- Immunity profiles of wild-type and recombinant Shiga-Like toxin-encoding bacteriophages and characterization of novel double lysogens. Allison, H. E., Sergeant, M. J., James, C. E., Saunders, J. R., Smith, D. L., Sharp, R. J., Marks, T. S., McCarthy, A. J. (2003). Infection and Immunity 71:3409-3418. Pathogenicity of Shiga-like toxin (stx)-producing Escherichia coli (STEC), notably serotype 0157, the causative agent of hemorrhagic colitis, hemolytic-uremic syndrome, and thrombotic thrombocytopenic purpura, is based partly on the presence of genes (stx1 and/or stx2) that are known to be carried on temperate lambdoid bacteriophages. Stx phages were isolated from different STEC strains and found to have genome sizes in the range of 48 to 62 kb and to carry either stx1 or stx2 genes. Restriction fragment length polymorphism patterns and sodium dodecyl sulfate-polyacrylamide gel electrophoresis protein profiles were relatively uninformative, but the phages could be differentiated according to their immunity profiles. Furthermore, these were sufficiently sensitive to enable the identification and differentiation of two different phages, both carrying the genes for Stx2 and originating from the same STEC host strain. The immunity profiles of the different Stx phages did not conform to the model established for bacteriophage lambda, in that the pattern of individual Stx phage infection of various lysogens was neither expected nor predicted. Unexpected differences were also observed among Stx phages in their relative lytic productivity within a single host. Two antibiotic resistance markers were used to tag a recombinant phage in which the stx genes were inactivated, enabling the first reported observation of the simultaneous infection of a single host with two genetically identical Stx phages. The data demonstrate that, although Stx phages are members of the lambdoid family, their replication and infection control strategies are not necessarily identical to the archetypical bacteriophage l, and this could be responsible for the widespread occurrence of stx genes across a diverse range of E. coli serotypes.coli.serotypes.
- Isolation and characterization of Campylobacter bacteriophages from retail poultry. Atterbury, R. J., Connerton, P. L., Dodd, C. E. R., Rees, C. E. D., Connerton, I. F. (2003). Applied and Environmental Microbiology 69:4511-4518. The ability of phages to survive processing is an important aspect of their potential use in the biocontrol of Campylobacter in poultry production. To this end, we have developed a procedure to recover Campylobacter bacteriophages from chilled and frozen retail poultry and have validated the sensitivity of the method by using a characterized Campylobacter phage (i.e., NCTC 12674). By using this method, we have shown that Campylobacter phages can survive on retail chicken under commercial storage conditions. Retail chicken portions purchased in the United Kingdom were screened for the presence of endogenous Campylobacter phages. Thirty-four Campylobacter bacteriophages were isolated from 300 chilled retail chicken portions, but none could be recovered from 150 frozen chicken portions. The phage isolates were characterized according to their lytic profiles, morphology, and genome size. The free-range products were significantly more likely to harbor phages (P < 0.001 by single-factor analysis of variance) than were standard or economy products. This study demonstrates that Campylobacter bacteriophages, along with their hosts, can survive commercial poultry processing procedures and that the phages exhibited a wide range of recovery rates from chicken skin stored at 4°C.
- Metagenomic analysis of an uncultured viral community from human feces. Breitbart, M., Hewson, I., Felts, B., Mahaffy, J. M., Nulton, J., Salamon, P., Rohwer, F. (2003). Journal of Bacteriology 185:6220-6223. Here we present the first metagenomic analyses of an uncultured viral community from human feces, using partial shotgun sequencing. Most of the sequences were unrelated to anything previously reported. The recognizable viruses were mostly siphophages, and the community contained an estimated 1,200 viral genotypes.
- In vivo lysogenic conversion of Tox(-) Streptococcus pyogenes to Tox(+) with Lysogenic Streptococci or free phage. Broudy, T. B., Fischetti, V. A. (2003). Infection and Immunity 71:3782-3786. Temperate bacteriophage can transfer toxin-encoding genes between bacteria, often resulting in acquired pathogenicity. However, little is known regarding the effects of the eukaryotic host on the phage-pathogen interaction. Using Streptococcus pyogenes as a model, we demonstrate, both in vitro and in vivo, that the eukaryote mediates the efficient induction of toxin-encoding temperate phage and the resultant conversion of Tox- flora to Tox+. Furthermore, we show that both phage induction and subsequent conversion need not happen in the same mammalian host, as host-to-host phage transmission can result in toxigenic conversion within the secondary host. Ultimately, our findings demonstrate that the eukaryotic host serves as an essential component in the phage-mediated evolution of virulence within the microbial population.
- The future of bacteriophage biology. Campbell, A. (2003). Nature reviews Genetics 4:471-477. After an illustrious history as one of the primary tools that established the foundations of molecular biology, bacteriophage research is now undergoing a renaissance in which the primary focus is on the phages themselves rather than the molecular mechanisms that they explain. Studies of the evolution of phages and their role in natural ecosystems are flourishing. Practical questions, such as how to use phages to combat human diseases that are caused by bacteria, how to eradicate phage pests in the food industry and what role they have in the causation of human diseases, are receiving increased attention. Phages are also useful in the deeper exploration of basic molecular and biophysical questions.
- Microbiological aspects of an urban river used for unrestricted irrigation in the semi-arid region of north-east Brazil. Ceballos, B. S. O., Soares, N. E., Moraes, M. R., Catao, R. M. R., Konig, A. (2003). Water Science and Technology 47:51-57. This study compared the behaviour of pathogenic bacteria (Salmonella and Listeria), faecal indicators (faecal coliforms FC and faecal streptococci FS), somatic coliphages and F-specific bacteriophages in an urban river contaminated with domestic sewage and surface run-off from agricultural and cattle grazing lands. The influence of physical and chemical parameters was also investigated as well as Salmonella and Listeria serotype diversity and drug resistance patterns. Faecal contamination was high (FC = 5 x 106 - 4 x 103 CFU/100 mL; FS = 4 x 105 - 2 x 102 CFU/100 mL) but decreased along the river by up to 99.5% following 47% reduction of BOD5 and 91% increase of DO, both associated with the self purification process. Somatic coliphages (6.9 x 105 - 1 x 103 PFU/100 mL) and F-specific bacteriophages (5.8 x 104 - 65 PFU/100 mL) behaved similarly with reductions of 99.85%. Salmonella and Listeria were isolated at all sampling points with highest frequencies (91-100%) at those with sewage discharge and rural water run-off. The lowest value (35%) occurred at the end of the river where it was (a) wider and shallower, (b) it ran slower and was warmer (29-33ºC), (c) the pH was alkaline (8.2-9.9), (d) electrical conductivity (2,200-5,800 microS/cm) and DO (6-13 mg/L) were highest. Pathogen decline did not follow exactly FC and FS reduction patterns, while physical and chemical parameters apparently did not interfere with Salmonella and Listeria survival to the same extent as they did with FC and FS. Somatic coliphages and F-specific bacteriophages did not show more resistance than bacterial indicators. Catchment area contribution seemed to be more significant for pathogens than for indicators and rainy periods increased pathogenic isolation frequency. Five Salmonella serotypes and five serogroups were identified. S. hadar and serogroup E were predominant (50%); both are increasing in Brazil apparently from animal sources. Nearly 25% of Salmonella strains were resistant to at least one of twelve antimicrobials tested. Resistance to tetracycline was common (17%) followed by cefalotine (3%). Five Listeria serogroups were isolated and L. grayi (43%) and L. monocytogenes (9%) were present at all points. Listeria drug resistance rates were 100% for oxaciline followed by clindamicine (97%), tetracycline (34%) and vancomycin (32%). Both pathogenic bacterial strains presented resistance to the same drugs observed in humans and warm blood animals but the high number of sensitive strains and the low numbers of strains resistant to more than one drug was not expected because of the heavy anthropogenic impact in this basin.
- Comparative electrochemical inactivation of bacteria and bacteriophage. Drees, K. P., Abbaszadegan, M., Maier, R. M. (2003). Water Research 37:2291-2300. Electric fields and currents have been shown to be capable of disinfecting drinking water and reducing the numbers of bacteria and yeast in food. However, little research has been conducted regarding the effectiveness of electric fields and currents in the inactivation of viruses. The objective of this study was to compare the ability of bacteria and bacteriophage to survive exposure to direct electric current in an electrochemical cell, where they would be subject to irreversible membrane permeabilization processes, direct oxidation of cellular/viral constituents by electric current, and disinfection by electrochemically generated oxidants. Suspensions of the bacteria Escherichia coli and Pseudomonas aeruginosa and bacteriophage MS2 and PRD1 at both high (approximately 1 x 106CFU or PFU/mL) and low (approximately 1 x 103CFU or PFU/mL) population densities were exposed to currents ranging from 25 to 350mA in 5 s pulses. Post-exposure plaque counts of the bacteriophage were proportionally higher than bacterial culturable counts at corresponding current exposures. E. coli and MS2 were then exposed to 5mA for 20 min at both high and low population densities. The inactivation rate of E. coli was 2.1-4.3 times greater than that of MS2. Both bacteria and bacteriophage were more resistant to exposure to direct current at higher population densities. Also, amelioration of inactivation within the electrochemical cell by the reducing agent glutathione indicates the major mechanism of inactivation in the electrochemical cell is disinfection by electrochemically generated oxidants. The implications of these results are that technologies relying upon direct current to reduce the numbers of microbes in food and water may not be sufficient to reduce the numbers of potentially pathogenic viruses and ensure the safety of the treated food or water.
- Application of a novel immunomagnetic separation-bacteriophage assay for the detection of Salmonella enteritidis and Escherichia coli O157:H7 in food. Favrin, S. J., Jassim, S. A., Griffiths, M. W. (2003). International Journal of Food Microbiology 85:63-71. Salmonella infection is the second most prevalent cause of foodborne illness in most developing countries. Meat, poultry, and dairy products are frequently implicated in outbreaks. The objective of this study was to apply a novel immunomagnetic separation (IMS)-bacteriophage assay to the detection of Salmonella enteritidis in artificially inoculated skimmed milk powder, chicken rinses, and ground beef. In all food types tested, the IMS-bacteriophage assay was able to detect an average of 3 CFU of S. enteritidis in 25 g or ml of food sample. Total assay time including pre-enrichment is about 20 h. The results indicate that the IMS-bacteriophage assay is a rapid and sensitive means of detecting S. enteritidis in these foods. The assay was successfully adapted to the detection of Escherichia coli O157:H7 and was able to detect E. coli in ground beef at the lowest inoculation level tested, 2 CFU/g. The assay was also adapted to the simultaneous detection of S. enteritidis and E. coli. The results indicate that the IMS-bacteriophage assay shows promise for the simultaneous detection of these pathogens, but further development work would be necessary to improve sensitivity and produce reliable results at low inoculation levels.
- Evaluation of potential indicators of viral contamination in shellfish and their applicability to diverse geographical areas. Formiga-Cruz, M., Allard, A. K., Conden-Hansson, A. C., Henshilwood, K., Hernroth, B. E., Jofre, J., Lees, D. N., Lucena, F., Papapetropoulou, M., Rangdale, R. E., Tsibouxi, A., Vantarakis, A., Girones, R. (2003). Applied and Environmental Microbiology 69:1556-1563. The distribution of the concentration of potential indicators of fecal viral pollution in shellfish was analyzed under diverse conditions over 18 months in diverse geographical areas. These microorganisms have been evaluated in relation to contamination by human viral pathogens detected in parallel in the analyzed shellfish samples. Thus, significant shellfish-growing areas from diverse countries in the north and south of Europe (Greece, Spain, Sweden, and the United Kingdom) were defined and studied by analyzing different physicochemical parameters in the water and the levels of Escherichia coli, F-specific RNA bacteriophages, and phages infecting Bacteroides fragilis strain RYC2056 in the shellfish produced, before and after depuration treatments. A total of 475 shellfish samples were studied, and the results were statistically analyzed. According to statistical analysis, the presence of human viruses seems to be related to the presence of all potential indicators in the heavily contaminated areas, where E. coli would probably be suitable as a fecal indicator. The F-RNA phages, which are present in higher numbers in Northern Europe, seem to be significantly related to the presence of viral contamination in shellfish, with a very weak predictive value for hepatitis A virus, human adenovirus, and enterovirus and a stronger one for Norwalk-like virus. However, it is important to note that shellfish produced in A or clean B areas can sporadically contain human viruses even in the absence of E. coli or F-RNA phages. The data presented here will be useful in defining microbiological parameters for improving the sanitary control of shellfish consumed raw or barely cooked.
- Improvement of phage defence in Lactococcus lactis by introduction of the plasmid encoded restriction and modification system LlaAI. Gabs, S., Josephsen, J. (2003). Letters in Applied Microbiology 36:332-336. AIMS: To study the ability of the plasmid-encoded restriction and modification (R/M) system LlaAI to function as a bacteriophage resistance mechanism in Lactococcus lactis during milk fermentations. METHODS AND RESULTS: Plasmid pAIcat4, carrying the R/M system LlaAI and a chloramphenicol resistance cassette, was introduced into the plasmid-free strain L. lactis MG1614 and the industrial strain L. lactis 964. By measuring changes in conductivity the influence of different phage on the growth was determined. CONCLUSIONS: The plasmid-encoded R/M system LlaAI significantly improves the bacteriophage resistance of L. lactis during milk fermentations. SIGNIFICANCE AND IMPACT OF THE STUDY: It is essential to determine the potential of a phage defence mechanism in L. lactis starter culture strains during growth in milk before steps are taken to improve starter cultures. This study shows that LlaAI is useful for improvement of starter cultures.
- Bacteriophages as viral indicators for radiation processing of water: a chemical approach. Gehringer, P., Eschweiler, H., Leth, H., Pribil, W., Pfleger, S., Cabaj, A., Haider, T., Sommer, R. (2003). Applied Radiation and Isotopes 58:651-656. Inactivation of the bacteriophages PHI X 174 (somatic coliphage), MS2 (F-specific coliphage) and B40-8 (phage infecting Bacteroides fragilis) suspended in tap water was studied applying gamma and electron beam irradiation as well. PHI X 174 phage was found to be a suitable viral indicator for water disinfection by means of ionizing radiation. The nutrient broths introduced simultaneously with the bacteriophages into the water when it is spiked with the phages for the experiments did not significantly change the scavenging capacity of the water matrix. No dose rate effect was observed with MS2 and B40-8 phages but PHI X 174 phage showed a clear dose rate effect. It was found that in water MS2 phage is significantly more sensitive to ionizing radiation than Escherichia coli.
- Reduction of experimental Salmonella and Campylobacter contamination of chicken skin by application of lytic bacteriophages. Goode, D., Allen, V. M., Barrow, P. A. (2003). Applied and Environmental Microbiology 69:5032-5036. Lytic bacteriophages, applied to chicken skin that had been experimentally contaminated with Salmonella enterica serovar Enteritidis or Campylobacter jejuni at a multiplicity of infection (MOI) of 1, increased in titer and reduced the pathogen numbers by less than 1 log10 unit. Phages applied at a MOI of 100 to 1,000 rapidly reduced the recoverable bacterial numbers by up to 2 log10 units over 48 h. When the level of Salmonella contamination was low (< log10 2 per unit area of skin) and the MOI was 105, no organisms were recovered. By increasing the number of phage particles applied (i.e., MOI of 107), it was also possible to eliminate other Salmonella strains that showed high levels of resistance because of restriction but to which the phages were able to attach.
- Morphological, host range, and genetic characterization of two coliphages. Goodridge, L., Gallaccio, A., Griffiths, M. W. (2003). Applied and Environmental Microbiology 69:5364-5371. Two coliphages, AR1 and LG1, were characterized based on their morphological, host range, and genetic properties. Transmission electron microscopy showed that both phages belonged to the Myoviridae; phage particles of LG1 were smaller than those of AR1 and had an isometric head 68 nm in diameter and a complex contractile tail 111 nm in length. Transmission electron micrographs of AR1 showed phage particles consisting of an elongated isometric head of 103 by 74 nm and a complex contractile tail 116 nm in length. Both phages were extensively tested on many strains of Escherichia coli and other enterobacteria. The results showed that both phages could infect many serotypes of E. coli. Among the enterobacteria, Proteus mirabilis, Shigella dysenteriae, and two Salmonella strains were lysed by the phages. The genetic material of AR1 and LG1 was characterized. Phage LG1 had a genome size of 49.5 kb compared to 150 kb for AR1. Restriction endonuclease analysis showed that several restriction enzymes could degrade DNA from both phages. The morphological, genome size, and restriction endonuclease similarities between AR1 and phage T4 were striking. Southern hybridizations showed that AR1 and T4 are genetically related. The wide host ranges of phages AR1 and LG1 suggest that they may be useful as biocontrol, therapeutic, or diagnostic agents to control and detect the prevalence of E. coli in animals and food.
- The complete sequence of marine bacteriophage VpV262 infecting Vibrio parahaemolyticus indicates that an ancestral component of a T7 viral supergroup is widespread in the marine environment. Hardies, S. C., Comeau, A. M., Serwer, P., Suttle, C. A. (2003). Virology 310:359-371. The 46,012-bp sequence of the marine bacteriophage VpV262 infecting the bacterium Vibrio parahaemolyticus is reported. The VpV262 sequence reveals that it is a distant relative of marine Roseophage SIO1, and an even more distant relative of coliphage T7. VpV262 and SIO1 appear to represent a widespread marine phage group that lacks an RNA polymerase gene and is ancestral to the T7-like phages. We propose that this group together with the T7-like phages be designated as the T 7 supergroup. The ancestral head structure gene module for the T7 supergroup was reconstructed by using sensitive biased Psi-blast searches supplemented by statistical support derived from gene order. In the early and replicative segments, these phages have participated in extensive interchange with the viral gene pool. VpV262 carries a different replicative module than SIO1 and the T7-like phages.
- Can an arbitrary sequence evolve towards acquiring a biological function? Hayashi, Y., Sakata, H., Makino, Y., Urabe, I., Yomo, T. (2003). Journal of Molecular Evolution 56:162-168. To explore the possibility that an arbitrary sequence can evolve towards acquiring functional role when fused with other pre-existing protein modules, we replaced the D2 domain of the fd-tet phage genome with the soluble random polypeptide RP3-42. The replacement yielded an fd-RP defective phage that is six-order magnitude lower infectivity than the wild-type fd-tet phage. The evolvability of RP3-42 was investigated through iterative mutation and selection. Each generation consists of a maximum of ten arbitrarily chosen clones, whereby the clone with highest infectivity was selected to be the parent clone of the generation that followed. The experimental evolution attested that, from an initial single random sequence, there will be selectable variation in a property of interest and that the property in question was able to improve over several generations. fd-7, the clone with highest infectivity at the end of the experimental evolution, showed a 240-fold increase in infectivity as compared to its origin, fd-RP. Analysis by phage ELISA using anti-M13 antibody and anti-T7 antibody revealed that about 37-fold increase in the infectivity of fd-7 was attributed to the changes in the molecular property of the single polypeptide that replaced the D2 domain of the g3p protein. This study therefore exemplifies the process of a random polypeptide generating a functional role in rejuvenating the infectivity of a defective bacteriophage when fused to some preexisting protein modules, indicating that an arbitrary sequence can evolve toward acquiring a functional role. Overall, this study could herald the conception of new perspective regarding primordial polypeptides in the field of molecular evolution.
- Evaluation of biotracers to monitor effluent retention time in constructed wetlands. Hodgson, C. J., Perkins, J., Labadz, J. C. (2003). Letters in Applied Microbiology 36:362-371. AIMS: With concern surrounding the environmental impact of chemical tracers on the aquatic environment, this paper presents the initial evaluation of biotracers used to determine the effluent retention time, an important performance indicator, in a Free Water Surface Constructed Wetland. METHODS AND RESULTS: Production of the biotracers, coliphage MS2, and the bacteriophage of Enterobacter cloacae and antibiotic resistant endospores of Bacillus globigii is described in detail. Their subsequent use in three separate tracer experiments - January, March and June (2000) - revealed the variability of retention time with respect to effluent flow. The biotracer MS2 showed the constructed wetland had a retention time of 8-9 h at a mean discharge of 0.9 l s-1, increasing to 10-12 h at a mean discharge 0.3 l s-1. A similar retention of 9-10 h at a mean discharge of 0.3 l s-1 was calculated for the Ent. cloacae phage. In contrast, use of endospores revealed considerably longer retention times at these mean discharge rates; 12-24 h and 36-48 h, respectively. CONCLUSION: Biotracers could provide a useful and environmentally friendly technique to monitor effluent retention in constructed wetlands. At this stage the phage tracers appear particularly promising due to ease of isolation and recovery. SIGNIFICANCE AND IMPACT OF THE STUDY: Initial results are encouraging and have highlighted the potential of biotracers as alternatives to chemical tracers, even in microbially-rich waters.
- Evaluation of aerosol spray and intramuscular injection of bacteriophage to treat an Escherichia coli respiratory infection. Huff, W. E., Huff, G. R., Rath, N. C., Balog, J. M., Donoghue, A. M. (2003). Poultry science 82:1108-1112. Two studies were conducted to determine the efficacy of either aerosol or i.m. injection of bacteriophage to treat an Escherichia coli respiratory infection in broiler chickens. An additional two studies were conducted to enumerate the bacteriophage in the blood of birds at 1, 2, 3, 4, 5, 6, 24, and 48 h after being sprayed or injected i.m. with bacteriophage. Five birds were bled at each period. In study 1, there were 10 treatments with three replicate pens of 10 birds. The treatments consisted of an untreated control, heat-killed bacteriophage spray, active bacteriophage spray, E. coli challenge at 7 d of age, and E. coli challenge followed by spraying the birds with heat-killed bacteriophage or active bacteriophage at 2, 24, or 48 h after challenge. In study 2 there were 11 treatments with three replicate pens of 10 birds per pen. The treatments were untreated controls, birds injected i.m. in the thigh with heat-killed or active bacteriophage, E. coli challenge at 7 d of age, PBS challenge, E. coli challenge followed by injection of heat-killed or active bacteriophage immediately after challenge or at 24 or 48 h after challenge. In both studies the E. coli challenge consisted of injecting 10(4) cfu into the thoracic air sac. Treatment of this severe E. coli infection with the bacteriophage aerosol spray significantly reduced mortality from 50 to 20% when given immediately after the challenge but had little treatment efficacy when administered 24 or 48 h after challenge. The i.m. injection of bacteriophage significantly reduced mortality from 53 to 17%, 46 to 10%, and 44 to 20% when given immediately, 24, or 48 h after challenge, respectively. Only a few birds sprayed with bacteriophage had detectable bacteriophage in their blood with an average of 96 pfu/mL 1 h after bacteriophage administration, and no bacteriophage was detected 24 and 48 h after bacteriophage administration. All birds injected i.m. with bacteriophage had detectable levels of bacteriophage in their blood at levels of 10(4) pfu/mL of blood up to 6 h after bacteriophage administration, and four of the five birds had detectable bacteriophage in their blood at an average level of 70 pfu/mL of blood 24 h after bacteriophage administration. The relative inefficiency of the spray treatment to the i.m. injection treatment may be due to the inability to get bacteriophage into the blood at high concentrations when the birds are sprayed versus the consistent high titers achieved with the i.m. injection of bacteriophage. These data provide support to the concept that bacteriophage may be an effective alternative to antibiotics in animal production when they are administered in a way that delivers high titers of the bacteriophage to the critical site of the bacterial infection.
- [Bacteriophage therapy]. Huovinen, P. (2003). Duodecim; laaketieteellinen aikakauskirja 119:581-583.
- Efficient release of overproduced gene products from Escherichia coli BL21(DE3) by lytic infection with newly isolated bacteriophages. Iida, Yuichiro, Matsuda, Yoshinori, Saito, Ryuichiro, Nakasato, Masanori, Nonomura, Teruo, Kakutani, Koji, Tosa, Yukio, Mayama, Shigeyuki, Toyoda, H. (2003). Bioscience Biotechnology and Biochemistry 67:198-202. Overproduced proteins from Escherichia coli BL21(DE3) were efficiently released with virulent bacteriophages. Leviviridae-like bacteriophages were isolated from soil and used to lyse BL21(DE3) cells transformed with beta-glucosidase, chitinase, or chitosanase genes. This method caused lysis of bacterial cells similar to that by conventional sonication and enabled us to effectively recover and purify the enzymes.
- The vertical distribution and diversity of marine bacteriophage at a station off Southern California. Jiang, S., Fu, W., Chu, W., Fuhrman, J. A. (2003). Microbial Ecology 45:399-410. Sixty-two bacteriophages were isolated on eight indigenous bacteria from a Pacific Ocean station spanning 887-m vertical depth, on two occasions between 1999 and 2000. On the basis of 16S rRNA sequences, six hosts were tentatively identified to be in the genus Vibrio and the other two were closely related to Altermonas macleodii (W9a) and Pseudoalteromonas spp. (W13a). Restriction fragment length polymorphism (RFLP) analysis of phage genomes using AccI and HapI showed that 16 phages infecting host C4a (Vibrio) displayed 14 unique RFLP patterns. However, identical phages infecting host C4b, C6a, and C6b (all Vibrio) were obtained from both the surface layer and the hypoxic zone at 850 m. Most phage isolates from the second year had a different RFLP pattern but shared genetic similarity to the phages infecting the same host from the previous year based on a hybridization study using phage genome probes. Cluster analysis of RFLP patterns and hybridization results also indicated that phages infecting the same or genetically related hosts, in general, shared higher degrees of homology in spite of the diverse RFLP patterns. Pulsed field gel electrophoresis (PFGE) analysis of native viral genomes indicated a range in genome size from less than 40 to 200 kb, and the dominant band shifted up by about 5-10 kb in the deep samples compared to the shallow ones. Hybridization of phage genome probes with total viral community DNA from various depths suggests these isolates, or at least some of their genes, represent a detectable portion of the natural viral community and were distributed throughout the water column. Thus, the results of this study demonstrated that the genetic diversity of bacteriophage in the ocean is far greater than that of their bacterial hosts. However, host range may have contributed to the evolution of the diverse phage population in the marine environment.
- Lack of correlation between O-serotype, bacteriophage susceptibility and genomovar status in the Burkholderia cepacia complex. Kenna, D. T., Barcus, V. A., Langley, R. J., Vandamme, P., Govan, J. R. W. (2003). FEMS Immunology and Medical Microbiology 35:87-92. The Burkholderia cepacia complex comprises at least nine phylogenetically related genomic species (genomovars) which cause lifethreatening infection in immunocompromised humans, particularly individuals with cystic fibrosis or chronic granulomatous disease. Prior to recognition that 'B. cepacia' comprise multiple species, in vitro studies revealed that the lipopolysaccharide (LPS) of these Gramnegative bacteria is strongly endotoxic. In this study, we used 117 B. cepacia complex isolates to determine if there is a correlation between O-antigen serotype and genomovar status. Isolates were also tested for their ability to act as bacterial hosts for the LPS-binding bacteriophages NS1 and NS2. The absence of genomovar II (Burkholderia multivorans) in 'historical B. cepacia' isolates was notable. Neither O-serotype nor phage susceptibility correlated with genomovar status. We conclude that variability in LPS may contribute to the success of these highly adaptable bacteria as human pathogens.
- The role of horizontal gene transfer by bacteriophages in the origin of pathogenic bacteria. Krylov, V. (2003). Russian Journal of Genetics 39:483-504. The review considers the involvement of bacteriophages in transferring genes, which determine bacterial pathogenicity, and the increasing role of comparative genomics and genetics of bacteria and bacteriophages in detecting new cases of horizontal gene transfer. Examples of phage participation in this process proved to a different extent are described. Emphasis is placed on the original work carried out in Russia and focused on bacteriophages (temperate transposable phages and giant virulent fKZ-like phages) of conditional pathogen Pseudomonas aeruginosa. Consideration is given to the possible lines of further research of the role of bacteriophages in the infection process and, in particular, the role of virulent phages, whose products are sim-ilar to those of pathogenic bacteria, in modification of clinical signs of infectious diseases and in evolution. An attempt is made to predict the possible direction of pathogen evolution associated with development of new treatment strategies and generation of new specific niches.
- [Role of horizontal gene transfer by bacteriophages in the origin of pathogenic bacteria]. Krylov, V. N. (2003). Genetika 39:595-620. The review considers the involvement of bacteriophages in transferring genes, which determine bacterial pathogenicity, and the increasing role of comparative genomics and genetics of bacteria and bacteriophages in detecting new cases of horizontal gene transfer. Examples of phage participation in this process proved to a different extent are described. Emphasis is placed on the original work carried out in Russia and focused on bacteriophages (temperate transposable phages and giant virulent phi KZ-like phages) of conditional pathogen Pseudomonas aeruginosa. Consideration is given to the possible lines of further research of the role of bacteriophages in the infection process and, in particular, the role of virulent phages, whose products are similar to those of pathogenic bacteria, in modification of clinical signs of infectious diseases and in evolution. An attempt is made to predict the possible direction of pathogen evolution associated with development of new treatment strategies and generation of new specific niches.
- Lysogeny and bacteriophage host range within the Burkholderia cepacia complex. Langley, R., Kenna, D. T., Vandamme, P., Ure, R., Govan, J. R. W. (2003). Journal of Medical Microbiology 52:483-490. The Burkholderia cepacia complex comprises a group of nine closely related species that have emerged as life-threatening pulmonary pathogens in immunocompromised patients, particularly individuals with cystic fibrosis or chronic granulomatous disease. Attempts to explain the genomic plasticity, adaptability and virulence of the complex have paid little attention to bacteriophages, particularly the potential contribution of lysogenic conversion and transduction. In this study, lysogeny was observed in 10 of 20 representative strains of the B. cepacia complex. Three temperate phages and five lytic phages isolated from soils, river sediments or the plant rhizosphere were chosen for further study. Six phages exhibited T-even morphology and two were lambda-like. The host range of individual phages, when tested against 66 strains of the B. cepacia complex and a representative panel of other pseudomonads, was not species-specific within the B. cepacia complex and, in some phages, included Burkholderia gladioli and Pseudomonas aeruginosa. These new data indicate a potential role for phages of the B. cepacia complex in the evolution of these soil bacteria as pathogens of plants, humans and animals, and as novel therapeutic agents.
- [Bacteriophages for treatment and prophylaxis of infectious diseases]. Lazareva, E. B. (2003). Antibiotiki i Khimioterapiya 48:36-40.
- Biocontrol of Listeria monocytogenes on fresh-cut produce by treatment with lytic bacteriophages and a bacteriocin. Leverentz, B., Conway, W. S., Camp, M. J., Janisiewicz, W. J., Abuladze, T., Yang, M., Saftner, R., Sulakvelidze, A. (2003). Applied and Environmental Microbiology 69:4519-4526. The fresh-cut produce industry has been the fastest-growing portion of the food retail market during the past 10 years, providing consumers with convenient and nutritious food. However, fresh-cut fruits and vegetables raise food safety concerns, because exposed tissue may be colonized more easily by pathogenic bacteria than intact produce. This is due to the higher availability of nutrients on cut surfaces and the greater potential for contamination because of the increased amount of handling. We found that applied Listeria monocytogenes populations survived and increased only slightly on fresh-cut Red Delicious apples stored at 10°C but increased significantly on fresh-cut honeydew melons stored at 10°C over 7 days. In addition, we examined the effect of lytic, L. monocytogenes-specific phages via two phage application methods, spraying and pipetting, on L. monocytogenes populations in artificially contaminated fresh-cut melons and apples. The phage mixture reduced L. monocytogenes populations by 2.0 to 4.6 log units over the control on honeydew melons. On apples, the reduction was below 0.4 log units. In combination with nisin (a bacteriocin), the phage mixture reduced L. monocytogenes populations by up to 5.7 log units on honeydew melon slices and by up to 2.3 log units on apple slices compared to the control. Nisin alone reduced L. monocytogenes populations by up to 3.2 log units on honeydew melon slices and by up to 2.0 log units on apple slices compared to the control. The phage titer was stable on melon slices, but declined rapidly on apple slices. The spray application of the phage and phage plus nisin reduced the bacterial numbers at least as much as the pipette application. The effectiveness of the phage treatment also depended on the initial concentration of L. monocytogenes.
- A role for bacteriophage T4 rI gene function in the control of phage development during pseudolysogeny and in slowly growing host cells. Los, M., Wegrzyn, G., Neubauer, P. (2003). Research in Microbiology 154:547-552. Although most studies on bacteriophages have been performed under laboratory conditions that are optimal for host cell growth, in nature, bacteria and bacteriophages coexist in different habitats. Here, by using different growth rates in carbon-limited chemostats, we investigated the development of phage T4 in its host Escherichia coli. Our results strongly suggest that T4 can form pseudolysogens not only when bacterial growth is completely inhibited, but also in growing host cells. The rI gene, previously known to be indispensable for lysis inhibition, seems to play an important role in optimization of phage development in slowly growing cells as well as during establishment and maintenance of pseudolysogeny.
- Isolation and characterization of a Lactobacillus plantarum bacteriophage, phiJL-1, from a cucumber fermentation. Lu, Z., Breidt, F. Jr, Fleming, H. P., Altermann, E., Klaenhammer, T. R. (2003). International Journal of Food Microbiology 84:225-235. A virulent Lactobacillus plantarum bacteriophage, PhiJL-1, was isolated from a commercial cucumber fermentation. The phage was specific for two related strains of L. plantarum, BI7 and its mutant (deficient in malolactate fermenting ability) MU45, which have been evaluated as starter cultures for controlled cucumber fermentation and as biocontrol microorganisms for minimally processed vegetable products. The phage genome of PhiJL-1 was sequenced to reveal a linear, double-stranded DNA (36.7 kbp). Sodium dodecyl sulfate-polyacryamide gel electrophoresis (SDS-PAGE) profiles indicated that PhiJL-1 contains six structural proteins (28, 34, 45, 50, 61, and 76 kDa). Electron microscopy revealed that the phage has an isometric head (59 nm in diameter), a long non-contractile tail (182 nm in length and 11 nm in width), and a complex base plate. The phage belongs to the Bradley group B1 or Siphoviridae family. One-step growth kinetics of the phage showed that the latent period was 35 min, the rise period was 40 min, and the average burst size was 22 phage particles/infected cell. Phage particles (90%) adsorbed to the host cells 20 min after infection. Calcium supplementation (up to 30 mM CaCl(2)) in MRS media did not affect the first cycle of phage adsorption, but promoted rapid phage propagation and cell lysis in the infection cycle subsequent to adsorption. The D values of PhiJL-1 at pH 6.5 were estimated to be 2.7 min at 70 °C and 0.2 min at 80 °C by a thermal inactivation experiment. Knowledge of the properties of L. plantarum bacteriophage PhiJL-1 may be important for the development of controlled vegetable fermentations.
- Bacteriophage ecology in commercial sauerkraut fermentations. Lu, Z., Breidt, F., Plengvidhya, V., Fleming, H. P. (2003). Applied and Environmental Microbiology 69:3192-3202. Knowledge of bacteriophage ecology in vegetable fermentations is essential for developing phage control strategies for consistent and high quality of fermented vegetable products. The ecology of phages infecting lactic acid bacteria (LAB) in commercial sauerkraut fermentations was investigated. Brine samples were taken from four commercial sauerkraut fermentation tanks over a 60- or 100-day period in 2000 and 2001. A total of 171 phage isolates, including at least 26 distinct phages, were obtained. In addition, 28 distinct host strains were isolated and identified as LAB by restriction analysis of the intergenic transcribed spacer region and 16S rRNA sequence analysis. These host strains included Leuconostoc, Weissella, and Lactobacillus species. It was found that there were two phage-host systems in the fermentations corresponding to the population shift from heterofermentative to homofermentative LAB between 3 and 7 days after the start of the fermentations. The data suggested that phages may play an important role in the microbial ecology and succession of LAB species in vegetable fermentations. Eight phage isolates, which were independently obtained two or more times, were further characterized. They belonged to the family Myoviridae or Siphoviridae and showed distinct host ranges and DNA fingerprints. Two of the phage isolates were found to be capable of infecting two Lactobacillus species. The results from this study demonstrated for the first time the complex phage ecology present in commercial sauerkraut fermentations, providing new insights into the bioprocess of vegetable fermentations.
- Phages of the marine cyanobacterial picophytoplankton. Mann, N. H. (2003). FEMS Microbiology Reviews 27:17-34. Cyanobacteria of the genera Synechococcus and Prochlorococcus dominate the prokaryotic component of the picophytoplankton in the oceans. It is still less than 10 years since the discovery of phages that infect marine Synechococcus and the beginning of the characterisation of these phages and assessment of their ecological impact. Estimations of the contribution of phages to Synechococcus mortality are highly variable, but there is clear evidence that phages exert a significant selection pressure on Synechococcus community structure. In turn, there are strong selection pressures on the phage community, in terms of both abundance and composition. This review focuses on the factors affecting the diversity of cyanophages in the marine environment, cyanophage interactions with their hosts, and the selective pressures in the marine environment that affect cyanophage evolutionary biology.
- Bacterial photosynthesis genes in a virus. Mann, N. H., Cook, A., Millard, A., Bailey, S., Clokie, M. (2003). Nature 424:741. A bacteriophage may protect itself and its host against a deadly effect of bright sunlight.
- [Coliphages as indicators of fecal contamination in sea water]. Meloni, P., Isola, D., Loi, N., Schintu, M., Contu, A. (2003). Annali di igiene : medicina preventiva e di comunita 15:111-116. Assessment of water quality has traditionally relied on faecal indicator organisms, which however do not necessarily correlate well with the presence of pathogenic organisms. Coliphages are regarded as possible alternative indicators. Although they can be detected in water by rapid, simple and reliable procedures, any agreement about a standard method has not yet been reached. Moreover guidelines for the levels of bacteriophages have not yet been set as for coliform bacteria, making difficult to evaluate results. In this work both bacteriophages anti E. coli and traditional indicators of fecal contamination were detected on 274 seawater samples taken from 23 sampling stations located along the coast of southern Sardinia (Italy). The results confirm the usefulness of coliphages as indicators of fecal contamination and suggest a level which could be considered a guideline value for their presence in seawater.
- [Development of cyanobacterial phages at the Institute of Microbiology and Virology of the National Academy of Sciences of Ukraine (History and perspectives)]. Mendzhul, M. I., Lysenko, T. G., Syrchin, S. A. (2003). Mikrobiologichnyi Zhurnal 65:133-140. The paper deals with the basic trends of fundamental investigations of the Department of Algae Viruses in the field of cyanophagia-ecology, biological and physico-chemical properties of cyanophages as well as interrelation with the host cells. Such problems as a possibility to use the system cyanophage-cyanobacteria as the experimental model for development of the unified functional model of productive infection, efficient methods of prophylaxis and therapy of virus infections as well as the solution of various biotechnological problems are discussed.
- Bacterial host strains that support replication of somatic coliphages. Muniesa, M., Moce-Llivina, L., Katayama, H., Jofre, J. (2003). Antonie van Leeuwenhoek 83:305-315. Somatic coliphages detected by Escherichia coli strain WG5 have been proposed as potential indicators of water quality. Their potential replication in the water environment is considered a drawback for their use as indicators. However, the contribution of replication outside the gut to the total numbers has never been quantified. It has not been determined either the fraction of bacterial strains that might support replication of phages detected by strain WG5 in the water environment. We examined the sensitivity of 291 host strains to 25 phages by streaking slants of the presumptive host strain onto an agar layer that contains bacteriophages, which gives a total of 7275 combinations (sensitivity tests). Only a 3.02% of the tests showed sensitivity. Additionally, six environmental strains were used as hosts to count phages in sewage and seawater. Phages isolated on these strains were used to infect strain WG5. The environmental strains detected 1 log10 fewer phages than strain WG5 in sewage and seawater. The fraction of phages that were detected by the six strains and that also infected strain WG5 ranged from < 0.07% to < 2.0% of the total amount of bacteriophages detected by strain WG5 in the same samples. Our results confirm that less than 3% of naturally occurring hosts support replication of phages infecting E. coli. We conclude that the contribution of replication to the number of somatic coliphages detected in the aquatic environment is negligible.
- Genomic sequence of C1, the first streptococcal phage. Nelson, D., Schuch, R., Zhu, S., Tscherne, D. M., Fischetti, V. A. (2003). Journal of Bacteriology 185:3325-3332. C(1), a lytic bacteriophage infecting group C streptococci, is one of the earliest-isolated phages, and the method of bacterial classification known as phage typing was defined by using this bacteriophage. We present for the first time a detailed analysis of this phage by use of electron microscopy, protein profiling, and complete nucleotide sequencing. This virus belongs to the Podoviridae family of phages, all of which are characterized by short, noncontractile tails. The C(1) genome consists of a linear double-stranded DNA molecule of 16,687 nucleotides with 143-bp inverted terminal repeats. We have assigned functions to 9 of 20 putative open reading frames based on experimental substantiation or bioinformatic analysis. Their products include DNA polymerase, holin, lysin, major capsid, head-tail connector, neck appendage, and major tail proteins. Additionally, we found one intron belonging to the HNH endonuclease family interrupting the apparent lysin gene, suggesting a potential splicing event yielding a functional lytic enzyme. Examination of the C(1) DNA polymerase suggests that this phage utilizes a protein-primed mechanism of replication, which is prominent in the phi29-like members of Podoviridae. Consistent with this evidence, we experimentally determined that terminal proteins are covalently attached to both 5' termini, despite the fact that no homology to known terminal proteins could be elucidated in any of our open reading frames. Likewise, comparative genomics revealed no close evolutionary matches, suggesting that the C(1) bacteriophage is a unique member of the Podoviridae
- [How do bacteria acquire the resistance to antibiotics]. Ohno, A. (2003). Nippon Rinsho - Japanese Journal of Clinical Medicine 61 Suppl 3:158-163.
- Yersiniophages. Special reference to phi YeO3-12. Pajunen, M. I., Molineux, I. J., Skurnik, M. (2003). Advances in experimental medicine and biology 529:233-240.
- Origins of highly mosaic mycobacteriophage genomes. Pedulla, M. L., Ford, M. E., Houtz, J. M., Karthikeyan, T., Wadsworth, C., Lewis, J. A., Jacobs-Sera, D., Falbo, J., Gross, J., Pannunzio, N. R., Brucker, W., Kumar, V., Kandasamy, J., Keenan, L., Bardarov, S., Kriakov, J., Lawrence, J. G., Jacobs, W. R., Jr., Hendrix, R. W., Hatfull, G. F. (2003). Cell 113:171-182. Bacteriophages are the most abundant organisms in the biosphere and play major roles in the ecological balance of microbial life. The genomic sequences of ten newly isolated mycobacteriophages suggest that the bacteriophage population as a whole is amazingly diverse and may represent the largest unexplored reservoir of sequence information in the biosphere. Genomic comparison of these mycobacteriophages contributes to our understanding of the mechanisms of viral evolution and provides compelling evidence for the role of illegitimate recombination in horizontal genetic exchange. The promiscuity of these recombination events results in the inclusion of many unexpected genes including those implicated in mycobacterial latency, the cellular and immune responses to mycobacterial infections, and autoimmune diseases such as human lupus. While the role of phages as vehicles of toxin genes is well established, these observations suggest a much broader involvement of phages in bacterial virulence and the host response to bacterial infections.
- Virus removal during simulated soil-aquifer treatment. Quanrud, D. M., Carroll, S. M., Gerba, C. P., Arnold, R. G. (2003). Water Research 37:753-762. Removals of indigenous coliphage and seeded poliovirus type 1 during simulated soil-aquifer treatment were evaluated during transport of secondary effluent under unsaturated flow conditions in 1-m soil columns. Independent variables included soil type (river sand or sandy loam) and infiltration rate. Removal of coliphage was in all cases less than removal of poliovirus type 1 (strain LSc-2ab), supporting contentions that indigenous coliphage can act as a conservative indicator of groundwater contamination by viral pathogens of human origin. Coliphage retention was significantly more efficient (p<0.001) in the finer-grained sandy loam (93%) than in sand (76%). Increasing reactor detention time from 5 to 20 h increased coliphage attenuation from 70% to 99% in a 1-m sand column. There was a significant linear correlation (p=0.012) between log-transformed (fractional) coliphage concentration [log(C/C(0))] and reactor detention time. Re-mobilization of attached coliphage occurred during simulated rainfall using low-ionic-strength water. Inhibition of aerobic respiration resulted in significantly less efficient coliphage attenuation (p=0.033), suggesting the involvement of aerobic microorganisms in the survival/retention of this virus.
- Inactivation of Lactobacillus delbrueckii bacteriophages by heat and biocides. Quiberoni, A., Guglielmotti, D. M., Reinheimer, J. A. (2003). International Journal of Food Microbiology 84:51-62. The effect of several biocides and thermal treatments on the viability of four Lactobacillus delbrueckii phages was investigated. Time to achieve 99% inactivation of phages at 63 and 72 degrees C in three suspension media (Tris Magnesium Gelatin (TMG) buffer, Man Rogosa Sharpe (MRS) broth and reconstituted nonfat dry skim milk (RSM)) was calculated. Thermal resistance depended on the phage considered, but a marked heat-resistance was exhibited by one phage (Ib(3)) since its high titre suspensions were completely inactivated only after 45 min at 72 degrees C or 15 min at 90 degrees C. A clear protective effect of the milk was revealed when the three suspension media were compared. As regards to the effects of biocides on phages, only peracetic acid was found to be effective for inactivating high titre suspensions. Ethanol, even at a concentration of 100%, was not suitable to assure no surviving phage particles and isopropanol turned out to be less effective than ethanol. Sodium hypochlorite at 200-400 ppm inactivated the phages completely, except phage Ib(3), which was only destroyed after treatments with 1200 ppm. The diversity observed in the heat and biocide resistance of L. delbrueckii phages is useful to establish a basis for adopting the most effective thermal and chemical treatments for inactivating them in dairy plants and laboratory environments.
- Bacteriophages and Clostridium spores as indicator organisms for removal of pathogens by passage through saturated dune sand. Schijven, J. F., de Bruin, H. A. M., Hassanizadeh, S. M., Roda Husman, A. M. (2003). Water Research 37:2186-2194. In a field study on the efficiency of dune recharge for drinking water production, bacteriophage MS2 was shown to be removed 8 log(10) by passage through the dune sand. The question of whether pathogenic viruses would be removed as much as MS2 was studied by comparing complete breakthrough curves of MS2 with those of the human viruses Coxsackievirus B4 (CB4) and Poliovirus 1 (PV1) in laboratory columns. The columns were designed to closely simulate the field conditions: same sand, water, porewater velocity and temperature. Employing a two-site kinetic model to simulate breakthrough curves, attachment/detachment to two types of kinetic sites as well as inactivation of free and attached viruses were evaluated. It was found that attachment to only one of the sites is of significance for determining overall removal. At field scale, removal of the less negatively charged PV1 was extrapolated to be about 30 times greater than that of MS2, but removal of CB4 would be only as much as that of MS2. Also, removal of spores of Clostridium perfringens D10, a potential surrogate for Cryptosporidium oocysts, was studied. The attachment rate coefficient of the spores was 7.5 times greater than that of MS2. However, this does not imply that the removal of the spores is 7.5 times greater than that of MS2. Due to negligible inactivation in combination with detachment of previously attached spores, the actual removal rate of the spores depends on the duration of contamination and eventually all spores will break through. Provided no irreversible attachment or physical straining occurs, this may also be the case for other persistent microorganisms, like oocysts of Cryptosporidium.
- Escherichia coli O157:H7 Shiga toxin-encoding bacteriophages: integrations, excisions, truncations, and evolutionary implications. Shaikh, N., Tarr, P. I. (2003). Journal of Bacteriology 185:3596-3605. As it descended from Escherichia coli O55:H7, Shiga toxin (Stx)-producing E. coli (STEC) O157:H7 is believed to have acquired, in sequence, a bacteriophage encoding Stx2 and another encoding Stx1. Between these events, sorbitol-fermenting E. coli O157:H- presumably diverged from this clade. We employed PCR and sequence analyses to investigate sites of bacteriophage integration into the chromosome, using evolutionarily informative STEC to trace the sequence of acquisition of elements encoding Stx. Contrary to expectations from the two currently sequenced strains, truncated bacteriophages occupy yehV in almost all E. coli O157:H7 strains that lack stx1 (stx1-negative strains). Two truncated variants were determined to contain either GTT or TGACTGTT sequence, in lieu of 20,214 or 18,895 bp, respectively, of the bacteriophage central region. A single-nucleotide polymorphism in the latter variant suggests that recombination in that element extended beyond the inserted octamer. An stx2 bacteriophage usually occupies wrbA in stx1+/stx2+ E. coli O157:H7, but wrbA is unexpectedly unoccupied in most stx1-negative/stx2+) E. coli O157:H7 strains, the presumed progenitors of stx1+)/stx2+ E. coli O157:H7. Trimethoprim-sulfamethoxazole promotes the excision of all, and ciprofloxacin and fosfomycin significantly promote the excision of a subset of complete and truncated stx bacteriophages from the E. coli O157:H7 strains tested; bile salts usually attenuate excision. These data demonstrate the unexpected diversity of the chromosomal architecture of E. coli O157:H7 (with novel truncated bacteriophages and multiple stx2 bacteriophage insertion sites), suggest that stx1 acquisition might
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