a reduced level of cell leakiness, a prominent feature of F+ cells abortively infected by T7, persists. The FxsA protein is shown to be a cytoplasmic membrane protein. How T7 avoids exclusion by F in cells that exhibit increased levels of FxsA is discussed in terms of its membrane localization. [TOP OF PAGE]

226. Resistance to DNA damage in natural viral communities from the Gulf of Mexico. Weinbauer, M.G., Wilhelm, S.W., Pledger, R.J., Mitchell, D.L., Suttle, C.A. (1999). Aquat. Microb. Ecol. 17:111-120. Using a highly specific radioimmunoassay, the sunlight-induced formation of cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6-4) pyrimidone photoproducts ([6-4] PPs) in viral DNA was investigated for natural virus communities in offshore and coastal waters of the western Gulf of Mexico as well as for clonal viral isolates. Concentrations of (6-4) PPs were consistently lower than CPD concentrations, and ranged from 1.5 to 17.0% of total measured photodamage. The accumulation of photoproducts varied among the natural viral community, the marine Vibrio phage PWH3a-P1 and the Synechococcus sp. DC2 (WH7803) cyanophage SYN-M3, which were deployed in situ from dawn until dark. Natural viral communities were more resistant to DNA damage than the cyanophage isolate SYN-M3, which was more resistant to damage than bacteriophage PWH3a-P1. Moreover, depth profiles revealed that photodamage in viral isolates deployed in the water column accumulated more rapidly at offshore stations than at coastal stations. In natural virus communities collected from offshore surface waters, photodamage accumulated during the solar day with maximum damage occurring between 15:00 and 18:00 h. Depth profiles obtained during calm seas showed that photodamageconcentrations were high in surface waters at the offshore stations and at 1 coastal station. Results at other coastal stations undergoing significant mixing demonstrated no photoproduct accumulations. Results demonstrate that natural virus communities were more tolerant to DNA damaging radiation than the laboratory isolates used in this study. Consequently, laboratory isolates can be poor proxies for UV impacts on natural viral communities. [TOP OF PAGE]

227. Sunlight-induced DNA damage and resistance in natural viral communities. Weinbauer, M.G., Wilhelm, S.W., Suttle, C.A., Pledger, R.J., Mitchell, D.L. (1999). Aquat. Microb. Ecol. 17:111-120. Using a highly specific radioimmunoassay, the sunlight-induced formation of cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6-4) pyrimidone photoproducts ([6-4] PPs) in viral DNA was investigated for natural virus communities in offshore and coastal waters of the western Gulf of Mexico as well as for clonal viral isolates. Concentrations of (6-4) PPs were consistently lower than CPD concentrations, and ranged from 1.5 to 17.0% of total measured photodamage. The accumulation of photoproducts varied among the natural viral community, the marine Vibrio phage PWH3a-P1 and the Synechococcus sp. DC2 (WH7803) cyanophage SYN-M3, which were deployed in situ from dawn until dark. Natural viral communities were more resistant to DNA damage than the cyanophage isolate SYN-M3, which was more resistant to damage than bacteriophage PWH3a-P1. Moreover, depth profiles revealed that photodamage in viral isolates deployed in the water column accumulated more rapidly at offshore stations than at coastal stations. In natural virus communities collected from offshore surface waters, photodamage accumulated during the solar day with maximum damage occurring between 15:00 and 18:00 h. Depth profiles obtained during calm seas showed that photodamage concentrations were high in surface waters at the offshore stations and at 1 coastal station. Results at other coastal stations undergoing significant mixing demonstrated no photoproduct accumulations. Results demonstrate that natural virus communities were more tolerant to DNA damaging radiation than the laboratory isolates used in this study. Consequently, laboratory isolates can be poor proxies for UV impacts on natural viral communities. [TOP OF PAGE]

228. Lysogeny and prophage induction in coastal and offshore bacterial communities. Weinbauer, M.G., Suttle, C.A. (1999). Aquat. Microb. Ecol. 18:217-225. The influence of solar radiation and hydrogen peroxide on induction of lysogens, and the resulting effect on bacteriophage production and bacterial mortality was investigated for coastal and oceanic marine bacterial communities at 6 stations in the western Gulf of Mexico. The percentage of lysogenic cells induced by mitomycin C was also determined. Solar radiation and hydrogen peroxide were not as effective as mitomycin C at inducing phage production. The burst size of cells induced by mitomycin C was estimated by transmission electron microscopy, assuming that cells completely filled with viral particles were on the verge of bursting. The smallest estimates of burst size were associated with oligotrophic oceanic stations and ranged from 15 to 28 viruses produced per lytic event, while in more productive coastal waters the estimated burst sizes ranged from 33 to 64. The mitomycin C-induced phage production and burst size were used to estimate the number of lysogenic bacterial cells. On average, the percentage of inducible lysogens was higher at offshore (1.5 to 11.4%) than at coastal (0.8 to 2.2%) stations. However, with the exception of 1 station, less than 5% of the bacteria could be induced to produce phage, suggesting that lysogens only occasionally comprised a significant component of these bacterial communities. The proportion of lysogens that could be induced by sunlight, relative to those that could be induced by mitomycin C, was lower at oceanic than coastal stations. This implies that prophages in optically transparent offshore waters were more resistant to induction by solar radiation, or that most lysogens that could be triggered by sunlight were already induced. Based on a steady-state model, induction of lysogenic bacteria by solar radiation or hydrogen peroxide could result in between 0 and 3.5% or 0.9 and 3.4% of the total bacterial mortality, respectively. Our results imply that solar radiation and hydrogen peroxide induced lysogenic phage production were not an important source of phage production or bacterial mortality in offshore or coastal waters of the western Gulf of Mexico. [TOP OF PAGE]

229. Family values in the age of genomics: comparative analyses of temperate bacteriophage HK022. Weisberg, R.A., Gottesmann, M.E., Hendrix, R.W., Little, J.W. (1999). Annual Review of Genetics 33:565-602. HK022 is a temperate coliphage related to phage lambda. Its chromosome has been completely sequenced, and several aspects of its life cycle have been intensively studied. In the overall arrangement, expression, and function of most of its genes, HK022 broadly resembles lambda and other members of the lambda family. Upon closer view, significant differences emerge. The differences reveal alternative strategies used by related phages to cope with similar problems and illuminate previously unknown regulatory and structural motifs. HK022 prophages protect lysogens from superinfection by producing a sequence-specific RNA binding protein that prematurely terminates nascent transcripts of infecting phage. It uses a novel RNA-based mechanism to antiterminate its own early transcription. The HK022 protein shell is strengthened by a complex pattern of covalent subunit interlinking to form a unitary structure that resembles chain-mail armour. Its integrase and repressor proteins are similar to those of lambda, but the differences provide insights into the evolution of biological specificity and the elements needed for construction of a stable genetic switch. [TOP OF PAGE]

230. The panda and the phage: Compensatory mutations and the persistence of small populations. Whitlock, M.C., Otto, S.P. (1999). Trends in Ecology & Evolution 14:295-296. Mutation is the ultimate source of all the genetic variation necessary for evolution by natural selection; without mutation evolution would soon cease. Unfortunately, this comes at a cost: most mutations that affect fitness are deleterious. For most large sexual populations, these less fit alleles are eventually eliminated from the population by natural selection. In small populations, however, new deleterious mutations can sometimes increase in frequency and even fix within the population. If harmful mutations fix repeatedly, the fitness of a population might eventually reach such a low level that the population is not capable of sustaining itself and may go extinct, the socalled 'mutational meltdown'. The most important genetic threat to small endangered populations is thought to be this accumulation of new deleterious mutations by genetic drift… But there is hope. Back mutations are only one type of beneficial mutation, and they are a limited subset of the variety of ways in which a genome can mutate to repair the effects of a new deleterious mutation. A fascinating new paper by Christina Burch and Lin Chao has demonstrated, using the bacteriophage f6 as a model system, the ready availability of novel mutations that are capable of compensating for the fitness effects of a fixed deleterious allele rather than simply reverting to the original genotype…. [TOP OF PAGE]

231. Different trajectories of parallel evolution during viral adaptation. Wichman, H.A., Badgett, M.R., Scott, L.A., Boulianne, C.M., Bull, J.J. (1999). Science 285:422-424. [TOP OF PAGE]

232. Purification of MS2 bacteriophage from complex growth media and resulting analysis by the integrated virus detection system (IVDS). Wick, C.H., McCubbin, P.E. (1999). Toxicology Methods 9:253-263. Purification and concentration of viruses from the background material is required whatever subsequent analysis methods are used. For the analysis of viruses it is essential and detection methods depend on this solution. This report demonstrates a methodology for the removal of growth media from a virus preparation. A sample of MS2 was purified using a new ultrafiltration (UF) technique with hollow fibers. A typical MS2 virus sample with a nominal stated concentration of 1.4 X 10¹² plaque-forming units (pfu)/mL in the original growth media was used to demonstrate this method. After UF, the growth media was removed and the virus counted using the integrated virus detection system (IVDS) instrument. This report further describes the use of this ultrafiltration procedure to remove other impurities, such as cesium chloride and albumin, from solutions containing a purified solution of MS2 bacteriophage. These solutions were also analyzed using the IVDS instrument. [TOP OF PAGE]

233. Characterization of purified MS2 bacteriophage by the physical counting methodology used in the integrated virus detection system (IVDS). Wick, C.H., McCubbin, P.E. (1999). Toxicology Methods 9:245-252. A new physically based methodology-the integrated virus detection system (IVDS)-was used to characterize a high-concentration, 10.2 mg protein/mL, sample preparation of MS2 bacteriophage with a reported 10¹⁴ plaque-forming units (pfu)/mL (DPM14) virus count in a common TNME buffer. Virus counts were made using the IVDS instrument following serial dilution. Results indicated virus counts of 1.5 X 105 for the neat sample (DPM14), followed by 6.5 X 10⁴ viruses (DPM13), 1.2 X 10⁴ viruses (DPM12), 9.3 X 10² viruses (DPM11), 88 viruses (DPM10), and 5 viruses (DPM9), respectively. Lower concentrations displayed a consistent multiplier and were consistent with target dilutions. Increases in virus concentration appear to decrease the multiplier, probably through aggregation. The results demonstrate a consistent and simple-to-use methodology. The results further indicate that the IVDS instrument can be used for characterization of other virus preparations with equal ease and similar results. [TOP OF PAGE]

234. Passage of MS2 bacteriophage through various molecular weight filters. Wick, C.H., McCubbin, P.E. (1999). Toxicology Methods 9:265-273. MS2 bacteriophage has a reported nominal molecular weight of 2M daltons. It would be expected that this phage would not pass through filters of various sizes with low molecular weight cutoff (MWCO) values of less than 1M daltons. It was discovered that MS2 bacteriophage will pass through filters with 750K-, 500K-, and 300K-dalton MWCO values. MS2 was retained on the 100K-dalton filter. A cross-flow hollow fiber apparatus was used for the 750K- and 500K-dalton analysis. Centrifuge filters of 1M and 300K and 100K daltons were used. The rate of passage of MS2 through the cross-flow filters is dependent on the tangential flow rate and pressure. Passage through the centrifuge filters depended on the gravitational force applied. [TOP OF PAGE]

235. Viruses and nutrient cycles in the sea. Wilhelm, S.W., Suttle, C.A. (1999). BioScience 49:781-788. Viruses play critical roles in the structure and function of aquatic food webs. [TOP OF PAGE]

236. Analysis of cyanophage diversity and population structure in a south-north transect of the Atlantic ocean. Wilson, W.H., Fuller, N.J., Joint, I.R., Mann, N.H. (1999). Bulletin de l'Institut Oceanographique (Monaco). 0:209-216. Cyanophages (viruses which infect cyanobacteria) are abundant in the marine environment and are thought to be a significant factor in determining the dynamics of Synechococcus spp. populations. In an effort to use molecular techniques to characterise cyanophage populations, we designed cyanophage-specific (CPS) PCR primers based on a gene found in three genetically distinct marine cyanophages (Fuller et al., 1998). CPS primers were used to amplify cyanophage DNA extracted from viral communities concentrated from sea-water samples obtained during a cruise transect between the Falkland Islands, in the south Atlantic ocean, to the UK. Following phylogenetic analysis of cloned and sequenced PCR products, it was revealed that genetic diversity of marine cyanophage clones within a single water sample was as great as clones and cyanophage isolates collected between different oceans. Denaturing gradient gel electrophoresis (DGGE) analysis confirmed this high diversity. DGGE analysis also revealed changes in cyanophage population structure in surface seawater over the south-north transect and throughout depth profiles in the water column. Maximum Synechococcus spp. concentrations, in a stratified water column, correlated with maximum cyanophage diversity. [TOP OF PAGE]

237. Population dynamics of Chesapeake Bay virioplankton: total-community analysis by pulsed-field gel electrophoresis. Wommack, K.E., Ravel, J., Hill, R.T., Colwell, R.R. (1999). Appl. Environ. Microbiol. 65:231-240. It has been hypothesized that, by specifically lysing numerically dominant host strains, the virioplankton may play a role in maintaining clonal diversity of heterotrophic bacteria and phytoplankton populations. If viruses selectively lyse only those host species that are numerically dominant, then the number of a specific virus within the virioplankton would be expected to change dramatically over time and space, in coordination with changes in abundance of the host. In this study, the abundances of specific viruses in Chesapeake Bay water samples were monitored, using nucleic acid probes and hybridization analysis. Total virioplankton in a water sample was separated by pulsed-field gel electrophoresis and hybridized with nucleic acid probes specific to either single viral strains or a group of viruses with similar genome sizes. The abundances of specific viruses were inferred from the intensity of the hybridization signal. By using this technique, a virus comprising 1/1,000 of the total virioplankton abundance (ca. 10(4) PFU/ml) could be detected. Titers of either a single virus species or a group of viruses changed over time, increasing to peak abundance and then declining to low or undetectable levels, and were geographically localized in the bay. Peak signal intensities, i.e., peak abundances of virus strains, were 10-fold greater than the low background level. Furthermore, virus species were found to be restricted to a particular depth, since probes specific to viruses from bottom water did not hybridize with virus genomes from surface water at the same geographical location. Overall, changes in abundances of specific viruses within the virioplankton were episodic, supporting the hypothesis that viral infection influences, if not controls, clonal diversity within heterotrophic bacteria and phytoplankton communities. [TOP OF PAGE]

238. Hybridization analysis of Chesapeake Bay Virioplankton. Wommack, K.E., Ravel, J., Hill, R.T., Colwell, R.R. (1999). Appl. Environ. Microbiol. 65:241-250. It has been hypothesized that, by specifically lysing numerically dominant host strains, the virioplankton may play a role in maintaining clonal diversity of heterotrophic bacteria and phytoplankton populations. If viruses selectively lyse only those host species that are numerically dominant, then the number of a specific virus within the virioplankton would be expected to change dramatically over time and space, in coordination with changes in abundance of the host. In this study, the abundances of specific viruses in Chesapeake Bay water samples were monitored, using nucleic acid probes and hybridization analysis. Total virioplankton in a water sample was separated by pulsed-field gel electrophoresis and hybridized with nucleic acid probes specific to either single viral strains or a group of viruses with similar genome sizes. The abundances of specific viruses were inferred from the intensity of the hybridization signal. By using this technique, a virus comprising 1/1,000 of the total virioplankton abundance (ca. 10(4) PFU/ml) could be detected. Titers of either a single virus species or a group of viruses changed over time, increasing to peak abundance and then declining to low or undetectable levels, and were geographically localized in the bay. Peak signal intensities, i.e., peak abundances of virus strains, were 10-fold greater than the low background level. Furthermore, virus species were found to be restricted to a particular depth, since probes specific to viruses from bottom water did not hybridize with virus genomes from surface water at the same geographical location. Overall, changes in abundances of specific viruses within the virioplankton were episodic, supporting the hypothesis that viral infection influences, if not controls, clonal diversity within heterotrophic bacteria and phytoplankton communities. [TOP OF PAGE]

239. An unexpected temporal pattern of coliphage isolation in groundwaters sampled from wells at varied distances from reclaimed water recharge sites. Yanko, W.A., Jackson, J.L., Williams, F.P., Walker, A.S., Castillo, M.S. (1999). Water Res. 33:53-64. Potable and monitoring wells located in close proximity to a large groundwater recharge project which utilizes a blend of surface water and reclaimed wastewater for recharge were tested for coliphage over a period of 6 months to assess the potential for virus migration. During the first 3 months FRNA phage were detected once at a shallow monitoring well. In late summer, an unexpected pulse of phage was detected in all wells, including control sites, suggesting an ecological phenomenon independent of recharge operations. Cubic and filamentous F-specific coliphage, consistent with the Leviviridae and Inoviridae groups, and a noncontractile tailed phage consistent with the Siphoviridae family were detected. There was no discernible relationship between recharge operations and the pattern of phage populations detected. Phage were detected using a host designated HS12, a variant of HS(pFamp)R (Debartolomeis, J. and Cabelli, V. J. (1991). Evaluation of an Escherichia coli host strain for enumeration of f male-specific bacteriophages. Appl. Environ. Microbiol. 57, 1301). During the study it was found that HS12 contained a temperate Myoviridae phage; Myoviridae phage were subsequently excluded from the results. A total of 26 production wells, including 3 control sites, were sampled monthly and 6 monitoring wells were sampled every two weeks. Water reclamation plant effluents and river water upstream of effluent discharges were randomly sampled. The concentration and distribution of phage isolated was quite different in chlorinated effluent compared to river water. The majority of isolates from reclaimed water were filamentous DNA F-specific phage suggesting this group was more resistant to chlorine. Groundwater samples were analyzed using a novel large volume enrichment technique that proved very sensitive for detecting low concentrations of phage. [TOP OF PAGE]

240. Biodistribution of filamentous phage-Fab in nude mice. Yip, Y.L., Hawkins, N.J., Smith, G., Ward, R.L. (1999). Journal of Immunological Methods 225:171-178. In vivo panning of peptide libraries in mice has allowed the isolation of peptides which target the vasculature of specific organs. The application of this approach to phage displaying Fab fragments (phage-Fab) could lead to the isolation of antibodies which recognize novel tumor antigens. In this study, we have evaluated the biodistribution of phage-Fab in nude mice. Balb/c nude mice were injected intravenously with 10(9) TU of phage displaying the anti-colon cancer Fab c30.6. Blood samples were collected at nine time points over a period of 72 h and three groups of four mice were sacrificed at 4 min, 24 h and 72 h. Normal tissues (liver, colon, spleen, kidneys, lungs, skeletal muscle) and faeces were collected at these time points and the number of viable phage in each sample was determined. The distribution of phage in tissues was also examined by immunohistochemical analysis of paraffin-embedded tissues. Regression analysis of plasma kinetic data showed that the half-life and the volume of distribution of phage was 3.6 h and 1 ml, respectively. Phage uptake occurred predominantly in lungs, kidneys, spleen and liver. Relatively few phage were distributed to colon and muscle, and phage were eliminated from the circulation by 72 h. Immunohistochemical analysis showed phage to be mainly within the vasculature at 4 min, whereas notable phage extravasation was observed at 24 h and 72 h. In conclusion, this study provides information on the in vivo behavior of phage-Fab which will be useful in the design of in vivo panning strategies. By choosing appropriate time points for tissue collection, it may be possible to isolate novel Fabs against both intra- and extravascular targets. [TOP OF PAGE]

241. Characterization of a Vibrio parahaemolyticus phage isolated from marine. Yoon, S.O., Ju, S.A., Heo, M.S., Jung, C.R., Ju, J.W. (1999). Journal of the Korean Society for Microbiology 34:423-433. A novel bacteriophage, designated as VPP97, that infects the strains of Vibiro parahaemolyticus (hallophilic, Gram-negative bacterium) isolated most commonly from marine environments, has been discovered, and several of its properties have been determined. The plaques were clear and sized 0.6-1.0 mm in diameter. The virion forms a single band on 70% sucrose gradient and p1.50 CsCl gradient by sucrose gradient centrifugation and CsCl gradient centrifugation respectively. It has a hexagonal head and a relatively long tail, as shown by electron microscopy. Vibrio alginolyticus, Vibrio fluvialis and Vibrio furnissii were also sensitive to this phage It was almost totally inactivated at 70ºC and at pH below 5 or over 10. The nucleic acid of VPP97 is composed of DNA. The VPP97 had 9 specific structural proteins sized between 21.5 kDa and 97.4 kDa on SDS-PAGE. When V. parahaemolyticus cultures were treated with either phage VPP97 or one of the several antibiotics for 2 hours, the viable number of V. parahaemolyticus treated with the phage VPP97 is lower than that treated with chloramphenicol, erythromycin or penicillin, but not lower than that treated with tetracycline. Mice that have responded to the phage treatment revealed the lower numbers of V. parahaemolyticus in small intestine and less damage on small intestine compared to the untreated mice. Therefore, we suggest that the phage treatment appears effective to the infection by V. parahaemolyticus. [TOP OF PAGE]

242. Amplification and spread of viruses in a growing plaque. You, L., Yin, J. (1999). J. Theor. Biol. 200:365-373. The two-dimensional propagation of viruses through a "lawn" of receptive hosts, commonly called plaque growth, reflects the dynamics of interactions between viruses and host cells. Here we treat the amplification of viruses during plaque growth as a reaction-diffusion system, where interactions among the virus, uninfected host cells, and virus-producing host-virus complexes are accounted for using rates of viral adsorption to and desorption from the host-cell surface, rates of reproduction and release of progeny viruses by lysis of the host, and by the coupling of these reactions with diffusion of free virus within the agar support. Numerical solution of the system shows the development of a traveling wave of reproducing viruses, where the velocity of the wave is governed by the kinetic and diffusion parameters. The model has been applied to predict the propagation velocity of a bacteriophage plaque. Different mechanisms may account for the dependence of this velocity on the host density during early stages of a growing plaque. The model provides a means to explore how changes in the virus-host interactions may be manifest in a growing plaque. [TOP OF PAGE]

243. Blue-green algal viruses (cyanophages). Zhao, Y., Shi, Z., Huang, G., Wang, X. (1999). Virologica Sinica 14:100-105. [TOP OF PAGE]

244. [Examining interaction of phages with microorganisms by fluorometry and electro-orientation spectroscopy]. Zhilenkov, E.L., Fomchenkov, V.M., Novikov, I.A., Sadomov, V.E., Oborotov, M.V., Gremiakova, T.A. (1999). Vestnik Rossiiskoi Akademii Medisinskiky Nauk 24-29. Bacterial sensitivity to different various phages was examined by electro-orientation spectroscopy, fluorometry, and electron microscopy. The strains of Pseudomonas aeruginosa, Staphylococcus aureus, Yersinia pestis, Mycobacterium smegmatis, and Xanthomonas campestris were used. The fluorescence intensity of a membranotropic agent in the ANS-cell-phage system was shown to depend on the interaction of a bacterial virus and a microorganism. Fluorometric data correlated with electro-orientation spectroscopic findings. An analysis of the low-frequency site makes it possible to determine phage adsorption on the bacterial surface. The changes in electro-orientation effects at high frequencies suggest that there are barrier dysfunctions in the external membranes and that there is cellular phage reproductions. Whether fluorometry and electro-orientation spectroscopy can be further used for rapid identification of microorganisms by using phages is discussed. [TOP OF PAGE]

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