Isolation and characterization of Arthrobacter bacteriophages and their application to phage typing of soil arthrobacters.

Seventeen bacteriophages, active against 19 Arthrobacter soil isolates, were isolated from concentrated samples of river water and sewage. Attempts to isolate Arthrobacter bacteriophages from filtrates of broth cultures of the soil isolates or from ultraviolet light-irradiated cultures were unsuccessful. Bacteriophages were not detected in either concentrated or unconcentrated soil extracts. Electron microscopic studies of 11 phages showed morphologies characteristic of Bradley's groups B (exhibited by 9 phages) and C (exhibited by 2 phages). Moles percent guanine plus cytosine, calculated from the deoxyribonucleic acid density of three phages, ranged from 60.2 to 65.3. The phages were characterized by their plague and virion morphology, host range, and serological specificity.     

Isolation and preliminary characterization of twenty bacteriophages infecting either brevibacterium or arthrobacter strains

Thirty-seven bacteriophages plaquing on Corynebacterium, Brevibacterium, or Arthrobacter strains were isolated from soil or vegetation samples. Restriction analysis of phage DNA indicated that 20 phages were unique; one of them produced entirely turbid plaques on Brevibacterium ketoglutamicum and was characterized as temperate. All these phages were assigned to group B of the classification of Bradley (Bacteriol. Rev. 31:230-314, 1967) and had relatively narrow host ranges.     

Diversity of temperate bacteriophages induced in bovine and ovine Mannheimia haemolytica isolates and identification of a new P2-like phage

The diversity of temperate bacteriophages was examined in 32 Mannheimia haemolytica, six Mannheimia glucosida and four Pasteurella trehalosi isolates. Phage particles were induced and identified by electron microscopy in 24 (75%) M. haemolytica isolates, but in only one (17%) M. glucosida and one (25%) P. trehalosi isolate. The M. haemolytica phages were relatively diverse as seven Siphoviridae, 15 Myoviridae and two Podoviridae-like phages were identified; the Myoviridae-type phages also exhibited structural variation of their tails. The bacteriophages induced in M. glucosida and P. trehalosi were of the Myoviridae type. Restriction endonuclease (RE) analysis identified nine distinct RE types among the M. haemolytica bacteriophages, providing further evidence of their relative diversity. A limited number of phages caused plaques on indicator strains and the phages exhibited a narrow host range. A subgroup of 11 bovine serotype A1 and A6 isolates contained Myoviridae-type phages of the same RE type (type A), but these differed in their abilities to infect and form plaques on the same panel of indicator strains. A P2-like phage (phiPHL213.1), representative of the RE type A phages, was identified from the incomplete M. haemolytica genome sequence. The phiPHL213.1 genome contains previously unidentified genes and represents a new member of the P2 phage family.     

Isolation and characterization of Campylobacter bacteriophages from retail poultry

The ability of phages to survive processing is an important aspect of their potential use in the biocontrol of Campylobacter in poultry production. To this end, we have developed a procedure to recover Campylobacter bacteriophages from chilled and frozen retail poultry and have validated the sensitivity of the method by using a characterized Campylobacter phage (i.e., NCTC 12674). By using this method, we have shown that Campylobacter phages can survive on retail chicken under commercial storage conditions. Retail chicken portions purchased in the United Kingdom were screened for the presence of endogenous Campylobacter phages. Thirty-four Campylobacter bacteriophages were isolated from 300 chilled retail chicken portions, but none could be recovered from 150 frozen chicken portions. The phage isolates were characterized according to their lytic profiles, morphology, and genome size. The free-range products were significantly more likely to harbor phages (P < 0.001 by single-factor analysis of variance) than were standard or economy products. This study demonstrates that Campylobacter bacteriophages, along with their hosts, can survive commercial poultry processing procedures and that the phages exhibited a wide range of recovery rates from chicken skin stored at 4 degrees C.     

Abundance in Sewage of Bacteriophages That Infect Escherichia coli O157:H7 and That Carry the Shiga Toxin 2 Gene

Shiga toxin-converting bacteriophages are involved in the pathogenicity of some enteric bacteria, such as Escherichia coli O157:H7, but data on the occurrence and distribution of such phages as free particles in nature were not available. An experimental approach has been developed to detect the presence of the Shiga toxin 2 (Stx 2)-encoding bacteriophages in sewage. The Stx 2 gene was amplified by PCR from phages concentrated from 10-ml samples of sewage. Moreover, the phages carrying the Stx 2 gene were detected in supernatants from bacteriophage enrichment cultures by using an Stx 2-negative E. coli O157:H7 strain infected with phages purified from volumes of sewage as small as 0.02 ml. Additionally, the A subunit of Stx 2 was detected in the supernatants of the bacteriophage enrichment cultures, which also showed cytotoxic activity for Vero cells. By enrichment of phages concentrated from different volumes of sewage and applying the most-probable-number technique, it was estimated that the number of phages infectious for E. coli O157:H7 and carrying the Stx 2 gene was in the range of 1 to 10 per ml of sewage from two different origins. These values were approximately 1% of all phages infecting E. coli O157:H7.     

Bacteriophages of Erwinia amylovora

Fifty bacteriophage isolates of Erwinia amylovora, the causal agent of fire blight, were collected from sites in and around the Niagara region of southern Ontario and the Royal Botanical Gardens, Hamilton, Ontario. Forty-two phages survived the isolation, purification, and storage processes. The majority of the phages in the collection were isolated from the soil surrounding trees exhibiting fire blight symptoms. Only five phages were isolated from infected aerial tissue in pear and apple orchards. To avoid any single-host selection bias, six bacterial host strains were used in the initial isolation and enrichment processes. Molecular characterization of the phages with a combination of PCR and restriction endonuclease digestions showed that six distinct phage types, described as groups 1 to 6, were recovered. Ten phage isolates were related to the previously characterized E. amylovora PEa1, with some divergence of molecular markers between phages isolated from different sites. A study of the host ranges of the phages revealed that certain types were unable to efficiently lyse some E. amylovora strains and that some isolates were able to lyse the epiphytic bacterium Pantoea agglomerans. Representatives from the six molecular groups were studied by electron microscopy to determine their morphology. The phages exhibited distinct morphologies when examined by an electron microscope. Group 1 and 2 phages were tailed and contractile, and phages belonging to groups 3 to 6 had short tails or openings with thin appendages. Based on morphotypes, the bacteriophages of E. amylovora were placed in the order Caudovirales, in the families Myoviridae and PODOVIRIDAE:     

The use of bacteriophages in eliminating polyresistant strains ofStaphylococcus aureus andStreptococcus agalactiae

Temperate bacteriophages were induced in and released from isolates of Staphylococcus aureus and Streptococcus agalactiae using mitomycin C. Various specific indicator cultures were tested for providing clear plaques after phage infection. Specific lytic mixture of bacteriophages was prepared using the induced, modified and laboratory variants of phages. Under laboratory conditions, the mixture eliminated all isolates from the tested collection of microorganisms. The restriction barrier of some bacterial isolates to bacteriophage infection was overcome either by UV irradiation or in vitro modification of bacteriophage DNA with specific methyltransferases. Conjugative R plasmids, capable of replication in G+ and G- bacteria, were detected and isolated from S. aureus and S. agalactiae antibiotic-resistant strains.     

Isolation and Characterization of Campylobacter Bacteriophages from Retail Poultry

The ability of phages to survive processing is an important aspect of their potential use in the biocontrol of Campylobacter in poultry production. To this end, we have developed a procedure to recover Campylobacter bacteriophages from chilled and frozen retail poultry and have validated the sensitivity of the method by using a characterized Campylobacter phage (i.e., NCTC 12674). By using this method, we have shown that Campylobacter phages can survive on retail chicken under commercial storage conditions. Retail chicken portions purchased in the United Kingdom were screened for the presence of endogenous Campylobacter phages. Thirty-four Campylobacter bacteriophages were isolated from 300 chilled retail chicken portions, but none could be recovered from 150 frozen chicken portions. The phage isolates were characterized according to their lytic profiles, morphology, and genome size. The free-range products were significantly more likely to harbor phages (P < 0.001 by single-factor analysis of variance) than were standard or economy products. This study demonstrates that Campylobacter bacteriophages, along with their hosts, can survive commercial poultry processing procedures and that the phages exhibited a wide range of recovery rates from chicken skin stored at 4°C.     

Bacteriophages of Erwinia amylovora

Fifty bacteriophage isolates of Erwinia amylovora, the causal agent of fire blight, were collected from sites in and around the Niagara region of southern Ontario and the Royal Botanical Gardens, Hamilton, Ontario. Forty-two phages survived the isolation, purification, and storage processes. The majority of the phages in the collection were isolated from the soil surrounding trees exhibiting fire blight symptoms. Only five phages were isolated from infected aerial tissue in pear and apple orchards. To avoid any single-host selection bias, six bacterial host strains were used in the initial isolation and enrichment processes. Molecular characterization of the phages with a combination of PCR and restriction endonuclease digestions showed that six distinct phage types, described as groups 1 to 6, were recovered. Ten phage isolates were related to the previously characterized E. amylovora PEa1, with some divergence of molecular markers between phages isolated from different sites. A study of the host ranges of the phages revealed that certain types were unable to efficiently lyse some E. amylovora strains and that some isolates were able to lyse the epiphytic bacterium Pantoea agglomerans. Representatives from the six molecular groups were studied by electron microscopy to determine their morphology. The phages exhibited distinct morphologies when examined by an electron microscope. Group 1 and 2 phages were tailed and contractile, and phages belonging to groups 3 to 6 had short tails or openings with thin appendages. Based on morphotypes, the bacteriophages of E. amylovora were placed in the order Caudovirales, in the families Myoviridae and Podoviridae.     

Bacteriophages and indicator bacteria in human and animal faeces

In an attempt to explain the presence of F-specific (RNA) bacteriophages in waste-water, faecal material from humans and a variety of animals was examined. The phages were detected in appreciable numbers only in faeces from pigs, broiler chickens, sheep and calves but not from dogs, cows, horses and humans. Parallel examinations for somatic coliphages, thermotolerant coliforms, faecal streptococci and spores of sulphite-reducing clostridia revealed the consistent presence of these organisms in all types of samples, albeit in variable numbers. The number of F-specific bacteriophages was related to the total number of somatic coliphages, but phage counts were unrelated to bacterial counts. F-specific RNA phages were grouped by serotyping and all animal isolates were found to belong to either group I (MS2 subtype) or IV (four different subtypes). Among the group IV isolates, most belonged to well-known subtypes SP (24 isolates) or FI (18 isolates) but five isolates were related to phage ID2 and one isolate was a new subtype. In contrast with animal isolates, 19 isolates from hospital wastewater belonged to serogroups II or III.     

Bacteriophages and indicator bacteria in human and animal faeces

In an attempt to explain the presence of F-specific (RNA) bacteriophages in waste-water, faecal material from humans and a variety of animals was examined. The phages were detected in appreciable numbers only in faeces from pigs, broiler chickens, sheep and calves but not from dogs, cows, horses and humans. Parallel examinations for somatic coliphages, thermotolerant coliforms, faecal streptococci and spores of sulphite-reducing clostridia revealed the consistent presence of these organisms in all types of samples, albeit in variable numbers. The number of F-specific bacteriophages was related to the total number of somatic coliphages, but phage counts were unrelated to bacterial counts. F-specific RNA phages were grouped by serotyping and all animal isolates were found to belong to either group I (MS2 subtype) or IV (four different subtypes). Among the group IV isolates, most belonged to well-known subtypes SP (24 isolates) or FI (18 isolates) but five isolates were related to phage ID2 and one isolate was a new subtype. In contrast with animal isolates, 19 isolates from hospital wastewater belonged to serogroups II or III.     

Novel Bacteriophages in Enterococcus spp.

Most of the bacteriophages (phages) currently reported in Enterococcus spp. belong to tailed families of bacteriophages Podoviridae, Siphoviridae, and Myoviridae. There is a little information on non-tailed bacteriophages isolated from enterococci. Samples of sewage and piggery effluents were tested on pig and chicken isolates of Enterococcus faecalis, E. faecium and E. gallinarum for lytic phages. In addition, isolates were exposed to mitomycin C to induce lysogenic phages. Bacteriophages that were detected were visualized by electron microscopy. Ten bacteriophages were of isometric shape with long flexible or non-flexible tails, while one had a long head with a long flexible tail; all contained double-stranded DNA molecules. Seven Polyhedral, filamentous, and pleomorphic-shaped phages containing DNA or RNA were also observed. The pleomorphic phages were droplet- or lemon-shaped in morphology. This study is the first report on polyhedral phages in Enterococcus spp. of animal origin and also the first report of filamentous and pleomorphic phages in enterococci.     

Can immobilization of Bacillus megaterium cells in alginate beads protect them against bacteriophages?

The ability of free and alginate-immobilized cells of Bacillus megaterium to dissolve tricalcium phosphate as well as their susceptibility to phages were compared in pure liquid cultures and in pot experiment with maize. In both liquid culture and cultivated soil, alginate-immobilized cells of B. megaterium exhibited much higher efficiency in increasing the availability of phosphorus than the free cells. Bacteriophages of B. megaterium were found to be common in soil. Specific bacteriophages, in the presence of free cells of B. megaterium, completely inhibited the phosphate-dissolving activity of the bacteria in pure liquid culture and markedly decreased their number in rhizosphere of maize plants. The phosphorus content of maize plants inoculated with free cells of B. megaterium decreased in the presence of their specific bacteriophages, whereas, when alginate-immobilized cells were used as inoculum, no effect of the presence of bacteriophages on phosphate-dissolving activity of bacterial cells was detected.     

Host range and lytic capability of four bacteriophages against bovine and clinical human isolates of Shiga toxin-producing Escherichia coli O157:H7

Aims:  To evaluate host range and lytic capability of four bacteriophages (rV5, wV7, wV8 and wV11) against Escherichia coli O157:H7 (STEC O157:H7) from cattle and humans.
Methods and Results:  Four hundred and twenty-two STEC O157:H7 isolates (297 bovine; 125 human) were obtained in Alberta, Canada. The four phages were serially diluted and incubated for 5 h with overnight cultures of STEC O157:H7 to estimate their multiplicity of infection (MOI). All bovine STEC O157:H7 were subjected to pulsed-field gel electrophoresis (PFGE) and phage typing (PT). Phage wV7 lysed all human and bovine isolates irrespective of PFGE genotype or PT phenotype and exhibited the lowest MOI (0·004–0·006, P  < 0·0001) of all phages. Phages rV5 and wV11 exhibited a lower MOI (0·002–0·04, P  < 0·0001) than did phage wV8 (25–29) and they had a narrower host range than wV7 or wV8. Phages rV5, wV11 and wV8 lysed 342 (81·0%), 321 (76·1%) and 407 (96·4%), respectively, of the 422 isolates. Susceptibility of bovine STEC O157:H7 to rV5, w11 and wV8 was influenced by PFGE genotype and/or PT phenotype.
Conclusions:  Phages exhibited activity against the majority of bovine and human STEC O157:H7 isolates. PFGE genotype and/or PT phenotype of the host-target influenced their vulnerability to phage attack. Susceptibility of bovine STEC O157:H7 to phage may also differ among farms. Both lytic capability and host range should be considered in the selection of therapeutic phage for on-farm control of STEC O157:H7.
Significance and Impact of the Study:  The present work indicates that a four-phage cocktail should be equally effective at mitigating STEC O157:H7 isolates both of bovine and of human origin. Given that some STEC O157:H7 exhibited resistance to some but not all phages, a phage cocktail is the logical approach to efficacious on-farm therapy.     

Large Pseudomonas phages isolated from barley rhizosphere

Abstract: Five bacteriophages infecting common fluorescent pseudomonads ( Pseudomonas fluorescens and Pseudomonas putida ) were isolated from barley rhizosphere soil. Morphological and molecular characteristics of the phages are described together with selected phage-host interactions. All phages belonged to the Myoviridae family with isometrical heads on contractile tails; 4 of them were unusually large and had complex protein and DNA profiles. The large phages had estimated genome sizes of 200 kb or more. Restriction enzyme analyses and DNA-DNA hybridizations showed that all isolates represented different phage species. None of the isolates were observed to establish lysogeny with the main host strain, P. putida MM1. The large phages multiplied slowly on their hosts, producing very small plaques; one-step growth experiments with one of the large phages (Psp 4) hence demonstrated a long latent period (2.5 h) and a very small burst size (10 particles). One of the large phages (Psp 3) was abundant in the rhizosphere (approx. 10⁴ pfu g⁻¹ soil) and had a particularly broad host range which extended to both fluorescent ( Pseudomonas aeruginosa, P. fluorescens, P. putida and Pseudomonas chlororaphis ) and non-fluorescent (Pseudomonas stutzeri) Pseudomonas spp. occurring in soil. The ecological importance of the large Pseudomonas phages must be further studied, but their slow multiplication rates suggested a possible mechanism of balanced phage-host co-existence in the rhizosphere.     

Diversity and Geographical Distribution of Flavobacterium psychrophilum Isolates and Their Phages: Patterns of Susceptibility to Phage Infection and Phage Host Range

Flavobacterium psychrophilum is an important fish pathogen worldwide that causes cold water disease (CWD) or rainbow trout fry syndrome (RTFS). Phage therapy has been suggested as an alternative method for the control of this pathogen in aquaculture. However, effective use of bacteriophages in disease control requires detailed knowledge about the diversity and dynamics of host susceptibility to phage infection. For this reason, we examined the genetic diversity of 49 F. psychrophilum strains isolated in three different areas (Chile, Denmark, and USA) through direct genome restriction enzyme analysis (DGREA) and their susceptibility to 33 bacteriophages isolated in Chile and Denmark, thus covering large geographical (>12,000 km) and temporal (>60 years) scales of isolation. An additional 40 phage-resistant isolates obtained from culture experiments after exposure to specific phages were examined for changes in phage susceptibility against the 33 phages. The F. psychrophilum and phage populations isolated from Chile and Denmark clustered into geographically distinct groups with respect to DGREA profile and host range, respectively. However, cross infection between Chilean phage isolates and Danish host isolates and vice versa was observed. Development of resistance to certain bacteriophages led to susceptibility to other phages suggesting that “enhanced infection” is potentially an important cost of resistance in F. psychrophilum, possibly contributing to the observed co-existence of phage-sensitive F. psychrophilum strains and lytic phages across local and global scales. Overall, our results showed that despite the identification of local communities of phages and hosts, some key properties determining phage infection patterns seem to be globally distributed.     

Shiga toxin gene loss and transfer in vitro and in vivo during enterohemorrhagic Escherichia coli O26 infection in humans

Escherichia coli serogroup O26 consists of enterohemorrhagic E. coli (EHEC) and atypical enteropathogenic E. coli (aEPEC). The former produces Shiga toxins (Stx), major determinants of EHEC pathogenicity, encoded by bacteriophages; the latter is Stx negative. We have isolated EHEC O26 from patient stools early in illness and aEPEC O26 from stools later in illness, and vice versa. Intrapatient EHEC and aEPEC isolates had quite similar pulsed-field gel electrophoresis (PFGE) patterns, suggesting that they might have arisen by conversion between the EHEC and aEPEC pathotypes during infection. To test this hypothesis, we asked whether EHEC O26 can lose stx genes and whether aEPEC O26 can be lysogenized with Stx-encoding phages from EHEC O26 in vitro. The stx2 loss associated with the loss of Stx2-encoding phages occurred in 10% to 14% of colonies tested. Conversely, Stx2- and, to a lesser extent, Stx1-encoding bacteriophages from EHEC O26 lysogenized aEPEC O26 isolates, converting them to EHEC strains. In the lysogens and EHEC O26 donors, Stx2-converting bacteriophages integrated in yecE or wrbA. The loss and gain of Stx-converting bacteriophages diversifies PFGE patterns; this parallels findings of similar but not identical PFGE patterns in the intrapatient EHEC and aEPEC O26 isolates. EHEC O26 and aEPEC O26 thus exist as a dynamic system whose members undergo ephemeral interconversions via loss and gain of Stx-encoding phages to yield different pathotypes. The suggested occurrence of this process in the human intestine has diagnostic, clinical, epidemiological, and evolutionary implications.     

Phage typing of Escherichia coli isolated from chickens.

Ten different bacteriophages were isolated from untreated city sewage water. These phages were stable at 57 degrees C for 40 min. A modified agar layer technique was used to obtain high titre phages. Ninety-four of a stock of 101 cultures of Escherichia coli, which were isolated from inflamed portions of intestines of chickens, were lysed by one or more of these phages. The E. coli of a known serological grouping were phage typed.     

Comparative resistance of phage isolates of four genotypes of f-specific RNA bacteriophages to various inactivation processes

The effect of natural inactivation in freshwater, chlorination, ammonia, extreme pHs, temperature, and salt content on phage inactivation was evaluated on mixtures of F-specific RNA bacteriophage isolates belonging to genotypes I, II, III, and IV. The bacteriophages studied were previously but recently isolated from natural samples, characterized as F-specific RNA bacteriophages and genotyped by plaque hybridization with genotype-specific probes. Natural inactivation in river water was modeled by in situ incubation of bacteriophages inside submerged dialysis tubes. After several days bacteriophages of genotype I showed the highest persistence, which was significantly different from that of bacteriophages of genotype II, IV, or III. The pattern of resistance of phages belonging to the various genotypes to extreme pHs, ammonia, temperature, salt concentration, and chlorination was similar. In all cases, phages of genotype I showed the highest persistence, followed by the phages of genotypes II, III, and IV. The phages of genotypes III and IV were the least resistant to all treatments, and resistance of genotypes III and IV to the treatments was similar. Bacteriophages of genotype II showed intermediate resistance to some of the treatments. The resistance of four phages of genotype I to natural inactivation and chlorination did not differ significantly. These results indicate that genotypes III and IV are much more sensitive to environmental stresses and to treatments than the other genotypes, especially than genotype I. This should be taken into consideration in future studies aimed at using genotypes of F-specific RNA bacteriophages to fingerprint the origin of fecal pollution.     

Comparative Resistance of Phage Isolates of Four Genotypes of F-Specific RNA Bacteriophages to Various Inactivation Processes

The effect of natural inactivation in freshwater, chlorination, ammonia, extreme pHs, temperature, and salt content on phage inactivation was evaluated on mixtures of F-specific RNA bacteriophage isolates belonging to genotypes I, II, III, and IV. The bacteriophages studied were previously but recently isolated from natural samples, characterized as F-specific RNA bacteriophages and genotyped by plaque hybridization with genotype-specific probes. Natural inactivation in river water was modeled by in situ incubation of bacteriophages inside submerged dialysis tubes. After several days bacteriophages of genotype I showed the highest persistence, which was significantly different from that of bacteriophages of genotype II, IV, or III. The pattern of resistance of phages belonging to the various genotypes to extreme pHs, ammonia, temperature, salt concentration, and chlorination was similar. In all cases, phages of genotype I showed the highest persistence, followed by the phages of genotypes II, III, and IV. The phages of genotypes III and IV were the least resistant to all treatments, and resistance of genotypes III and IV to the treatments was similar. Bacteriophages of genotype II showed intermediate resistance to some of the treatments. The resistance of four phages of genotype I to natural inactivation and chlorination did not differ significantly. These results indicate that genotypes III and IV are much more sensitive to environmental stresses and to treatments than the other genotypes, especially than genotype I. This should be taken into consideration in future studies aimed at using genotypes of F-specific RNA bacteriophages to fingerprint the origin of fecal pollution.