Isolation and Characterization of the New Mosaic Filamentous Phage VFJ Φ of Vibrio cholerae

Filamentous phages have distinguished roles in conferring many pathogenicity and survival related features to Gram-negative bacteria including the medically important Vibrio cholerae, which carries factors such as cholera toxin on phages. A novel filamentous phage, designated VFJΦ, was isolated in this study from an ampicillin and kanamycin-resistant O139 serogroup V. cholerae strain ICDC-4470. The genome of VFJΦ is 8555 nucleotides long, including 12 predicted open reading frames (ORFs), which are organized in a modular structure. VFJΦ was found to be a mosaic of two groups of V. cholerae phages. A large part of the genome is highly similar to that of the fs2 phage, and the remaining 700 bp is homologous to VEJ and VCYΦ. This 700 bp region gave VFJΦ several characteristics that are not found in fs2 and other filamentous phages. In its native host ICDC-4470 and newly-infected strain N16961, VFJΦ was found to exist as a plasmid but did not integrate into the host chromosome. It showed a relatively wide host range but did not infect the classical biotype O1 V. cholerae strains. After infection, the host strains exhibited obvious inhibition of both growth and flagellum formation and had acquired a low level of ampicillin resistance and a high level of kanamycin resistance. The antibiotic resistances were not directly conferred to the hosts by phage-encoded genes and were not related to penicillinase. The discovery of VFJΦ updates our understanding of filamentous phages as well as the evolution and classification of V. cholerae filamentous phage, and the study provides new information on the interaction between phages and their host bacteria.     

Bacteriophage of Haemophilus influenzae III. Morphology, DNA Homology, and Immunity Properties of HP1c1, S2, and the Defective Bacteriophage from Strain Rd

The phages HP1c1 and S2 and a defective phage of Haemophilus influenzae have been compared. The morphology of the phages and the mol wt of their DNAs are similar, although the defective phage appears to have a different tail plate region. Electron microscope observation indicates that the defective phage does not attach to the cell surface, and its DNA appears to lack cohesive ends. The homology of the DNAs of the phages has been measured by hydridization. DNA from the defective phage shows little or no homology with the other phage DNAs. HP1c1 and S2 DNAs show a high level of homology. Each of these phages can form plaques on lawns of the lysogen of the other phage but at reduced plating efficiencies, suggesting that the two phages have related but not identical immunity systems.     

The isolation and characterization of two Stenotrophomonas maltophilia bacteriophages capable of cross-taxonomic order infectivity

Background

A rapid worldwide increase in the number of human infections caused by the extremely antibiotic resistant bacterium Stenotrophomonas maltophilia is prompting alarm. One potential treatment solution to the current antibiotic resistance dilemma is “phage therapy”, the clinical application of bacteriophages to selectively kill bacteria.

Results

Towards that end, phages DLP1 and DLP2 (vB_SmaS-DLP_1 and vB_SmaS-DLP_2, respectively) were isolated against S. maltophilia strain D1585. Host range analysis for each phage was conducted using 27 clinical S. maltophilia isolates and 11 Pseudomonas aeruginosa strains. Both phages exhibit unusually broad host ranges capable of infecting bacteria across taxonomic orders. Transmission electron microscopy of the phage DLP1 and DLP2 morphology reveals that they belong to the Siphoviridae family of bacteriophages. Restriction fragment length polymorphism analysis and complete genome sequencing and analysis indicates that phages DLP1 and DLP2 are closely related but different phages, sharing 96.7 % identity over 97.2 % of their genomes. These two phages are also related to P. aeruginosa phages vB_Pae-Kakheti_25 (PA25), PA73, and vB_PaeS_SCH_Ab26 (Ab26) and more distantly related to Burkholderia cepacia complex phage KL1, which together make up a taxonomic sub-family. Phages DLP1 and DLP2 exhibited significant differences in host ranges and growth kinetics.

Conclusions

The isolation and characterization of phages able to infect two completely different species of bacteria is an exciting discovery, as phages typically can only infect related bacterial species, and rarely infect bacteria across taxonomic families, let alone across taxonomic orders.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1848-y) contains supplementary material, which is available to authorized users.     

Vibrio vulnificus Phage PV94 Is Closely Related to Temperate Phages of V. cholerae and Other Vibrio Species

Background

Vibrio vulnificus is an important pathogen which can cause serious infections in humans. Yet, there is limited knowledge on its virulence factors and the question whether temperate phages might be involved in pathogenicity, as is the case with V. cholerae. Thus far, only two phages (SSP002 and VvAW1) infecting V. vulnificus have been genetically characterized. These phages were isolated from the environment and are not related to Vibrio cholerae phages. The lack of information on temperate V. vulnificus phages prompted us to isolate those phages from lysogenic strains and to compare them with phages of other Vibrio species.

Results

In this study the temperate phage PV94 was isolated from a V. vulnificus biotype 1 strain by mitomycin C induction. PV94 is a myovirus whose genome is a linear double-stranded DNA of 33,828 bp with 5′-protruding ends. Sequence analysis of PV94 revealed a modular organization of the genome. The left half of the genome comprising the immunity region and genes for the integrase, terminase and replication proteins shows similarites to V. cholerae kappa phages whereas the right half containing genes for structural proteins is closely related to a prophage residing in V. furnissii NCTC 11218.

Conclusion

We present the first genomic sequence of a temperate phage isolated from a human V. vulnificus isolate. The sequence analysis of the PV94 genome demonstrates the wide distribution of closely related prophages in various Vibrio species. Moreover, the mosaicism of the PV94 genome indicates a high degree of horizontal genetic exchange within the genus Vibrio, by which V. vulnificus might acquire virulence-associated genes from other species.     

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.     

Defective pages as an antagonistic factor in closely-related bacilli

The antagonistic effect produced by the detective phage PBSX during cocultivation of the mutant strain B. subtilis 168, in which this phage is heat-inducible, and strain B. subtilis NRS231, which also bears a defective phage, was investigated. As soon as in the first hours of cocultivation under conditions of PBSX induction, the number of viable cells of strain NRS231 decreased by two orders of magnitude. However, the effect was not observed if the temperature of cocultivation was noninducing. The results confirm the supposition that defective phages may play a role in the competition between closely related bacilli.     

The diversity and host interactions of Propionibacterium acnes bacteriophages on human skin

The viral population, including bacteriophages, is an important component of the human microbiota, yet is poorly understood. We aim to determine whether bacteriophages modulate the composition of the bacterial populations, thus potentially playing a role in health or disease. We investigated the diversity and host interactions of the bacteriophages of Propionibacterium acnes, a major human skin commensal implicated in acne pathogenesis. By sequencing 48 P. acnes phages isolated from acne patients and healthy individuals and by analyzing the P. acnes phage populations in healthy skin metagenomes, we revealed that P. acnes phage populations in the skin microbial community are often dominated by one strain. We also found phage strains shared among both related and unrelated individuals, suggesting that a pool of common phages exists in the human population and that transmission of phages may occur between individuals. To better understand the bacterium–phage interactions in the skin microbiota, we determined the outcomes of 74 genetically defined Propionibacterium strains challenged by 15 sequenced phages. Depending on the Propionibacterium lineage, phage infection can result in lysis, pseudolysogeny, or resistance. In type II P. acnes strains, we found that encoding matching clustered regularly interspaced short palindromic repeat spacers is insufficient to confer phage resistance. Overall, our findings suggest that the prey–predator relationship between bacteria and phages may have a role in modulating the composition of the microbiota. Our study also suggests that the microbiome structure of an individual may be an important factor in the design of phage-based therapy.     

Bacteriophages ofBacillus subtilis: Comparison of different isolation techniques and possible use for classification ofBacillus subtilis strains

When different techniques are used for the isolation of bacteriophages ofBacillus subtilis a number of different phages may be obtained. Furthermore defective phages are found in old cultures of all strains ofB. subtilis tested so far. The possible use of the phages and the defective phages for classifyingB. subtilis strains into a number of groups according to their susceptibility to different phages and according to the presence of certain defective phages in the cells is discussed.     

Genetic Variation of Lactobacillus delbrueckii subsp. lactis Bacteriophages Isolated from Cheese Processing Plants in Finland

The genomes of four Lactobacillus delbrueckii subsp. lactis bacteriophages were characterized by restriction endonuclease mapping, Southern hybridization, and heteroduplex analysis. The phages were isolated from different cheese processing plants in Finland between 1950 and 1972. All four phages had a small isometric head and a long noncontractile tail. Two different types of genome (double-stranded DNA) organization existed among the different phages, the pac type and the cos type, corresponding to alternative types of phage DNA packaging. Three phages belonged to the pac type, and a fourth was a cos-type phage. The pac-type phages were genetically closely related. In the genomes of the pac-type phages, three putative insertion/deletions (0.7 to 0.8 kb, 1.0 kb, and 1.5 kb) and one other region (0.9 kb) containing clustered base substitutions were discovered and localized. At the phenotype level, three main differences were observed among the pac-type phages. These concerned two minor structural proteins and the efficiency of phage DNA packaging. The genomes of the pac-type phages showed only weak homology with that of the cos-type phage. Phage-related DNA, probably a defective prophage, was located in the chromosome of the host strain sensitive to the cos-type phage. This DNA exhibited homology under stringent conditions to the pac-type phages.     

Rapid detection by multiplex PCR of Genomic Islands, prophages and Integrative Conjugative Elements in V. cholerae 7th pandemic variants

Vibrio cholerae poses a threat to human health, and new epidemic variants have been reported so far. Seventh pandemic V. cholerae strains are characterized by highly related genomic sequences but can be discriminated by a large set of Genomic Islands, phages and Integrative Conjugative Elements. Classical serotyping and biotyping methods do not easily discriminate among new variants arising worldwide, therefore the establishment of new methods for their identification is required. We developed a multiplex PCR assay for the rapid detection of the major 7th pandemic variants of V. cholerae O1 and O139. Three specific genomic islands (GI-12, GI-14 and GI-15), two phages (Kappa and TLC), Vibrio Seventh Pandemic Island 2 (VSP-II), and the ICEs of the SXT/R391 family were selected as targets of our multiplex PCR based on a comparative genomic approach. The optimization and specificity of the multiplex PCR was assessed on 5 V. cholerae 7th pandemic reference strains, and other 34 V. cholerae strains from various epidemic events were analyzed to validate the reliability of our method. This assay had sufficient specificity to identify twelve different V. cholerae genetic profiles, and therefore has the potential to be used as a rapid screening method.     

Molecular evidence for the lysogenic state of microorganisms belonging to the genus Bordetella and characterization of Bordetella parapertussis temperate bacteriophage 662-2

A new bacteriophage ?K of microorganisms belonging to the genus Bordetella was isolated from cells of the earlier characterized strains 66_2-2 (1 and 2) obtained upon phage conversion of B. parapertussis 17 903 cells by B. pertussis bacteriophage ?134. Bacteriophage ?K is identical to previously described Bordetella bacteriophages ?T, ?134, and ?214 in morphology and some biological properties but has a permuted genome different from all other phages. DNA of bacteriophage ?K is not integrated in the chromosome of B. parapertussis 17 903, similar to DNA of bacteriophages ?T, ?134, and ?214 that are not integrated into B. pertussis and B. bronchiseptica chromosomes, but may be present in a small part of the bacterial population as linear plasmids. Sequences homologous to DNA of bacteriophage ?K were detected in the chromosome of strain 66_2-2 (1 and 2) and in chromosomes of all tested strains B. pertussis and B. bronchiseptica. Prophage integration in chromosomes of microorganisms of the genus Bordetella may vary in different bacterial strains and species. An assumption about abortive lysogeny of B. parapertussis bacteria for ?K phage and of B. pertussis and B. bronchiseptica for closely related phages ?T, ?134, and ?214 has been advanced. The possibility of involvement of B. pertussis insertion sequences in the formation of the chromosomal structure in 66_2-2 convertants and in phage genomes is considered.     

Alteration of Host Specificity to Lytic Bacteriophages in Streptococcus cremoris

A mutant of Streptococcus cremoris strain ML1 was isolated based on its resistance to acriflavine. The mutant strain showed resistance to the growth of virulent bacteriophages to which the parental strain was sensitive whereas it became sensitive to a number of other virulent phages to which the parental strain was resistant. At the same time, infection of the mutant strain by another bacteriophage sc607 resulted in killing of cells without production of progeny phages. The phage adsorption appeared normal, suggesting that the killing was a postadsorption event. Such killing of bacterial cells was prevented by chloramphenicol treatment, indicating that involvement of some protein either synthesized by phage or phage-induced cellular protein. Synthesis of ribonucleic acid was abruptly terminated after infection of the mutant strain by phage sc607 but not of the parental strain. The alteration of host specificity in the mutant to different lytic bacteriophages and especially abortive infection by phage sc607 resembles the prophage-mediated interference observed in other bacteria.     

Isolation of Yersinia ruckeri Bacteriophages

Eight bacteriophages effective against Yersinia ruckeri, the enteric redmouth disease bacterium, were isolated. Phage YerA41, a tailed icosahedral virus isolated from sewage enrichments, lysed 34 of 35 strains of Y. ruckeri serovar I, but was inactive against 15 strains belonging to three other serological groups. Six other phages lysed strains of serovars II, V, and I′, a subgroup of serovar I. YerL62, a phage obtained by mitomycin C induction, was specific for one of three serovar V strains. These bacteriophages, particularly YerA41, have potential value for fish disease diagnostic work.     

Lysis to Kill: Evaluation of the Lytic Abilities,and Genomics of Nine Bacteriophages Infective for Gordonia spp. and Their Potential Use in Activated Sludge Foam Biocontrol

Nine bacteriophages (phages) infective for members of the genus Gordonia were isolated from wastewater and other natural water environments using standard enrichment techniques. The majority were broad host range phages targeting more than one Gordonia species. When their genomes were sequenced, they all emerged as double stranded DNA Siphoviridae phages, ranging from 17,562 to 103,424 bp in size, and containing between 27 and 127 genes, many of which were detailed for the first time. Many of these phage genomes diverged from the expected modular genome architecture of other characterized Siphoviridae phages and contained unusual lysis gene arrangements. Whole genome sequencing also revealed that infection with lytic phages does not appear to prevent spontaneous prophage induction in Gordonia malaquae lysogen strain BEN700. TEM sample preparation techniques were developed to view both attachment and replication stages of phage infection.     

Lysogeny in Bradyrhizobium japonicum and Its Effect on Soybean Nodulation

Rhizobiophage V, isolated from soil in the vicinity of soybean roots, was strongly lytic on Bradyrhizobium japonicum 123B (USDA 123) but only mildly lytic on strain L4-4, a chemically induced small-colony mutant of 123. Numerous bacteriophage-resistant variants were isolated from L4-4 infected with phage V; two were studied in detail and shown to be lysogenic. The two, L4-4 (V5) and L4-4 (V12), are the first reported examples of temperate-phage infection in B. japonicum. Phage V and its derivative phages V5 and V12 were closely related on the basis of common sensitivity to 0.01 M sodium citrate and phage V antiserum, phage immunity tests, and apparently identical morphology when examined by electron microscopy. However, the three phages differed in host range and in virulence. Lysogens L4-4 (V5) and L4-4 (V12) were immune to infection by phages V and V5 but not to infection by V12. Southern hybridization analysis confirmed the incorporation of phage V into the genomes of strains L4-4(V5) and L4-4(V12) and also demonstrated the incorporation of phage V into the genome of a phage V-resistant derivative of USDA 123 designated 123 (V2). None of the three lysogens, L4-4(V5), L4-4(V12), or 123B(V2), was able to nodulate soybean plants. However, Southern hybridization profile data indicated that phage V had not incorporated into any of the known B. japonicum nodulation genes.     

Phenomena in mixed cultures ofSalmonella paratyphi B; serological changes and adaptations of the bacteriophage; incomplete natural phages; transformation of phage types

Summary The phenomenon that natural phages are only released in mixed cultures and are not found in pure cultures of bacterial strains has been discussed. It was described how an infecting phage absorbs material from the natural phage in the bacterium and the other way round, that the natural phage absorbs material from the infecting phage. Hereby the bacteriophages can change serologically. Simultaneously the bacteriophage can be adapted. The phenomenon that natural phages are only released in mixed cultures is in some cases explained by assuming that the prophage is incompletely present in the bacterium and is completed by material from an infecting phage.     

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.     

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.     

Ambivalent bacteriophages of different species active on Escherichia coli K12 and Salmonella sp. 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 bongori 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 temperature 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.     

A Study of 33 Bacteriophages of Rhizobium meliloti

A total of 33 Rhizobium meliloti bacteriophages were studied. Of those, 21 were isolated in northern France from field soil in which Medicago sativa L. was grown. The other 12 phages were obtained by UV light and mitomycin C induction from 46 R. meliloti strains. Rhizobiophages were characterized by their morphology, host range, serological properties, restriction endonuclease patterns, DNA-DNA homologies, and DNA molecular weights. Five morphotypes were observed showing tailed phages with icosahedral heads. The categories of morphotypes included the Myoviridae (11 phages), Siphoviridae (3 morphotypes and 20 phages), and Podoviridae (2 phages). Type NM1 phage (Siphoviridae) is highly unusual because of the presence of transverse bars on the phage tail. Soil phages had broad host ranges, whereas phages isolated from bacterial cultures showed more or less narrow host ranges. Restriction endonuclease patterns and DNA-DNA hybridization experiments showed that the five phage type genomes were unrelated. Molecular weights of phage type DNAs were estimated, and they corresponded to values expected for capsid sizes, except for phage NM8. Type M11S (Siphoviridae) did not correspond to any other described Rhizobium phages and represents a new species.