Comparative Study of 35 Bacteriophages of Lactobacillus helveticus: Morphology and Host Range

This survey included 23 phages isolated from cheese whey and 12 temperate phages induced with mitomycin from their lysogenic host strains. All of the phages had an isometric head and a tail with a contractile sheath. In addition, short-tailed (160-nm-long) and long-tailed (260-nm-long) phages were distinguished. Short-tailed phages were by far the most widespread in French cheese factories (32 of the 35 phages studied). The study of phage relationships enabled two large groups of strains to be distinguished: those not or slightly sensitive to phages and those very sensitive to phages. There was an obvious relationship in the first group between phage sensitivity (or resistance) and the geographic origin of the strains. The second group contained primarily strains from large international collections and those isolated from commercial starters. The relationships among short-tailed phages, either temperate or isolated as lytic, suggest that lysogenic strains could be the major source of phages in French cheese factories.     

Phage typing of the coagulase-positive staphylococci isolated from various species of animals

More than 200 coagulase-positive strains of animal origin have been studied by means of Staphylococcus aureus typing phages, belonging to two international sets and intended for typing staphylococci isolated from large cattle and humans, and experimental "chicken" phage A 1591. Among S. aureus strains the cultures isolated from swine, cows, chickens, and belonging to biotypes B1, C1, B2, respectively, have been mostly (in 78.5-90.0% of cases) determined by phage typing. The strains belonging to one biotype have proved to be sensitive predominantly to the same phages. In this connection further differentiation of staphylococci within individual biotypes by means of the phages used in these experiments seems to be impracticable. S. intermedius strains have been found to be completely resistant to the above phages, which confirms that S. intermedius is rightly considered to be an independent species of coagulase-positive staphylococci.     

Analysis of Lactobacillus phages and bacteriocins in American dairy products and characterization of a phage isolated from yogurt.

Yogurt and acidophilus milk that contain Lactobacillus acidophilus could promote human health because L. acidophilus can inhibit enteric and food-borne microbial pathogens. To evaluate the stability of diary L. acidophilus cultures, we studied whether some diary lactobacilli could be inhibited by phages or bacteriocins released by other dairy lactobacilli. From 20 yogurts and two acidophilus milks purchased at local food markets, 38 Lactobacillus strains were isolated. Eight Lactobacillus type strains were used as controls. With mitomycin induction and agar spot assay, phages and bacteriocins were isolated from these strains and their activities were analyzed. Lactobacillus strains from 11 yogurts released phages, while the strains from most of the remaining products released bacteriocins. One phage, designated phi y8, was characterized. It was spontaneously released from its host strain L. acidophilus Y8, at a rate of about 10(4)/ml. This phage lysed nine other dairy Lactobacillus strains tested. It had a burst size of 100, an elongated prolate head of 39 by 130 nm, a long, flexible but noncontractile tail of 300 nm, and a 54.3-kb linear double-stranded DNA. DNA fingerprinting analysis indicated that L. acidophilus phages of nine yogurts in this study belonged to the same type as phi y8. Although they may be sensitive to bacteriocins, all lysogens resisted further phage attacks, whereas most nonlysogens were sensitive to both phages and bacteriocins. Therefore, Lacotbacillus cultures of some American yogurts and acidophilus milks may be unstable or unsafe because they can either be inhibited by phages or bacteriocins or release them to inhibit lactobacilli or other diary products.     

Ambivalent bacteriophages of different species active on Escherichia coli K12 and Salmonella sps. strains

A study was made of several bacteriophages (including phages U2 and LB related to T-even phages of Escherichia coli) that grow both on E. coli K12 and on some Salmonella strains. Such phages were termed ambivalent. T-even ambivalent phages (U2 and LB) are rare and have a limited number of hosts among Salmonella strains. U2 and LB are similar to canonical E. coli-specific T-even phages in morphological type and size of the phage particle and in reaction with specific anti-T4 serum. Phages U2 and LB have identical sets of structural proteins, some of which are similar in size to structural proteins of phages T2 and T4. DNA restriction patterns of phages U2 and LB differ from each other and from those of T2 and T4. Still, DNAs of all four phages have considerable homology. Unexpectedly, phages U2 and LB grown on Salmonella bungori were unstable during centrifugation in a CsCl gradient. Ambivalent bacteriophages were found in species other than T-even phages and were similar in morphotype to lambdoid and other E. coli phages. One of the ambivalent phages was highly similar to well-known Felix01, which is specific for Salmonella. Ambivalent phages can be used to develop a new set for phage typing in Salmonella. An obvious advantage is that ambivalent phages can be reproduced in the E. coli K12 laboratory strain, which does not produce active temperate phages. Consequently, the resulting typing phage preparation is devoid of an admixture of temperate phages, which are common in Salmonella. The presence of temperate phages in phage-typing preparations may cause false-positive results in identifying specific Salmonella strains isolated from the environment or salmonellosis patients. Ambivalent phages are potentially useful for phage therapy and prevention of salmonellosis in humans and animals.     

Primary Isolation Strain Determines Both Phage Type and Receptors Recognised by Campylobacter jejuni Bacteriophages

In this study we isolated novel bacteriophages, infecting the zoonotic bacterium Campylobacter jejuni. These phages may be used in phage therapy of C. jejuni colonized poultry to prevent spreading of the bacteria to meat products causing disease in humans. Many C. jejuni phages have been isolated using NCTC12662 as the indicator strain, which may have biased the selection of phages. A large group of C. jejuni phages rely on the highly diverse capsular polysaccharide (CPS) for infection and recent work identified the O-methyl phosphoramidate modification (MeOPN) of CPS as a phage receptor. We therefore chose seven C. jejuni strains each expressing different CPS structures as indicator strains in a large screening for phages in samples collected from free-range poultry farms. Forty-three phages were isolated using C. jejuni NCTC12658, NCTC12662 and RM1221 as host strains and 20 distinct phages were identified based on host range analysis and genome restriction profiles. Most phages were isolated using C. jejuni strains NCTC12662 and RM1221 and interestingly phage genome size (140 kb vs. 190 kb), host range and morphological appearance correlated with the isolation strain. Thus, according to C. jejuni phage grouping, NCTC12662 and NCTC12658 selected for CP81-type phages, while RM1221 selected for CP220-type phages. Furthermore, using acapsular ∆kpsM mutants we demonstrated that phages isolated on NCTC12658 and NCTC12662 were dependent on the capsule for infection. In contrast, CP220-type phages isolated on RM1221 were unable to infect non-motile ∆motA mutants, hence requiring motility for successful infection. Hence, the primary phage isolation strain determines both phage type (CP81 or CP220) as well as receptors (CPS or flagella) recognised by the isolated phages.     

Phage types of Staphylococcus aureus isolated in Poland in 1999-2004

The aim of study was to determine how population of Staphylococcus aureus strains from hospital environment forms itself with respect to phage types and frequency of their occurrence recently. 1157 S. aureus isolated from various clinical materials between 1999 and 2004 have been investigated. The basic set of phages and the three additional ones were used for typing strains according to Blair and Williams method. The results proved that population of S. aureus strains isolated from hospital environment differentiates with respect to phage types, like in the past. Strains belonging to phage group II dominates among them, but a considerable increase in number of phage group III have been remarked lately. A differential value of an individual phages from basic set remains on their usual level for years.     

Bacteriophage Typing as an Epidemiological Tool for Urinary Escherichia coli

Phage typing was used to identify strains of Escherichia coli isolated from urinary and nonurinary sources. When eight phages isolated in Pennsylvania were used to type 717 cultures from Missouri, 50.3% of 624 urinary isolates and 34.4% of 93 nonurinary isolates were typable. Strains from nonurinary sources were not found commonly in urine. When five additional phages isolated in Missouri were added to the original set of eight phages, 80.4% of 331 urinary isolates were typable. When this set of phages was used to type 552 urinary cultures isolated in California, Minnesota, Ohio, Pennsylvania, Virginia, and West Virginia, 82.0% of the cultures were typable. Some common phage types were found in high incidence among cultures from the different regions. No correlation was found between phage type and the pattern of resistance to antibiotics. Phage typing data were presented also on the number of strains in individual urine specimens and the recurrences of strains in patients with chronic bacteriuria. Of 97 fecal isolates, 75.2% of the cultures were typable, and the most common phage type was observed in high incidence among the urinary isolates from this region. When 75 cultures from nine other genera of enteric bacteria were typed, only the shigellae were lysed. In view of the information obtained by phage typing and the ease and speed with which it can be done, it is suggested that phage typing be considered a new tool in epidemiological studies of urinary tract infections by E. coli.     

Susceptibility of Pseudomonas aeruginosa veterinary isolates to Pbunavirus PB1-like phages

In recent years, antimicrobial-resistant Pseudomonas aeruginosa strains have increased in the veterinary field. Therefore, phage therapy has received significant attention as an approach for overcoming antimicrobial resistance. In this context, we isolated and characterized four Pseudomonas bacteriophages. Phylogenetic analysis showed that the isolated phages are novel Myoviridae Pbunavirus PB1-like phages with ØR12 belonging to a different clade compared with the other three. These phages had distinct lytic activity against 22 P. aeruginosa veterinary isolates. The phage cocktail composed from the PB1-like phages clearly inhibited the occurrence of the phage-resistant variant, suggesting that these phages could be useful in phage therapy.     

Isolation and characterization of five Erwinia amylovora bacteriophages and assessment of phage resistance in strains of Erwinia amylovora

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 phiEa1 and phiEa7 and 3 novel phages named phiEa100, phiEa125, and phiEa116C, were identified based on differences in genome size and restriction fragment pattern. phiEa1, the phage distributed most widely, had an approximately 46-kb genome which exhibited some restriction site variability between isolates. Phages phiEa100, phiEa7, and phiEa125 each had genomes of approximately 35 kb and could be distinguished by their EcoRI restriction fragment patterns. phiEa116C contained an approximately 75-kb genome. phiEa1, phiEa7, phiEa100, phiEa125, and 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. 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 10(5) CFU.     

Bacteriophages of lactobacilli

Lactobacilli are members of the bacterial flora of lactic starter cultures used to generate lactic acid fermentation in a number of animal or plant products used as human or animals foods. They can be affected by phage outbreaks, which can result in faulty and depreciated products. Two groups of phages specific of Lactobacillus casei have been thoroughly studied. 1. The first group is represented by phage PL-1. This phage behaves as lytic in its usual host L. casei ATCC 27092, but can lysogenize another strain, L. casei ATCC 334. Bacterial receptors of this phage are located in a cell-wall polysaccharide and rhamnose is the main component of the receptors. Ca2+ and adenosine triphosphate (ATP) are indispensable to ensure the injection of the phage DNA into the bacterial cell. The phage DNA is double-stranded, mostly linear, but with cohesive ends which enables it to be circularized. The vegetative growth of PL-1 proceeds according to the classical mode. Cell lysis is produced by an N-acetyl-muramidase at the end of vegetative growth. 2. The second group is represented by the temperate phage phi FSW of L. casei ATCC27139. It has been shown how virulent phages originate from this temperate phage in Japanese dairy plants. The lysogenic state of phi FSW can be altered either by point mutations or by the insertion of a mobile genetic element called ISL 1, which comes from the bacterial chromosome. This is the first transposable element that has been described in lactobacilli. Lysogeny appears to be widespread among lactobacilli since one study showed that 27% of 148 strains studied, representing 15 species, produced phage particles after induction by mitomycin C. Similarly, 23 out of 30 strains of Lactobacillus salivarius are lysogenic and produce, after induction by mitomycin C, temperate phages, killer particles, or defective phages. Temperate phages have also been found in 10 out of 105 strains of Lactobacillus bulgaricus or Lactobacillus lactis after induction by mitomycin C. Phages so far studied of the latter 2 and closely related lactobacilli, either temperate or isolated as lytic, may be divided into 4 unrelated groups called a, b, c and d. Most of these phages are found in group a and an unquestionable relationship has already been shown between lytic phages and temperate phages that belong to this group. Lytic phage LL-H of L. lactis LL 23, isolated in Finland, is one of the most representative of those of group a and has been extensively studied on the molecular level.     

Identification of adsorption inhibition, restriction/modification and abortive infection type phage resistance systems in Lactococcus lactis strains

98 Lactococcus lactis strains were isolated from traditional fermented milk products in Turkey tested against 60 lactococcal lytic phages to determine their resistance levels. While 82 L. lactis strains were sensitive against lactic phages at different levels, 16 L. lactis strains showed resistance to all phages tested. Types of phage resistance among 16 L. lactis strains were identified as phage adsorption inhibition in eight strains, restriction/modification in six strains and abortive infection (heat sensitive phage resistance) in two strains, using three broad-spectrum phages phi pll 98-32, phi pld 67-42 and phi pld 67-44.     

Bacteriophage types of Salmonella enteritidis occurring in Poland in 1986-1995

The Lalko phages collection was used to phage type a total of 517 Salmonella Enteritidis strains isolated from food-poisoning outbreaks (312 strains) and other common sources (205 strains) in Poland, during the years 1986-1995. Above 99.0% of all strains tested were recognized as belonging to definitive phage type. Phage types 1, 6 and 7 were predominant. The strains of type 1 and 7 were most numerous. Of the 517 examined strains 312 were isolated from 46 food-poisoning outbreaks. Most of them came from the one phage type outbreaks; 8 mixed outbreaks were noted. The greatest number of the food-poisoning outbreaks was caused by Salmonella Enteritidis phage types 1, 6 and 7. Phage type 16 was isolated from persons for the first time.     

Interconversion of Type C and D Strains of Clostridium botulinum by Specific Bacteriophages

These studies show that Clostridium botulinum types C and D cultures can be cured of their prophages and converted to either type C or D depending on the specific phage used. Strains of types C and D were cured of their prophages and simultaneously ceased to produce their dominant toxins designated as C(1) and D, respectively. Cured nontoxigenic cultures derived from type C strain 162 were sensitive to the phages from the toxigenic type C strain 162 and type D strain South African. When cured nontoxigenic cultures derived from strain 162 were infected with the tox(+) phages from the 162 strain of type C and the South African strain of type D, they then produced toxin neutralized by types C and D antisera, respectively. Cured nontoxigenic cultures isolated from the type D South African strain were only sensitive to the parent phage, and, when reinfected with the tox(+) phage, they produced toxin neutralized by type D antiserum. Type C strain 153 and type D strain 1873, when cured of their respective prophages, also ceased to produce toxins C(1) and D, but, unlike strain 162 and the South African strain, they continued to produce a toxin designated as C(2). When the cured cultures from strains 153 and 1873 were infected with the tox(+) phage from type D strain 1873, the cultures simultaneously produced toxin that was neutralized by type D antiserum. When these cured cultures were infected with the tox(+) phage from type C strain 153, the cultures produced toxin that was neutralized by type C antiserum. These studies with the four strains of C. botulinum confirm that the toxigenicity of types C and D strains requires the continued participation of tox(+) phages. Evidence is presented that types C and D cultures may arise from a common nontoxigenic strain.     

Specificity patterns of Agrobacterium tumefaciens phages

Summary Lysogeny was not detected in 10 strains of A. tumefaciens by plating techniques or ultra-violet induction. Fifteen phages were isolated from raw sewage against 13 cultures of A. tumefaciens and purified by single-plaque selections. No phage lysed all of the strains of A. tumefaciens tested; one phage lysed only a single strain; 2 other phages attacked 7 strains. Ten of the 15 phages lysed no more than 3 strains. Three host strains showed identical phage susceptibilities. No relationship was noted between susceptibility to phage and ability of a strain to incite crown galls.Thirteen phages lysed at least 1 of 4 strains of A. radiobacter, but none attacked single strains of A. rubi or A. pseudotsugae. Eleven phages lysed the one strain of A. rhizogenes used. None of the phages had identical host ranges with respect to all the Agrobacterium spp. tested. Similarly none of 5 selected phages attacked any one of 59 strains of bacteria from 12 different genera including 35 strains of rhizobia. Within the limits of this study the phages used were genus-specific.Published with approval of the Director, Wisconsin Agricultural Experiment Station, Madison, Wisconsin, U.S.A. 53706.     

Differentiation of methicillin-resistant strains of Staphylococcus aureus by prophage specificity]

By inducing with mitomycin C the following phages were isolated from all the tested 32 methicillin resistant strains of S. aureus: the serogroup B phage was isolated from 2 strains, the serogroup B and F phages were isolated from 5 strains and the serogroup F phage was isolated from 25 strains. The phages were divided into 5 groups by the antiphage immunity. In group 1 of the phages 4 additional phages were specified. By the specificity of the prophages in the cultures all the strains were divided into 5 groups. Group 1 of the cultures was divided into 5 subgroups (A, B, C, D and E).     

Evaluation of the usefulness of three collections of bacteriophages for typing methicillin-resistant Staphylococcus aureus,isolated in Moscow hospitals

The typing of S. aureus methicillin-resistant strains, isolated in different hospitals of Moscow; was carried out with the use of three collections of phages: the International Set of Phages; the set of phages of the International Center of S. aureus phage typing in London (L); and the experimental collection of phages of the Gamaleya Institute of Epidemiology and Microbiology in Moscow (M). In this study made with the use of both the phages of the International Diagnostic Set and phages L in the standard typing dose of 1 TP about 6% of the cultures under study proved to be sensitive. When the typing dose was increased to 100 TP the phages of the international diagnostic set lyzed 75.5% of the cultures. The typed strains were found to belong to phage types 77 (71.7%), 77/84/85 (19.6%) and 94/96 (6.5%). At a concentration of 100 TP phages L lyzed 83.7% of the cultures, but the dominating phage types could not be determined due to a great variety of phage markers. In contrast to the two preceding collections, the third phage collection M was composed in such a way that in the study of the investigated culture the specificity of its restriction modification was primarily evaluated and only then the presence of antiphage immunity was determined. This latter collection was used in the evaluation of 93.1% of the cultures. By the specificity of their restriction specification system the majority of them were classified with two new groups, heretofore not described. Only this collection M made it possible to differentiate epidemic and sporadic strains and to evaluate the epidemic situation in all 6 hospitals.     

Synthesis of Indicator Strains and Density of Ribonucleic Acid-Containing Coliphages in Sewage

Escherichia coli strains freshly isolated from natural sources are inefficient indicators of coliphages present in sewage. Four E. coli strains recently isolated from clinical specimens were mutagenized to obtain lac(-) mutants. Such mutants were infected with an F'lac(+) sex factor of E. coli K-12. Pairs of isogenic lac(-) and lac(-)/F'lac(+) strains were used as indicators of coliphages present in sewage, and it was found that such strains can be effectively used for a direct and almost selective enumeration of F-specific coliphage contents of sewage samples. Serological tests were applied to a number of F-specific phages isolated. All the isolates that were tested fell into two distinguishable antigenic classes: members of one class being related to ribonucleic acid (RNA) phage MS2 and those of the other being related to another RNA phage, namely, Qbeta. MS2-related phages have been found to be more widely distributed than the Qbeta related phages. Most habitats sampled were found to yield only one or the other kind of phage. Single-stranded deoxyribonucleic acid-containing F-specific phages were not detectable by the methods employed by us.     

Comparative study of temperate bacteriophages isolated from Yersinia

170 Yersinia strains belonging to various species were investigated for the presence of temperate bacteriophages. By induction with mitomycin C seven phages were isolated from Y. enterocolitica strains and one phage from a Y. frederiksenii strain. The phages were characterized on the basis of their morphology, host range, genome size, DNA homology, and protein composition. They belong to different phage families and reveal narrow to moderate wide host ranges. Some of the isolated phages were able to infect pathogenic as well as nonpathogenic strains of Y. enterocolitica. The genomes of all isolated phages were found to be composed of double stranded DNA ranging from about 40 to 60 kb. In addition to the analysed phages, a number of putative phages were induced in strains of Y. frederiksenii, Y. kristensenii, Y. intermedia, and Y. mollaretii. The putative phages were identified by isolation of phage DNA from cell free lysates but could not be propagated on indicator strains. Southern hybridization experiments revealed relationships between phages belonging to different families. Moreover, DNA homologies were observed between phages isolated from nonpathogenic Yersinia strains and a phage which was isolated from a pathogenic Y. enterocolitica serogroup O:3 strain.     

Genomic sequence and receptor for the Vibrio cholerae phage KSF-1phi: evolutionary divergence among filamentous vibriophages mediating lateral gene transfer

KSF-1phi, a novel filamentous phage of Vibrio cholerae, supports morphogenesis of the RS1 satellite phage by heterologous DNA packaging and facilitates horizontal gene transfer. We analyzed the genomic sequence, morphology, and receptor for KSF-1phi infection, as well as its phylogenetic relationships with other filamentous vibriophages. While strains carrying the mshA gene encoding mannose-sensitive hemagglutinin (MSHA) type IV pilus were susceptible to KSF-1phi infection, naturally occurring MSHA-negative strains and an mshA deletion mutant were resistant. Furthermore, d-mannose as well as a monoclonal antibody against MSHA inhibited infection of MSHA-positive strains by the phage, suggesting that MSHA is the receptor for KSF-1phi. The phage genome comprises 7,107 nucleotides, containing 14 open reading frames, 4 of which have predicted protein products homologous to those of other filamentous phages. Although the overall genetic organization of filamentous phages appears to be preserved in KSF-1phi, the genomic sequence of the phage does not have a high level of identity with that of other filamentous phages and reveals a highly mosaic structure. Separate phylogenetic analysis of genomic sequences encoding putative replication proteins, receptor-binding proteins, and Zot-like proteins of 10 different filamentous vibriophages showed different results, suggesting that the evolution of these phages involved extensive horizontal exchange of genetic material. Filamentous phages which use type IV pili as receptors were found to belong to different branches. While one of these branches is represented by CTXphi, which uses the toxin-coregulated pilus as its receptor, at least four evolutionarily diverged phages share a common receptor MSHA, and most of these phages mediate horizontal gene transfer. Since MSHA is present in a wide variety of V. cholerae strains and is presumed to express in the environment, diverse filamentous phages using this receptor are likely to contribute significantly to V. cholerae evolution.     

Evaluation of the possibility of using genetic and microbiological research methods for resolving current problems in the epidemiology of salmonellosis

S. typhimurium strains isolated in 14 regions of the USSR have, parallel with considerable similarity in their biological characteristics, a number of essential differences. These differences become manifest in the determination of the plasmid spectra of the above organisms. The transfer of R-plasmid PLE518 F1 me fin+ with a molecular weight of 96 Md to strains, sensitive to the action of typing bacteriophages, renders these strains resistant to the lytic action of a number of phages, which leads to the conversion of their phage type. In some cases it may deteriorate the validity of the method of phage typing, used for epidemiological purposes.