Incidence and properties of temperate bacteriophages induced from lactic streptococci.

Sixty-three strains of lactic streptococci isolated from commercial lactic streptococcal starter cultures were examined for lysogeny by treatment with ultraviolet light or mitomycin C. After treatment with the inducing agent, all strains, whether or not they lysed, were examined for evidence of phage release by electron microscopy. Thirty-eight strains yielded intact phages or phage particles of varying morphology. All the temperate phages had isometric heads and noncontractile tails; some had collars and structurally distinctive baseplates. Indicator host strains were found for phages induced from seven different strains. Three strains that released phages spontaneously yielded titers of 10(3) to 10(4) plaque-forming units per ml. When strains that spontaneously released phages were grown in mixed culture with indicator strains, increased phage titers of 10(6) to 10(7) plaque-forming units per ml were observed. These findings indicate that lysogenic lactic streptococcal strains may serve as a reservoir for phages that attack sensitive strains in mixed- or multiple-strain lactic starter cultures.     

Incidence and properties of temperate bacteriophages induced from lactic streptococci.

Sixty-three strains of lactic streptococci isolated from commercial lactic streptococcal starter cultures were examined for lysogeny by treatment with ultraviolet light or mitomycin C. After treatment with the inducing agent, all strains, whether or not they lysed, were examined for evidence of phage release by electron microscopy. Thirty-eight strains yielded intact phages or phage particles of varying morphology. All the temperate phages had isometric heads and noncontractile tails; some had collars and structurally distinctive baseplates. Indicator host strains were found for phages induced from seven different strains. Three strains that released phages spontaneously yielded titers of 10(3) to 10(4) plaque-forming units per ml. When strains that spontaneously released phages were grown in mixed culture with indicator strains, increased phage titers of 10(6) to 10(7) plaque-forming units per ml were observed. These findings indicate that lysogenic lactic streptococcal strains may serve as a reservoir for phages that attack sensitive strains in mixed- or multiple-strain lactic starter cultures.     

Bacteriophage typing of Listeria species.

A bacteriophage typing scheme for differentiating Listeria isolates from dairy products and various other foodstuffs was developed. Sixteen selected phages isolated from both environmental sources and lysogenic strains were used for typing and, according to their lytic spectra, divided into four groups. Thus far, 41 distinct patterns of lysis were seen when this set was used in typing 57 defined reference strains, representing all five confirmed species and 16 serotypes in addition to 454 Listeria isolates of primarily foodborne origin. Overall, typability was 84.5%; i.e., a strain was lysed by at least one phage at 100x routine test dilution. Strains belonging to serovar 3 were mostly resistant to lysis by the phages employed. The results were highly reproducible, as determined in retyping trials several weeks later. Some phages isolated from environmental sources showed a wider lytic spectrum than did those isolated from lysogenic strains. In accordance with this, the phages were found in different clusters within a computer-generated linkage map. Species specificity and serovar specificity of the lytic reaction were not found. None of the phages was able to lyse strains of Listeria grayi, Listeria murrayi or Jonesia denitrificans. This phage typing system may provide important information for a means of recognizing and eliminating sources of contamination by Listeria spp. within dairy plant equipment.     

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.     

Isolation and Preliminary Characterization of Bacteriophages for Bdellovibrio bacteriovorus

Ten bacteriophages that attack and lyse saprophytic strains of Bdellovibrio bacteriovorus were isolated. Morphological, serological, and host-range studies revealed that there were four different bdellovibrio phages present among the isolates. One of the phages lysed a strain of B. bacteriovorus that requires the presence of a suitable bacterial host for growth. The phage attached to the bdellovibrio cells in the absence of the bacterial host cells; lysis occurred only in the presence of host cells. The 19 saprophytic bdellovibrio strains employed in the phage host-range studies were grouped on the basis of their susceptibility to phage lysis.     

Some Properties of Five New Salmonella Bacteriophages

Five bacteriophages were isolated from lysogenic strains of Salmonella potdam. On the basis of plaque morphology, thermostability, serology, host range, one-step growth parameters, and phage morphology, they were divided into three groups: group A, phages P4 and P9c; group B, phages P3 and P9a; and group C, phage P10. Group A phages had a hexagonal head 55 nm in diameter with a short tail 15 nm long. These phages were particularly characterized by high thermostability, lack of serological relationship with any of the other phages, and restriction of lysis to other Salmonella strains of Kauffmann-White group C(1). Group B phages had a head identical in size and shape to that of the A phages, but they possessed a tail 118 nm long with a contractile sheath. A unique feature was the occurrence of tail fibers at the end of the core rather than at the base of the sheath. These phages were considerably less thermostable, had extended host ranges, and were serologically distinct from each other but unrelated to the A phages. The group C phage, P10, had a head identical to that of the A and B phages. It had a tail 95 nm in length, with tail fibers attached to a base plate at the end of a contractile sheath. P10 was highly sensitive to heat, lysed only smooth strains of Salmonella, and showed a degree of serological relationship to both B phages. The relationship of these phage groups to previous Salmonella phage grouping schemes is discussed.     

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.     

枯草芽孢杆菌BF7658噬菌体的研究

从一些工厂分离到6株感染枯草芽孢杆菌BF7658的噬菌体,对它们的形态学和生物学特性以及DNA和结构蛋白进行了比较,电镜观察指出,所有噬菌体的头部外形都呈现椭园形,但其大小和尾部长度有所不同,它们的宿主范围较窄,用限制酶分析,噬菌体DNA分子量在29.9kb和98kb之间,根据解链温度计算出噬菌体DNA的G+C含量在45.1和60.2mol％之间,6株噬菌体的蛋白经SDS-聚丙烯酰胺凝胶电泳测定呈现四条主带和五条次带。     

Molecular characterization and module composition of P22-related Salmonella phage genomes.

Genomes of newly isolated Salmonella phages were analysed by comparison of their EcoRI restriction patterns and by hybridization. Characteristic hybridization probes from reference phages P22, ES18 and E. coli phage lambda were chosen. Four probes selected from the lysis region examined the dispersal of the lambdoid lysis genes. Other probes characterized were the replication genes and part of the structural genes. The complex immunity region was investigated by means of hybridization as well as biological tests. The results showed the relationship of the isolated phages to the P22 branch of the lambdoid phages and revealed their modular genome organization consisting of different proportions of P22-related sequences. DNA restriction patterns of phages released from Salmonella strains sampled in limited geographical areas were significantly less heterogeneous than those of phages released from the worldwide sampled SARA collection. The use of prophage restriction patterns as a tool for the typing of Salmonellae to support the epidemiologic classification of pathogenic strains is discussed.     

Observations on bacteriophages of Clostridium botulinum type C isolates from different sources and the role of certain phages in toxigenicity.

Twenty strains of Clostridium botulinum type C, including 12 isolates from avian sources with varying toxigenic properties, were examined by electron microscope for the presence of bacteriophages. All toxigenic strains were infected with one or two types of phages. Three types of phages designated large, small, and intermediate were observed. Most of the strains carried the large and small phage, with the large phage being present in much greater numbers. Since there is evidence that highly toxigenic strains of C. botulinum type C are responsible for large outbreaks of botulism in wild birds, the phenomenon of toxigenic variation among the type C strains was investigated. Experiments were carried out employing a broth medium on a phagefree nontoxigenic strain for elucidating the role of bacteriophages in toxigenicity. All phage suspensions contained large phages, with the exception of one that caused conversion. The exception was a preparation containing an intermediate type of phage. Phages from different strains produced cultures of varying toxigenic characteristics. By employing a tube-lytic test and an agar-overlay-phage assay technique, it was determined that whenever the phage-bacterium relationship resulted in an initial high degree of lysis, the potency of toxin in the culture was weak. It appeared that in highly toxigenic strains, the phage-bacterium relationship is characterized by a stable lysogenic type of association. It was also found that in a highly toxigenic converted culture the percentage of toxigenic cells was 100, whereas in hypotoxigenic culture the percentage was only 20.     

Observations on bacteriophages of Clostridium botulinum type C isolates from different sources and the role of certain phages in toxigenicity.

Twenty strains of Clostridium botulinum type C, including 12 isolates from avian sources with varying toxigenic properties, were examined by electron microscope for the presence of bacteriophages. All toxigenic strains were infected with one or two types of phages. Three types of phages designated large, small, and intermediate were observed. Most of the strains carried the large and small phage, with the large phage being present in much greater numbers. Since there is evidence that highly toxigenic strains of C. botulinum type C are responsible for large outbreaks of botulism in wild birds, the phenomenon of toxigenic variation among the type C strains was investigated. Experiments were carried out employing a broth medium on a phagefree nontoxigenic strain for elucidating the role of bacteriophages in toxigenicity. All phage suspensions contained large phages, with the exception of one that caused conversion. The exception was a preparation containing an intermediate type of phage. Phages from different strains produced cultures of varying toxigenic characteristics. By employing a tube-lytic test and an agar-overlay-phage assay technique, it was determined that whenever the phage-bacterium relationship resulted in an initial high degree of lysis, the potency of toxin in the culture was weak. It appeared that in highly toxigenic strains, the phage-bacterium relationship is characterized by a stable lysogenic type of association. It was also found that in a highly toxigenic converted culture the percentage of toxigenic cells was 100, whereas in hypotoxigenic culture the percentage was only 20.     

New bacteriophages active on strains of Hyphomicrobium

Fifty-five lytic bacteriophages isolated from water and soil samples were active on many strains of the genus Hyphomicrobium. The optimal isolation procedure was an adsorption method in which samples from a habitat similar to that of the respective host bacterium were used as the phage inoculum. According to the morphology and nucleic acid type these bacteriophages belonged to different families: Myoviridae (type A1: five phages); Styloviridae (type B1: 33 phages; type B2: eight phages) and Podoviridae (type C1: nine phages). The Styloviridae (type B1) appeared in two morphological variants (tails flexible or rigid). All phages investigated were specific for the genus Hyphomicrobium and were unable to lyse members of other genera of hyphal, budding bacteria (e.g. Hyphomonas, Pedomicrobium, genus D, genus T). The host specificity of 42 phages was tested with 156 Hyphomicrobium strains: 122 strains were lysed by at least one of these phages, but 34 Hyphomicrobium strains were not susceptible. Morphotype B1 phages with identical morphology could be distinguished according to their host-range properties on prophage-containing Hyphomicrobium strains. With regard to differences in morphology and host range, 25 phages were selected for more detailed investigations. From these phages DNA was isolated; the melting transition midpoints (Tm) ranged from 67 to 93 degrees C. The upper and higher values suggested the presence of DNA modifications. Six different adsorption patterns could be distinguished among the Hyphomicrobium phages. Preferred attachment sites were the proximal pole of the mother cell, the hyphal tip, the distal pole of the bud, and the distal pole of the swarmer cell.     

Biodiversity of Lactococcus lactis bacteriophages in Polish dairy environment

We present here the results of an exploration of the bacteriophage content of dairy wheys collected from milk plants localized in various regions of Poland. Thirty-three whey samples from 17 regions were analyzed and found to contain phages active against L. lactis strains. High phage titer in all whey samples suggested phage-induced lysis to be the main cause of fermentation failures. In total, over 220 isolated phages were examined for their restriction patterns, genome sizes, genetic groups of DNA homology, and host ranges. Based on DNA digestions the identified phages were classified into 34 distinct DNA restriction groups. Phage genome sizes were estimated at 14-35 kb. Multiplex PCR analysis established that the studied phages belong to two out of the three main lactococcal phage types--c2 and 936, while P335-type phages were not detected. Yet, analyses of bacterial starter strains revealed that the majority of them are lysogenic and carry prophages of P335-type in their chromosome. Phage geographical distribution and host range are additionally discussed.     

Observations on lysogeny in glutamic acid bacteria.

Induced lysis occurred in a number of different strains of glutamic acid bacteria. Mixed culture experiments indicated that induced cultures produce phages or bacteriocins. Temperate phages were isolated from two related strains of Brevibacterium divaricatum.     

Molecular taxonomy of Lactobacillus phages

Forty-eight strains of lactobacilli used as starter strains in the dairy industry were examined for lysogeny after treatment with mitomycin C. Two strains of L. delbrueckii subsp. bulgaricus were able to produce active phages. These temperate phages as well as 4 virulent phages isolated during abnormal fermentations were compared to a previously characterized phage mv4 which is temperate. All these phages were shown to be partially homologous by DNA-DNA hybridization. Genes that code for viral proteins seem to be well conserved since 2 major virion polypeptides of 18 (or 19) kD and 34 kD could be detected in the protein composition of each phage. Immunoblotting studies of the 7 phages using serum raised against phage mv4 confirmed that the proteins of the different phages were related. All these phages can be classified in the previously constituted group a, which now comprises 4 temperate and 15 virulent phages. These results show that some virulent phages appearing during abnormal fermentations and some temperate phages isolated by appearing during abnormal fermentations and some temperate phages isolated by induction of starter strains can be closely related genetically. Five virulent phages of L. helveticus were also compared according to their restriction pattern and their DNA homology. They were shown to be related to one another, but unrelated to phages of other lactic acid bacteria species.     

Characterization of temperate bacteriophages of Leuconostoc oenos and evidence for two prophage attachment sites in the genome of starter strain PSU-1

The close relatedness between 17 Leuconostoc oenos bacteriophages, induced with mitomycin C from strains isolated in different geographic regions, was inferred from their morphology, DNA homology and protein composition. The genome of all the phages had cohesive end termini and ranged in size from 36.4 to 40.9 kb. According to the restriction patterns obtained by digestion with five enzymes, the phages were divided in six groups. Lysogenization of a spontaneous phage-cured derivative of Leuc. oenos strain PSU-1 was achieved with 16 phages and the analysis of the lysogens showed that the phage DNA integrates in the host chromosome in one or two sites. The att B loci were located on the macrorestriction Asc I and Not I fragments of the recipient strain. A survey of Leuc. oenos strains with a phage DNA probe confirmed the lysogenic nature of several, but not all of the original phage hosts. These results are discussed in the light of evidence for the instability of some lysogenic PSU-1 derivatives.     

Characterization and classification of virulent lactococcal bacteriophages isolated from a Cheddar cheese plant

C.N. CASEY, E. MORGAN, C. DALY AND G.F. FITZGERALD, 1993. Twenty-two bacteriophages, isolated from cheese-vat whey samples over a period of 4 years, were found to be active against one or more of four different strains of Lactococcus lactis subsp. cremoris used in a defined strain starter system in an Irish Cheddar cheese factory. All the phages were small isometric-headed with non-contractile tails, a baseplate and a collar; they had genome sizes of approximately 30 kb, and belonged to a single DNA hybridization group. All 22 phages could be classified into four distinct groups based on restriction analysis which overlapped perfectly with those based on host range. Each group of phage examined showed cross-reactive host ranges. None of the phage DNAs hybridized to the chromosomes of any of the seven cultures used in the factory during the four cheesemaking seasons, and neither could phages be induced from any of the strains by mitomycin-C or ultraviolet light treatment.     

Bacteriophages of methanotrophs isolated from fish.

Bacteriophages of methanotrophic bacteria were isolated from 67 fish. Only two phages isolated from two fish species specifically lysed Methylocystis sp. and Flavobacterium gasotypicum. The phages lysing these species were designated 63-F and CMF-1-F, respectively. The isolated phages differed greatly in the fine structure of the virion, plaque morphology, spectrum of lytic action, serological properties, and UV sensitivity. At the same time, they had identical one-step growth characteristics: their latent period equalled 5 h, lysis time was 3 to 4 h, and burst size was about 240 virions. The phages had guanine- and cytosine-rich double-stranded DNAs consisting of common nitrogen bases. The molecular masses of the DNAs as determined by the sums of restriction endonuclease cleavage fragments were 28 X 10(6) daltons for phage 63-F and 31 X 10(6) daltons for phage CMF-1-F.     

Bacteriophages of methanotrophs isolated from fish

Bacteriophages of methanotrophic bacteria were isolated from 67 fish. Only two phages isolated from two fish species specifically lysed Methylocystis sp. and Flavobacterium gasotypicum. The phages lysing these species were designated 63-F and CMF-1-F, respectively. The isolated phages differed greatly in the fine structure of the virion, plaque morphology, spectrum of lytic action, serological properties, and UV sensitivity. At the same time, they had identical one-step growth characteristics: their latent period equalled 5 h, lysis time was 3 to 4 h, and burst size was about 240 virions. The phages had guanine- and cytosine-rich double-stranded DNAs consisting of common nitrogen bases. The molecular masses of the DNAs as determined by the sums of restriction endonuclease cleavage fragments were 28 X 10(6) daltons for phage 63-F and 31 X 10(6) daltons for phage CMF-1-F.     

Early Abortive Lysis by Phage BF23 in Escherichia coli K-12 Carrying the Colicin Ib Factor

Growth of phage BF23 was restricted in Escherichia coli K-12 strains carrying a colicin I factor (ColIb); most infected cells lysed early without producing progeny phages. Either addition of chloramphenicol before phage infection or ultraviolet irradiation of phage prevented early abortive lysis, an indication that certain phage functions are required for this phenomenon. Very little or no phage-induced lysozyme was synthesized in the infected ColI(+) cells. This result suggests that early abortive lysis was not due to the lysozyme action. A small fraction (0.05) of BF23-infected ColI(+) cells showed normal phage growth. This "escaped growth" may reflect the physiological state of the host bacteria rather than the heterogeneity of the infecting phage. Host-controlled modification was not observed. A phage mutant, BF23hI, able to grow on ColI(+) cells, was isolated and was characterized to be recessive to the wild-type BF23 in its ability to undergo early abortive lysis. Among the T series phages, T5 induced early abortive lysis, and growth of T5 was restricted upon infection to ColI(+) cells. These results and the other observations, including the occurrence of phenotypic mixing between BF23 and T5, suggest that these two phages are related to each other even though the receptor sites for BF23 and T5 are apparently different.