Subfamily Autographivirinae Research Articles

Characterization of Pseudomonas aeruginosa Bacteriophage L5 Which Requires Type IV Pili for Infection.

Pseudomonas aeruginosa is a common opportunistic human pathogen. With the emergence of multidrug-resistant (MDR) clinical infection of P. aeruginosa, phage therapy has received renewed attention in treating P. aeruginosa infections. Moreover, a detailed understanding of the host receptor of lytic phage is crucial for selecting proper phages for therapy. Here, we describe the characterization of the P. aeruginosa bacteriophage L5 with a double-stranded DNA genome of 42,925 bp. The genomic characteristics indicate that L5 is a lytic bacteriophage belonging to the subfamily Autographivirinae. In addition, the phage receptors for L5 were also identified as type IV pili, because the mutation of pilZ, which is involved in pili synthesis, resists phage infection, while the complementation of pilZ restored its phage sensitivity. This research reveals that L5 is a potential phage therapy candidate for the treatment of P. aeruginosa infection.

Isolation and Characterization of Bacteriophage Against Wastewater Isolates of Methicillin-resistant Staphylococcus aureus

Background: Methicillin-resistant Staphylococcus aureus (MRSA) not only is a notorious pathogen in clinical settings but also is an environmental issue that its presence in environmental wastewater is highlighted by several reports. Due to the negative impacts of antibiotics, alternatives like bacteriophages, as biocontrol, are considered safe. However, not all bacteriophages are safe. Thus, the characterization of bacteriophages is necessary. Objectives: This study aimed to, firstly isolate MRSA from wastewater and, secondly to perform bacteriophage isolation from the water samples to investigate its physical and genomic characteristics. Methods: Water samples were collected from seven locations across Nagpur city, India, bacteria were isolated on the S. aureus specific agar. For detecting MRSA, we followed the disc diffusion method. Isolation of bacteriophage against MRSA was performed by a modified enrichment method. We investigated its physical characteristics by the one-step growth rate, adsorption rate, host range, survivability, electron microscopy, and genomic sequencing for bioinformatics analysis. Results: Four MRSA were isolated from wastewater samples. We got a bacteriophage against an MRSA from the river Ganga. The bacteriophage belongs to the Podoviridae family, subfamily Autographivirinae. It was stable till 40°C and could survive at a highly alkaline pH. It is specific to its host. The bacteriophage DNA encodes 52 ORF, and all predicted genes are on the same strand; it also encodes a phage RNA polymerase. Conclusions: It is the first report of an S. aureus bacteriophage that belongs to the sub-family Autographivirinae. Our study and literature survey conclude that S. aureus bacteriophages of the Podoviridae family are safe for various downstream applications.

Characterization and genome analysis of a novel Vibrio parahaemolyticus phage vB_VpP_DE17

A novel phage vB_VpP_DE17, which infects Vibrio parahaemolyticus, was isolated from the sewer of the Huangsha aquatic market in Guangzhou. Transmission electron microscopy indicated that DE17 had an icosahedral head (47 ± 2 nm diameter) and a short, non-contractile tail (17 ± 2 nm). The genome of DE17 was a double-stranded linear DNA with a length of 43,397 bp and GC content of 49.23%. In total, 49 putative open reading frames (ORFs) were predicted and could be divided into six modules: DNA metabolism, lysis, packaging, structure, additional function, and hypothetical proteins. Taxonomic analysis revealed that the phage belonging to the genus of Maculvirus, Autographivirinae subfamily, Podoviridae family. DE17 had a short latent period of 5 min with burst size of 80 pfu/cell. Its optimum temperature and pH ranges were 4 °C–50 °C and 5–10, respectively; it was completely inactivated after 20 min of ultraviolet irradiation. No transfer RNA (tRNA), virulence associated, or antibiotic resistance genes were identified. Bacterial challenge test revealed that DE17 had a certain inhibitory effect on V. parahaemolyticus within 6 h. Characterization, genomic analysis and in vitro antibacterial assays of DE17 will further enhance our understanding of phage biology and diversity.

Characterization of vB_VpaP_MGD2, a newly isolated bacteriophage with biocontrol potential against multidrug-resistant Vibrio parahaemolyticus.

Vibrio parahaemolyticus is a major foodborne pathogen and is also pathogenic to shrimp. Due to the emergence of multidrug-resistant V. parahaemolyticus strains, bacteriophages have shown promise as antimicrobial agents that could be used for controlling antibiotic-resistant strains. Here, a V. parahaemolyticus phage, vB_VpaP_MGD2, was isolated from a clam (Meretrix meretrix) and further characterized to evaluate its potential capability for biocontrol. Podophage vB_VpaP_MGD2 had a wide host range and was able to lyse 27 antibiotic-resistant V. parahaemolyticus strains. A one-step growth curve showed that vB_VpaP_MGD2 has a short latent period of 10 min and a large burst size of 244 phages per cell. Phage vB_VpaP_MGD2 was able to tolerate a wide range of temperature (30°C-50°C) and pH (pH 3-pH 10). Two multidrug-resistant strains (SH06 and SA411) were suppressed by treatment with phagevB_VpaP_MGD2 at a multiplicity of infection of 100 for 24 h without apparent regrowth of bacterial populations. The frequency of mutations causing bacteriophage resistance was relatively low (3.1 × 10-6). Phage vB_VpaP_MGD2 has a double-stranded DNA with a genome size of 45,105 bp. Among the 48 open reading frames annotated in the genome, no lysogenic genes or virulence genes were detected. Sequence comparisons suggested that vB_VpaP_MGD2 is a member of a new species in the genus Zindervirus within the subfamily Autographivirinae. This is the first report of a member of the genus Zindervirus that can infect V. parahaemolyticus. These findings suggest that vB_VpaP_MGD2 may be a candidate biocontrol agent against early mortality syndrome/acute hepatopancreatic necrosis disease (EMS/AHPND) caused by multidrug-resistant V. parahaemolyticus in shrimp production.

Application of a novel phage ZPAH7 for controlling multidrug-resistant Aeromonas hydrophila on lettuce and reducing biofilms

Aeromonas hydrophila is an important pathogenic bacterium that causes foodborne illness worldwide. In this study, virulent phages from the sediment of a fish farm were propagated and isolated on a multidrug-resistant strain of A. hydrophila, ZYAH75. One phage, designated as ZPAH7, featured a unique turbid halo around a clear plaque on the bacterial lawn (indicative of potential depolymerase activity), and was selected for further analysis. ZPAH7 was classified as podophage by morphological and genomic methods. Further comparisons of genome nucleotide similarity, ratios of homologous proteins and phylogenetic relatedness among the terminase large subunit and major capsid proteins of similar phage deposited in GENBANK, led us to propose a new genus, ZPAH7virus, in the Autographivirinae subfamily of Podoviridae. ZPAH7 had an adsorption rate of 79% in 5 min, an eclipse period of 15 min, a latent period of 25 min, and a burst size of 148 ± 9 PFU/cell. Antimicrobial application experiments showed that ZPAH7 lead to significantly reduction on A. hydrophila on lettuce. Additionally, ZPAH7 was able to inhibit biofilm formation, as well as degrade and kill bacteria in established biofilms. Furthermore, lytic activity of ZPAH7 remained stable across a wide range of temperatures and pH measurements. These results suggest ZPAH7 could be used as a potential biological control agent against A. hydrophila on food and/or biofilms on food contact surfaces.

The Podovirus ϕ80-18 Targets the Pathogenic American Biotype 1B Strains of Yersinia enterocolitica.

We report here the complete genome sequence and characterization of Yersinia bacteriophage vB_YenP_ϕ80-18. ϕ80-18 was isolated in 1991 using a Y. enterocolitica serotype O:8 strain 8081 as a host from a sewage sample in Turku, Finland, and based on its morphological and genomic features is classified as a podovirus. The genome is 42 kb in size and has 325 bp direct terminal repeats characteristic for podoviruses. The genome contains 57 predicted genes, all encoded in the forward strand, of which 29 showed no similarity to any known genes. Phage particle proteome analysis identified altogether 24 phage particle-associated proteins (PPAPs) including those identified as structural proteins such as major capsid, scaffolding and tail component proteins. In addition, also the DNA helicase, DNA ligase, DNA polymerase, 5′-exonuclease, and the lytic glycosylase proteins were identified as PPAPs, suggesting that they might be injected together with the phage genome into the host cell to facilitate the take-over of the host metabolism. The phage-encoded RNA-polymerase and DNA-primase were not among the PPAPs. Promoter search predicted the presence of four phage and eleven host RNA polymerase –specific promoters in the genome, suggesting that early transcription of the phage is host RNA-polymerase dependent and that the phage RNA polymerase takes over later. The phage tolerates pH values between 2 and 12, and is stable at 50°C but is inactivated at 60°C. It grows slowly with a 50 min latent period and has apparently a low burst size. Electron microscopy revealed that the phage has a head diameter of about 60 nm, and a short tail of 20 nm. Whole-genome phylogenetic analysis confirmed that ϕ80-18 belongs to the Autographivirinae subfamily of the Podoviridae family, that it is 93.2% identical to Yersinia phage fHe-Yen3-01. Host range analysis showed that ϕ80-18 can infect in addition to Y. enterocolitica serotype O:8 strains also strains of serotypes O:4, O:4,32, O:20 and O:21, the latter ones representing similar to Y. enterocolitica serotype O:8, the American pathogenic biotype 1B strains. In conclusion, the phage ϕ80-18 is a promising candidate for the biocontrol of the American biotype 1B Y. enterocolitica.

Autographivirinae Bacteriophage Arno 160 Infects Pectobacterium carotovorum via Depolymerization of the Bacterial O-Polysaccharide.

Phytopathogenic bacteria belonging to the Pectobacterium and Dickeya genera (soft-rot Pectobacteriaceae) are in the focus of agriculture-related microbiology because of their diversity, their substantial negative impact on the production of potatoes and vegetables, and the prospects of bacteriophage applications for disease control. Because of numerous amendments in the taxonomy of P. carotovorum, there are still a few studied sequenced strains among this species. The present work reports on the isolation and characterization of the phage infectious to the type strain of P. carotovorum. The phage Arno 160 is a lytic Podovirus representing a potential new genus of the subfamily Autographivirinae. It recognizes O-polysaccahride of the host strain and depolymerizes it in the process of infection using a rhamnosidase hydrolytic mechanism. Despite the narrow host range of this phage, it is suitable for phage control application.

Characterization and genome sequencing of a novel T7-like lytic phage, kpssk3, infecting carbapenem-resistant Klebsiella pneumoniae.

Carbapenem-resistant Klebsiella pneumoniae (CRKP) has spread globally and emerged as an urgent public health threat. Bacteriophages are considered an effective weapon against multidrug-resistant pathogens. In this study, we report a novel lytic phage, kpssk3, which is able to lyse CRKP and degrade exopolysaccharide (EPS). The morphological characteristics of kpssk3 observed by transmission electron microscopy, including a polyhedral head and a short tail, indicate that it belongs to the family Podoviridae. A one-step growth curve revealed that kpssk3 has a latent period of 10 min and a burst size of 200 plaque-forming units (pfu) per cell. kpssk3 was able to lyse 25 out of 27 (92.59%) clinically isolated CRKP strains, and it also exhibited high stability to changes in temperature and pH. kpssk3 has a linear dsDNA genome of 40,539 bp with 52.80% G+C content and 42 putative open reading frames (ORFs). No antibiotic resistance genes, virulence factors, or integrases were identified in the genome. Based on bioinformatic analysis, the tail fiber protein of phage kpssk3 was speculated to possess depolymerase activity towards EPS. By comparative genomics and phylogenetic analysis, it was determined that kpssk3 is a new T7-like virus and belongs to the subfamily Autographivirinae. The characterization and genomic analysis of kpssk3 will promote our understanding of phage biology and diversity and provide a potential strategy for controlling CRKP infection.

Characterization of Pectobacterium carotovorum subsp. carotovorum Bacteriophage PP16 Prospective for Biocontrol of Potato Soft Rot

Phages of the phytopathogenic Pectobacteriaceae species causing black leg and soft rot of potato were investigated. These phages are promising as biocontrol agents to prevent the loss of seed and ware potato tubers. The present work characterizes a new podovirus PP16, infecting a broad range of Pectobacterium carotovorum strains. Based on its genomic composition, phage PP16 was assigned to a separate phylogenetic branch of the genus Phimunavirus, subfamily Autographivirinae. Bacteriophage PP16 efficiently inhibited development of bacterial infection both in vitro and in planta. The field experiment demonstrated a substantial increase of plant germination after the treatment of seed potato with phage PP16.

New Bacteriophages against Emerging Lineages ST23 and ST258 of Klebsiella pneumoniae and Efficacy Assessment in Galleria mellonella Larvae.

Klebsiella pneumoniae is a bacterial pathogen of high public health importance. Its polysaccharide capsule is highly variable but only a few capsular types are associated with emerging pathogenic sublineages. The aim of this work is to isolate and characterize new lytic bacteriophages and assess their potential to control infections by the ST23 and ST258 K. pneumoniae sublineages using a Galleria mellonella larvae model. Three selected bacteriophages, targeting lineages ST258 (bacteriophages vB_KpnP_KL106-ULIP47 and vB_KpnP_KL106-ULIP54) and ST23 (bacteriophage vB_KpnP_K1-ULIP33), display specificity for capsular types KL106 and K1, respectively. These podoviruses belong to the Autographivirinae subfamily and their genomes are devoid of lysogeny or toxin-associated genes. In a G. mellonella larvae model, a mortality rate of 70% was observed upon infection by K. pneumoniae ST258 and ST23. This number was reduced to 20% upon treatment with bacteriophages at a multiplicity of infection of 10. This work increases the number of characterized bacteriophages infecting K. pneumoniae and provides information regarding genome sequence and efficacy during preclinical phage therapy against two prominent sublineages of this bacterial species.

Characterization and genome analysis of a novel bacteriophage vB_SpuP_Spp16 that infects Salmonella enterica serovar pullorum.

A novel virulent bacteriophage vB_SpuP_Spp16 (hereafter designated Spp16) that infects Salmonella enterica serovar pullorum was isolated. Transmission electron microscopy showed that Spp16 possessed an isometric polyhedral head (60nm in diameter) and a short tail (10nm in length) belonging to the family Podoviridae. Its complete genome was determined to be 41,832bp, with a 39.46% GC content by next-generation sequencing. The genome contains 53 proposed open reading frames that are involved in DNA replication and modification, transcriptional regulation, phage structural and packaging proteins and bacterial lysis. No transfer RNA genes were identified. The termini of genome were determined using our previously proposed termini identification method, which suggests that this phage has redundant termini with 421bp direct terminal repeats. BLASTn analysis revealed the highest sequence similarity with Yersinia phage phi80-18, with a genome coverage of 33% and highest sequence identity of 69%. The phylogenetic analysis indicated that Spp16 forms a distinct branch of the subfamily Autographivirinae. Comparative genomics analysis showed that the phage Spp16 should be regarded as a new subcluster within the GAP227-like cluster in the Autographivirinae subfamily. The phage Spp16 has an obligate lytic life cycle demonstrated by experimental data and genomic analysis. These results suggest that Spp16 may be a proper candidate to control diseases caused by Salmonella enterica serovar pullorum.

Characterization of Dev-CD-23823 and Dev-CT57, new Autographivirinae bacteriophages infecting Cronobacter spp.

Cronobacter spp. are opportunistic pathogenic bacteria responsible for severe infections in neonates. Powdered infant formula has been confirmed to be the source of infection in some cases. Bacteriophages offer a safe means for eliminating this pathogen. In the present study, we characterized two closely related Cronobacter-specific bacteriophages of the proposed genus "GAP227virus". The phages Dev-CD-23823 and Dev-CT57 possessed broad host specificity, as they infected 88% and 80% of the Cronobacter strains tested. Genome sequence comparisons of phages Dev-CD-23823 and Dev-CT57 showed different levels of similarity to the prototype GAP227 phage. The Dev-CT57 phage was highly similar, whereas the Dev-CD-23823 phage showed only 75% sequence identity. A phylogenic tree based on the RNA polymerase (RNAP) gene from selected representatives of the subfamily Autographivirinae confirmed the grouping of Dev-CD-23823, Dev-CT57 and GAP227 in one cluster together with phages PP2, Phi80-18 and PhiR8-01. A common conserved motif was also detected in the RNAP promoters of these phages. The functional activity of these RNAP promoters was confirmed experimentally using a promoter probe vector, and a phage-specific signal was observed; however, some cross-specificity of Dev-CD-23823 and Dev-CT57 promoters was also detected. These results will contribute to our understanding of the biology and evolution of Autographivirinae phages.

Characterization and Genomic Analysis of SFPH2, a Novel T7virus Infecting Shigella.

Shigellosis, caused by Shigella, is a major global health concern, with nearly 164.7 million cases and over a million deaths occurring annually worldwide. Shigella flexneri is one of the most common subgroups of Shigella with a high incidence of multidrug-resistance. The phage therapy approach is an effective method for controlling multidrug-resistant bacteria. However, only a few Shigella phages have been described to date. In this study, a novel lytic bacteriophage SFPH2 was isolated from a sewage sample obtained from a hospital in Beijing, China, using a multidrug-resistant S. flexneri 2a strain (SF2) isolated from the fecal sample of a dysentery patient. SFPH2 is a member of the Podoviridae virus family with an icosahedral capsid and a short, non-contractile tail. It was found to be stable over a wide range of temperatures (4–50°C) and pH values (pH 3–11). Moreover, SFPH2 could infect two other S. flexneri serotypes (serotypes 2 variant and Y). High-throughput sequencing revealed that SFPH2 has a linear double-stranded DNA genome of 40,387 bp with 50 open reading frames. No tRNA genes were identified in the genome. Comparative analysis of the genome revealed that the SFPH2 belongs to the subfamily Autographivirinae and genus T7virus. The genome shows high similarity with other enterobacterial T7virus bacteriophages such as Citrobacter phage SH4 (95% identity and 89% coverage) and Cronobacter phage Dev2 (94% identity and 92% coverage). A comparison of the fiber proteins showed that minor differences in the amino acid residues might specify different protein binding regions and determine host species. In conclusion, this is the first report of a T7virus that can infect Shigella; SFPH2 has a functional stability under a wide range of temperatures and pH values, showing the potential to be widely applied to control Shigella–associated clinical infections and reduce the transmission rates of S. flexneri serotype 2a and its variants in the environment.

Complete genomic sequence of the Vibrio alginolyticus bacteriophage Vp670 and characterization of the lysis-related genes, cwlQ and holA

BackgroundBiocontrol of bacterial pathogens by bacteriophages (phages) represents a promising strategy. Vibrio alginolyticus, a gram-negative bacterium, is a notorious pathogen responsible for the loss of economically important farmed marine animals. To date, few V. alginolyticus phages have been successfully isolated, and only three complete genome sequences of them have been released. The limited available phage resources and poor genomic data hamper research on V. alginolyticus phages and their applications for the biocontrol of V. alginolyticus.ResultsWe isolated a phage, Vp670, against the V. alginolyticus strain E06333 and obtained its full genomic sequence. It contains 43,121 nucleotides with a GC content of 43.4%, and codes for 49 predicted open reading frames. Observation by electron microscope combined with phylogenetic analysis of DNA polymerase indicates that Vp670 belongs to the subfamily Autographivirinae in the family Podoviridae. orf3 (designated holA) and orf8 (designated cwlQ) are predicted to encode a holin (HolA) and an endolysin (CwlQ), respectively. Expression of holA alone or coexpression of holA and cwlQ from within arrested the growth of Escherichia coli and V. alginolyticus while the expression of cwlQ alone had no effect on the growth of them. Further observation by transmission electron microscopy revealed that the expression of holA vanished the outer membrane and caused the release of cellular contents of V. alginolyticus and the coexpression of holA and cwlQ directly burst the cells and caused a more drastic release of cellular contents. Expression of cwlQ alone in V. alginolyticus did not cause cytomorphological changes.ConclusionsPhage Vp670 is a V. alginolyticus phage belonging to the family of Podoviridae. The genome of Vp670 contains a two-component lysis module, which is comprised of holA and cwlQ. holA is predicted to encode for the holin protein, HolA, and cwlQ is predicted to encode for the endolysin protein, CwlQ. Both holA and cwlQ likely play important roles during the release of phage progeny.

Identification of a bacteriophage from an environmental multidrug-resistant E. coli isolate and its function in horizontal transfer of ARGs

Horizontal transfer of ARGs was generally considered to be mediated by three methods – transformation, conjugation and transduction through phages – during which the contribution of bacteriophages to gene transfer in the environment is unclear or even questioned. In this study, a multiple-antibiotic-resistant Escherichia coli strain and its phage (YZ1) were isolated from a municipal wastewater treatment system. The results of the morphological and genomic analyses of phage YZ1 showed that it is a member of the T7 viral genus in the subfamily Autographivirinae. Its genome is similar to that of the E. coli phage K1F in both organization and sequence and does not encode ARGs. However, 28 paired reads in the raw sequencing data aligned to ARGs, including those promoting β-lactam, aminoglycoside, and fluoroquinolone resistance, among others. Quantitative PCR showed that ARGs were present in bacteriophage DNA (approximately 103 copies/mL) and were also detected in the bacterial host DNA. The results suggested that while infrequent, some ARG-carrying transducing phages were presumably generated by erroneous packaging during infection of antibiotic-resistant bacteria, which may create the possibility of horizontal transfer of ARGs.

Isolation and characterization of a group of new Proteus bacteriophages.

Four lytic Proteus bacteriophages, PM75, PM85, PM93, and PM116, which are active against multi-drug-resistant strains of P. mirabilis, were isolated from cattle and poultry samples. According to electron microscopy data, all of the investigated phages belonged to the family Podoviridae. They all demonstrated lytic activity against sensitive strains of P. mirabilis, and three of the phages, PM85, PM93, and PM116, are potential candidates for use in antibacterial treatment. The genomes and putative proteins of bacteriophages PM85, PM93, and PM116 were similar to those of Proteus phage vB_PmiP_Pm5460 [KP890822], and the investigated phages formed a distinct clade within the genus Sp6virus, subfamily Autographivirinae. The genome sequence of phage PM75 was similar to that of a previously described Proteus phage, PM16 [KF319020], and both of them demonstrated low nucleotide sequence identity to the genomes of the other most similar phages, namely, Vibrio phage VP93, Pantoea phage LIMElight, and KP34-like bacteriophages. According to cluster analysis of the complete genome sequences and phylogenetic analysis of the proteins essential for their life cycle, phages PM75 and PM16 are distinct from other similar phages from the phiKMV supergroup and should be recognized as constituting a new genus, "Pm16virus", within the subfamily Autographivirinae.

Genomic characteristics of vB_PpaP_PP74, a T7-like Autographivirinae bacteriophage infecting a potato pathogen of the newly proposed species Pectobacterium parmentieri.

Bacteriophage vB_PpaP_PP74 (PP74) is a novel virulent phage that infects members of the species Pectobacterium parmentieri, a newly established species of soft-rot-causing bacteria in the family Pectobacteriaceae, derived from potato-specific Pectobacterium wasabiae. vB_PpaP_PP74 was identified as a member of the family Podoviridae by transmission electron microscopy. The phage has a 39,790-bp dsDNA genome containing 50 open reading frames (ORFs). Because of the absence of genes encoding toxins or lysogeny factors, PP74 may be considered a candidate phage for pathogen biocontrol applications. The genome layout is similar to genomes of T7-like phages within the subfamily Autographivirinae, and therefore, functions can be attributed to most of ORFs. However, the closest nucleotide sequence homologs of phage PP74 are unclassified Escherichia phages. Based on phylogenetic analysis, vB_PpaP_PP74 is a sensu lato T7-like phage, but it forms a distant subgenus group together with homologous enterobacterial phages.

The O-specific polysaccharide lyase from the phage LKA1 tailspike reduces Pseudomonas virulence

Pseudomonas phage LKA1 of the subfamily Autographivirinae encodes a tailspike protein (LKA1gp49) which binds and cleaves B-band LPS (O-specific antigen, OSA) of Pseudomonas aeruginosa PAO1. The crystal structure of LKA1gp49 catalytic domain consists of a beta-helix, an insertion domain and a C-terminal discoidin-like domain. The putative substrate binding and processing site is located on the face of the beta-helix whereas the C-terminal domain is likely involved in carbohydrates binding. NMR spectroscopy and mass spectrometry analyses of degraded LPS (OSA) fragments show an O5 serotype-specific polysaccharide lyase specificity. LKA1gp49 reduces virulence in an in vivo Galleria mellonella infection model and sensitizes P. aeruginosa to serum complement activity. This enzyme causes biofilm degradation and does not affect the activity of ciprofloxacin and gentamicin. This is the first comprehensive report on LPS-degrading lyase derived from a Pseudomonas phage. Biological properties reveal a potential towards its applications in antimicrobial design and as a microbiological or biotechnological tool.

Comparative genome analysis of novel Podoviruses lytic for hypermucoviscous Klebsiella pneumoniae of K1, K2, and K57 capsular types

Hypermucoviscous (HV) strains of capsular types K1, K2 and K57 are the most virulent representatives of the Klebsiella pneumoniae species. Eight novel bacteriophages lytic for HV K. pneumoniae were isolated and characterized. Three bacteriophages, KpV41, KpV475, and KpV71 were found to have a lytic activity against mainly K. pneumoniae of capsular type K1. Two phages, KpV74, and KpV763 were lytic for K2 capsular type K. pneumoniae, and the phage KpV767 was specific to K57-type K. pneumoniae only. Two more phages, KpV766, and KpV48 had no capsular specificity. The phage genomes consist of a linear double-stranded DNA of 40,395–44,623bp including direct terminal repeats of 180–246 bp. The G + C contents are 52.3–54.2 % that is slightly lower than that of genomes of K. pneumoniae strains being used for phage propagation. According to the genome structures, sequence similarity and phylogenetic data, the phages are classified within the genus Kp32virus and Kp34virus of subfamily Autographivirinae, family Podoviridae. In the phage genomes, genes encoding proteins with putative motifs of polysaccharide depolymerase were identified. Depolymerase genes of phages KpV71 and KpV74 lytic for hypermucoviscous K. pneumoniae of K1 and K2 capsular type, respectively, were cloned and expressed in Escherichia coli, and the recombinant gene products were purified. The specificity and polysaccharide-degrading activity of the recombinant depolymerases were demonstrated.

Biology and Genomics of an Historic Therapeutic Escherichia coli Bacteriophage Collection.

We have performed microbiological and genomic characterization of an historic collection of nine bacteriophages, specifically infecting a K1 E. coli O18:K1:H7 ColV+ strain. These phages were isolated from sewage and tested for their efficacy in vivo for the treatment of systemic E. coli infection in a mouse infection model by Smith and Huggins (1982). The aim of the study was to identify common microbiological and genomic characteristics, which co-relate to the performance of these phages in in vivo study. These features will allow an informed selection of phages for use as therapeutic agents. Transmission electron microscopy showed that six of the nine phages were Podoviridae and the remaining three were Siphoviridae. The four best performing phages in vivo belonged to the Podoviridae family. In vitro, these phages exhibited very short latent and rise periods in our study. In agreement with their microbiological profiles, characterization by genome sequencing showed that all six podoviruses belong to the Autographivirinae subfamily. Of these, four were isolates of the same species (99% identity), whereas two had divergent genomes compared to other podoviruses. The Siphoviridae phages, which were moderate to poor performers in vivo, exhibited longer latent and rise periods in vitro. Two of the three siphoviruses were closely related to each other (99% identity), but all can be associated with the Guernseyvirinae subfamily. Genome sequence comparison of both types of phages showed that a gene encoding for DNA-dependent RNA polymerase was only present in phages with faster replication cycle, which may account for their better performance in vivo. These data define a combination of microbiological, genomic and in vivo characteristics which allow a more rational evaluation of the original in vivo data and pave the way for the selection of phages for future phage therapy trails.