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.     

Comparative whole genome analysis of six diagnostic brucellaphages

Whole genome sequencing of six diagnostic brucellaphages, Tbilisi (Tb), Firenze (Fz), Weybridge (Wb), S708, Berkeley (Bk) and R/C, was followed with genomic comparisons including recently described genomes of the Tb phage from Mexico (Tb_M) and Pr phage to elucidate genomic diversity and candidate host range determinants. Comparative whole genome analysis revealed high sequence homogeneity among these brucellaphage genomes and resolved three genetic groups consistent with defined host range phenotypes. Group I was composed of Tb and Fz phages that are predominantly lytic for Brucella abortus and Brucella neotomae; Group II included Bk, R/C, and Pr phages that are lytic mainly for B. abortus, Brucella melitensis and Brucella suis; Group III was composed of Wb and S708 phages that are lytic for B. suis, B. abortus and B. neotomae. We found that the putative phage collar protein is a variable locus with features that may be contributing to the host specificities exhibited by different brucellaphage groups. The presence of several candidate host range determinants is illustrated herein for future dissection of the differential host specificity observed among these phages.     

Contrasting genomic patterns and infection strategies of two co‐existing Bacteroidetes podovirus genera

Bacterial viruses (phages) are abundant, ecologically important biological entities. However, our understanding of their impact is limited by model systems that are primarily not well represented in nature, e.g. Enterophages and their hosts. Here, we investigate genomic characteristics and infection strategies among six aquatic Bacteroidetes phages that represent two genera of exceptionally large (～70–75 kb genome) podoviruses, which were isolated from the same seawater sample using Cellulophaga baltica as host. Quantitative host range studies reveal that these genera have contrasting narrow (specialist) and broad (generalist) host ranges, with one‐step growth curves revealing reduced burst sizes for the generalist phages. Genomic comparisons suggest candidate genes in each genus that might explain this host range variation, as well as provide hypotheses about receptors in the hosts. One generalist phage, φ38:1, was more deeply characterized, as its infection strategy switched from lytic on its original host to either inefficient lytic or lysogenic on an alternative host. If lysogenic, this phage was maintained extrachromosomally in the alternative host and could not be induced by mitomycin C. This work provides fundamental knowledge regarding phage‐host ranges and their genomic drivers while also exploring the ‘host environment’ as a driver for switching phage replication mode.     

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.     

Morphological, Host Range, and Genetic Characterization of Two Coliphages

Two coliphages, AR1 and LG1, were characterized based on their morphological, host range, and genetic properties. Transmission electron microscopy showed that both phages belonged to the Myoviridae; phage particles of LG1 were smaller than those of AR1 and had an isometric head 68 nm in diameter and a complex contractile tail 111 nm in length. Transmission electron micrographs of AR1 showed phage particles consisting of an elongated isometric head of 103 by 74 nm and a complex contractile tail 116 nm in length. Both phages were extensively tested on many strains of Escherichia coli and other enterobacteria. The results showed that both phages could infect many serotypes of E. coli. Among the enterobacteria, Proteus mirabilis, Shigella dysenteriae, and two Salmonella strains were lysed by the phages. The genetic material of AR1 and LG1 was characterized. Phage LG1 had a genome size of 49.5 kb compared to 150 kb for AR1. Restriction endonuclease analysis showed that several restriction enzymes could degrade DNA from both phages. The morphological, genome size, and restriction endonuclease similarities between AR1 and phage T4 were striking. Southern hybridizations showed that AR1 and T4 are genetically related. The wide host ranges of phages AR1 and LG1 suggest that they may be useful as biocontrol, therapeutic, or diagnostic agents to control and detect the prevalence of E. coli in animals and food.     

Core and accessory genome architecture in a group of Pseudomonas aeruginosa Mu-like phages

Background

Bacteriophages that infect the opportunistic pathogen Pseudomonas aeruginosa have been classified into several groups. One of them, which includes temperate phage particles with icosahedral heads and long flexible tails, bears genomes whose architecture and replication mechanism, but not their nucleotide sequences, are like those of coliphage Mu. By comparing the genomic sequences of this group of P. aeruginosa phages one could draw conclusions about their ontogeny and evolution.

Results

Two newly isolated Mu-like phages of P. aeruginosa are described and their genomes sequenced and compared with those available in the public data banks. The genome sequences of the two phages are similar to each other and to those of a group of P. aeruginosa transposable phages. Comparing twelve of these genomes revealed a common genomic architecture in the group. Each phage genome had numerous genes with homologues in all the other genomes and a set of variable genes specific for each genome. The first group, which comprised most of the genes with assigned functions, was named “core genome”, and the second group, containing mostly short ORFs without assigned functions was called “accessory genome”. Like in other phage groups, variable genes are confined to specific regions in the genome.

Conclusion

Based on the known and inferred functions for some of the variable genes of the phages analyzed here, they appear to confer selective advantages for the phage survival under particular host conditions. We speculate that phages have developed a mechanism for horizontally acquiring genes to incorporate them at specific loci in the genome that help phage adaptation to the selective pressures imposed by the host.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1146) contains supplementary material, which is available to authorized users.     

Three Prochlorococcus cyanophage genomes: signature features and ecological interpretations

The oceanic cyanobacteria Prochlorococcus are globally important, ecologically diverse primary producers. It is thought that their viruses (phages) mediate population sizes and affect the evolutionary trajectories of their hosts. Here we present an analysis of genomes from three Prochlorococcus phages: a podovirus and two myoviruses. The morphology, overall genome features, and gene content of these phages suggest that they are quite similar to T7-like (P-SSP7) and T4-like (P-SSM2 and P-SSM4) phages. Using the existing phage taxonomic framework as a guideline, we examined genome sequences to establish “core” genes for each phage group. We found the podovirus contained 15 of 26 core T7-like genes and the two myoviruses contained 43 and 42 of 75 core T4-like genes. In addition to these core genes, each genome contains a significant number of “cyanobacterial” genes, i.e., genes with significant best BLAST hits to genes found in cyanobacteria. Some of these, we speculate, represent “signature” cyanophage genes. For example, all three phage genomes contain photosynthetic genes (psbA, hliP) that are thought to help maintain host photosynthetic activity during infection, as well as an aldolase family gene (talC) that could facilitate alternative routes of carbon metabolism during infection. The podovirus genome also contains an integrase gene (int) and other features that suggest it is capable of integrating into its host. If indeed it is, this would be unprecedented among cultured T7-like phages or marine cyanophages and would have significant evolutionary and ecological implications for phage and host. Further, both myoviruses contain phosphate-inducible genes (phoH and pstS) that are likely to be important for phage and host responses to phosphate stress, a commonly limiting nutrient in marine systems. Thus, these marine cyanophages appear to be variations of two well-known phages—T7 and T4—but contain genes that, if functional, reflect adaptations for infection of photosynthetic hosts in low-nutrient oceanic environments.     

Genomic,Proteomic, Morphological,and Phylogenetic Analyses of vB_EcoP_SU10, a Podoviridae Phage with C3 Morphology

A recently isolated phage, vB_EcoP_SU10 (SU10), with the unusual elongated C3 morphotype, can infect a wide range of Escherichia coli strains. We have sequenced the genome of this phage and characterized it further by mass spectrometry based proteomics, transmission electron microscopy (TEM), scanning electron microscopy (SEM), and ultra-thin section electron microscopy. The genome size is 77,327 base pairs and its genes, and genome architecture, show high similarity to the phiEco32 phage genes and genome. The TEM images reveal that SU10 have a quite long tail for being a Podoviridae phage, and that the tail also changes conformation upon infection. The ultra-thin section electron microscopy images of phages at the stage of replication within the host cell show that the phages form a honeycomb-like structure under packaging of genomes and assembly of mature capsids. This implies a tight link between the replication and cutting of the concatemeric genome, genome packaging, and capsid assembly. We have also performed a phylogenetic analysis of the structural genes common between Podoviridae phages of the C1 and C3 morphotypes. The result shows that the structural genes have coevolved, and that they form two distinct groups linked to their morphotypes. The structural genes of C1 and C3 phages appear to have diverged around 280 million years ago applying a molecular clock calibrated according to the presumed split between the Escherichia – Salmonella genera.     

Silage Collected from Dairy Farms Harbors an Abundance of Listeriaphages with Considerable Host Range and Genome Size Diversity

Since the food-borne pathogen Listeria monocytogenes is common in dairy farm environments, it is likely that phages infecting this bacterium (“listeriaphages”) are abundant on dairy farms. To better understand the ecology and diversity of listeriaphages on dairy farms and to develop a diverse phage collection for further studies, silage samples collected on two dairy farms were screened for L. monocytogenes and listeriaphages. While only 4.5% of silage samples tested positive for L. monocytogenes, 47.8% of samples were positive for listeriaphages, containing up to >1.5 × 10⁴ PFU/g. Host range characterization of the 114 phage isolates obtained, with a reference set of 13 L. monocytogenes strains representing the nine major serotypes and four lineages, revealed considerable host range diversity; phage isolates were classified into nine lysis groups. While one serotype 3c strain was not lysed by any phage isolates, serotype 4 strains were highly susceptible to phages and were lysed by 63.2 to 88.6% of phages tested. Overall, 12.3% of phage isolates showed a narrow host range (lysing 1 to 5 strains), while 28.9% of phages represented broad host range (lysing ≥11 strains). Genome sizes of the phage isolates were estimated to range from approximately 26 to 140 kb. The extensive host range and genomic diversity of phages observed here suggest an important role of phages in the ecology of L. monocytogenes on dairy farms. In addition, the phage collection developed here has the potential to facilitate further development of phage-based biocontrol strategies (e.g., in silage) and other phage-based tools.     

Genome sequence of a novel deep-sea vent epsilonproteobacterial phage provides new insight into the co-evolution of Epsilonproteobacteria and their phages

Epsilonproteobacteria are among the predominant primary producers in deep-sea hydrothermal vent ecosystems. However, phages infecting deep-sea vent Epsilonproteobacteria have never been isolated and characterized. Here, we successfully isolated a novel temperate phage, NrS-1, that infected a deep-sea vent chemolithoautotrophic isolate of Epsilonproteobacteria, Nitratiruptor sp. SB155-2, and its entire genome sequence was obtained and analyzed. The NrS-1 genome is linear, circularly permuted, and terminally redundant. The NrS-1 genome is 37,159 bp in length and contains 51 coding sequences. Five major structural proteins including major capsid protein and tape measure protein were identified by SDS-PAGE and mass spectrometry analysis. NrS-1 belongs to the family Siphoviridae, but its sequence and genomic organization are distinct from those of any other previously known Siphoviridae phages. Homologues of genes encoded in the NrS-1 genome were widely distributed among the genomes of diverse Epsilonproteobacteria. The distribution patterns had little relation to the evolutionary traits and ecological and physiological differentiation of the host epsilonproteobacterial species. The widespread occurrence of phage genes in diverse Epsilonproteobacteria supports early co-evolution between temperate phages and Epsilonproteobacteria prior to the divergence of their habitats and physiological adaptation.     

Phage morphology recapitulates phylogeny: the comparative genomics of a new group of myoviruses

Among dsDNA tailed bacteriophages (Caudovirales), members of the Myoviridae family have the most sophisticated virion design that includes a complex contractile tail structure. The Myoviridae generally have larger genomes than the other phage families. Relatively few "dwarf" myoviruses, those with a genome size of less than 50 kb such as those of the Mu group, have been analyzed in extenso. Here we report on the genome sequencing and morphological characterization of a new group of such phages that infect a diverse range of Proteobacteria, namely Aeromonas salmonicida phage 56, Vibrio cholerae phages 138 and CP-T1, Bdellovibrio phage φ1422, and Pectobacterium carotovorum phage ZF40. This group of dwarf myoviruses shares an identical virion morphology, characterized by usually short contractile tails, and have genome sizes of approximately 45 kb. Although their genome sequences are variable in their lysogeny, replication, and host adaption modules, presumably reflecting differing lifestyles and hosts, their structural and morphogenesis modules have been evolutionarily constrained by their virion morphology. Comparative genomic analysis reveals that these phages, along with related prophage genomes, form a new coherent group within the Myoviridae. The results presented in this communication support the hypothesis that the diversity of phages may be more structured than generally believed and that the innumerable phages in the biosphere all belong to discrete lineages or families.     

Genomic analysis of cold-active Colwelliaphage 9A and psychrophilic phage�Chost interactions

The 104 kb genome of cold-active bacteriophage 9A, which replicates in the marine psychrophilic gamma-proteobacterium Colwellia psychrerythraea strain 34H (between ?12 and 8 °C), was sequenced and analyzed to investigate elements of molecular adaptation to low temperature and phage?Chost interactions in the cold. Most characterized ORFs indicated closest similarity to gamma-proteobacteria and their phages, though no single module provided definitive phylogenetic grouping. A subset of primary structural features linked to psychrophily suggested that the majority of annotated phage proteins were not psychrophilic; those that were, primarily serve phage-specific functions and may also contribute to 9A??s restricted temperature range for replication as compared to host. Comparative analyses suggest ribonucleotide reductase genes were acquired laterally from host. Neither restriction modification nor the CRISPR-Cas system appeared to be the predominant phage defense mechanism of Cp34H or other cold-adapted bacteria; we hypothesize that psychrophilic hosts rely more on the use of extracellular polymeric material to block cell surface receptors recognized by phages. The relative dearth of evidence for genome-specific defenses, genetic transfer events or auxiliary metabolic genes suggest that the 9A-Cp34H system may be less tightly coupled than are other genomically characterized marine phage?Chost systems, with possible implications for phage specificity under different environmental conditions.     

Widespread phages of endosymbionts: Phage WO genomics and the proposed taxonomic classification of Symbioviridae

Wolbachia are the most common obligate, intracellular bacteria in animals. They exist worldwide in arthropod and nematode hosts in which they commonly act as reproductive parasites or mutualists, respectively. Bacteriophage WO, the largest of Wolbachia’s mobile elements, includes reproductive parasitism genes, serves as a hotspot for genetic divergence and genomic rearrangement of the bacterial chromosome, and uniquely encodes a Eukaryotic Association Module with eukaryotic-like genes and an ensemble of putative host interaction genes. Despite WO’s relevance to genome evolution, selfish genetics, and symbiotic applications, relatively little is known about its origin, host range, diversification, and taxonomic classification. Here we analyze the most comprehensive set of 150 Wolbachia and phage WO assemblies to provide a framework for discretely organizing and naming integrated phage WO genomes. We demonstrate that WO is principally in arthropod Wolbachia with relatives in diverse endosymbionts and metagenomes, organized into four variants related by gene synteny, often oriented opposite the putative origin of replication in the Wolbachia chromosome, and the large serine recombinase is an ideal typing tool to distinguish the four variants. We identify a novel, putative lytic cassette and WO’s association with a conserved eleven gene island, termed Undecim Cluster, that is enriched with virulence-like genes. Finally, we evaluate WO-like Islands in the Wolbachia genome and discuss a new model in which Octomom, a notable WO-like Island, arose from a split with WO. Together, these findings establish the first comprehensive Linnaean taxonomic classification of endosymbiont phages, including non-Wolbachia phages from aquatic environments, that includes a new family and two new genera to capture the collective relatedness of these viruses.     

Genome-informed approach to identify genetic determinants of Flavobacterium psychrophilum phage susceptibility

The fish pathogen Flavobacterium psychrophilum infects farmed salmonids worldwide, and application of bacteriophages has been suggested for controlling disease outbreaks in aquaculture. Successful application of phages requires detailed knowledge about the variability in phage susceptibility of the host communities. In this study, we analysed the genetic diversity of F. psychrophilum hosts and phages from the Baltic Sea area to identify genetic determinants of phage-host interaction patterns. A host range analysis of 103 phages tested against 177 F. psychrophilum strains (18 231 phage–host interactions) identified nine phage clusters, infecting from 10% to 91% of the strain collection. The core genome-based comparison of 35 F. psychrophilum isolates revealed an extremely low overall genomic diversity (>99.5% similarity). However, a small subset of 16 ORFs, including genes involved in the type IX secretion system (T9SS), gliding motility and hypothetical cell-surface related proteins, exhibited a highly elevated genetic diversity. These specific genetic variations were linked to variability in phage infection patterns obtained from experimental studies, indicating that these genes are key determinants of phage susceptibility. These findings provide novel insights on the molecular mechanisms determining phage susceptibility in F. psychrophilum and emphasizes the importance of phages as drivers of core genomic diversity in this pathogen.     

Phage response to CRISPR-encoded resistance in Streptococcus thermophilus

Clustered regularly interspaced short palindromic repeats (CRISPR) and their associated genes are linked to a mechanism of acquired resistance against bacteriophages. Bacteria can integrate short stretches of phage-derived sequences (spacers) within CRISPR loci to become phage resistant. In this study, we further characterized the efficiency of CRISPR1 as a phage resistance mechanism in Streptococcus thermophilus. First, we show that CRISPR1 is distinct from previously known phage defense systems and is effective against the two main groups of S. thermophilus phages. Analyses of 30 bacteriophage-insensitive mutants of S. thermophilus indicate that the addition of one new spacer in CRISPR1 is the most frequent outcome of a phage challenge and that the iterative addition of spacers increases the overall phage resistance of the host. The added new spacers have a size of between 29 to 31 nucleotides, with 30 being by far the most frequent. Comparative analysis of 39 newly acquired spacers with the complete genomic sequences of the wild-type phages 2972, 858, and DT1 demonstrated that the newly added spacer must be identical to a region (named proto-spacer) in the phage genome to confer a phage resistance phenotype. Moreover, we found a CRISPR1-specific sequence (NNAGAAW) located downstream of the proto-spacer region that is important for the phage resistance phenotype. Finally, we show through the analyses of 20 mutant phages that virulent phages are rapidly evolving through single nucleotide mutations as well as deletions, in response to CRISPR1.     

A Virulent Phage Infecting Lactococcus garvieae,with Homology to Lactococcus lactis Phages

A new virulent phage belonging to the Siphoviridae family and able to infect Lactococcus garvieae strains was isolated from compost soil. Phage GE1 has a prolate capsid (56 by 38 nm) and a long noncontractile tail (123 nm). It had a burst size of 139 and a latent period of 31 min. Its host range was limited to only two L. garvieae strains out of 73 tested. Phage GE1 has a double-stranded DNA genome of 24,847 bp containing 48 predicted open reading frames (ORFs). Putative functions could be assigned to only 14 ORFs, and significant matches in public databases were found for only 17 ORFs, indicating that GE1 is a novel phage and its genome contains several new viral genes and encodes several new viral proteins. Of these 17 ORFs, 16 were homologous to deduced proteins of virulent phages infecting the dairy bacterium Lactococcus lactis, including previously characterized prolate-headed phages. Comparative genome analysis confirmed the relatedness of L. garvieae phage GE1 to L. lactis phages c2 (22,172 bp) and Q54 (26,537 bp), although its genome organization was closer to that of phage c2. Phage GE1 did not infect any of the 58 L. lactis strains tested. This study suggests that phages infecting different lactococcal species may have a common ancestor.     

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.     

Life-Style and Genome Structure of Marine Pseudoalteromonas Siphovirus B8b Isolated from the Northwestern Mediterranean Sea

Marine viruses (phages) alter bacterial diversity and evolution with impacts on marine biogeochemical cycles, and yet few well-developed model systems limit opportunities for hypothesis testing. Here we isolate phage B8b from the Mediterranean Sea using Pseudoalteromonas sp. QC-44 as a host and characterize it using myriad techniques. Morphologically, phage B8b was classified as a member of the Siphoviridae family. One-step growth analyses showed that this siphovirus had a latent period of 70 min and released 172 new viral particles per cell. Host range analysis against 89 bacterial host strains revealed that phage B8b infected 3 Pseudoalteromonas strains (52 tested, >99.9% 16S rRNA gene nucleotide identity) and 1 non-Pseudoaltermonas strain belonging to Alteromonas sp. (37 strains from 6 genera tested), which helps bound the phylogenetic distance possible in a phage-mediated horizontal gene transfer event. The Pseudoalteromonas phage B8b genome size was 42.7 kb, with clear structural and replication modules where the former were delineated leveraging identification of 16 structural genes by virion structural proteomics, only 4 of which had any similarity to known structural proteins. In nature, this phage was common in coastal marine environments in both photic and aphotic layers (found in 26.5% of available viral metagenomes), but not abundant in any sample (average per sample abundance was 0.65% of the reads). Together these data improve our understanding of siphoviruses in nature, and provide foundational information for a new ‘rare virosphere’ phage–host model system.     

Characterization of the Newly Isolated Lytic Bacteriophages KTN6 and KT28 and Their Efficacy against Pseudomonas aeruginosa Biofilm

We here describe two novel lytic phages, KT28 and KTN6, infecting Pseudomonas aeruginosa, isolated from a sewage sample from an irrigated field near Wroclaw, in Poland. Both viruses show characteristic features of Pbunalikevirus genus within the Myoviridae family with respect to shape and size of head/tail, as well as LPS host receptor recognition. Genome analysis confirmed the similarity to other PB1-related phages, ranging between 48 and 96%. Pseudomonas phage KT28 has a genome size of 66,381 bp and KTN6 of 65,994 bp. The latent period, burst size, stability and host range was determined for both viruses under standard laboratory conditions. Biofilm eradication efficacy was tested on peg-lid plate assay and PET membrane surface. Significant reduction of colony forming units was observed (70-90%) in 24 h to 72 h old Pseudomonas aeruginosa PAO1 biofilm cultures for both phages. Furthermore, a pyocyanin and pyoverdin reduction tests reveal that tested phages lowers the amount of both secreted dyes in 48-72 h old biofilms. Diffusion and goniometry experiments revealed the increase of diffusion rate through the biofilm matrix after phage application. These characteristics indicate these phages could be used to prevent Pseudomonas aeruginosa infections and biofilm formation. It was also shown, that PB1-related phage treatment of biofilm caused the emergence of stable phage-resistant mutants growing as small colony variants.     

Characterization of Novel Phages Isolated in Coagulase-Negative Staphylococci Reveals Evolutionary Relationships with Staphylococcus aureus Phages

Despite increasing interest in coagulase-negative staphylococci (CoNS), little information is available about their bacteriophages. We isolated and sequenced three novel temperate Siphoviridae phages (StB12, StB27, and StB20) from the CoNS Staphylococcus hominis and S. capitis species. The genome sizes are around 40 kb, and open reading frames (ORFs) are arranged in functional modules encoding lysogeny, DNA metabolism, morphology, and cell lysis. Bioinformatics analysis allowed us to assign a potential function to half of the predicted proteins. Structural elements were further identified by proteomic analysis of phage particles, and DNA-packaging mechanisms were determined. Interestingly, the three phages show identical integration sites within their host genomes. In addition to this experimental characterization, we propose a novel classification based on the analysis of 85 phage and prophage genomes, including 15 originating from CoNS. Our analysis established 9 distinct clusters and revealed close relationships between S. aureus and CoNS phages. Genes involved in DNA metabolism and lysis and potentially in phage-host interaction appear to be widespread, while structural genes tend to be cluster specific. Our findings support the notion of a possible reciprocal exchange of genes between phages originating from S. aureus and CoNS, which may be of crucial importance for pathogenesis in staphylococci.