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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

  82. Identification of a diagnostic marker to detect freshwater cyanophages of filamentous cyanobacteria. Baker,A.C., Goddard,V.J., Davy,J., Schroeder,D.C., Adams,D.G., Wilson,W.H. (2006). Appl. Environ. Microbiol. 72:5713-5719. Cyanophages are viruses that infect the cyanobacteria, globally important photosynthetic microorganisms. Cyanophages are considered significant components of microbial communities, playing major roles in influencing host community diversity and primary productivity, terminating cyanobacterial water blooms, and influencing biogeochemical cycles. Cyanophages are ubiquitous in both marine and freshwater systems; however, the majority of molecular resear