Bacteriophage Ecology Group - Reference Abstracts (2001)

	Bacteriophage Ecology Group Reference Abstracts (2001)
	Dedicated to the ecology and evolutionary biology of the parasites of unicellular organisms (UOPs)
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© Phage et al.	last updated on Wednesday, December 26, 2001

Bacteriophage latent-period evolution as a response to resource availability. Abedon, S.T., Herschler, T.D., Stopar, D. (2001). Appl. Environ. Microbiol. 67:4233-4241. Bacteriophages (phages) modify microbial communities by lysing hosts, transferring genetic material, and effecting lysogenic conversion. To understand how natural communities are affected it is important to develop predictive models. Here we consider how variation between models in eclipse period, latent period, adsorption constant, burst size, the handling of differences in host quantity and host quality, and in modeling strategy can affect predictions. First we compare two published models of phage growth, which differ primarily in terms of how they model the kinetics of phage adsorption; one is a computer simulation and the other is an explicit calculation. At higher host quantities (~10⁸ cells/ml), both models closely predict experimentally determined phage population growth rates. At lower host quantities (10⁷ cells/ml), the computer simulation continues to closely predict phage growth rates, but the explicit model does not. Next we concentrate on predictions of latent-period optima. A latent-period optimum is the latent period that maximizes the population growth of a specific phage growing in the presence of a specific quantity and quality of host cells. Both models predict similar latent-period optima at higher host densities (e.g., 17 min at 10⁸ cells/ml). At lower host densities, however, the computer simulation predicts latent-period optima that are much shorter than those suggested by explicit calculations (e.g., 90 versus 1,250 min at 10⁵ cells/ml). Finally, we consider the impact of host quality on phage latent-period evolution. By taking care to differentiate latent-period phenotypic plasticity from latent-period evolution, we argue that the impact of host quality on phage latent-period evolution may be relatively small. [TOP OF PAGE]
Le matin des bactériophages. Ackermann, H.-W. (2001). Virologie 5:35-43. With about 5 150 electron microscopic observations, phages contstitute the larges of all viral groups. The International Committee on Taxonomy of Viruses (ICTV) persently recognized one order, 13 families, and 30 genera. The order Caudovirales includes three families of tailed phages and about 5 000 members (96.4%). The 10 families of icosahedral, filamentous, or pleomorphic phages totalize 186 viruses. Phages are found in all the bacterial world and, with up to 10¹⁰ particles/ml in seawater, seem to be the most frequent microbes of Earth. Phages are polyphyletic in origin. Tailed phages appear to be the msot ancient viruses and recombination with exchange of genes or gene blocs (modular evolution) seems to be their preferred way of evolution. Tailed phages and herpesviruses present multiple analogies. Harmful phages can create havoc in bacterial fermentations, especially in the dairy industry. By contrast, phages are very useful in general bacteriology, therapy of infectious diseases is making a come-back and phages are likely to have a brilliant future in research. [TOP OF PAGE]
Frequency of morphological phage descriptions in the year 2000. Brief Review. Ackermann, H.-W. (2001). Archives of Virology 146:843-857. Over 5100 bacteria viruses have been examined in the electron microscope since 1959. About 4950 phages (96%) are tailed and only 186 phages (3.6%), are cubic, filamentous, or pleomorphic. Phages belong to 13 virus families and occur in over 140 bacterial genera. Phages are listed by morphotypes and host genera. Siphoviridae or phages with long, noncontractile tails compromise 61% of tailed phages. The distribution of phages in different bacterial phylogenetic divisions is shown. [TOP OF PAGE]
The Vibrio cholerae VPI?/CTX?/TCP: Interactions of PHAGE-PHAGE-bacterium. Ai, Y.-C., Meng, F. (2001). Acta Microbiologica Sinica 41:[TOP OF PAGE]
Distribution of virus-like particles in an oligotrophic marine environment (Alboran Sea, Western Mediterranean). Alonso, M.C., Jimenez-Gomez, F., Rodriguez, J., Borrego, J.J. (2001). Microb. Ecol. 42:407-415. Viruses are abundant in a variety of aquatic environments, often exceeding bacterial abundance by one order of magnitude. In the present study, the spatial distribution of viruses in offshore waters of the Alboran Sea (Western Mediterranean) have been studied to determine the relationships between viruses and host communities in this oligotrophic marine environment. Viral abundance was determined using two methods: (i) epifluorescence light microscopy using the dsDNA binding fluorochrome DAPI, and (ii) direct counts by transmission electron microscopy (TEM). The results obtained were significantly different; the highest viral counts were obtained by mean of TEM analyses. In all the samples tested the number of viruses was exceeded by the bacterial concentrations, with a ratio between viral and bacterial titers varying between 1.4 and 20. VLP (virus-like particle) counts were not significantly correlated (p>0.001) with chlorophyll a concentration or the abundance of cyanobacteria. However, there was a positive and significant correlation with bacterial abundance (p<0.001). The analysis of size and morphology of viral particles by TEM and the correlation obtained between the numbers of VLP and bacteria suggest that the majority of the viral particles in the Alboran Sea are bacteriophages. None of the indirect evidence suggested that eukaryotic algae or cyanobacteria were important host organisms in these waters. [TOP OF PAGE]
The bacteriophages of ruminal prevotellas. Ambrozic, J., Ferme, D., Grabnar, M., Ravnikar, M., Avgustin, G. (2001). Folia Microbiologica 46:37-39. Rumen bacteriophage-lyzed bacterial strains of the genus Prevotella were isolated and preliminarily characterized. The strain TCl-1 the species P. bryantii was the only prevotella strain successfully infected with filter sterilized rumen fluid from a black-and-white Holstein cow. Two types of plaques were observed, both rather small and turbid. Preliminary electron microscopy observation showed that several morphologically different bacteriophages were present in these plaques. The plaque eluates were further used for the infection of other prevotella strains. The plaques produced by the bacteriophages were observed with two strains, i.e. P. bryantii B(1)4 and P. brevis GA33. The bacteriophages from both strains were examined by transmission electron microscopy and several morphologically different bacteriophages were observed, among others also a large virion with an icosahedral head with the diameter of approximately 120 nm. The bacteriophage was identified in plaques of bacterial cells of the strain GA33 and has an approximately 800 nm long helical tail, which places it among the largest ruminal bacteriophages described to date. Other bacteriophages from the same indicator strain as well as from P. bryantii B(1)4 strain were smaller and tail structures were not observed in all of them. [TOP OF PAGE]
Optimisation and standardisation of a method for detecting and enumerating bacteriophages infecting Bacteroides fragilis. Araujo, R., Muniesa, M., Mendez, J., Puig, A., Queralt, N., Lucena, F., Jofre, J. (2001). Journal of Virological Methods 93:127-136. A method for the detection and enumeration of bacteriophages infecting Bacteroides fragilis has been standardised. The recommended host strain is RYC2056 (ATCC 700786) because of the relatively high counts (10(4)-10(5) PFU/100ml) that it recovers in sewage from very different geographical areas. The addition of 0.25% bile to the culture and assay media and the manipulation of the host strain under strict anaerobic conditions resulted in a significant increase (more than 100%) in the number of phages detected. No other changes in the media and culture conditions resulted in changes in the phage counts detected. However, these increases do not justify changing the culture conditions and media described, taking into consideration that bile renders the media cloudy making it difficult to follow the host growth and that most laboratories do not have the facilities to work under strict anaerobic conditions. Nalidixic acid (100 microg/ml) and kanamycin (100 microg/ml) in the assay medium significantly reduce the background flora from polluted samples without affecting the phage counts. Freezing cultures just before the end of the log-phage growth at (-70+/-10) degrees C with BSA-sucrose as cryoprotector, storing of 1-2 ml in glass vials at (-70+/-10) degrees C and using them directly to inoculate fresh broth allows the obtention of cultures ready for phage enumeration in about 2.5 h. All these developments have been incorporated into a procedure that makes the method for detecting phages infecting B. fragilis as workable as the standardised methods available for the detection of coliphages. [TOP OF PAGE]
Isolation and characterization of bacteriophage-resistant mutants of Vibrio cholerae O139. Attridge, S.R., Fazeli, A., Manning, P.A., Stroeher, U.H. (2001). Microbial Pathogenesis 30:237-246. Vibrio cholerae O139 strains produce a capsule which is associated with complement resistance and is used as a receptor by bacteriophage JA1. Spontaneous JA1-resistant mutants were found to have several phenotypes, with loss of capsule and/or O-antigen from the cell surface. Determination of the residual complement resistance and infant mouse colonization potential of each mutant suggested that production of O-antigen is of much greater significance than the presence of capsular material for both of these properties. Two different in vitro assays of complement resistance were compared and the results of one shown to closely reflect the comparative recoveries of bacteria from the colonization experiments. Preliminary complementation studies implicated two rfb region genes, wzz and wbfP, as being essential for the biosynthesis of capsule but not O-antigen. [TOP OF PAGE]
Bacteriological and virological quality of seawater bathing areas along the Tyrrhenian coast. Aulicino, F.A., Orsini, P., Carere, M., Mastrantonio, A. (2001). International Journal of Environmental Health Research 11:5-11. Monitoring was carried out during summer 1997 along a selected area of the Tyrrhenian coast near the Tiber river mouth. Fifty-eight seawater samples, collected from 19 stations, were examined for coliforms, streptococci, Enteroviruses, Salmonellae, coliphages, Bacteroides fragilis phages, Pseudomonas, alophilic Vibrios, Aeromonas and yeasts. Salmonellae and coliphages were isolated in 3 and 12 out of 58 samples, respectively. Enteroviruses and Bacteroides fragilis phages were not isolated. Reoviruses were isolated only from 2 out of 58 samples. A limited number of samples of the northern stations located near the Tiber and other river mouths exceeded the guide values for bathing water by the EU Directive. All the southern stations, located near canals, were of very good microbiological quality. Pseudomonas, Vibrio, Aeromonas and yeasts were isolated from all stations and their values in 100 ml of seawater were 10-10⁶, 10-10⁶, 0-10⁶ and 1-10³, respectively. An extensive disinfection practice carried out on domestic wastes, which are discharged in rivers and canals, probably brought pollution levels of most stations to values within the bacterial standards. The spread of Pseudomonas, Aeromonas, etc. showed that all the coastal area studied was characterized by the presence of organic matter coming from land that can support the presence of opportunistic pathogens and other microbial flora. [TOP OF PAGE]
The use of bacteriophages for treatment and prevention of bacterial disease in animals and animal models of human infection. Barrow, P.A. (2001). Journal of Chemical Technology and Biotechnology. 76:677-682. A brief history of the use of lytic bacteriophages in bacterial disease therapy is presented. After early disillusionment with the idea following poor experimental work, control of phages and field trials, studies were set up in the 1980's in the UK to study their use in farm animal infections. Work with E. coli septicaemia and diarrhoea has shown that phages can be highly effective prophylactically and therapeutically and more effective than antibiotics. There is considerable potential for their use in a limited number of infection types in both man and animals. [TOP OF PAGE]
Phage treatment: can we utilise it for certain infective diseases in India? Bhatia, R.S. (2001). Journal of the Association of Physicians of India 49:590 [TOP OF PAGE]
The genome of the archael virus SIRV1 has features in common with genomes of eukaryal viruses. Blum, H., Zillig, W., Mallock, S., Domdey, H., Prangishvili, D. (2001). Virology 281:6-9. The virus SIRV1 of the extremely thermophilic archaeon Sulfolobus has a double-stranded DNA genome similar in architecture to the genomes of eukaryal viruses of the families Poxviridae, Pycodnaviridae, and Asfarviridae: the two strands of the 32,301 bp long linear genome are covalently connected forming a continuous polynucleotide chain and 2029 kb long inverted repeats are present at the termini. Very likely it also shares with these viruses mechanisms of initiation of replication and resolution of replicative intermediates. [TOP OF PAGE]
Phylogeny, genome evolution, and host specificity of single-stranded RNA bacteriophage (family Leviviridae). Bollback, J.P., Huelsenbeck, J.P. (2001). J. Mol. Evol. 52:117-128. Bacteriophage of the family Leviviridae have played an important role in molecular biology where representative species, such as Qbeta and MS2, have been studied as model systems for replication, translation, and the role of secondary structure in gene regulation. Using nucleotide sequences from the coat and replicase genes we present the first statistical estimate of phylogeny for the family Leviviridae using maximum-likelihood and Bayesian estimation. Our analyses reveal that the coliphage species are a monophyletic group consisting of two clades representing the genera Levivirus and Allolevivirus. The Pseudomonas species PP7 diverged from its common ancestor with the coliphage prior to the ancient split between these genera and their subsequent diversification. Differences in genome size, gene composition, and gene expression are shown with a high probability to have changed along the lineage leading to the Allolevivirus through gene expansion. The change in genome size of the Allolevivirus ancestor may have catalyzed subsequent changes that led to their current genome organization and gene expression. [TOP OF PAGE]
Phages of Lactococcus lactis: an ecological and economical equilibrium. Boucher, I., Moineau, S. (2001). Recent Research Developments in Virology 3:243-256. Lactic acid bacteria (LAB) are a group of organisms widely used in food fermentation. Interests in these microorganisms have increased sharply in the last decade; these organisms have even been dubbed the bugs of the new millennium. One distinctive fact about LAB-fermented foods is that they are produced in non-sterile conditions. Thus, LAB are susceptible to infection by lytic bacteriophages naturally present in these environments. Recent developments (by our group and others) in the field of bacteriophages of Lactococcus, the most studied LAB, are investigated and presented in the review. [TOP OF PAGE]
Bacteriophage-bacteriophage interactions in the evolution of pathogenic bacteria. Boyd, E.F., Davis, B.M., Hochhut, B. (2001). Trends in Microbiology 9:137-144. Many bacteriophages carry virulence genes encoding proteins that play a major role in bacterial pathogenesis. Recently, investigators have identified bacteriophage-bacteriophage interactions in the bacterial host cell that also contribute significantly to the virulence of bacterial pathogens. The relationships between the bacteriophages pertain to one bacteriophage providing a helper function for another, unrelated bacteriophage in the host cell. Accordingly, these interactions can involve the mobilization of bacteriophage DNA by another bacteriophage, for example in Escherichia coli, Vibrio coli and Staphylococcus aureus; the host receptor for one bacteriophage being encoded by another, as found in V. cholerae; and the presence of one bacteriophage potentiating the virulence properties of another bacteriophage, as found in V. cholerae and Salmonella enterica. [TOP OF PAGE]
Phage-related DNA polymorphism in dairy and probiotic Lactobacillus. Brandt, K., Tilsala-Timisjarvi, A., Alatossava, T. (2001). Micron 32:59-65. Various DNA-based methods are presently being applied for identification of industrial bacterial cultures including dairy starter and probiotic strains of Lactobacillus. The success of strain-specific identification depends on the power of the DNA-based methods to reveal intraspecies DNA polymorphism. This study reveals that all eleven arbitrarily chosen Lactobacillus rhamnosus starter, laboratory and probiotic strains contain Lb. rhamnosus phage Lc-Nu related nucleotide sequences. One of these highly homologous regions in the genome of phage Lc-Nu was the 2.4 kb HindIII fragment, which has been sequenced. Nucleotide sequence analysis suggested that one side of the 2.4 kb HindIII fragment encodes a phage Lc-Nu helicase and accordingly represents an early gene region of phage Lc-Nu genome. Five forward and five reverse primers were derived from the nucleotide sequence of the 2.4 kb HindIII fragment of phage Lc-Nu DNA for PCR-based identification of the eleven Lb. rhamnosus strains included in this study. Six different types of PCR product patterns were obtained. Among the patterns three were unique to particular Lb. rhamnosus strains. The results suggest that phage-related DNA sequences are, surprisingly, distributed widely among the Lb. rhamnosus strains, and that these sequences could also be a source of DNA polymorphism to apply for DNA-based identification of bacterial strains. Phage Lc-Nu related DNA homology was also found in the chromosome of Lb. casei, the species closely related to Lb. rhamnosus. [TOP OF PAGE]
Analysis of the complete DNA sequence of the temperate bacteriophage TP901-1: Evolution, structure, and genome organization of lactococcal bacteriophages. Brondsted, L., Ostergaard, S., Pedersen, M., Hammer, K., Vogensen, F.K. (2001). Virology 283:93-109. A complete analysis of the entire genome of the temperate lactococcal bacteriophage TP901-1 has been performed and the function of 21 of 56 TP901-1-encoded ORFs has been assigned. This knowledge has been used to propose 10 functional modules each responsible for specific functions during bacteriophage TP901-1 proliferation. Short regions of microhomology in intergenic regions present in several lactococcal bacteriophages and chromosomal fragments of Lactococcus lactis are suggested to be points of exchange of genetic material through homologous recombination. Our results indicate that TP901-1 may have evolved by homologous recombination between the host chromosome and a mother phage and support the observation that phage remnants as well as prophages located in the Lactococcus chromosome contribute significantly to bacteriophage evolution. Some proteins encoded in the early transcribed region of the TP901-1 genome were more homologous to proteins encoded by phages infecting gram-positive hosts other than L. lactis. This protein homology argues for the occurrence of horizontal genetic exchange among these bacteriophages and indicates that they have access to a common gene pool. [TOP OF PAGE]
Induction of lysogenic bacteriophage and phage-associated toxin from group A streptococci during coculture with human pharyngeal cells. Broudy, T.B., Pancholi, V., Fischetti, V.A. (2001). Infect. Immun. 69:1440-1443. We found that when group A streptococci are cocultured with human pharyngeal cells, they upregulate and secrete a 25-kDa toxin, determined to be the bacteriophage-encoded streptococcal pyrogenic exotoxin C (SpeC). This prompted us to determine if the bacteriophage themselves are induced during coculture conditions. We found that bacteriophage induction does occur, resulting in the release of apprx10⁵ phage particles during the 3-h coculture. Furthermore, we show that the bacteriophage induction event is mediated by a pharyngeal cell soluble factor for which we provide an initial characterization. [TOP OF PAGE]
Comparative phage genomics and the evolution of Siphoviridae: Insights from dairy phages. Brussow, H., Desiere, F. (2001). Molecular Microbiology 39:213-222. Comparative phage genomics can retrace part of the evolutionary history of phage modules encoding phage-specific functions such as capsid building or establishment of the lysogenic state. The diagnosis of relatedness is not based exclusively on sequence similarity, but includes topological considerations of genome organization. The gene maps from the lambda-, psiM2-, L5-, Sfi21-, Sfi11-, phiC31-, sk1- and TM4-like phages showed a remarkable synteny of their structural genes defining a lambda supergroup within Siphoviridae (Caudovirales with long non-contractile tails). A hierarchy of relatedness within the lambda supergroup suggested elements of vertical evolution in the capsid module of Siphoviridae. Links to P22-like Podoviridae and P2-like Myoviridae were also detected. Numerous cases of horizontal gene transfer were observed, but recent transfers were limited to interbreeding phage populations. We suggest that tailed phages are the result of both vertical and horizontal evolution and are thus a good model system for web-like phylogenies. [TOP OF PAGE]
Generalized transduction in Streptomyces coelicolor. Burke, J., Schneider, D., Westpheling, J. (2001). Proc. Natl. Acad. Sci. USA 98:6289-6294. We report the isolation of generalized transducing phages for Streptomyces species able to transduce chromosomal markers or plasmids between derivatives of Streptomyces coelicolor, the principal genetic model system for this important bacterial genus. We describe four apparently distinct phages (DAH2, DAH4, DAH5, and DAH6) that are capable of transducing multiple chromosomal markers at frequencies ranging from 10^-5 to 10^-9 per plaque-forming unit. The phages contain DNA ranging in size from 93 to 121 kb and mediate linked transfer of genetic loci at neighboring chromosomal sites sufficiently close to be packaged within the same phage particle. The key to our ability to demonstrate transduction by these phages was the establishment of conditions expected to severely reduce superinfection killing during the selection of transductants. The host range of these phages, as measured by the ability to form plaques, extends to species as distantly related as Streptomyces avermitilis and Streptomyces verticillus, which are among the most commercially important species of this genus. Transduction of plasmid DNA between S. coelicolor and S. verticillus was observed at frequencies of approximately 10^-4 transductants per colony-forming unit. [TOP OF PAGE]
The origins and evolution of viruses. Campbell, A. (2001). Trends in Microbiology 9:61-61. [TOP OF PAGE]
Chemical and microbial characterization of household graywater. Casanova, L.M., Gerba, C.P., Karpiscak, M. (2001). J Environ Sci Health Part A Tox Hazard Subst Environ Eng 36:395-401. In arid areas, the search for efficient methods to conserve water is of paramount importance. One of the methods of water conservation available today is graywater recycling--the reuse of water from the sinks, showers, washing machine, and dishwasher in a home. The purpose of this project was to characterize the chemical and microbial quality of graywater from a single-family home with two adults. Water samples from a graywater holding tank were analyzed over a seven-month period for total coliforms, fecal coliforms, fecal streptococci, Staphylococcus aureus (S. aureus), Pseudomonas aeruginosa (P. aeruginosa), and coliphages. The pH, turbidity, biological oxygen demand (BOD), suspended solids (SS), electrical conductivity (EC), sulfates (SO4), and chlorides (Cl) were also measured. The mean numbers of total coliforms, fecal coliforms, fecal streptococci, and P. aeruginosa were 8.03 x 10⁷, 5.63 x 10⁵, 2.38 x 10², and 1.99 x 10⁴ CFU/100 mL, respectively. S. aureus and coliphages were not detected. In the chemical analysis, mean values of 7.47 for pH, 43 nephelometric turbidity units (NTU) for turbidity, 64.85 mg/L for BOD, 35.09 mg/L for SS, 0.43 mS/cm for EC, 59.59 mg/L for SO4, and 20.54 mg/L for Cl were measured. These data were compared to data taken in 1986 and 1987, when two adults and one child lived in the household. Analysis showed no statistically significant difference in levels of total coliforms and suspended solids between the two data sets. There were statistically significant differences in levels of fecal coliforms, pH, turbidity, chlorides, sulfates, and BOD between the two households. Fecal coliforms, turbidity, and BOD were higher in the household with two adults and one child. Levels of Cl, SO4, and pH were higher in the household with two adults. [TOP OF PAGE]
Microbial population dynamics and diversity during a bloom of the marine coccolithophorid Emiliania huxleyi (Haptophyta). Castberg, T., Larsen, A., Sandaa, R.A., Brussaard, C.P.D., Egge, J.K., Heldal, M., Thyrhaug, R., van Hannen, E.J., Bratbak, G. (2001). Mar. Ecol. Prog. Ser. 221:39-46. Several previous studies have shown that Emiliania huxleyi blooms and terminations have been succeeded by an increase in large virus-like particles (LVLP), strongly suggesting the bloom collapse was caused by viral lysis. However, due to methodological limitations, knowledge of how such blooms affect the rest of the microbial community is limited. In the current study we induced a bloom of E. huxleyi in seawater enclosures and applied methods enabling us to describe the algae, bacteria and virus communities with greater resolution than has been done previously, The development of the dominating algal, viral and bacterial populations in the nutrient-amended seawater enclosures was followed by flow cytometry (FCM). Light microscopy (LM), PCR-denaturing gradient gel electrophoresis (PCR-DGGE) and pulsed-field gel electrophoresis (PFGE) were used to describe the changes in community composition in greater detail. The algal community was dominated by E. huxleyi until termination of the bloom by viral lysis, After bloom termination the additional algal populations present in the enclosures increased in abundance. A marked increase in viruses other than the one infecting E. huxleyi was also observed. Total bacterial number and community composition were also greatly influenced by the bloom and its collapse. [TOP OF PAGE]
Phages and their application against drug-resistant bacteria. Chanishvili, N., Chanishvili, T., Tediashvili, M., Barrow, P.A. (2001). Journal of Chemical Technology and Biotechnology. 76:689-699. At the beginning of the 20th century the phenomenon of spontaneous bacterial lysis was discovered independently by Twort and d'Herelle. Despite the suggestion at that time by d'Herelle that these agents might be applied to the control of bacterial diseases in the west this idea was explored in a desultory fashion only and was eventually discarded largely due to the advent of extensive antibiotic usage. However, interest was maintained in countries of the former Soviet Union where bacteriophage therapy has been applied extensively since that time. Central to this work was the Eliava Institute of Bacteriophage, Microbiology and Virology in Tbilisi, Georgia, which was founded in 1923 through the joint efforts of d'Herelle and the Georgian George Eliava. Ironically, given his contributions to public health in the Soviet Union, Eliava was branded as an enemy of the people in 1937 and executed. d'Herelle never again returned to Georgia. In spite of these tragic events this institute remained the focus for phage therapy in the world and despite being continuously active in this field for 75 years, now struggles for its financial life. In the Eliava Institute, phages were sought for bacterial pathogens implicated in disease outbreaks in different parts of the Soviet Union and were dispatched for use in hospitals throughout the country. Although infections caused by a wide variety of bacterial pathogens have been treated, much of this has been published in Russian and is not readily available in the west. Work has also been carried out in Poland over many years and this has only recently been published in English. By contrast, interest in the west has been limited to a small number of enthusiasts and academics and until very recently little interest has been shown. The main reason that the medical and scientific communities are now beginning to take notice, is the continuing world-wide rise in the incidence of multiply-antibiotic-resistant bacterial pathogens and the absence of effective means for their control. Recent publicity over the work of the Eliava Institute has concentrated the minds of the western world on the potential for infectious disease control that bacteriophage offer, a procedure that is biologically more acceptable than antibiotic use and which has been in use for several decades already. [TOP OF PAGE]
Application of digital image analysis and flow cytometry to enumerate marine viruses stained with SYBR gold. Chen, F., Lu, J.R., Binder, B.J., Liu, Y.C., Hodson, R.E. (2001). Appl. Environ. Microbiol. 67:539-545. A novel nucleic acid stain, SYBR Gold, was used to stain marine viral particles in various types of samples. Viral particles stained with SYBR Gold yielded bright and stable fluorescent signals that could be detected by a cooled charge-coupled device camera or by flow cytometry. The fluorescent signal strength of SYBR Gold-stained viruses was about twice that of SYBR Green I-stained viruses. Digital images of SYBR Gold-stained viral particles were processed to enumerate the concentration of viral particles by using digital image analysis software. Estimates of viral concentration based on digitized images were 1.3 times higher than those based on direct counting by epifluorescence microscopy. Direct epifluorescence counts of SYBR Gold-stained viral particles were in turn about 1.34 times higher than those estimated by the transmission electron microscope method. Bacteriophage lysates stained with SYBR Gold formed a distinct population in flow cytometric signatures. Flow cytometric analysis revealed at least four viral subpopulations for a Lake Erie sample and two subpopulations for a Georgia coastal sample. Flow cytometry-based viral counts for various types of samples averaged 1.1 times higher than direct epifluorescence microscopic counts. The potential application of digital image analysis and flow cytometry for rapid and accurate measurement of viral abundance in aquatic environments is discussed. [TOP OF PAGE]
Analysis of six prophages in Lactococcus lactis IL1403: Different genetic structure of temperate and virulent phage populations. Chopin, A., Bolotin, A., Sorokin, A., Ehrlich, S.D., Chopin, M.C. (2001). Nucleic Acids Research 29:644-651. We report the genetic organisation of six prophages present in the genome of Lactococcus lactis IL1403. The three larger prophages (36-42 kb), belong to the already described P335 group of temperate phages, whereas the three smaller ones (13-15 kb) are most probably satellites relying on helper phage(s) for multiplication. These data give a new insight into the genetic structure of lactococcal phage populations. P335 temperate phages have variable genomes, sharing homology over only 10-33% of their length. In contrast, virulent phages have highly similar genomes sharing homology over >90% of their length. Further analysis of genetic structure in all known groups of phages active on other bacterial hosts such as Escherichia coli, Bacillus subtilis, Mycobacterium and Streptococcus thermophilus confirmed the existence of two types of genetic structure related to the phage way of life. This might reflect different intensities of horizontal DNA exchange: low among purely virulent phages and high among temperate phages and their lytic homologues. We suggest that the constraints on genetic exchange among purely virulent phages reflect their optimal genetic organisation, adapted to a more specialised and extreme form of parasitism than temperate/lytic phages. [TOP OF PAGE]
Nucleotide sequence of coliphage HK620 and the evolution of lambdoid phages. Clark, A.J., Inwood, W., Cloutier, T., Dhillon, T.S. (2001). J. Mol. Biol. 311:657-679. HK620 is a temperate lambdoid bacteriophage that adsorbs to the O-antigen of its host, Escherichia coli H. The genome of a temperature-sensitive clear-plaque mutant consists of 38,297 nucleotides in which we recognize 60 open reading frames (orfs). Eighteen of these lie in a region of the genome that we call the virion structure domain. The other 42 orfs lie in what we call the metabolic domain.Virions of HK620 resemble those of phage P22. The virion structural orfs encode three kinds of putative proteins relative to the virion proteins of P22: (1) those that are nearly (about 90 %) identical; (2) those that are weakly (about 30 %) identical; and (3) those composed of nearly and weakly identical segments. We hypothesize that these composite proteins form bridges between the virion proteins of the other two kinds.Three of the putative virion proteins that are only weakly identical to P22 proteins are 71, 60 and 79 % identical to proteins encoded by the phage APSE-1, whose virions also resemble those of P22. Because the hosts of APSE-1 and HK620 have been separated from each other by an estimated 200 My, we propose using the amino acid differences that have accumulated in these proteins to estimate a biological clock for temperate lambdoid phages.The putative transcriptional regulatory gene circuitry of HK620 seems to resemble that of phage lambda. Integration, on the other hand, resembles that of satellite phage P4 in that the attP sequence lies between the leftward promoter and int rather than downstream of int.Comparing the metabolic domains of several lambdoid phage genomes reveals seven short conserved sequences roughly defining boundaries of functional modules. We propose that these boundary sequences are foci of genetic recombination that serve to assort the modules and make the metabolic domain highly mosaic genetically. [TOP OF PAGE]
Bacteriophage T4 multiplication in an Escherichia coli biofilm. Corbin, B.D., Aron, G.M., McLeon, R.J.C. (2001). Canadian Journal of Microbiology 47:680-684. An Escherichia coli K-12 biofilm was grown at a dilution rate of 0.028 h^-1 for 48 h in a glucose-limited chemostat coupled to a modified Robbins' device to determine its susceptibility to infection by bacteriophage T4. Bacteriophage T4 at a multiplicity of infection (MOI) of 10 caused a log reduction in biofilm density (expressed as colony forming units (CFU) per cm(2)) at 90 min postinfection. After 6 h, a net decrease and equilibrium in viral titer was seen. When biofilms were exposed to T4 phage at a MOI of 100, viral titer doubled after 90 min. After 6 h, viral titers (expressed as plaque forming units (PFU) per cm(2)) stabilized at levels approximately one order of magnitude higher than seen at a MOI of 10. Scanning confocal laser microscopy images also indicated disruption of biofilm morphology following T4 infection with the effects being more pronounced at a MOI of 100 than at a MOI of 10. These results imply that biofilms under carbon limitation can act as natural reservoirs for bacteriophage and that bacteriophage can have some influence on biofilm morphology. [TOP OF PAGE]
Bacteriophage versus bacteria. Davies, M.J. (2001). Trends in Biotechnology 19:164-164. No Abstract. [TOP OF PAGE]
Estimation of the average burst size of Phix174 am3, cs70 for use in mutation assays with transgenic mice. Delongchamp, R.R., Valentine, C.R., Malling, H.V. (2001). Environmental and Molecular Mutagenesis 37:356-360. In mutation assays using transgenic mice, with recoverable vectors such as PhiX174 am3, cs70, mutations originate from two sources: (1) in vivo mutations, that is, mutations that were fixed in the mouse, or (2) ex vivo mutations, that is, mutations that were fixed during recovery or plating. When a bacteriophage infects a bacterium, it multiplies and bursts the cell, releasing a number of phages referred to as the burst size. Our method for distinguishing between in vivo mutations and ex vivo mutations estimates the average number of bursts, the denominator of in vivo mutant frequencies, by dividing the total plaque-forming units (PFU) by the average number of phages in a burst. Herein, we outline a probability model relating observed plaque counts to the burst size and present the statistical method used to estimate the burst size. The average size of a single burst from nonrevertant phages was estimated in eight studies under the conditions of our mutation assay. The average burst size was stable across studies at 182.5 plaques per burst (standard error, 14.25). The probability that a burst is a specific size was approximated by a negative binomial distribution, which implies a Poisson-Pascal distribution for the observed plaque counts. The observed plaque counts were adequately fit by this approximation. [TOP OF PAGE]
Progeny of the phage school. Dixon, B. (2001). ASM News 69:432-433. Frederick Twort, the eccentric polymath who discovered bacterial viruses, would have robustly welcomed the applications of bacteriophages now emergy, from therapeutics to environmental protection. [TOP OF PAGE]
Direct and quantitative detection of bacteriophage by "hearing" surface detachment using a quartz crystal microbalance. Dultsev, F.N., Speight, R.E., Fiorini, M.T., Blackburn, J.M., Abell, C., Ostanin, V.P., Klenerman, D. (2001). Analytical Chemistry 73:3935-3939. We show that it is possible to detect specifically adsorbed bacteriophage directly by breaking the interactions between proteins displayed on the phage coat and ligands immobilized on the surface of a quartz crystal microbalance (QCM). This is achieved through increasing the amplitude of oscillation of the QCM surface and sensitively detecting the acoustic emission produced when the bacteriophage detaches from the surface. There is no interference from nonspecifically adsorbed phage. The detection is quantitative over at least 5 orders of magnitude and is sensitive enough to detect as few as 20 phage. The method has potential as a sensitive and low-cost method for virus detection. [TOP OF PAGE]
Transduction by phiBB-1, a bacteriophage of Borrelia burgdorferi. Eggers, C.H., Kimmel, B.J., Bono, J.L., Elias, A.F., Rosa, P., Samuels, D.S. (2001). J. Bacteriol. 183:4771-4778. We previously described a bacteriophage of the Lyme disease agent Borrelia burgdorferi designated phiBB-1. This phage packages the host complement of the 32-kb circular plasmids (cp32s), a group of homologous molecules found throughout the genus Borrelia. To demonstrate the ability of phiBB-1 to package and transduce DNA, a kanamycin resistance cassette was inserted into a cloned fragment of phage DNA, and the resulting construct was transformed into B. burgdorferi CA-11.2A cells. The kan cassette recombined into a resident cp32 and was stably maintained. The cp32 containing the kan cassette was packaged by phiBB-1 released from this B. burgdorferi strain. phiBB-1 has been used to transduce this antibiotic resistance marker into naive CA-11.2A cells, as well as two other strains of B. burgdorferi. This is the first direct evidence of a mechanism for lateral gene transfer in B. burgdorferi. [TOP OF PAGE]
Diminished diarrheal response to Vibrio cholerae strains carrying the replicative form of the CTXf genome instead of CTXf lysogens in adult rabbits. Faruque, S.M., Rahman, M.M., Hasan, A.K., Nair, G.B., Mekalanos, J.J., Sack, D.A. (2001). Infect. Immun. 69:6084-6090. Toxigenic Vibrio cholerae strains are lysogens of CTXf, a filamentous bacteriophage which encodes cholera toxin (CT). Following infection of recipient V. cholerae cells by CTX(Phi), the phage genome either integrates into the host chromosome at a specific attachment site (attRS) or exists as a replicative-form (RF) plasmid. We infected naturally occurring attRS-negative nontoxigenic V. cholerae or attenuated (CTX(-) attRS negative) derivatives of wild-type toxigenic strains with CTX(Phi) and examined the diarrheagenic potential of the strains carrying the RF of the CTXf genome using the adult rabbit diarrhea model. Under laboratory conditions, strains carrying the RF of CTX(Phi) produced more CT than corresponding lysogens as assayed by a G(M1)-based enzyme-linked immunosorbent assay and by fluid accumulation in ligated ileal loops of rabbits. However, when tested for diarrhea in rabbits, the attRS-negative strains (which carried the CTXf genome as the RF) were either negative or produced mild diarrhea, whereas the attRS-positive strains with integrated CTXf produced severe fatal diarrhea. Analysis of the strains after intestinal passage showed that the attRS-negative strains lost the phage genome at approximately a fivefold higher frequency than under in vitro conditions, and 75 to 90% of cells recovered from challenged rabbits after 24 h were CT negative. These results suggested that strains carrying the RF of CTXf are unable to cause severe disease due to rapid loss of the phage in vivo, and the gastrointestinal environment thus provides selection of toxigenic strains with an integrated CTXf genome. These results may have implications for the development of live V. cholerae vaccine candidates impaired in chromosomal integration of CTX(Phi). These findings may also contribute to understanding of the etiology of diarrhea occasionally associated with nontoxigenic V. cholerae strains. [TOP OF PAGE]
Development and optimization of a novel immunomagnetic separation- bacteriophage assay for detection of Salmonella enterica serovar enteritidis in broth. Favrin, S.J., Jassim, S.A., Griffiths, M.W. (2001). Appl. Environ. Microbiol. 67:217-224. Salmonella is the second-leading cause of food-borne illness in most developed countries, causing diarrhea, cramps, vomiting, and often fever. Many rapid methods are available for detection of Salmonella in foods, but these methods are often insensitive or expensive or require a high degree of technical ability to perform. In this paper we describe development and characterization of a novel assay that utilizes the normal infection cycle of bacteriophage SJ2 for detection of Salmonella enterica serovar Enteritidis in broth. The assay consists of four main stages: (i) capture and concentration of target cells by using immunomagnetic separation (IMS); (ii) infection of the target bacterium with phage; (iii) amplification and recovery of progeny phage; and (iv) assay of progeny phage on the basis of their effect on a healthy population of host cells (signal-amplifying cells). The end point of the assay can be determined by using either fluorescence or optical density measurements. The detection limit of the assay in broth is less than 10⁴ CFU/ml, and the assay can be performed in 4 to 5 h. The results of this study demonstrate that the IMS-bacteriophage assay is a rapid, simple, and sensitive technique for detection of Salmonella serovar Enteritidis in broth cultures which can be applied to preenriched food samples. [TOP OF PAGE]
Variable assortment of prophages provides a transferable repertoire of pathogenic determinants in Salmonella. Figueroa-Bossi, N., Uzzau, S., Maloriol, D., Bossi, L. (2001). Molecular Microbiology 39:260-271. Gene transfer between separate lineages of a bacterial pathogen can promote recombinational divergence and the emergence of new pathogenic variants. Temperate bacteriophages, by virtue of their ability to carry foreign DNA, are potential key players in this process. Our previous work has shown that representative strains of Salmonella typhimurium (LT2, ATCC14028 and SL1344) are lysogenic for two temperate bacteriophages: Gifsy-1 and Gifsy-2. Several lines of evidence suggested that both elements carry genes that contribute to Salmonella virulence. One such gene, on the Gifsy-2 prophage, codes for the (Cu, Zn) superoxide dismutase SodCI. Other putative pathogenicity determinants were uncovered more recently. These include genes for known or presumptive type III-translocated proteins and a locus, duplicated on both prophages, showing sequence similarity to a gene involved in Salmonella enteropathogenesis (pipA). In addition to Gifsy-1 and Gifsy-2, each of the above strains was found to harbour a specific set of prophages also carrying putative pathogenicity determinants. A phage released from strain LT2 and identified as phage Fels-1 carries the nanH gene and a novel sodC gene, which was named sodCIII. Strain ATCC14028 releases a lambdoid phage, named Gifsy-3, which contains the phoP/phoQ-activated pagJ gene and the gene for the secreted leucine-rich repeat protein SspH1. Finally, a phage specifically released from strain SL1344 was identified as SopEPHI. Most phage-associated loci transferred efficiently between Salmonella strains of the same or different serovars. Overall, these results suggest that lysogenic conversion is a major mechanism driving the evolution of Salmonella bacteria. [TOP OF PAGE]
Phage antibacterials make a comeback. Fischetti, V.A. (2001). Nature Biotechnology 19:734-735. [no abstract?]. [TOP OF PAGE]
H-mutant bacteriophages as a potential biocontrol of bacterial blight of geranium. Flaherty, J.E., Harbaugh, B.K., Jones, J.B., Somodi, G.C., Jackson, L.E. (2001). Hortscience 36:98-100. Bacteriophages specific to Xanthomonas campestris pv. pelargonii (Xcp), the causal agent of bacterial blight of geranium, Pelargonium Xhortorum L.H. Bailey, were isolated from soil and sludge samples from Florida, California, Minnesota, and Utah. Sixteen phages were evaluated for their potential to lyse 21 Xcp strains collected from around the world. The Xcp strains varied in their susceptibility to the phage isolates with 4 to 14 phages producing a lytic or highly virulent reaction. A mixture of five h-mutants was developed from phages that exhibited the broadest host-ranges and tested against the same Xcp strains. The h-mutant phage mixture lysed all 21 Xcp strains. Three experiments were designed to determine the efficacy of using a mixture of four h-mutant phages to control the spread of the bacterial blight pathogen on potted and seedling geraniums under greenhouse conditions. Plants surrounding diseased inoculated plants were treated with a phage mixture at 5 X 108 pfu/mL daily, biweekly, or triweekly, or treated with Phyton-27(R), at 2.0 mLcntdotL-1 every 10 or 14 days. In potted geraniums, daily foliar sprays of the phage mixture had reduced disease incidence and severity by 50% and 75%, respectively, relative to control plants after 6 weeks. In two plug experiments, the phage mixture applied daily also had reduced disease incidence and severity by 69% and 86%, and 85% and 92%, respectively, when compared with controls after 5 weeks. In all three experiments, disease incidence and severity were less for plants treated daily with phages than for those treated less frequently with phages or with Phyton-27(R). Chemical name used: copper sulfate pentahydrate (Phyton-27(R)). [TOP OF PAGE]
Bacteriophage lambda: Alive and well and still doing its thing. Friedman, D.I., Court, D.L. (2001). Current Opinion in Microbiology 4:201-207. The lambda (lambda) family of bacteriophages continues to provide significant insights into the understanding of basic biological processes, as well as useful technological innovations. Areas in which recent advances have occurred include transcription elongation, repressor interactions, genomics and post-transcriptional regulation. The homologous lambda recombination functions have been exploited as an efficient in vivo recombinant engineering system for functional genomic studies. The virulence of some pathogenic strains of Escherichia coli is enhanced by the expression of Shiga toxin (stx) genes encoded on a resident lambdoid prophage. Recent work suggests that the phage regulatory network may be a significant contributor to toxin production and release by these pathogenic E. coli. [TOP OF PAGE]
Reviewing efficacy of alternative water treatment techniques. Hambidge, A. (2001). Health Estate 55:23-25. This section is designed to provide a brief summary of some of the findings. A good deal of work has been conducted by Mr N. L. Pavey and the team at BSRIA, Bracknell. The BSRIA publications are an excellent source of further information. Ultraviolet radiation: UV radiation of wavelength 254 nm destroys bacteria by a mechanism of damaging nucleic acids by producing thymine dimers which disrupt DNA replication [Gavdy and Gavdy, 1980]. L. pneumophila has been reported as sensitive to UV dosages of 2,500-7,000 uW.s/cm2 [Antopol & Ellner, 1979; Knudson, 1985]. Antopol and Ellner [1979] examined the susceptibility of L. pneumophila to UV dosage. Their results indicated that 50% of the organisms were killed by 380 uWs/cm2 and 90% were killed by 920 uWs/cm2. Kills of 99 and 99.9% were obtained using 1,840 and 2,760 uWs/cm2 respectively. Muraca et al [1987] showed that continuous UV irradiation resulted in a 5 logarithm decrease in waterborne L. pneumophila in a circulating system. Gilpin [1984] reported that in laboratory buffer solutions, exposure to 1 uW of UV radiation per cm2 achieved a 50% kill of L longbeachae in 5 minutes, L. gormanii in 2-30 minutes and L pneumophila in 17 minutes. Exposure times for 99% kills for L. longbeachae, L pneumophila and L. Gormanii were 33, 48 and 63 minutes respectively. The same research worker conducted experiments using a 3 litre circulating water system, connected to a stainless steel housing containing a UV source. The UV lamp output was 7 ergs/mm2 per second per 100 cm at 254 nm. L. pneumophila was killed within 15 seconds, that is within their first pass through the system. Continuous disinfection with UV has the advantages of imparting no taste, odour or harmful chemical by-products and requires minimal operation and maintenance [Muraca et al 1988]. Keevil et al [1989] state that UV irradiation fails to clear systems of biofilm because of poor penetration into microflocs of the micro-organisms. Copper/silver ionisation: A recent study of full scale hot water test rigs incorporating copper-silver ionisation systems has been reported by Pavey, 1996. Copper and silver ions were introduced into the water by electrolysis. One of the principal mechanisms of biocidal action of these ions is thought to be cell penetration. The positively charged copper ions form electrostatic bonds with negatively charged sites on the cell wall. The cell membrane is thus distorted, allowing ingress of silver ions which attack the cell by binding at specific sites to DNA, RNA, respiratory enzymes and cellular protein, causing catastrophic failure of the life support systems of the cell. Silver and copper ion concentrations of 40 and 400 ug/L respectively were effective against planktonic Legionellae in cold water systems and hot water systems containing soft water. In hard water, the ionisation was ineffective due to the inability to control silver ion concentrations. This was caused by scaling of the electrodes and silver ion complexation by the high concentration of dissolved solids. Bosch et al [1993] had earlier extended the application of copper-silver disinfection to human enteric viruses in water, such as adenovirus, rotavirus, hepatitis A virus, and poliovirus. Their work showed that copper and silver ions in the presence of reduced levels of free chlorine did not ensure the total elimination of viral pathogens from water. In the case of an amoeba, Naegleria fowleria [responsible for primary amoebic meningoencephalitis], Cassells et al [1995] have demonstrated that a combination of silver and copper ions were ineffective at inactivating the amoebae at 80 and 800 ug/L respectively. However addition of 1.0 mg/L free chlorine produced a synergistic effect, with superior inactivation relative to either chlorine or silver-copper in isolation. A similar synergy was reported by Yahya et al [1989] in their study of Staphylococcus sp. and Pseudomonas aeruginosa. Yahya et al [1992] also suggested an additive or synergistic effect in the inactivation of coliphage MS-2 and poliovirus. Other techniques: There are a number of other techniques. We have conducted trials of most of these in the control of Legionella sp., but these fall out of the scope of this article, and as such less emphasis has been placed on them here. Ozonation: Ozone [O3] is an oxidising gas, generated electrically from oxygen [O2]. L. pneumophila can be killed at < 1 mg/L of ozone [Edelstien et al 1982]. Muraca et al [1987] found that 1-2 mg/L of continuous ozone over a six hour contact time, produced a 5 logarithm decrease of L. pneumophila. The effectiveness of ozone treatment against a range of bacteria and coliphages has been studied Botzenhart et al [1993]. E. coli was least resistant to ozone, followed by MS 2-coliphage and PhiX 174-coliphage, with L. pneumophila and Bacillus subtilis spores being the most resistant. (ABSTRACT TRUNCATED). [TOP OF PAGE]
A conserved genetic module that encodes the major virion components in both the coliphage T4 and the marine cyanophage S-PM2. Hambly, E., Tétart, F., Desplats, C., Wilson, H., Krisch, H.M., Mann, N.H. (2001). Proc. Natl. Acad. Sci. USA 98:11411-11416. Sequence analysis of a 10-kb region of the genome of the marine cyanomyovirus S-PM2 reveals a homology to coliphage T4 that extends as a contiguous block from gene (g)18 to g23. The order of the S-PM2 genes in this region is similar to that of T4, but there are insertions and deletions of small ORFs of unknown function. In T4, g18 codes for the tail sheath, g19, the tail tube, g20, the head portal protein, g21, the prohead core protein, g22, a scaffolding protein, and g23, the major capsid protein. Thus, the entire module that determines the structural components of the phage head and contractile tail is conserved between T4 and this cyanophage. The significant differences in the morphology of these phages must reflect the considerable divergence of the amino acid sequence of their homologous virion proteins, which uniformly exceeds 50%. We suggest that their enormous diversity in the sea could be a result of genetic shuffling between disparate phages mediated by such commonly shared modules. These conserved sequences could facilitate genetic exchange by providing partially homologous substrates for recombination between otherwise divergent phage genomes. Such a mechanism would thus expand the pool of phage genes accessible by recombination to all those phages that share common modules. [TOP OF PAGE]
Reduction in exopolysaccharide viscosity as an aid to bacteriophage penetration through Pseudomonas aeruginosa biofilms. Hanlon, G.W., Denyer, S.P., Olliff, C.J., Ibrahim, L.J. (2001). Appl. Environ. Microbiol. 67:2746-2753. To cause an infection, bacteriophages must penetrate the alginate exopolysaccharide of Pseudomonas aeruginosa to reach the bacterial surface. Despite a lack of intrinsic motility, phage were shown to diffuse through alginate gels at alginate concentrations up to 8% (wt/vol) and to bring about a 2-log reduction in the cell numbers in 20-day-old biofilms of P. aeruginosa. The inability of alginate to act as a more effective diffusional barrier suggests that phage may cause a reduction in the viscosity of the exopolysaccharide. Samples (n = 5) of commercial alginate and purified cystic fibrosis (CF) alginate were incubated with 2 x 10⁸ purified phage per ml for 24 h at 37 degrees C. After incubation the samples and controls were subjected to rheological analysis with a Carrimed controlled stress rheometer. The viscosities of phage-treated samples were reduced by up to 40% compared to those of controls incubated in the absence of phage. The experiment was repeated by using phage concentrations of 10¹⁰ and 10¹² phage per ml and samples taken for analysis at intervals up to 4 h. The results indicated that there was a time- and concentration-dependent reduction in viscosity of up to 40% compared to the viscosities of the controls. Commercial and purified CF alginate samples, both phage treated and untreated, were subjected to gel filtration chromatography by using Sephacryl High Resolution S-400 medium in order to obtain evidence of degradation. The results demonstrated that alginate treated with phage had a lower molecular weight than untreated alginate. The data suggest that bacteriophage migration through P. aeruginosa biofilms may be facilitated by a reduction in alginate viscosity brought about by enzymic degradation and that the source of the enzyme may be the bacterial host itself. [TOP OF PAGE]
Isolation and characterization of a temperature-sensitive generalized transducing bacteriophage for Vibrio cholerae. Hava, D.L., Camilli, A. (2001). J Microbiol Methods 46:217-225. CP-T1 is the only described generalized transducing bacteriophage for the intestinal pathogen Vibrio cholerae, yet many of its basic biological parameters remain unknown. Due to low frequencies of transduction and pseudolysogen formation, CP-T1 has not been widely used as a genetic tool. To overcome these limitations, we have isolated a conditional mutant of CP-T1 that exhibits temperature-sensitive plaque formation. Several biological properties of CP-T1ts were determined, including its restrictive temperature, adsorbance profile to host cells, burst time, and burst size. Based on these properties, an optimized transduction protocol was designed which resulted in several fold higher transduction frequencies for a variety of genetic markers from a number of chromosomal loci. Generalized transduction was also demonstrated between classical and E1 Tor biotype strains of V. cholerae. [TOP OF PAGE]
First evidence of lysogeny in Propionibacterium freudenreichii subsp. shermanii. Herve, C., Coste, A., Rouault, A., Fraslin, J.M., Gautier, M. (2001). Appl. Environ. Microbiol. 67:231-238. Dairy propionic acid bacteria, particularly the species Propionibacterium freudenreichii, play a major role in the ripening of Swiss type cheese. Isometric and filamentous bacteriophages infecting P. freudenreichii have previously been isolated from cheese. In order to determine the origin of these bacteriophages, lysogeny of P. freudenreichii was determined by isometric bacteriophage type analysis. The genomic DNA of 76 strains were hybridized with the DNA of nine bacteriophages isolated from Swiss type cheeses, and the DNA of 25 strains exhibited strong hybridization. Three of these strains released bacteriophage particules following UV irradiation (254 nm) or treatment with low concentrations of mitomycin C. A prophage-cured derivative of P. freudenreichii was readily isolated and subsequently relysogenized. Lysogeny was therefore formally demonstrated in P. freudenreichii. [TOP OF PAGE]
Effects of concentrated viral communities on photosynthesis and community composition of co-occurring benthic microalgae and phytoplankton. Hewson, I., O'Neil, J.M., Heil, C.A., Bratbak, G., Dennison, W.C. (2001). Aquat. Microb. Ecol. 25:1-10. Marine viruses have been shown to affect phytoplankton productivity; however, there are no reports on the effect of viruses on benthic microalgae (microphytobenthos). Hence, this study investigated the effects of elevated concentrations of virus-like particles on the photosynthetic physiology and community composition of benthic microalgae and phytoplankton. Virus populations were collected near the sediment surface and concentrated by tangential flow ultrafiltration, and the concentrate was added to benthic and water column samples that were obtained along a eutrophication gradient in the Brisbane River/Moreton Bay estuary, Australia. Photosynthetic and community responses of benthic microalgae, phytoplankton and bacteria were monitored over 7 d in aquaria and in situ. Benthic microalgal communities responded to viral enrichment in both eutrophic and oligotrophic sediments. In eutrophic sediments, Euglenophytes (Euglena sp.) and bacteria decreased in abundance by 20 to 60 and 26 to 66%, respectively, from seawater controls. In oligotrophic sediments, bacteria decreased in abundance by 30 to 42% from seawater controls but the dinoflagellate Gymnodinium sp. increased in abundance by 270 to 3600% from seawater controls, The increased abundance of Gymnodinium sp. may be related to increased availability of dissolved organic matter released from lysed bacteria. Increased (140 to 190% from seawater controls) initial chlorophyll a fluorescence measured with a pulse-amplitude modulated fluorometer was observed in eutrophic benthic microalgal incubations following virus enrichment, consistent with photosystem II damage. Virus enrichment in oligotrophic water significantly stimulated carbon fixation rates, perhaps due to increased nutrient availability by bacterial lysis. The interpretation of data from virus amendment experiments is difficult due to potential interaction with unidentified bioactive compounds within seawater concentrates. However, these results show that viruses are capable of influencing microbial dynamics in sediments. [TOP OF PAGE]
Bacteriophage therapy for bacterial infections: Rekindling a memory from the pre-antibiotics era. Ho, K. (2001). Perspectives in Biology and Medicine 44:1-16. [TOP OF PAGE]
Filamentous phage associated with recent pandemic strains of Vibrio parahaemolyticus. Iida, T., Hattori, A., Tagomori, K., Nasu, H., Naim, R., Honda, T. (2001). Emerging Infectious Diseases 7:477-478. A group of pandemic strains of Vibrio parahaemolyticus has recently appeared in Asia and North America. We demonstrate that a filamentous phage is specifically associated with the pandemic V. parahaemolyticus strains. An open reading frame unique to the phage is a useful genetic marker to identify these strains. [TOP OF PAGE]
Practical evaluation of molecular subtyping and phage typing in outbreaks of infection due to Salmonella enterica serotype typhimurium. Jeoffreys, N.J., James, G.S., Chiew, R., Gilbert, G.L. (2001). Pathology 33:66-72. Identification and control of food-poisoning outbreaks due to salmonellosis depend on prompt microbiological diagnosis and subtyping to identify the causative strain. In Australia, Salmonella enterica subspecies enterica serotype typhimurium (S. typhimurium) is responsible for 40-70% of cases of human salmonellosis. Phage typing is the usual method of subtyping S. typhimurium, but on its own, has limitations. We compared it with three molecular subtyping methods using 100 isolates of S. typhimurium, representing four different phage types (PT 1, 9, 126 and 135) and comprising 74 isolates from three presumed outbreaks, 25 isolates from sporadic cases of salmonellosis and S. typhimurium ATCC 10428 (phage type 126). The isolates were divided into 11 subtypes by IS200 restriction fragment length polymorphism (RFLP) typing, four each by ribotyping and pulsed-field gel electrophoresis (PFGE) and 17 distinct strains using a combination of phage and molecular typing. Isolates from two presumed outbreaks were resolved into multiple strains, possibly explaining the failure to identify a common source for either during the original investigations. IS200 RFLP analysis was the most discriminatory and reproducible typing method. Several strains were identifiable within and shared between phage types 1, 9 and 126. Phage and IS200 RFLP typing together, would provide improved definition of S. typhimurium outbreaks. [TOP OF PAGE]
Human adenoviruses and coliphages in urban runoff-impacted coastal waters of Southern California. Jiang, S., Noble, R., Chu, W. (2001). Appl. Environ. Microbiol. 67:179-184. A nested-PCR method was used to detect the occurrence of human adenovirus in coastal waters of Southern California. Twenty- to forty-liter water samples were collected from 12 beach locations from Malibu to the border of Mexico between February and March 1999. All sampling sites were located at mouths of major rivers and creeks. Two ultrafiltration concentration methods, tangential flow filtration (TFF) and vortex flow filtration (VFF), were compared using six environmental samples. Human adenoviruses were detected in 4 of the 12 samples tested after nucleic acid extraction of VFF concentrates. The most probable number of adenoviral genomes ranged from 880 to 7,500 per liter of water. Coliphages were detected at all sites, with the concentration varying from 5.3 to 3332 PFU/liter of water. F-specific coliphages were found at 5 of the 12 sites, with the concentration ranging from 5.5 to 300 PFU/liter. The presence of human adenovirus was not significantly correlated with the concentration of coliphage (r = 0.32) but was significantly correlated (r = 0.99) with F-specific coliphage. The bacterial indicators (total coliforms, fecal coliforms, and enterococci) were found to exceed California recreational water quality daily limits at 5 of the 12 sites. However, this excess of bacterial indicators did not correlate with the presence of human adenoviruses in coastal waters. The results of this study call for both a reevaluation of our current recreational water quality standards to reflect the viral quality of recreational waters and monitoring of recreational waters for human viruses on a regular basis. [TOP OF PAGE]
Mosaic structure of shiga-toxin-2-encoding phages isolated from Escherichia coli O157:H7 indicates frequent gene exchange between lambdoid phage genomes. Johansen, B.K., Wasteson, Y., Granum, P.E., Brynestad, S. (2001). Microbiology 147:1929-1936. Shiga-toxin-2 (stx 2 )-encoding bacteriophages were isolated from Norwegian Escherichia coli O157: H7 isolates of cattle and human origin. The phages were characterized by restriction enzyme analysis, hybridization with probes for toxin genes and selected phage DNA such as the P gene, integrase gene and IS1203, and by PCR studies and partial sequencing of selected DNA regions in the integrase to stx 2 region of the phages. The stx 2 -phage-containing bacteria from which inducible phages were detected produced similar amounts of toxin, as shown by a Vero cell assay. The results indicate that the population of stx 2 -carrying phages is heterogeneous, although the phages from epidemiologically linked E. coli O157: H7 isolates were similar. There appears to have been frequent recombination of stx 2 phages with other lambdoid phages. [TOP OF PAGE]
Mechanism of the photocatalytic inactivation of Lactobacillus casei phage PL-1 by titania thin film. Kashige, N., Kakita, Y., Nakashima, Y., Miake, F., Watanabe, K. (2001). Current Microbiology 42:184-189. The mechanism of the inactivation of Lactobacillus casei phage PL-1 suspended in a phosphate buffer by black-light (BL)-catalytic titanium dioxide (TiO₂) thin film was studied. Generation of both superoxide anions (O^2-) and hydroxyl radicals (•OH) was confirmed in the aqueous medium in which TiO₂ film was settled with BL irradiation under gentle shaking. With BL-irradiation alone without TiO₂ film, only O₂- was generated to some extent. The genome DNA inside the phage particles was found to be fragmented by the treatment of PL-1 phages with BL-catalytic TiO₂ film. The phage inactivation by BL-catalytic TiO₂ film was inhibited by the addition of albumin in a concentration-dependent manner. BL-catalytic TiO₂ film was considered to cause primarily the damage to the capsid protein through the generation of active oxygen species such as •OH, followed by damage to the genome DNA inside the phage particles. [TOP OF PAGE]
Elimination of fecal coliforms and F-specific RNA coliphage from oysters (Crassostrea virginica) relaid in floating containers. Kator, H., Rhodes, M. (2001). Journal of Food Protection 64:796-801. Declining oyster (Crassostrea virginica) production in the Chesapeake Bay has stimulated aquaculture based on floats for off-bottom culture. While advantages of off-bottom culture are significant, the increased use of floating containers raises public health and microbiological concerns, because oysters in floats may be more susceptible to fecal contamination from storm runoff compared to those cultured on-bottom. We conducted four commercial-scale studies with market-size oysters naturally contaminated with fecal coliforms (FC) and a candidate viral indicator, F-specific RNA (FRNA) coliphage. To facilitate sampling and to test for location effects, 12 replicate subsamples, each consisting of 15 to 20 randomly selected oysters in plastic mesh bags, were placed at four characteristic locations within a 0.6- by 3.0-m "Taylor" float, and the remaining oysters were added to a depth not exceeding 15.2 cm. The float containing approximately 3,000 oysters was relaid in the York River, Virginia, for 14 days. During relay, increases in shellfish FC densities followed rain events such that final mean levels exceeded initial levels or did not meet an arbitrary product end point of 50 FC/100 ml. FRNA coliphage densities decreased to undetectable levels within 14 days (16 to 28 degrees C) in all but the last experiment, when temperatures fell between 12 and 16 degrees C. Friedman (nonparametric analysis of variance) tests performed on FC/Escherichia coli and FRNA densities indicated no differences in counts as a function of location within the float. The public health consequences of these observations are discussed, and future research and educational needs are identified. [TOP OF PAGE]
Comparative study of vaginal Lactobacillus phages isolated from women in the United States and Turkey: prevalence, morphology, host range, and DNA homology. Kilic, A.O., Pavlova, S.I., Alpay, S., Kilic, S.S., Tao, L. (2001). Clin. Diag. Lab. Immunol. 8:31-39. Lactobacilli play an important role in maintaining vaginal health. However, during bacterial vaginosis lactobacilli decrease for unknown reasons. Our preliminary study showed that phages could infect vaginal lactobacilli. Therefore, the aim of this study was to analyze the distribution, virulence, and types of vaginal Lactobacillus phages isolated from women of two countries: the United States and Turkey. A total of 209 vaginal lactobacilli were isolated from reproductive-aged women in the United States (n = 107) and Turkey (n = 102). By analysis of 16S rRNA gene sequence and by comparison of protein profiles, most lactobacilli were identified as L. crispatus, L. gasseri, and L. jensenii. After mitomycin C induction, 28% of American lactobacilli and 36% of Turkish lactobacilli released phages. A total of 67 phages were isolated and further characterized by their host range, electron microscopy, and DNA homology. All 67 phages were infective against lactobacilli from both collections. The host ranges of most phages were broad, including multiple Lactobacillus species. Even though the phages were all temperate, they were able to cause lytic infection in various strains. The electron micrographs of these phages showed a hexagon-shaped head and a long tail with or without a contractile tail sheath. Based on their morphology, these phages belonged to Bradley's phage groups A and B, and could be further classified into four morphotypes. All four types were found among American phages, but only three were found among Turkish isolates. DNA hybridization with labeled probes of the four types of phages revealed that additional genetic types existed within each morphotype among these phages. The phage genomic sizes ranged between 34 and 55 kb. Many of the lysogenic Lactobacillus strains released phages spontaneously at a high frequency of 10^-3 to 10^-4 PFU/cell. In conclusion, lysogeny in vaginal lactobacilli is widely spread. Some lysogenic lactobacilli spontaneously release phages with a broad host range. [TOP OF PAGE]
Octamer-based genome scanning distinguishes a unique subpopulation of Escherichia coli O157:H7 strains in cattle. Kim, J., Nietfeldy, J., Benson, A.K. (2001). Proc. Natl. Acad. Sci. USA 96:13288-13293. Multilocus-genotyping methods have shown that Escherichia coli O157:H7 is a geographically disseminated clone. However, high-resolution methods such as pulse-field gel electrophoresis demon-strate significant genomic diversity among different isolates. To assess the genetic relationship of human and bovine isolates of E. coli O157:H7 in detail, we have developed an octamer-based genome-scanning methodology, which compares the distance be-tween over-represented, strand-biased octamers that occur in the genome. Comparison of octamer-based genome-scanning prod-ucts derived from >1 megabase of the genome demonstrated the existence of two distinct lineages of E. coli O157:H7 that are disseminated within the United States. Human and bovine isolates are nonrandomly distributed among the lineages, suggesting that one of these lineages may be less virulent for humans or may not be efficiently transmitted to humans from bovine sources. Restric-tion fragment length polymorphism analysis with lambdoid phage genomes indicates that phage-mediated events are associated with divergence of the lineages, thereby providing one explana-tion for the degree of diversity that is observed among E. coli O157:H7 by other molecular-fingerprinting methods. [TOP OF PAGE]
Phage facts. Konforti, B. (2001). Nature Structural Biology 8:19-20. [TOP OF PAGE]
Antacid increases survival of Vibrio vulnificus and Vibrio vulnificus phage in a gastrointestinal model. Koo, J., Marshall, D.L., Depaola, A. (2001). Appl. Environ. Microbiol. 67:2895-2902. Viable counts of three strains of Vibrio vulnificus and its phage were determined during exposure to a mechanical gastrointestinal model with or without antacid for 9 h at 37 degrees C. V. vulnificus was eliminated (>4-log reduction) within 30 min in the gastric compartment (pH decline from 5.0 to 3.5). Viable V. vulnificus cells delivered from the gastric compartment during the first 30 min of exposure reached 10⁶ to 10⁸ CFU/ml in the intestinal compartment after 9 h (pH 7.0). Phages were eliminated within 45 min in the gastric compartment (pH decline from 5.1 to 2.5). Less than a 2-log reduction of phage was observed in the intestinal compartment after 9 h (pH 7.0). When the gastric compartment contained antacid V. vulnificus counts decreased slightly (<2 log) during 2 h of exposure (pH decline from 7.7 to 6.0), while counts in the intestinal compartment (pH 7.5) reached 10⁷ to 10⁹ CFU/ml. Phage numbers decreased 1 log after 2 h in the gastric compartment (pH decline from 7.7 to 5.7) containing antacid and decreased 1 log in the intestinal compartment (pH 7.6) after 9 h. Presence of antacid in the gastric compartment of the model greatly increased the ability of both V. vulnificus and its phage to survive simulated gastrointestinal transit and may be a factor involved with oyster-associated illness. [TOP OF PAGE]
Cloning of genomic DNA of Lactococcus lactis that restores phage sensitivity to an unusual bacteriophage sk1-resistant mutant. Kraus, J., Geller, B.L. (2001). Appl. Environ. Microbiol. 67:791-798. An unusual, spontaneous, phage sk1-resistant mutant (RMSK1/1) of Lactococcus lactis C2 apparently blocks phage DNA entry into the host. Although no visible plaques formed on RMSK1/1, this host propagated phage at a reduced efficiency. This was evident from center-of-infection experiments, which showed that 21% of infected RMSK1/1 formed plaques when plated on its phage-sensitive parental strain, C2. Moreover, viable cell counts 0 and 4 h after infection were not significantly different from those of an uninfected culture. Further characterization showed that phage adsorption was normal, but burst size was reduced fivefold and the latent period was increased from 28.5 to 36 min. RMSK1/1 was resistant to other, but not all, similar phages. Phage sensitivity was restored to RMSK1/1 by transformation with a cloned DNA fragment from a genomic library of a phage-sensitive strain. Characterization of the DNA that restored phage sensitivity revealed an open reading frame with similarity to sequences encoding lysozymes (beta-1,4-N-acetylmuramidase) and lysins from various bacteria, a fungus, and phages of Lactobacillus and Streptococcus and also revealed DNA homologous to noncoding sequences of temperate phage of L. lactis, DNA similar to a region of phage sk1, a gene with similarity to tRNA genes, a prophage attachment site, and open reading frames with similarities to sun and to sequences encoding phosphoprotein phosphatases and protein kinases. Mutational analyses of the cloned DNA showed that the region of homology with lactococcal temperate phage was responsible for restoring the phage-sensitive phenotype. The region of homology with DNA of lactococcal temperate phage was similar to DNA from a previously characterized lactococcal phage that suppresses an abortive infection mechanism of phage resistance. The region of homology with lactococcal temperate phage was deleted from a phage-sensitive strain, but the strain was not phage resistant. The results suggest that the cloned DNA with homology to lactococcal temperate phage was not mutated in the phage-resistant strain. The cloned DNA apparently suppressed the mechanism of resistance, and it may do so by mimicking a region of phage DNA that interacts with components of the resistance mechanism. [TOP OF PAGE]
[Vibrio cholerae O139 bacteriophages]. Kudriakova, T.A., Makedonova, L.D., Kachkina, G.V., Saiamov, S.R. (2001). Zhurnal Mikrobiologii, Epidemiologii i Immunobiologii 28-30. Cholera bacteriophages have been isolated from 27 lysogenic cultures of V. cholerae O139. As shown the pages under study belong to two morphological groups A1 and F1 and serological types II and XII. The use of prophage typing and the sensitivity test to specific phage made it possible to differentiate V. cholerae strains, serogroup O139. [TOP OF PAGE]
Use of bacteriophage therapy in surgical practice. Lakhno, V.M., Bordunovskii, V.N. (2001). Vestnik Khirurgii Imeni I. I. Grekova 160:122-125. [TOP OF PAGE]
Population dynamics and diversity of phytoplankton, bacteria and viruses in a seawater enclosure. Larsen, A., Castberg, T., Sandaa, R.A., Brussaard, C.P.D., Egge, J.K., Heldal, M., Paulino, A., Thyrhaug, R., van Hannen, E.J., Bratbak, G. (2001). Mar. Ecol. Prog. Ser. 221:47-57. We now know that the abundance of free viruses in most marine environments is high. There is still, however, a lack of understanding of their occurrence and distribution and of in situ relationships between viral and host communities in natural environments. This may be partly due to methodological limitations. Our main aim was therefore to perform a case study in which a variety of methods were applied in order to give an improved, high-resolution description of the microbial communities in a natural environment, In order to do this we combined light microscopy (LM), transmission electron microscopy (TEM), flow cytometry (FCM), PCR denaturing gradient gel electrophoresis (PCR-DGGE) and pulsed-field gel electrophoresis (PFGE) and studied the diversity and succession of algae, bacteria and viruses in a nutrient enriched seawater enclosure. In the enclosure we experienced a situation where the development of the dominating algal population, which consisted of several flagellate species, was followed by proliferation of several different size-classes of viruses. The total bacterial number decreased markedly during the flagellate bloom but the community composition was maintained and the diversity remained high. Our results indicate a close linkage between various algal, bacterial and viral populations and show that virioplankton do not necessarily terminate algal and bacterial blooms but that they keep the host populations at non-blooming levels. [TOP OF PAGE]
A novel virus (HaNIV) causes lysis of the toxic bloom-forming alga Heterosigma akashiwo (Raphidophyceae). Lawrence, J.E., Chan, A.M., Suttle, C.A. (2001). Journal of Phycology 37:216-222. We describe a previously unknown virus that causes lysis of the toxic blopm-forming alga Heterosigma akashiwo (Hada) Hara et Chihara (Raphidophyceae). Heterosigma akashiwo nuclear inclusion virus (HaNIV) does not resemble other algal viruses described to date. HaNIV is small (ca. 30 nm diameter), is assembled in the nucleus, and forms crystalline arrays. We estimate that approximately 10⁵ HaNIV particles are released during lysis of a cell. During a time-course experiment, TEM revealed the first signs of HaNIV infection 24 h after viral addition, and by 74 h 98% of observed cells were visibly infected. The onset of cell lysis, as indicated by a decrease in the relative fluorescence of the cultures, was apparent by 42 h postinfection. The heterochromatin of infected cells is frequently found at the margin of the nucleoplasm, which is consistent with virus-mediated programmed cell death, or apoptosis. HaNIV is clearly different from other described viruses that infect alg ae, including other viral pathogens of H, akashiwo. These results indicate that viruses other than Phycodnaviridae are pathogens and cause mortality of microalgae in marine systems. It is Likely that HaNIV plays an integral role in the population dynamics of H. akashiwo. [TOP OF PAGE]
Viruses in the plankton of freshwater and saline Antarctic lakes. Laybourn-Parry, J., Hofer, J.S., Sommaruga, R. (2001). Freshwater Biology 46:1279-1287. 1. Virus-like particle (VLP) abundances in nine freshwater to saline lakes in the Vestfold Hills, Eastern Antarctica (68degree S) were determined in December 1999. In the ultra-oligotrophic to oligotrophic freshwater lakes, VLP abundances ranged from 1.01 to 3.28 X 106 mL-1 in the top 6 m of the water column. In the saline lakes the range was between 6.76 and 36.5 X 10⁶ mL-1. The lowest value was found in meromictic Ace Lake and the highest value in hypersaline Lake Williams. Virus to bacteria ratios (VBR) were lowest in the freshwater lakes and highest in the saline lakes, with a maximum of 23.4 in the former and 50.3 in the latter. 2. A range of morphologies among VLP was observed, including phages with short (Podoviridae) and long tails, icosahedric viruses of up to 300 nm and star-like particles of about 80 nm diameter. 3. In these microbially dominated ecosystems there was no correlation between VLP and either bacterial numbers of chlorophyll a. There was a significant correlation between VLP abundances and dissolved organic carbon concentration (r = 0.845, P < 0.01). 4. The data suggested that viruses probably attack a spectrum of bacteria and protozoan species. Virus-like particle numbers in the freshwater lakes were lower than values reported for lower latitude systems. Those in the saline lakes were comparable with abundances reported from other Antarctic lakes, and were higher than most values published for lower latitude lakes and many marine systems. Across the salinity spectrum from freshwater through brackish to hypersaline, VLP concentrations increased roughly in relation to increasing trophy. 5. Given that Antarctic lakes have a plankton almost entirely made up of bacteria and protists, and that VLP abundances are high, it is likely that viruses play a pivotal role in carbon cycling in these extreme ecosystems. [TOP OF PAGE]
Efficacy of bacteriophage use in complex treatment of the patients with burn wounds. Lazareva, E.B., Smirnov, S.V., Khvatov, V.B., Spiridonova, T.G., Bitkova, E.E., Darbeeva, O.S., Mayskaya, L.M., Parphenyuk, R.L., Menshikov, D.D. (2001). Antibiotiki i Khimioterapiya 46:10-14. Results of clinical and laboratory evaluation of the treatment with pyobacteriophage in tablets of the patients with burn wounds are presented. It was shown that phagotherapy provided more rapid cure of pyoseptic complications, temperature normalization, wounds purification and lower lethality Bacteriological analysis of wound secretions revealed that after the treatment staphylococci and streptococci were cultured 2 times rarely, Proteus spp. Were isolated 1.5 times rarely, E. coli was not isolated. The amount of positive haemocultures also diminished. Investigation of immunologic status demonstrated statistically significant normalization of immunity on cell level. Phagocytosis level didn't change while in control group (without bacteriophage use) it became lower. Antibody level enhanced but less extensively than in control group. The results of trial demonstrates positive effect of phagotherapy use at the patients with burns. [TOP OF PAGE]
Examination of bacteriophage as a biocontrol method for salmonella on fresh-cut fruit: a model study. Leverentz, B., Conway, W.S., Alavidze, Z., Janisiewicz, W.J., Fuchs, Y., Camp, M.J., Chighladze, E., Sulakvelidze, A. (2001). Journal of Food Protection 64:1116-1121. The preparation and distribution of fresh-cut produce is a rapidly developing industry that provides the consumer with convenient and nutritious food. However, fresh-cut fruits and vegetables may represent an increased food safety concern because of the absence or damage of peel and rind, which normally help reduce colonization of uncut produce with pathogenic bacteria. In this study, we found that Salmonella enteritidis populations can (i) survive on fresh-cut melons and apples stored at 5 degrees C, (ii) increase up to 2 log units on fresh-cut fruits stored at 10 degrees C, and (iii) increase up to 5 log units at 20 degrees C during a storage period of 168 h. In addition, we examined the effect of lytic, Salmonella-specific phages on reducing Salmonella numbers in experimentally contaminated fresh-cut melons and apples stored at various temperatures. We found that the phage mixture reduced Salmonella populations by approximately 3.5 logs on honeydew melon slices stored at 5 and 10 degrees C and by approximately 2.5 logs on slices stored at 20 degrees C, which is greater than the maximal amount achieved using chemical sanitizers. However, the phages did not significantly reduce Salmonella populations on the apple slices at any of the three temperatures. The titer of the phage preparation remained relatively stable on melon slices, whereas on apple slices the titer decreased to nondetectable levels in 48 h at all temperatures tested. Inactivation of phages, possibly by the acidic pH of apple slices (pH 4.2 versus pH 5.8 for melon slices), may have contributed to their inability to reduce Salmonella contamination in the apple slices. Higher phage concentrations and/or the use of low-pH-tolerant phage mutants may be required to increase the efficacy of the phage treatment in reducing Salmonella contamination of fresh-cut produce with a low pH. [TOP OF PAGE]
Colloidal interactions in suspensions of rods. Lin, K., Crocker, J.C., Zeri, A.C., Yodh, A.G. (2001). Phys Rev Lett 87:088301 We report direct measurements of entropic interactions of colloidal spheres in suspensions of rodlike fd bacteriophage. We investigate the influence of sphere size, rod concentration, and ionic strength on these interactions. Although the results compare favorably with a recent calculation, small discrepancies reveal entropic effects due to rod flexibility. At high salt concentrations, the potential turns repulsive as a result of viral adsorption on the spheres and viral bridging between the spheres. [TOP OF PAGE]
Mechanism of host cell death induced by infection of Escherichia coli with the c2 clear-plaque mutant of phage f1. Lin, S.H., Chen, W.P., Kuo, T.T. (2001). Botanical Bulletin of Academia Sinica (Taipei) 42:45-52. The c2 clear-plaque mutant arose spontaneously from the turbid plaque-inducing wild-type strain of bacteriophage f1. The mechanism of host cell death induced by infection of Escherichia coli with c2 has now been investigated. A marked decrease in cell membrane potential was apparent as early as 30 min after infection with c2, and leakage of cell contents was apparent after 4 h. Transmission electron microscopy also revealed the accumulation of granular membrane-like structures within cells at early stages of c2 infection. Electrophoretic analysis showed that the abundance of several bacterial outer membrane proteins was markedly reduced 2 h after infection with c2. Furthermore, substantial amounts of the phage coat protein (gpVIII) and single-stranded DNA-binding protein (gpV) were apparent in the inner membrane of c2-infected cells 2 h after infection. These data support the hypothesis that the death of c2-infected cells results from phage-induced damage to the bacterial cell membrane. [TOP OF PAGE]
Depolymerization of the capsular polysaccharide from Vibrio cholerae O139 by a lyase associated with the bacteriophage JA1. Linnerborg, M., Weintraub, A., Albert, M.J., Widmalm, G. (2001). Carbohydrate Research 333:263-269. We have studied the interaction between the Vibrio cholerae O139 specific phage JA1, belonging to the Podoviridae family, and the capsular polysaccharide (CPS) of the parent strain from which the phage was isolated. Upon incubation of the JA1 phage with the CPS, oligosaccharides were isolated and purified. The oligosaccharides derived from one (shown below) and two repeating units of the CPS were characterized using NMR spectroscopy, mass spectrometry and sugar analysis (structure: see text). The cleavage was found to occur by beta-elimination at the 4-substituted alpha-linked galacturonic acid, which results in a 4-deoxy-beta-L-threo-hex-4-enopyranosyl uronic acid group (Sug). The enzyme associated with the JA1 phage responsible for the depolymerization of the V. cholerae O139 CPS is thus a lyase. [TOP OF PAGE]
Physiological function of exopolysaccharides produced by Lactococcus lactis. Looijesteijn, P.J., Trapet, L., de, V.E., Abee, T., Hugenholtz, J. (2001). Int. J. Food Microbiol. 64:71-80. The physiological function of EPS produced by Lactococcus lactis was studied by comparing the tolerance of the non-EPS producing strain L. lactis ssp. cremoris MG1614 and an EPS producing isogenic variant of this strain to several anti-microbial factors. There was no difference in the sensitivity of the strains to increased temperatures, freezing or freeze-drying and the antibiotics, penicillin and vancomycin. A model system showed that EPS production did not affect the survival of L. lactis during passage through the gastrointestinal tract although the EPS itself was not degraded during this passage. The presence of cell associated EPS and EPS in suspension resulted in an increased tolerance to copper and nisin. Furthermore, cell associated EPS also protected the bacteria against bacteriophages and the cell wall degrading enzyme lysozyme. However, it has not been possible, so far, to increase EPS production using the presence of copper, nisin, lysozyme or bacteriophages as inducing factors. [TOP OF PAGE]
Distribution, isolation, host specificity, and diversity of cyanophages infecting marine Synechococcus spp. in river estuaries. Lu, J., Chen, F., Hodson, R.E. (2001). Appl. Environ. Microbiol. 67:3285-3290. The abundance of cyanophages infecting marine Synechococcus spp. increased with increasing salinity in three Georgia coastal rivers. About 80% of the cyanophage isolates were cyanomyoviruses. High cross-infectivity was found among the cyanophages infecting phycoerythrin-containing Synechococcus strains. Cyanophages in the river estuaries were diverse in terms of their morphotypes and genotypes. [TOP OF PAGE]
Elution, detection, and quantification of polio I, bacteriophages, Salmonella montevideo, and Escherichia coli O157:H7 from seeded strawberries and tomatoes. Lukasik, J., Bradley, M.L., Scott, T.M., Hsu, W.Y., Farrah, S.R., Tamplin, M.L. (2001). Journal of Food Protection 64:292-297. This study compared the effect of different physical and chemical treatments of strawberries and tomatoes to determine their ability to recover seeded viral and bacterial pathogens from produce surfaces. Solutions of salts, amino acids, complex media, and detergents were compared as eluants. Phosphate-buffered saline (PBS) containing 0.1% Tween 80 eluted the highest number of seeded microorganisms. Elution with this defined solution was then compared under different conditions of physical agitation. Rotary shaking for 20 min at 36 degrees C eluted higher numbers of viruses and bacteria than did low- or high-speed stomaching. Commercially available and laboratory prepared bacteriological differential media were compared for their ability to recover and distinguish eluted Salmonella montevideo and Escherichia coli O157:H7 strains from seeded produce. The recovery of seeded bacterial pathogens was low when differential media containing selective ingredients were used (MacConkey sorbitol agar, XLD agar, MacConkey agar). Highest recoveries were obtained on a medium consisting of tryptic soy agar supplemented with sodium thiosulfate and ferric ammonium citrate compared with selective media that inhibited up to 50% of the growth of the eluted microorganisms. [TOP OF PAGE]
The genome of archaeal prophage PsiM100 encodes the lytic enzyme responsible for autolysis of Methanothermobacter wolfeii. Luo, Y., Pfister, P., Leisinger, T., Wasserfallen, A. (2001). J. Bacteriol. 183:5788-5792. Methanothermobacter wolfeii (formerly Methanobacterium wolfei), a thermophilic methanoarchaeon whose cultures lyse upon hydrogen starvation, carries a defective prophage called PsiM100 on its chromosome. The nucleotide sequence of PsiM100 and its flanking regions was established and compared to that of the previously sequenced phage PsiM2 of Methanothermobacter marburgensis (formerly Methanobacterium thermoautotrophicum Marburg). The PsiM100 genome extends over 28,798 bp, and its borders are defined by flanking 21-bp direct repeats of a pure-AT sequence, which very likely forms the core of the putative attachment site where the crossing over occurred during integration. A large fragment of 2,793 bp, IFa, apparently inserted into PsiM100 but is absent in the genome of PsiM2. The remaining part of the PsiM100 genome showed 70.8% nucleotide sequence identity to the whole genome of PsiM2. Thirty-four open reading frames (ORFs) on the forward strand and one ORF on the reverse strand were identified in the PsiM100 genome. Comparison of PsiM100-encoded ORFs to those encoded by phage PsiM2 and to other known protein sequences permitted the assignment of putative functions to some ORFs. The ORF28 protein of PsiM100 was identified as the previously known autolytic enzyme pseudomurein endoisopeptidase PeiW produced by M. wolfeii. [TOP OF PAGE]
Analysis of the genetic switch and replication region of a P335-type bacteriophage with an obligate lytic lifestyle on Lactococcus lactis. Madsen, S.M., Mills, D., Djordjevic, G., Israelsen, H., Klaenhammer, T.R. (2001). Appl. Environ. Microbiol. 67:1128-1139. The DNA sequence of the replication module, part of the lysis module, and remnants of a lysogenic module from the lytic P335 species lactococcal bacteriophage variant phi31 was determined, and its regulatory elements were investigated. The identification of a characteristic genetic switch including two divergent promoters and two cognate repressor genes strongly indicates that variant phi31 was derived from a temperate bacteriophage. Regulation of the two early promoters was analyzed by primer extension and transcriptional promoter fusions to a lacLM reporter. The regulatory behavior of the promoter region differed significantly from the genetic responses of temperate Lactococcus lactis phages. The cro gene homologue regulates its own production and is an efficient repressor of cI gene expression. No detectable cI gene expression could be measured in the presence of cro. cI gene expression in the absence of cro exerted minor influences on the regulation of the two promoters within the genetic switch. Homology comparisons revealed a replication module which is most likely expressed from the promoter located upstream of the cro gene homologue. The replication module encoded genes with strong homology to helicases and primases found in several Streptococcus thermophilus phages. Downstream of the primase homologue, an AT-rich noncoding origin region was identified. The characteristics and location of this region and its ability to reduce the efficiency of plaquing of variant phi31 106-fold when present at high copy number in trans provide evidence for identification of the phage origin of replication. Phage variant phi31 is an obligately lytic phage that was isolated from commercial dairy fermentation environments. Neither a phage attachment site nor an integrase gene, required to establish lysogeny, was identified, explaining its lytic lifestyle and suggesting its origin from a temperate phage ancestor. Several regions showing extensive DNA and protein homologies to different temperate phages of Lactococcus, Lactobacillus, and Streptococcus were also discovered, indicating the likely exchange of DNA cassettes through horizontal gene transfer in the dynamic ecological environment of dairy fermentations. [TOP OF PAGE]
Sequence analysis and molecular characterization of the Lactococcus lactis temperate bacteriophage BK5-T. Mahanivong, C., Boyce, J.D., Davidson, B.E., Hillier, A.J. (2001). Appl. Environ. Microbiol. 67:3564-3576. The Lactococcus lactis temperate bacteriophage BK5-T is one of twelve type phages that define L. lactis phage species. This paper describes the nucleotide sequence and analysis of a 21-kbp region of the BK5-T genome and completes the nucleotide sequence of the genome of this phage. The 40,003-nucleotide linear genome encodes 63 open reading frames. Sequence runoff experiments showed that the cohesive ends of the BK5-T genome contained a 12-bp 3' single-stranded overhang with the sequence 5'-CACACACATAGG-3'. Two major BK5-T structural proteins, of approximately 30 and 20 kDa, were identified, and N-terminal sequence analysis determined that they were encoded by orf7 and orf12, respectively. A 169-bp fragment containing a 37-bp direct repeat and several smaller repeat sequences conferred resistance to BK5-T infection when introduced in trans to the host cell and is likely a part of the BK5-T origin of replication (ori). [TOP OF PAGE]
Growth and survival of clinical vs. environmental species of Aeromonas in tap water. Mary, P., Buchet, G., Defives, C., Hornez, J.P. (2001). Int. J. Food Microbiol. 69:191-198. The ability of four species of Aeromonas (two of clinical and two of environmental origin) to survive and/or grow in tap water microcosms supplemented with sodium thiosulphate was tested. After bottling, the autochthonous microflora reached 6 x 10⁵ cfu ml^-1 after a 5-day incubation period in tap water unfiltered and which was non-autoclaved. In filtered tap water, "ultramicrocells" were detected and final populations of ca. 10⁶ cfu ml^-1 after 7 days were obtained. Aeromonas was inoculated at an initial cell concentration of ca. 10⁴ cfu ml^-1. All strains were able to grow in tap water samples, which were filtered and autoclaved, and a final concentration of 10⁵-10⁶ cfu ml^-1 was observed. Any inherent capability of Aeromonas to grow in tap water was eliminated by the presence of autochthonous microflora and "ultramicrocells" bacteria. Survival rates were strain- and microcosm-dependent. In unfiltered-non-autoclaved water, viable counts declined to below the detection limit (i.e. 1 log cfu ml^-1) in 1.5 to 20 days. The declines in viable counts were even more pronounced in the filtered microcosm. Although inoculation ratios (100/1 in unfiltered-non-autoclaved and 1,000/1 in filtered microcosms) were favourable for aeromonads, at least for I to 3 days, the organisms disappeared in these microcosms. Thus, competition for nutrients was an unlikely cause of the limitation of aeromonads. The bacteriolytic effect of enzymes released by membrane vesicles from the autochthonous microflora and of "tail phage-like particles" bacteriocins were suggested as an in situ control of aeromonad populations. The present study showed that environmental strains of Aeromonas had no ecological advantage over clinical isolates. Thus, waterborne infections and contaminations of foods by pathogenic Aeromonas species could not be discounted. [TOP OF PAGE]
Mu-like prophage in serogroup B Neisseria meningitidis coding for surface-exposed antigens. Masignani, V., Giuliani, M.M., Tettelin, H., Comanducci, M., Rappuoli, R., Scarlato, V. (2001). Infect. Immun. 69:2580-2588. Sequence analysis of the genome of Neisseria meningititdis serogroup B revealed the presence of an apprx35-kb region inserted within a putative gene coding for an ABC-type transporter. The region contains 46 open reading frames, 29 of which are colinear and homologous to the genes of Escherichia coli Mu phage. Two prophages with similar organizations were also found in serogroup A meningococcus, and one was found in Haemophilus influenzae. Early and late phage functions are well preserved in this family of Mu-like prophages. Several regions of atypical nucleotide content were identified. These likely represent genes acquired by horizontal transfer. Three of the acquired genes are shown to code for surface-associated antigens, and the encoded proteins are able to induce bactericidal antibodies. [TOP OF PAGE]
Bacteriophage WO and virus-like particles in Wolbachia, an endosymbiont of arthropods. Masui, S., Kuroiwa, H., Sasaki, T., Inui, M., Kuroiwa, T., Ishikawa, H. (2001). Biochemical & Biophysical Research Communications 283:1099-1104. Wolbachia are intracellular symbionts mainly found in arthropods, causing various sexual alterations on their hosts by unknown mechanisms. Here we report the results that strongly suggest that Wolbachia have virus-like particles of phage WO, which was previously identified as a prophage-like element in the Wolbachia genome. Wolbachia (strain wTai) infection in an insect was detected with the antibody against Wsp, an outer surface protein of Wolbachia, by fluorescence microscopy and immunoelectron-microscopy for the first time. Virus-like particles in Wolbachia were observed by electron-microscopy. The 0.22-&mgr;m filtrate of insect ovary contained DAPI-positive particles, and PCR analysis demonstrated that a phage WO DNA passed through the filter while Wolbachia DNA were eliminated, suggesting that the DAPI-positive particles were phage WO. [TOP OF PAGE]
Improvement and optimization of two engineered phage resistance mechanisms in Lactococcus lactis. McGrath, S., Fitzgerald, G.F., van Sinderen, D. (2001). Appl. Environ. Microbiol. 67:608-616. Homologous replication module genes were identified for four P335 type phages. DNA sequence analysis revealed that all four phages exhibited more than 90% DNA homology for at least two genes, designated rep2009 and orf17. One of these genes, rep2009, codes for a putative replisome organizer protein and contains an assumed origin of phage DNA replication (ori2009), which was identical for all four phages. DNA fragments representing the ori2009 sequence confer a phage-encoded resistance (Per) phenotype on lactococcal hosts when they are supplied on a high-copy-number vector. Furthermore, cloning multiple copies of the ori2009 sequence was found to increase the effectiveness of the Per phenotype conferred. A number of antisense plasmids targeting specific genes of the replication module were constructed. Two separate plasmids targeting rep2009 and orf17 were found to efficiently inhibit proliferation of all four phages by interfering with intracellular phage DNA replication. These results represent two highly effective strategies for inhibiting bacteriophage proliferation, and they also identify a novel gene, orf17, which appears to be important for phage DNA replication. Furthermore, these results indicate that although the actual mechanisms of DNA replication are very similar, if not identical, for all four phages, expression of the replication genes is significantly different in each case. [TOP OF PAGE]
Livestock deaths associated with Clavibacter toxicus/Anguina sp. infection in seedheads of Agrostis avenacea and Polypogon monspeliensis. McKay, A.C., Ophel, K.M., Reardon, T.B., Gooden, J.M. (2001). Plant Disease 77:635-641. Flood plain staggers, a recently discovered poisoning of livestock, has been linked to Clavibacter toxicus infection in the seedheads of blown grass, Agrostis avenacea, in northern New South Wales and annual beardgrass, Polypogon monspeliensis, in the southeast of South Australia (Australia). The same bacterium on annual ryegrass, Lolium rigidum, causes the poisoning of livestock known as annual ryegrass toxicity. Strains of C. toxicus from A. avenacea and P. monspeliensis were indistinguishable from strains from L. rigidum based on colony morphology, serological reactions, and bacteriophage specificity. Bacteriophages isolated from C. toxicus on the three hosts were indistinguishable from each other based on DNA restriction patterns. In allozyme studies, considerable variation was observed between the C. toxicus strains from the three hosts, but the variation was within the range exhibited by a single species. C. toxicus is carried into L. rigidum by a seed gall-forming nematode, Anguina funesta. Anguina nematodes are also associated with C. toxicus infection of A. avenacea and P. monspeliensis. Allozyme studies indicate that the same Anguina species probably infects both grasses, and that it is not Anguina funesta, Anguina agrostis, Anguina tritici, or the species found on velvetgrass (Holcus lanatus). This is the first recording of a nematode other than Anguina funesta as a vector for C. toxicus. The new vector broadens the range of grasses that the bacterium can infect. [TOP OF PAGE]
Phenotype characterization of genetically defined microorganisms and growth of bacteriophage in biofilms. McLean, R.J., Corbin, B.D., Balzer, G.J., Aron, G.M. (2001). Methods in Enzymology 336:163-174. Phenotypic characterization will be a pivotal aspect of future research in understanding the biofilm mode of growth. We hope that the concepts and techniques presented in this chapter will benefit other investigators in this field. Although initial studies will necessarily involve monocultures, eventually mixed culture work will have to be performed to understand biofilm growth in the natural environment. As the study of biofilm-phage interactions is new, there is considerable fundamental work that needs to be addressed. Here, we anticipate that some phage are better adapted to growth in biofilms, some are adept in growing in mixed culture biofilms, and others are better adapted to infecting planktonic organisms. Whereas biofilms are now widely accepted as a fundamental aspect of microbial growth in nature, the field of phage ecology is quite new and an exciting challenge for the future. [TOP OF PAGE]
Phi29 family of phages. Meijer, W.J., Horcajadas, J.A., Salas, M. (2001). Microbiology and Molecular Biology Reviews 65:261-287. Continuous research spanning more than three decades has made the Bacillus bacteriophage phi29 a paradigm for several molecular mechanisms of general biological processes, such as DNA replication, regulation of transcription, phage morphogenesis, and phage DNA packaging. The genome of bacteriophage phi29 consists of a linear double-stranded DNA (dsDNA), which has a terminal protein (TP) covalently linked to its 5' ends. Initiation of DNA replication, carried out by a protein-primed mechanism, has been studied in detail and is considered to be a model system for the protein-primed DNA replication that is also used by most other linear genomes with a TP linked to their DNA ends, such as other phages, linear plasmids, and adenoviruses. In addition to a continuing progress in unraveling the initiation of DNA replication mechanism and the role of various proteins involved in this process, major advances have been made during the last few years, especially in our understanding of transcription regulation, the head-tail connector protein, and DNA packaging. Recent progress in all these topics is reviewed. In addition to phi29, the genomes of several other Bacillus phages consist of a linear dsDNA with a TP molecule attached to their 5' ends. These phi29-like phages can be divided into three groups. The first group includes, in addition to phi29, phages PZA, phi15, and BS32. The second group comprises B103, Nf, and M2Y, and the third group contains GA-1 as its sole member. Whereas the DNA sequences of the complete genomes of phi29 (group I) and B103 (group II) are known, only parts of the genome of GA-1 (group III) were sequenced. We have determined the complete DNA sequence of the GA-1 genome, which allowed analysis of differences and homologies between the three groups of phi29-like phages, which is included in this review. [TOP OF PAGE]
Characterization of two novel Rhizobium leguminosarum bacteriophages from a field release site of genetically-modified rhizobia. Mendum, T.A., Clark, I.M., Hirsch, P.R. (2001). Antonie van Leeuwenhoek 79:189-197. Two Rhizobium leguminosarum biovar viceae bacteriophages with contrasting properties were isolated from a field site in which the survival of genetically modified R. leguminosarum inoculants had been monitored for several years. Inoculant strain RSM2004 was used as the indicator for phage isolation and propagation. One phage, RL1RES, was temperate and could not replicate in any of the 42 indigenous R. leguminosarum field isolates tested although nested PCR indicated that phage sequences were present in six of the isolates. The second phage, RL2RES, was virulent, capable of generalised transduction, contained DNA with modified cytosine residues, and was capable of infecting all field isolates tested although the GM inoculant strain CT0370 was resistant. Sequence with homology to RL2RES was detected by nested PCR in six of the 42 field-isolates. These were not the same isolates that showed homology to RL1RES. The implication of these findings for the survival of rhizobial inoculants, and the ecology of phages and their host bacteria, are discussed. [TOP OF PAGE]
Multiple infection dynamics has pronounced effects on the fitness of RNA viruses. Miralles, R., Ferrer, R., Sole, R.V., Moya, A., Elena, S.F. (2001). Journal of Evolutionary Biology 14:654-662. Several factors play a role during the replication and transmission of RNA viruses. First, as a consequence of their enormous mutation rate, complex mixtures of genomes are generated immediately after infection of a new host. Secondly, differences in growth and competition rates drive the selection of certain genetic variants within an infected host. Thirdly, but not less important, a random sampling occurs at the moment of viral infectious passage from an infected to a healthy host. In addition, the availability of hosts also influences the fate of a given viral genotype. When new hosts are scarce, different viral genotypes might infect the same host, adding an extra complexity to the competition among genetic variants. We have employed a two-fold approach to analyse the role played by each of these factors in the evolution of RNA viruses. First, we have derived a model that takes into account all the preceding factors. This model employs the classic Lotka-Volterra competition equations but it also incorporates the effect of mutation during RNA replication, the effect of the stochastic sampling at the moment of infectious passage among hosts and, the effect of the type of infection (single, coinfection or superinfection). Secondly, the predictions of the model have been tested in an in vitro evolution experiment. Both theoretical and experimental results show that in infection passages with coinfection viral fitness increased more than in single infections. In contrast, infection passages with superinfection did not differ from the single infection. The coinfection frequency also affected the outcome: the larger the proportion of viruses coinfecting a host, the larger increase in fitness observed. [TOP OF PAGE]
Effect of phage on survival of Salmonella enteritidis during manufacture and storage of cheddar cheese made from raw and pasteurized milk. Modi, R., Hirvi, Y., Hill, A., Griffiths, M.W. (2001). Journal of Food Protection 64:927-933. The ability of Salmonella enteritidis to survive in the presence of phage, SJ2, during manufacture, ripening, and storage of Cheddar cheese produced from raw and pasteurized milk was investigated. Raw milk and pasteurized milk were inoculated to contain 10⁴ CFU/ml of a luminescent strain of Salmonella enteritidis (lux) and 10⁸ PFU/ml SJ2 phage. The milks were processed into Cheddar cheese following standard procedures. Cheese samples were examined for Salmonella enteritidis (lux), lactic acid bacteria, molds and yeasts, coliforms, and total counts, while moisture, fat, salt, and pH values were also measured. Salmonella enteritidis (lux) was enumerated in duplicate samples by surface plating on MacConkey novobiocin agar. Bioluminescent colonies of Salmonella enteritidis were identified in the NightOwl molecular imager. Samples were taken over a period of 99 days. Counts of Salmonella enteritidis (lux) decreased by 1 to 2 log cycles in raw and pasteurized milk cheeses made from milk containing phage. In cheeses made from milks to which phage was not added, there was an increase in Salmonella counts of about 1 log cycle. Lower counts of Salmonella enteritidis (lux) were observed after 24 h in pasteurized milk cheese containing phage compared to Salmonella counts in raw milk cheese with phage. Salmonella enteritidis (lux) survived in raw milk and pasteurized milk cheese without phage, reaching a final concentration of 10³ CFU/g after 99 days of storage at 8 degrees C. Salmonella did not survive in pasteurized milk cheese after 89 days in the presence of phage. However, Salmonella counts of approximately 50 CFU/g were observed in raw milk cheese containing phage even after 99 days of storage. In conclusion, this study demonstrates that the addition of phage may be a useful adjunct to reduce the ability of Salmonella to survive in Cheddar cheese made from both raw and pasteurized milk. [TOP OF PAGE]
Characterization of VPI pathogenicity island and CTXphi prophage in environmental strains of Vibrio cholerae. Mukhopadhyay, A.K., Chakraborty, S., Takeda, Y., Nair, G.B., Berg, D.E. (2001). J. Bacteriol. 183:4737-4746. Environmental isolates of Vibrio cholerae of eight randomly amplified polymorphic DNA (RAPD) fingerprint types from Calcutta, India, that were unusual in containing toxin-coregulated pilus or cholera toxin genes but not O1 or O139 antigens of epidemic strains were studied by PCR and sequencing to gain insights into V. cholerae evolution. We found that each isolate contained a variant form of the VPI pathogenicity island. Distinguishing features included (i) four new alleles of tcpF (which encodes secreted virulence protein; its exact function is unknown), 20 to 70% divergent (at the protein level) from each other and canonical tcpF; (ii) a new allele of toxT (virulence regulatory gene), 36% divergent (at the protein level) in its 5' half and nearly identical in its 3' half to canonical toxT; (iii) a new tcpA (pilin) gene; and (iv) four variant forms of a regulatory sequence upstream of toxT. Also found were transpositions of an IS903-related element and function-unknown genes to sites in VPI. Cholera toxin (ctx) genes were found in isolates of two RAPD types, in each case embedded in CTXphi-like prophages. Fragments that are inferred to contain only putative repressor, replication, and integration genes were present in two other RAPD types. New possible prophage repressor and replication genes were also identified. Our results show marked genetic diversity in the virulence-associated gene clusters found in some nonepidemic V. cholerae strains, suggest that some of these genes contribute to fitness in nature, and emphasize the potential importance of interstrain gene exchange in the evolution of this species. [TOP OF PAGE]
Phage conversion of Panton-Valentine leukocidin in Staphylococcus aureus: molecular analysis of a PVL-converting phage, phiSLT. Narita, S., Kaneko, J., Chiba, J., Piemont, Y., Jarraud, S., Etienne, J., Kamio, Y. (2001). Gene 268:195-206. Staphylococcal Panton-Valentine leukocidin (PVL) is an important virulence factor, which causes leukocytolysis and tissue necrosis. Our previous report on the existence of the PVL genes (lukS-PV and lukF-PV) on the genome of prophage phiPVL in the Staphylococcus aureus strain V8 suggested the horizontal transmission of PVL genes by temperate bacteriophage among S. aureus (Kaneko, et al., 1998. Gene 215, 57-67). Here, we demonstrated the phage conversion of S. aureus leading to the production of PVL by discovery of a novel PVL-carrying phage, phiSLT (Staphylococcal Leukocytolytic Toxin) from a clinical isolate of S. aureus. phiSLT was able to lysogenize several clinical isolates of PVL-negative S. aureus strains as well as strain RN4220 at the conserved 29-bp sequence (attB) and all the lysogenized S. aureus strains had the ability to produce PVL. phiSLT had an elongated head of about 100x50 nm and a flexible tail of 400 nm long, that was quite different from phiPVL which had an isometric hexagonal head of about 60 nm diameter. The linear double-stranded phiSLT genome comprised 42,942 bp with 29-bp attachment core sequences and contained 62 open reading frames. Only 6.4 kbp region containing lysis cassette, PVL genes, attP, integrase, and orf204 of phiSLT was identical to that of phiPVL, while other regions were different from those of phiPVL. Thus, it can be concluded that PVL genes are carried by different temperate phages, which have the same attachment site. [TOP OF PAGE]
Prevention and elimination of upper respiratory colonization of mice by group A streptococci by using a bacteriophage lytic enzyme. Nelson, D., Loomis, L., Fischetti, V.A. (2001). Proc. Natl. Acad. Sci. USA 98:4107-4112. Bacteriophage lytic enzymes quickly destroy the cell wall of the host bacterium to release progeny phage. Because such lytic enzymes specifically kill the species in which they were produced, they may represent an effective way to control pathogenic bacteria without disturbing normal microflora. In this report, we studied a murein hydrolase from the streptococcal bacteriophage C1 termed lysin. This enzyme is specific for groups A, C, and E streptococci, with little or no activity toward several oral streptococci or other commensal organisms tested. Using purified lysin in vitro, we show that 1,000 units (10 ng) of enzyme is sufficient to sterilize a culture of apprxeq10⁷ group A streptococci within 5 seconds. When a single dose of lysin (250 units) is first added to the oral cavity of mice, followed by 10⁷ live group A streptococci, it provides protection from colonization (28.5% infected, n = 21) compared with controls without lysin (70.5% infected, n = 17) (P < 0.03). Furthermore, when lysin (500 units) was given orally to 9 heavily colonized mice, no detectable streptococci were observed 2 h after lysin treatment. In all, these studies show that lysin represents a unique murein hydrolase that has a rapid lethal effect both in vitro and in vivo on group A streptococci, without affecting other indigenous microorganisms analyzed. This general approach may be used to either eliminate or reduce streptococci from the upper respiratory mucosal epithelium of either carriers or infected individuals, thus reducing associated disease. [TOP OF PAGE]
Three-component-mediated serotype conversion in Pseudomonas aeruginosa by bacteriophage D3. Newton, G.J., Daniels, C., Burrows, L.L., Kropinski, A.M., Clarke, A.J., Lam, J.S. (2001). Molecular Microbiology 39:1237-1247. Bacteriophage D3 is capable of lysogenizing Pseudomonas aeruginosa PAO1 (serotype O5), converting the O-antigen from O5 to O16 and O-acetylating the N-acetylfucosamine moiety. To investigate the mechanism of lysogenic conversion, a 3.6 kb fragment from the D3 genome was isolated capable of mediating serotypic conversion identical to the D3 lysogen strain (AK1380). The PAO1 transformants containing this 3.6 kb of D3 DNA exhibited identical lipopolysaccharide (LPS) banding patterns to serotype O16 in silver-stained SDS-PAGE gels and displayed reactivity to an antibody specific for O-acetyl groups. Further analysis led to the identification of three open reading frames (ORFs) required for serotype conversion: an alpha-polymerase inhibitor (iap); an O-acetylase (oac); and a beta-polymerase (wzybeta). The alpha-polymerase inhibitor (lap) is capable of inhibiting the assembly of the serotype-specific O5 B-band LPS and allows the phage-encoded beta-polymerase (Wzybeta) to form new beta-linked B-band LPS. The D3 phage also alters the LPS by the addition of O-acetyl groups to the FucNAc residue in the O-antigen repeat unit by the action of the D3 O-acetylase (Oac). These three components form a simple yet elegant system by which bacteriophage D3 is capable of altering the surface of P. aeruginosa PAO1. [TOP OF PAGE]
Increased mutation rate of E. coli K12 lambda cultures maintained in continuous logarithmic growth. Northrop, J.H. (2001). Journal of General Physiology 50:369-377. Continuous logarithmic growth of E. coli K12 lambda in an automatic culture cell resulted in marked increases in the proportion of several mutants. The P1 phage-resistant cells increased 10 to 3000 times, the T2 phage-resistant cells 1 to 1000 times, the neomycin-resistant cells 1 to 10 times, and the virus-producing cells 30 to 70 times. No change occurred in the penicillin-resistant cells. Calculation of the growth curves and direct determination of the mutation rates by the null fraction method showed that the increases in the proportion of mutants were due to increases in the mutation rates. [TOP OF PAGE]
Pathogenicity and resistance islands of staphylococci. Novick, R.P., Schlievert, P., Ruzin, A. (2001). Microbes Infect 3:585-594. Variable genetic elements including plasmids, transposons and prophages are involved in pathogenesis and antibiotic resistance, and are an important component of the staphylococcal genome. This review covers a set of newly described variable chromosomal elements, pathogenicity and resistance islands, carrying superantigen and resistance genes, especially toxic shock and methicillin resistance, respectively. [TOP OF PAGE]
Naturally occurring lactococcal plasmid pAH90 links bacteriophage resistance and mobility functions to a food-grade selectable marker. O' Sullivan, D., Ross, R.P., Twomey, D.P., Fitzgerald, G.F., Hill, C., Coffey, A. (2001). Appl. Environ. Microbiol. 67:929-937. The bacteriophage resistance plasmid pAH90 (26,490 bp) is a natural cointegrate plasmid formed via homologous recombination between the type I restriction-modification specificity determinants (hsdS) of two smaller lactococcal plasmids, pAH33 (6,159 bp) and pAH82 (20,331 bp), giving rise to a bacteriophage-insensitive mutant following phage challenge (D. O'Sullivan, D. P. Twomey, A. Coffey, C. Hill, G. F. Fitzgerald, and R. P. Ross, Mol. Microbiol. 36:866-876; 2000). In this communication we provide evidence that the recombination event is favored by phage infection. The entire nucleotide sequence of plasmid pAH90 was determined and found to contain 24 open reading frames (ORFs) responsible for phenotypes which include restriction-modification, phage adsorption inhibition, plasmid replication, cadmium resistance, cobalt transport, and conjugative mobilization. The cadmium resistance property, encoded by the cadA gene, which has an associated regulatory gene (cadC), is of particular interest, as it facilitated the selection of pAH90 in other phage-sensitive lactococci after electroporation. In addition, we report the identification of a group II self-splicing intron bounded by two exons which have the capacity to encode a relaxase implicated in conjugation in gram-positive bacteria. The functionality of this intron was evident by demonstrating splicing in vivo. Given that pAH90 encodes potent phage defense systems which act at different stages in the phage lytic cycle, the linkage of these with a food-grade selectable marker on a replicon that can be mobilized among lactococci has significant potential for natural strain improvement for industrial dairy fermentations which are susceptible to phage inhibition. [TOP OF PAGE]
Diversification of Escherichia coli genomes: are bacteriophages the major contributors? Ohnishi, M., Kurokawa, K., Hayashi, T. (2001). Trends in Microbiology 9:481-485. Determination of the genome sequence of enterohemorrhagic Escherichia coli O157 Sakai and genomic comparison with the laboratory strain K-12 has revealed that the two strains share a highly conserved 4.1-Mb sequence and that each also contains a larger amount of strain-specific sequence. The analysis also revealed the presence of a surprisingly larger number of prophages in O157, most of which are lambda-like phages that resemble each other. Based on these results, we discuss how bacteriophages contributed to this process. We also describe possible mechanisms by which O157 acquired many closely related phages, and raise the possibility that such bacteria might function as 'phage factories', releasing a variety of chemeric or mosaic prophages into the environment. [TOP OF PAGE]
Bacteriophage P4282, a parasite of Ralstonia solanacearum, encodes a bacteriolytic protein important for lytic infection of its host. Ozawa, H., TANAKA, H., Ichinose, Y., Shiraishi, T., Yamada, T. (2001). MGG Molecular Genetics and Genomics 265:95-101. To enhance bacterial wilt resistance in tobacco expressing a foreign protein, we isolated the bacteriolytic gene from a bacteriophage that infects Ralstonia solanacearum. The bacteriolytic protein of phage P4282 isolated in Tochigi Prefecture was purified from a lysate of R. solanacearum M4S cells infected with the phage, and its bacteriolytic activity was assayed by following the decrease in the turbidity of suspensions of R. solanacearum M4S cells. The molecular weight of the bacteriolytic protein was approximately 71 kDa, and the sequence of the N-terminal 13 amino acids was determined. We used oligonucleotide probes based on this amino acid sequence to isolate the bacteriolytic gene from phage P4282 DNA. This gene of 2061 bp encodes a product of 687 amino acids, whose calaculated molecular weight was 70.12 kDa. The bacteriolytic gene was placed under the control of an inducible promoter, and the plasmid was transformed into Escherichia coli NM522. The soluble proteins extracted from E. coli NM522 cells harboring the plasmid with the bacteriolytic gene showed obvious bacteriolytic activities against several strains of R. solanacearum isolated in various districts in Japan. DNA fragments from five phages, isolated in Niigata, Aomori, Okinawa, Fukushima and Yamaguchi Prefectures, hybridized to the bacteriolytic gene of phage P4282. These observations indicate that the bacteriolytic protein shows nonspecific activity against R. solanacearum strains, and a sequence similar to that of the bacteriolytic gene is conserved in the DNA of other bacteriophages. These results indicate that the generation of transgenic (tobacco) plants expressing the bacteriolytic gene of phage P4282 might result in enhanced resistance to bacterial wilt in tobacco. [TOP OF PAGE]
Complete genomic sequence of the lytic bacteriophage fYeO3-12 of Yersinia enterocolitica serotype O:3. Pajunen, M.I., Kiljunen, S.J., Soderholm, M.E.L., Skurnik, M. (2001). J. Bacteriol. 183:1928-1937. fYeO3-12 is a T3-related lytic bacteriophage of Yersinia enterocolitica serotype O:3. The nucleotide sequence of the 39,600-bp linear double-stranded DNA (dsDNA) genome was determined. The phage genome has direct terminal repeats of 232 bp, a GC content of 50.6%, and 54 putative genes, which are all transcribed from the same DNA strand. Functions were assigned to 30 genes based on the similarity of the predicted products to known proteins. A striking feature of the phiYeO3-12 genome is its extensive similarity to the coliphage T3 and T7 genomes; most of the predicted fYeO3-12 gene products were >70% identical to those of T3, and the overall organizations of the genomes were similar. In addition to an identical promoter specificity, fYeO3-12 shares several common features with T3, nonsubjectibility to F exclusion and growth on Shigella sonnei D2371-48 (M. Pajunen, S. Kiljunen, and M. Skurnik, J. Bacteriol. 182:5114-5120, 2000). These findings indicate that phiYeO3-12 is a T3-like phage that has adapted to Y. enterocolitica O:3 or vice versa. This is the first dsDNA yersiniophage genome sequence to be reported. [TOP OF PAGE]
Survival of bacteriophages of Lactococcus lactis in sodium hypochlorite and during storage. Parada, J.L., De Fabrizio, S.V. (2001). Revista Argentina de Microbiologia 33:89-95. Survival of lytic bacteriophages active against Lactococcus lactis ssp. lactis and ssp. cremoris was determined after treatment with sodium hypochlorite and during at 4degreeC. Three phages were isolated from dairy plants in Argentina (ARG) and the other phages were isolated in the United States of America (US). All of them represent phages that infected cheese manufacture industries and belong to different morphological or serological groups. These phages showed higher survival in M17 broth, buffered with sodium glycerophosphate, than in trypteine soy broth (TSB). Phage populations did not decrease significantly during 14 weeks in M17 broth, whereas in TSB the titers of phage suspensions began to decline around 9 days. In addition, the effect of sodium hypochlorite was more marked in broth than in milk. A higher surviving fraction was obtained in milk, even when tenfold higher concentrations of chloride were used. The effect of hypochlorite on phages of the same serological group was quite similar and independent of phage morphology. However, phage 137-1, which belongs to other serological group, showed lower resistance to sodium hypochlorite. Comparing the hypochlorite inactivation for ARG and US phages, it was observed that they have their own inactivation values, independently of their origin and morphological group. Long periods of time and high concentrations of chlorine were necessary to reduce the surviving fraction in milk. This indicates that hypochlorite concentrations and times of contact can be critical for the efficiency of the operative sanitization processes. [TOP OF PAGE]
Understanding bacteriophage therapy as a density-dependent kinetic process. Payne, R.J.H., Jansen, V.A.A. (2001). J. Theor. Biol. 208:37-48. Studies of bacteriophage as therapeutic agents have had mixed and unpredictable outcomes. We argue that interpretation of these apparently paradoxical results requires appreciation of various density-dependent threshold effects. We use a mathematical model to delineate different categories of outcome, including therapy by simple inundation, by active biocontrol, and by delayed active biocontrol. Counter-intuitively, there are situations in which earlier inoculation can be less efficacious, and simultaneous inoculation with antibiotics can be detrimental. Predictions of therapeutic responses are made using formulae dependent on biologically meaningful parameters; experimental measurement of the parameters will be a prerequisite of application of the model to particular study systems. Such modelling can point to which aspects of phage biology might most fruitfully be engineered so as to enhance the viability of bacteriophage therapy. [TOP OF PAGE]
Comparative study of nine Lactobacillus fermentum bacteriophages. Picozzi, C., Galli, A. (2001). Journal of Applied Microbiology 91:394-403. Aims: To investigate the basic properties of six temperate and three virulent phages, active on Lactobacillus fermentum, on the basis of morphology, host ranges, protein composition and genome characterization. Methods and Results: All phages belonged to the Siphoviridae family; two of them showed prolate heads. The host ranges of seven phages contained a common group of strains. SDS-PAGE protein profiles, restriction analysis of DNA and Southern blot hybridization revealed a high degree of homology between four temperate phages; partial homologies were also detected among virulent and temperate phages. Clustering derived from host range analysis was not related to the results of the DNA hybridizations. Conclusions: The phages investigated have common characteristics with other known phages active on the genus Lactobacillus. Sensitivity to viral infection is apparently enhanced by the presence of a resident prophage. Significance and Impact of the Study: These relationships contribute to the explanation for the origin of phage infection in food processes where Lact. fermentum is involved, such as sourdough fermentation. [TOP OF PAGE]
Viruses of the extremely therophilic archaeon Sulfolobus. Prangishvili, D., Stedman, K., Zillig, W. (2001). Trends Microbiol. 9:39-42. Viruses of Sulfolobus are highly unusual in their morphology, and genome structure and sequence. Certain characteristics of the replication strategies of these viruses and the virus-host interactions suggest relationships with eukaryal and bacterial viruses. Moreover, studying these viruses led to the discovery of archaeal promoters and has provided tools for the development of the molecular genetics of these organisms. The Sulfolobus viruses contain unique regulatory features and structures that undoubtedly hold surprises for researchers in the future. [TOP OF PAGE]
Contingent neutrality in competing viral populations. Quer, J., Hershey, C.L., Domingo, E., Holland, J.J., Novella, I.S. (2001). J. Virol. 75:7315-7320. The replicative fitness of a genetically marked (MARM-C) population of vesicular stomatitis virus was examined in competition assays in BHK-21 cells. In standard fitness assays involving up to eight competition passages of the mixed populations, MARM-C competes equally with the wild type (wt), but very prolonged competitions always led to the wt gaining dominance over MARM-C in a very slowed, nonlinear manner (J. Quer et al., J. Mol. Biol. 264:465-471, 1996). In the present study we show that a number of quite unrelated environmental perturbations, which decreased virus replication during competitions, all led to an accelerated dominance of the wt over MARM-C. These perturbations were (i) the presence of added (or endogenously generated) defective interfering particles, (ii) the presence of the chemical mutagen 5-fluorouracil (5-FU), or (iii) an increase in temperature to 40.5 degreesC. Thus, the "neutral fitness" of the MARM-C population is contingent. We have determined the entire genomic consensus sequence of MARM-C and have identified only six mutations. Clearly, some or all of these mutations allowed the MARM-C quasispecies population to compete equally with wt in a defined constant host environment, but the period of neutrality was shortened when the environment was perturbed during competitions. Interestingly, when four passages of each population were carried out independently in the presence of 5-FU (but in the absence of competition), no significant differences were detected in the fitness changes of wt and MARM-C, nor was there a difference in their subsequent abilities to compete with each other in a standard fitness assay. We propose a model for this contingent neutrality. The conditions employed to generate the MARM-C quasispecies population selected a small number of mutations in the consensus sequence. It appears that the MARM-C quasispecies population has moved into a segment of sequence space in which the average fitness value is neutral but, under environmental stress, beneficial mutations cannot be generated rapidly enough to compete with those being generated concurrently by competing wt virus quasispecies populations. [TOP OF PAGE]
Evolutionary role of restriction/modification systems as revealed by comparative genome analysis. Rocha, E.P., Danchin, A., Viari, A. (2001). Genome Research 11:946-958. Type II restriction modification systems (RMSs) have been regarded either as defense tools or as molecular parasites of bacteria. We extensively analyzed their evolutionary role from the study of their impact in the complete genomes of 26 bacteria and 35 phages in terms of palindrome avoidance. This analysis reveals that palindrome avoidance is not universally spread among bacterial species and that it does not correlate with taxonomic proximity. Palindrome avoidance is also not universal among bacteriophage, even when their hosts code for RMSs, and depends strongly on the genetic material of the phage. Interestingly, palindrome avoidance is intimately correlated with the infective behavior of the phage. We observe that the degree of palindrome and restriction site avoidance is significantly and consistently less important in phages than in their bacterial hosts. This result brings to the fore a larger selective load for palindrome and restriction site avoidance on the bacterial hosts than on their infecting phages. It is then consistent with a view where type II RMSs are considered as parasites possibly at the verge of mutualism. As a consequence, RMSs constitute a nontrivial third player in the host-parasite relationship between bacteria and phages. [TOP OF PAGE]
Seasonal variations in the microbial population density present in biological sludge. Saleem, M., Al-Malack, M.H., Bukhari, A.A. (2001). Environmental Technology 22:255-259. Sludge produced during the treatment of wastewater is being used as fertilizer in several Gulf countries. The Water and Sewage Authority of Saudi Arabia has targeted the reuse of the total amount of sludge in the future. However, these sludges should be properly treated before reuse as they contain a large number of pathogens and parasites. Little information is available on the microbial characteristics of sludge produced in wastewater treatment plants operating in this region. Variations in the population densities measured by Standard Plate Count, total coliform, fecal coliform, coliphage, and Clostridium perfringens present in the sludge, were monitored during a one year study at Al-Khobar wastewater treatment plant so that the effect of seasonal variations on the fate of these five indicator microorganisms could be investigated. This paper covers an evaluation of the fate of indicator microorganisms in the drying sludge. Insight gained in this study will be helpful in establishing guidelines for the use of sludge as fertilizer for agriculture purposes. [TOP OF PAGE]
Method for host-independent detection of generalized transducing bacteriophages in natural habitats. Sander, M., Schmieger, H. (2001). Appl. Environ. Microbiol. 67:1490-1493. Despite an increasing interest in horizontal gene transfer in bacteria, the role of generalized transduction in this process has not been well investigated yet. Certainly one of the reasons is that only a small fraction of general transducing bacteriophages have been characterized, because many bacterial hosts needed for propagation and identification are not culturable or are simply unknown. A method for host-independent detection of transducing bacteriophages was developed. Phage-encapsulated DNA was used as a template for PCR amplification of 16S ribosomal DNA using primers specific for the 16S rRNA genes of most eubacteria. Sequencing of the cloned amplification products permits the identification of the host bacteria. The Salmonella phage P22 was used as an example. Applying this method to a sample of the supernatant of the mixed liquor in the aeration tank of an activated sludge treatment works revealed the presence of transducing phages infecting several bacterial species for which such phages have not yet been described. This method is suitable for estimating the contribution of generalized transduction to horizontal gene transfer in different habitats. [TOP OF PAGE]
Phage-mediated transfer of virulence genes. Saunders, J.R., Allison, H., James, C.E., McCarthy, A.J., Sharp, R. (2001). Journal of Chemical Technology and Biotechnology. 76:662-666. Bacteriophages as accessory genetic elements play a crucial role in the dissemination of genes and the promotion of genetic diversity within bacterial populations. Such horizontal transfer of DNA is critical in the emergence of new pathogenic organisms, through the dissemination of genes encoding virulence factors such as toxins, adhesins and agressins. Phages can transfer genes that are not necessary for bacteriophage persistence and are generally recognised by their ability to convert their host bacteria to new phenotypes. This phenomenon is known as phage conversion. If such converting genes encode for virulence factors, the consequences of phage infection may include increased virulence of the host bacteria, and the conversion of a non-pathogenic strain to a potentially dangerous pathogen. A number of virulence factors in bacteria causing diseases in plants, animals and humans are encoded by converting phages, the vast majority of which are temperate as opposed to lytic in nature. [TOP OF PAGE]
Isolation and characterization of five Erwinia amylovora bacteriophages and assessment of phage resistance in strains of Erwinia amylovora. Schnabel, E.L., Jones, A.L. (2001). Appl. Environ. Microbiol. 67:59-64. Phages able to infect the fire blight pathogen Erwinia amylovora were isolated from apple, pear, and raspberry tissues and from soil samples collected at sites displaying fire blight symptoms. Among a collection of 50 phage isolates, 5 distinct phages, including relatives of the previously described phages variant phiEa1 and variant phiEa7 and 3 novel phages named variant phiEa100, variant phiEa125, and variant phiEa116C, were identified based on differences in genome size and restriction fragment pattern. variant phiEa1, the phage distributed most widely, had an approximately 46-kb genome which exhibited some restriction site variability between isolates. Phages variant phiEa100, variant phiEa7, and variant phiEa125 each had genomes of approximately 35 kb and could be distinguished by their EcoRI restriction fragment patterns. variant phiEa116C contained an approximately 75-kb genome. variant phiEa1, variant phiEa7, variant phiEa100, variant phiEa125, and variant phiEa116C were able to infect 39, 36, 16, 20, and 40, respectively, of 40 E. amylovora strains isolated from apple orchards in Michigan and 8, 12, 10, 10, and 12, respectively, of 12 E. amylovora strains isolated from raspberry fields (Rubus spp.) in Michigan. Only 22 of 52 strains were sensitive to all five phages, and 23 strains exhibited resistance to more than one phage. variant phiEa116C was more effective than the other phages at lysing E. amylovora strain Ea110 in liquid culture, reducing the final titer of Ea110 by >95% when added at a ratio of 1 PFU per 10 CFU and by 58 to 90% at 1 PFU per 105 CFU. [TOP OF PAGE]
Bacteriophage K1-5 encodes two different tail fiber proteins, allowing it to infect and replicate on both K1 and K5 strains of Escherichia coli. Scholl, D., Rogers, S., Adhya, S., Merril, C.R. (2001). J. Virol. 75:2509-2515. A virulent double-stranded DNA bacteriophage, PHIK1-5, has been isolated and found to be capable of infecting Escherichia coli strains that possess either the K1 or the K5 polysaccharide capsule. Electron micrographs show that the virion consists of a small icosohedral head with short tail spikes, similar to members of the Podoviridae family. DNA sequence analysis of the region encoding the tail fiber protein showed two open reading frames encoding previously characterized hydrolytic phage tail fiber proteins. The first is the K5 lyase protein gene of PHIK5, which allows this phage to specifically infect K5 E. coli strains. A second open reading frame encodes a protein almost identical in amino acid sequence to the N-acetylneuraminidase (endosialidase) protein of PHIK1E, which allows this phage to specifically infect K1 strains of E. coli. We provide experimental evidence that mature phage particles contain both tail fiber proteins, and mutational analysis indicates that each protein can be independently inactivated. A comparison of the tail gene regions of PHIK5, PHIK1E, and PHIK1-5 shows that the genes are arranged in a modular or cassette configuration and suggests that this family of phages can broaden host range by horizontal gene transfer. [TOP OF PAGE]
Reaction kinetics of protease with substrate phage: Kinetic model developed using stromelysin. Sharkov, N.A., Davis, R.M., Reidhaar-Olson, J.F., Navre, M., Cai, D. (2001). Journal of Biological Chemistry 276:10788-10793. Peptide libraries generated using phage display have been widely applied to proteolytic enzymes for substrate selection and optimization, but the reaction kinetics between the enzyme and substrate phage are not well understood. Using a quantitative ELISA assay to monitor the disappearance of substrate, we have been able to follow the course of reaction between stromelysin, a metalloprotease, and its substrate phage. We found that under the proteolytic conditions where the enzyme was present in nanomolar concentration or higher, in excess over the substrate, the proteolysis of substrate phage was a single exponential event and the observed rate linear with respect to enzyme concentration. The enzyme concentration dependence could be described by pseudo first-order kinetic equations. Our data suggest that substrate binding is slow relative to the subsequent hydrolysis step, implying that the phage display selection process enriches clones that have high binding affinity to the protease, and the selection may not discriminate those of different chemical reactivity toward the enzyme. Considering that multiple substrate molecules may be present on a single phage particle, we regard the substrate phage reaction kinetic model as empirical. The validity of the model was ascertained when we successfully applied it to determine the binding affinity of a competitive inhibitor of stromelysin. [TOP OF PAGE]
Bacteriophages: biology and history. Sharp, R. (2001). Journal of Chemical Technology and Biotechnology. 76:667-672. Bacteriophages were initially considered to offer the key to the control of bacterial infections; early studies, however, proved largely unsuccessful. In the 1940s and 1950s, pioneering studies into the structure and physiology of host/phage interactions laid the basis for the development of molecular biology and a spectrum of new biotechnologically-based industries. Bacteriophages able to infect most procaryotic groups of organisms have been isolated, and are readily isolated from soil, water, and sewage and most environments colonised by bacteria. Ecologically, phages are as varied and as versatile as their hosts with some able to survive extremes of temperature (up to 95 °C) and extremes of pH as low as pH 1. [TOP OF PAGE]
Antibody responses to bacteriophage variant phiX-174 in human subjects exposed to the Antarctic winter-over model of spaceflight. Shearer, W.T., Lugg, D.J., Rosenblatt, H.M., Nickolls, P.M., Sharp, R.M., Reuben, J.M., Ochs, H.D. (2001). Journal of Allergy and Clinical Immunology 107:160-164. Background: It has been proposed that exposure to long-term spaceflight conditions (stress, isolation, sleep disruption, containment, microbial contamination, and solar radiation) or to ground-based models of spaceflight will alter human immune responses, but specific antibody responses have not been fully evaluated. Objective: We sought to determine whether exposure to the 8-month Antarctic winter-over model of spaceflight would alter human antibody responses. Methods: During the 1999 Australian National Antarctic Research Expeditions, 11 adult study subjects at Casey, Antarctica, and 7 control subjects at Macquarie Island, sub-Antarctica, received primary and secondary immunizations with the T cell-dependent neoantigen bacteriophage variant phiX-174. Periodic plasma samples were analyzed for specific antibody function. Results: All of the subjects from Casey, Antarctica, cleared bacteriophage variant phiX-174 normally by 1 week after primary immunization, and all had normal primary and secondary antibody responses, including immunologic memory amplification and switch from IgM to IgG antibody production. One subject showed a high normal pattern, and one subject had a low normal pattern. The control subjects from Macquarie Island also had normal immune responses to bacteriophage variant phiX-174. Conclusions: These data do not support the hypothesis that de novo specific antibody responses of subjects become defective during the conditions of the Antarctic winter-over. Because the Antarctic winter-over model of spaceflight lacks the important factors of microgravity and solar radiation, caution must be used in interpreting these data to anticipate normal antibody responses in long-term spaceflight. [TOP OF PAGE]
Changes in bacterial community composition and dynamics and viral mortality rates associated with enhanced flagellate grazing in a mesoeutrophic reservoir. Simek, K., Weinbauer, M.G., Hornak, K., Dolan, J.R., Nedoma, J., Masin, M., Amann, R. (2001). Appl. Environ. Microbiol. 67:2723-2733. Bacterioplankton from a meso-eutrophic dam reservoir was size fractionated to reduce (<0.8-mum treatment) or enhance (<5-mum treatment) protistan grazing and then incubated in situ for 96 h in dialysis bags. Time course samples were taken from the bags and the reservoir to estimate bacterial abundance, mean cell volume, production, protistan grazing, viral abundance, and frequency of visibly infected cells. Shifts in bacterial community composition (BCC) were examined by denaturing gradient gel electrophoresis (DGGE), cloning and sequencing of 16S rDNA genes from the different treatments, and fluorescence in situ hybridization (PISH) with previously employed and newly designed oligonucleotide probes, Changes in bacterioplankton characteristics were clearly linked to changes in mortality rates. In the reservoir, where bacterial production about equaled protist grazing and viral mortality, community characteristics were nearly invariant, In the "grazer-free" (0.8-mum-filtered) treatment, subject only to a relatively low mortality rate (similar to 17% day(-1)) from viral lysis, bacteria increased markedly in concentration. While the mean bacterial cell volume was invariant, DGGE indicated a shift in BCC and FISH revealed an increase in the proportion of one lineage within the beta proteobacteria, In the grazing-enhanced treatment (5-mum filtrate), grazing mortality was similar to 200% and viral lysis resulted in mortality of 30% of daily production. Cell concentrations declined, and grazing-resistant flocs and filaments eventually dominated the biomass, together accounting for > 80% of the total bacteria by the end of the experiment. Once again, BCC changed strongly and a significant fraction of the large filaments was detected using a FISH probe targeted to members of the Flectobacillus lineage, Shifts of BCC were also reflected in DGGE patterns and in the increases in the relative importance of both beta proteobacteria and members of the Cytophaga-Flavobacterium cluster, which consistently formed different parts of the bacterial flocs. Viral concentrations and frequencies of infected cells were highly significantly correlated with grazing rates, suggesting that protistan grazing may stimulate viral activity. [TOP OF PAGE]
The place of viruses in the "tree of life". Sinkovics, J.G. (2001). Acta Microbiol. Immunol. Hungarica 48:115-127. Ribozymal entry into vesicle containing autocatalytically replicating oligopeptides engendered RNA proliferation and enzyme synthesis within units whose RNA genomes derived from ancestors of viroids. There is good reason to consider the coexistence of proto- or spheroplastic forms of ancient prokaryotes and archaeons. Predecessors of extant mycoplasmavirus L3 or archaeal fuselloviruses could induce cell fusions among these entities. The possibility that the first eukaryotic cells arose consequentially to virally mediated fusions of prokaryotic and archaeal proto- or spheroplasts is presented. Retrotransposons and endogenous retroviruses might have emerged in theropod dinosaurs when Aves evolved; and directed the development of syncytiotrophoblasts in the placentae of the first mammals. As viruses co-evolved with their hosts descendants of ancient viruses diverged from one another. Certain phenotypical features could connect extant phages and eukaryotic viruses to common ancestors. [TOP OF PAGE]
The social evoltuion of bacterial pathogenesis. Smith, J. (2001). Proc. R. Soc. Lond. B 268:61-69. Many of the genes responsible for the virulence of bacterial pathogens are carried by mobile genetic elements that can be transferred horizontally between different bacterial lineages. Horizontal transfer of virulence-factor genes has played a profound role in the evolution of bacterial pathogens, but it is poorly understood why these genes are so often mobile. Here, I present a hypothetical selective mechanism maintaining virulence-factor genes on horizontally transmissible genetic elements. For virulence factors that are secreted extracellularly, selection within hosts may favour mutant 'cheater' strains of the pathogen that do not produce the virulence factor themselves but still benefit from factors produced by other members of the pathogen population within a host. Using simple mathematical models, I show that if this occurs then selection for infectious transmission between hosts favours pathogen strains that can reintroduce functional copies of virulence-factor genes into cheaters via horizontal transfer, forcing them to produce the virulence factor. Horizontal gene transfer is thus a novel mechanism for the evolution of cooperation. I discuss predictions of this hypothesis that can be tested empirically and its implications for the evolution of pathogen virulence. [TOP OF PAGE]
Quorum sensing is a global regulatory mechanism in enterohemorrhagic Escherichia coli O157:H7. Sperandio, V., Torres, A.G., Giron, J.A., Kaper, J.B. (2001). J. Bacteriol. 183:5187-5197. Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is responsible for outbreaks of bloody diarrhea and hemolytic-uremic syndrome in many countries. EHEC virulence mechanisms include the production of Shiga toxins (Stx) and formation of attaching and effacing (AE) lesions on intestinal epithelial cells. We recently reported that genes involved in the formation of the AE lesion were regulated by quorum sensing through autoinducer-2, which is synthesized by the product of the luxS gene. In this study we hybridized an E. coli gene array with cDNA synthesized from RNA that was extracted from EHEC strain 86-24 and its isogenic luxS mutant. We observed that 404 genes were regulated by luxS at least fivefold, which comprises approximately 10% of the array genes; 235 of these genes were up-regulated and 169 were down-regulated in the wild-type strain compared to in the luxS mutant. Down-regulated genes included several involved in cell division, as well as ribosomal and tRNA genes. Consistent with this pattern of gene expression, the luxS mutant grows faster than the wild-type strain (generation times of 37.5 and 60 min, respectively, in Dulbecco modified Eagle medium). Up-regulated genes included several involved in the expression and assembly of flagella, motility, and chemotaxis. Using operon::lacZ fusions to class I, II, and III flagellar genes, we were able to confirm this transcriptional regulation. We also observed fewer flagella by Western blotting and electron microscopy and decreased motility halos in semisolid agar in the luxS mutant. The average swimming speeds for the wild-type strain and the luxS mutant are 12.5 and 6.6 microm/s, respectively. We also observed an increase in the production of Stx due to quorum sensing. Genes encoding Stx, which are transcribed along with lambda-like phage genes, are induced by an SOS response, and genes involved in the SOS response were also regulated by quorum sensing. These results indicate that quorum sensing is a global regulatory mechanism for basic physiological functions of E. coli as well as for virulence factors. [TOP OF PAGE]
Rapid coliphage detection assay. Stanek, J.E., Falkinham, J.O., III (2001). Journal of Virological Methods 91:93-98. A rapid coliphage detection assay was developed, based on the phage-induced release of beta-galactosidase from cells of Escherichia coli. The assay could detect as few as five coliphage per sample without an overnight incubation period. The range of acceptable assay parameters was identified. [TOP OF PAGE]
Application of Streptococcus thermophilus DPC1842 as an adjunct to counteract bacteriophage disruption in a predominantly lactococcal Cheddar cheese starter: use in bulk starter culture systems. Stokes, D., Ross, R.P., Fitzgerald, G.F., Coffey, A. (2001). Lait 81:327-334. A significant amount of Cheddar cheese manufactured world-wide relies on bulk starter cultures instead of direct vat set (DVS) cultures. While the inclusion of S. thermophilus is sometimes used to counteract failure due to lactococcal phage in the latter system, it is considered difficult to implement in bulk starter systems and is normally avoided. This stems from the problem in controlling the ratio of S. thermophilus to lactococci during the bulk starter preparation such that suitable acidification rates can be achieved. The current study demonstrates how S. thermophilus numbers can be controlled during growth in the bulk starter medium prior to inoculation of a culture, based on three lactococcal strains and S. thermophilus DPC1842, into the cheese vat. The concentration of inorganic phosphate necessary to inhibit the growth of strain DPC1842 in a whey-based bulk starter medium was found to be 0.18 mol.L^-1. Since higher levels of phosphate exist in different commercial bulk starter media used for mesophilic cultures, a number of these media can be used for propagation of this starter blend without domination of strain DPC1842 over its lactococcal counterparts. Strain DPC1842 is highly phage resistant and is particularly acid-fast in the cheese milk and reduces the pH efficiently at very low inocula. Cheddar cheese was manufactured in a commercial plant with this system and the resulting cheese had good flavour characteristics. This study demonstrates the effectiveness of the S. thermophilus DPC1842 component in rescuing the fermentation in the event of severe lactococcal phage attack. [TOP OF PAGE]
Bacteriophage therapy. Sulakvelidze, A., Alavidze, Z., Morris, J.G. (2001). Antimicrobial Agents and Chemotherapy 45:649-659. [TOP OF PAGE]
Community Structure: Viruses. Suttle, C.A. (2001). p. ???-??? In Hurst, C.J., Knudson, G.R., McInerney, M.J., Stezenbach, L.D., and Walter, M.V. (eds.), Manual of Environmental Microbiology (2nd Edition). ASM Press, Washington, DC. [TOP OF PAGE]
Evidence for holin function of tcdE gene in the pathogenicity of Clostridium difficile. Tan, K.S., Wee, B.Y., Song, K.P. (2001). J. Med. Microbiol. 50:613-619. Toxigenic strains of Clostridium difficile produce two large bacterial toxins called toxins A (TcdA) and B (TcdB). tcdA and tcdB genes are located on the pathogenicity locus of C. difficile, a unique characteristic of toxigenic strains of this species. Intergenic to the two toxin genes is tcdE, a small 501-bp open reading frame of unknown function. Expression of the tcdE gene in Escherichia coli caused bacterial cell death. Computational analysis of the amino acid sequence of TcdE revealed structural features that are strikingly similar to a class of bacteriophage proteins called holins. Holins are cytolytic proteins that cause lysis of bacterial hosts to effect the release of progeny phages. Further analysis of the recombinant clone expressing TcdE by transmission electron microscopy confirmed that the site of action of TcdE is on the bacterial cell membrane. The results provide evidence that TcdE is structurally and functionally similar to holin proteins. TcdE may function as a lytic protein to facilitate the release of TcdA and TcdB to the extracellular environment, as these toxins lack signal peptide. [TOP OF PAGE]
Phylogeny of the major head and tail genes of the wide-ranging T4-type bacteriophages. Tétart, F., Desplats, C., Kutateladze, M., Monod, C., Ackermann, H.-W., Krisch, H.M. (2001). J. Bacteriol. 183:358-366. We examined a number of bacteriophages with M-type morphology that propagate in different genera of enterobacteria, Aeromonas, Burkholderia, and Vibrio. Most of these phages had a prolate icosahedral head, a contractile tail, and a genome size that was similar to that of T4. A few of them had more elongated heads and larger genomes. All these phages are phylogenetically related, since they each had sequences homologous to the capsid gene (gene 23), tail sheath gene (gene 18), and tail tube gene (gene 19) of T4. On the basis of the sequence comparison of their virion genes, the T4-type phages can be classified into three subgroups with increasing divergence from T4: the T-evens, pseudoT-evens, and schizoT-evens. In general, the phages that infect closely related host species have virion genes that are phylogenetically closer to each other than those of phages that infect distantly related hosts. However, some of the phages appear to be chimeras, indicating that, at least occasionally, some genetic shuffling has occurred between the different T4-type subgroups. The compilation of a number of gene 23 sequences reveals a pattern of conserved motifs separated by sequences that differ in the M-type subgroups. Such variable patches in the gene 23 sequences may determine the size of the virion head and consequently the viral genome length. This sequence analysis provides molecular evidence that phages related to T4 are widespread in the biosphere and diverged from a common ancestor in acquiring the ability to infect different host bacteria and to occupy new ecological niches. [TOP OF PAGE]
Fate of indicator microorganisms, Giardia and Cryptosporidium in subsurface flow constructed wetlands. Thurston, J.A., Gerba, C.P., Foster, K.E., Karpiscak, M.M. (2001). Water Res. 35:1547-1551. Limited information is available on the ability of subsurface flow wetlands to remove enteric pathogens. Two multi-species wetlands, one receiving secondary sewage effluent and the other potable (disinfected) groundwater were studied from February 1995 to August 1996, at the Pima County Constructed Ecosystems Research Facility in Tucson, Arizona. Each wetland had a retention time of approximately 4 days. The objectives of this study were (1) to evaluate the ability of multi-species subsurface wetlands to physically remove Giardia cysts; Cryptosporidium oocysts, total and fecal coliforms, and coliphages; and (2) to determine the likely impact of local wildlife on the occurrence of these indicators and pathogens. In the wetland receiving secondary sewage effluent, total coliforms were reduced by an average of 98.8% and fecal coliforms by 98.2%. Coliphage were reduced by an average of 95.2%. Both Giardia cysts and Cryptosporidium oocysts were reduced by an average of 87.8 and 64.2%, respectively. In the wetland receiving disinfected groundwater, an average of 1.3 X 10² total coliforms/100 mL and 22.3 fecal coliforms/100 mL were most likely contributed by both flora and fauna. No parasites or coliphages were detected. [TOP OF PAGE]
Complete nucleotide sequence of the mycoplasma virus P1 genome. Tu, A.H., Voelker, L.L., Shen, X., Dybvig, K. (2001). Plasmid 45:122-126. Mycoplasma virus P1 is one of only four viruses isolated from the genus Mycoplasma. The host for P1, Mycoplasma pulmonis, possesses complex, phase-variable restriction and modification enzymes and the Vsa family of phase-variable surface proteins. The ability of P1 virus to infect host cells is influenced by these phase-variable systems, rendering P1 a valuable tool for assessing host properties. The double-stranded P1 DNA genome was sequenced (11,660 bp) and 11 ORFs were identified. The predicted P1 DNA polymerase is similar to that of phages that are known to have terminal protein (TP) attached to the 5' end of their genome, consistent with previous studies indicating that P1 DNA has covalently attached TP. Most of the other predicted P1 proteins have little sequence similarity to known proteins, and P1 virus is unrelated to the other mycoplasma virus, MAV1, for which the genome sequence is known. One of the predicted P1 proteins, the ORF 8 gene product, contains a repetitive collagen-like motif characteristic of some bacteriophage tail fiber proteins and is a candidate for interacting with the Vsa proteins. [TOP OF PAGE]
The clc element of Pseudomonas sp. strain B13 and other mobile degradative elements employing phage-like integrases. van der Meer, J.R., Ravatn, R., Sentchilo, V. (2001). Archives of Microbiology 175:79-85. Genes for metabolic pathways in bacteria that degrade aromatic or aliphatic pollutants have mostly been confined to either plasmid DNAs or to the chromosome. For a few pathways, including classical pathways for chlorocatechol and biphenyl degradation, recent evidence has been obtained for location of the pathway genes on mobile DNA elements which employ phage-like integrases. This enables the DNA elements to integrate into specific sites on the chromosome and yet to excise and transfer to other host bacteria. This mini-review gives an overview of those elements and their relationship to an increasing number of phage-like elements associated with bacterial virulence. [TOP OF PAGE]
Induction of hepatitis B virus-specific cytotoxic T lymphocytes response in vivo by filamentous phage display vaccine. Wan, Y., Wu, Y., Bian, J., Wang, X.Z., Zhou, W., Jia, Z.C., Tan, Y., Zhou, L. (2001). Vaccine 19:2918-2923. The ability of inducing MHC class I restricted cytotoxic T lymphocytes response in vivo via recombinant filamentous phage was investigated. The recombinant filamentous phage particles that displayed the Hepatitis B virus epitope S(28--39) were injected into BALB/c (H-2d) mice without adjuvants. A MHC class I restricted HBs specific CTL response was found 8 days after injection. The potentiality of using the recombinant filamentous phage as anti-virus vaccine was discussed. [TOP OF PAGE]
Therapy of infections in cancer patients with bacteriophages. Weber-Dabrowska, B., Mulczyk, M., Górski, A. (2001). CLIN APPL IMMUNOL REV 1:131-134. Cancer patients are known to be immunocompromised and susceptible to infections. We have used bacteriophages matched for specific bacterial isolates to treat antibiotic-resistant infections in those patients. Cure of infection was achived in all cases indicating very highefficacy of BP therapy. [TOP OF PAGE]
Predation in the presence of decoys: An inhibitory factor on pathogen control by bacteriophages or bdellovibrios in dense and diverse ecosystems. Wilkinson, M.H.F. (2001). J. Theor. Biol. 208:27-36. Several attempts have been made at the removal of specific pathogens from the intestinal microflora using either bacteriophages or "predatory" bacteria such as Bdellovibrio spp. To date these attempts have had mixed success. A mechanism explaining these findings based on competitive hindrance by non-prey, or decoy species is put forward. It is shown that this hindrance tends to damp out predator-prey oscillations, and therefore reduces the probability of prey extinction. Possible experiments to verify this theory are discussed. The decoy effect may play a role in any system with high densities of bacteria or other particulate matter, such as activated sludge or biofilms. [TOP OF PAGE]
Interaction of the PhiHSIC virus with its host: lysogeny or pseudolysogeny? Williamson, S.J., McLaughlin, M.R., Paul, J.H. (2001). Appl. Environ. Microbiol. 67:1682-1688. The marine phage PhiHSIC has been previously reported to enter into a lysogenic relationship with its host, HSIC, identified as Listonella pelagia. This phage produces a variety of plaques on its host, including turbid and haloed plaques, from which lysogens were previously isolated. These lysogens were unstable during long-term storage at -80° C and were lost. When HSIC was reinfected with phage PhiHSIC, pseudolysogen-like interactions between the phage and its host were observed. The cells (termed HSIC-2 or HSIC-2e) produced high viral titers (10¹¹ ml^-1) in the absence of inoculating phage and yet reached culture densities of nearly 10⁹ ml^-1. Prophages were not induced by mitomycin C or the polyaromatic hydrocarbon naphthalene in cells harboring such infections. However, such cells were homoimmune to superinfection. Colonies hybridized strongly with a gene probe from a 100-bp fragment of the PhiHSIC genome, while the host did not. Analysis of chromosomal DNA preparations suggested the presence of a chromosomally integrated prophage. Phage adsorption experiments suggested that HSIC-2 was adsorption impaired. Because of the chromosomal prophage integration and homoimmunity, we interpret these results to indicate that PhiHSIC establishes a lysogenic relationship with its host that involves an extremely high level of spontaneous induction. This could be caused by a weak repressor of phage production. Additionally, poor phage adsorption of HSIC-2 compared to the wild type probably helped maintain this pseudolysogen-like relationship. In many ways, pseudolysogenic phage-host interactions may provide a paradigm for phage-host interactions in the marine environment. [TOP OF PAGE]
Regulatory issues for phage-based clinical products. Withington, R. (2001). Journal of Chemical Technology and Biotechnology. 76:673-676. Phage-based therapeutic products are members of a growing and diverse group of products categorised as biologics, biologicals or biotechnological products. They are regulated in much the same way as conventional drugs although in America they have their own division at FDA, the Centre for Biologics Evaluation and Research, CBER (as opposed to CDER, the corresponding drugs division). The distinction is important because there are significant differences between the two divisions in the amount of toxicological characterisation, clinical testing and manufacturing data that must be submitted for approval. Also, there are important differences in the extent to which multiparty manufacturing arrangements are permitted. There are a number of regulatory issues surrounding phage-based clinical products that, if addressed early during product development, will not become blocks to progress later on. The regulatory issues arise in part because of the unique nature of phage-based clinical products and in part because of their intended clinical use. [TOP OF PAGE]
Quantifying viral propagation in vitro: toward a method for characterization of complex phenotypes. Yin, J., Duca, K., Lam, V., Keren, I., Endler, E.E., Letchworth, G.J., Novella, I.S. (2001). Biotechnology Progress [TOP OF PAGE]
Characterization of a lytic Lactobacillus plantarum bacteriophage and molecular cloning of a lysin gene in Escherichia coli. Yoon, S.S., Kim, J.W., Breidt, F., Fleming, H.P. (2001). Int. J. Food Microbiol. 65:63-74. Bacteriophage SC921, which can infect Lactobacillus plantarum specifically, was isolated from a fermented vegetable source, Kimchi. This phage is active against six of 11 strains of L. plantarum tested as hosts. Morphologically, it has an isometric head (60 nm in diameter) and a non-contractile tail (260 nm long and 9-11 nm wide), indicating that it belongs to Bradley's group B or the Siphoviridae family according to the International Committee on Taxonomy of Viruses (ICTV). The bouyant density was 1.58 g/cm3. SDS-PAGE experimentation indicated that the phage particle contains two major structural proteins and several minor proteins. The genome was a double stranded linear DNA molecule with cohesive ends and 66.5 kb long by mapping genomic DNA digested with the restriction endonucleases: KpnI, SmaI, and XbaI. The (G + C) content of the phage DNA is 39.4%. For this lysin gene study, 9.4 kb of KpnI-digested DNA fragment was cloned into pUC19 and expressed in Escherichia coli. The KpnI fragment was considered as the genetic element responsible for the lysis gene of L. plantarum bacteriophage. The cloned fragment in pUC19 was hybridized to a 9.4-kb fragment generated by KpnI digestion of SC 921 as a probe. This confirmed that the fragment in pUC19 originated from phage DNA. The lysin gene was near the middle of the phage genome. [TOP OF PAGE]
Simulating the growth of viruses. You, L., Yin, J. (2001). Pacific Symposium on Biocomputing 532-543. To explore how the genome of an organism defines its growth, we have developed a computer simulation for the intracellular growth of phage T7 on its E. coli host. Our simulation, which incorporates 30 years of genetic, biochemical, physiological, and biophysical data, is used here to study how the intracellular resources of the host, determined by the specific growth rate of the host, contribute toward phage development. It is also used to probe how changes in the linear organization of genetic elements on the T7 genome can affect T7 development. Further, we show how time-series trajectories of T7 mRNA and protein levels generated by the simulation may be used as raw data to test data-mining strategies, specifically, to identify partners in protein-protein interactions. Finally, we suggest how generalization of this work can lead to a knowledge-driven simulation for the growth of any virus. [TOP OF PAGE]
Purification of Piscirickettsia salmonis and associated phage particles. Yuksel, S.A., Thompson, K.D., Ellis, A.E., Adams, A. (2001). Diseases of Aquatic Organisms 44:231-235. Piscirickettsia salmonis was isolated from cell culture using differential centrifugation and purified on a 30% Percol gradient. The purity of the preparation was assessed by transmission electron microscopy and phage-like particles were found to be associated with some of the P. salmonis isolates examined. This is believed to be the first report of a phage associated with rickettsia from fish. [TOP OF PAGE]

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