Complete Genome Sequence of Klebsiella pneumoniae Bacteriophage KpS110, Encoding Five Tail-Associated Proteins with Putative Polysaccharide Depolymerase Domains.

We report the genome sequence of bacteriophage KpS110, which infects Klebsiella pneumoniae, a multidrug-resistant encapsulated bacterium that causes severe community-acquired and hospital-acquired infections. The phage genome is 156,801 bp, with 201 open reading frames. KpS110 is most closely related to phages of the family Ackermannviridae at the genome and proteome levels.

Comparison of the therapeutic potential of bacteriophage KpV74 and phage-derived depolymerase (ß-glucosidase) against Klebsiella pneumoniae capsular type K2.

Bacteriophages and phage polysaccharide-degrading enzymes (depolymerases) are garnering attention as possible alternatives to antibiotics. Here, we describe the antimicrobial properties of bacteriophage KpV74 and phage depolymerase Dep_kpv74 specific to the hypervirulent Klebsiella pneumoniae of the K2 capsular type. The depolymerase Dep_kpv74 was identified as a specific glucosidase that cleaved the K2 type capsular polysaccharides of the K. pneumoniae by a hydrolytic mechanism. This depolymerase was effective against thigh soft tissue K. pneumoniae infection in mice without inducing adverse behavioral effects or toxicity. The depolymerase efficiency was similar to or greater than the bacteriophage efficiency. The phage KpV74 had a therapeutic effect only for treating the infection caused by the phage-propagating K. pneumoniae strain and was completely inactive against the infection caused by the K. pneumoniae strain that did not support phage multiplication. The depolymerase was effective in both cases. A mutant resistant to phage and depolymerase was isolated during the treatment of mice with bacteriophage. A confirmed one-base deletion in the flippase-coding wzx gene of this mutant is assumed to affect the polysaccharide capsule, abolishing the KpV74 phage adsorption and reducing the K. pneumoniae virulence.

Novel Klebsiella pneumoniae K23-Specific Bacteriophages From Different Families: Similarity of Depolymerases and Their Therapeutic Potential.

Antibiotic resistance is a major public health concern in many countries worldwide. The rapid spread of multidrug-resistant (MDR) bacteria is the main driving force for the development of novel non-antibiotic antimicrobials as a therapeutic alternative. Here, we isolated and characterized three virulent bacteriophages that specifically infect and lyse MDR Klebsiella pneumoniae with K23 capsule type. The phages belonged to the Autographiviridae (vB_KpnP_Dlv622) and Myoviridae (vB_KpnM_Seu621, KpS8) families and contained highly similar receptor-binding proteins (RBPs) with polysaccharide depolymerase enzymatic activity. Based on phylogenetic analysis, a similar pattern was also noted for five other groups of depolymerases, specific against capsule types K1, K30/K69, K57, K63, and KN2. The resulting recombinant depolymerases Dep622 (phage vB_KpnP_Dlv622) and DepS8 (phage KpS8) demonstrated narrow specificity against K. pneumoniae with capsule type K23 and were able to protect Galleria mellonella larvae in a model infection with a K. pneumoniae multidrug-resistant strain. These findings expand our knowledge of the diversity of phage depolymerases and provide further evidence that bacteriophages and phage polysaccharide depolymerases represent a promising tool for antimicrobial therapy.

Structure and gene cluster of the capsular polysaccharide of multidrug resistant carbapenemase OXA-48-producing Klebsiella pneumoniae strain KPB536 of the genetic line ST147.

The Gram-negative opportunistic pathogen Klebsiella pneumoniae is a significant cause of community-acquired and healthcare-associated infections for which multidrug resistance is a concern worldwide. A major virulence determinant of K. pneumoniae is a polysaccharide capsule (CPS) which forms a barrier around the bacterial cell wall, providing protection from environmental pressures and immune responses of eukaryotic organisms. More than 70 chemical capsule structures of serologically typeable K. pneumoniae strains are known. However, there are little data on the CPS structure and cps gene cluster organization of clinical multidrug resistant K. pneumoniae strains. Our investigation of multidrug resistant carbapenemase OXA-48-producing K. pneumoniae strain KPB536 identified a capsular type that was structurally similar to K. pneumoniae K10 but different from any K. pneumoniae CPS reported so far. The content and organization of the cps gene cluster in K. pneumoniae KPB536 also was determined. The catalytic functions of glycosyltransferases coded by the cps_KPB536 gene cluster were assigned by comparison with those responsible for the synthesis of glycoside linkages in the CPSs of K. pneumoniae types K10 and K61.

Complete Genome Sequences of Two Salmonella Viruses, VSe11 and VSe102 (Family Myoviridae, Subfamily Ounavirinae), with a Very High Degree of Similarity.

Two lytic double-stranded DNA bacteriophages, VSe11 and VSe102, infecting broad-spectrum Salmonella enterica were isolated from the sewage of two different poultry farms. The phage genomes comprise 86,360 bp and 86,365 bp, respectively, with a G+C content of 39.0%, and both contain 129 putative coding sequences.

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.

Molecular characterization of podoviral bacteriophages virulent for Clostridium perfringens and their comparison with members of the Picovirinae.

Clostridium perfringens is a Gram-positive, spore-forming anaerobic bacterium responsible for human food-borne disease as well as non-food-borne human, animal and poultry diseases. Because bacteriophages or their gene products could be applied to control bacterial diseases in a species-specific manner, they are potential important alternatives to antibiotics. Consequently, poultry intestinal material, soil, sewage and poultry processing drainage water were screened for virulent bacteriophages that lysed C. perfringens. Two bacteriophages, designated ΦCPV4 and ΦZP2, were isolated in the Moscow Region of the Russian Federation while another closely related virus, named ΦCP7R, was isolated in the southeastern USA. The viruses were identified as members of the order Caudovirales in the family Podoviridae with short, non-contractile tails of the C1 morphotype. The genomes of the three bacteriophages were 17.972, 18.078 and 18.397 kbp respectively; encoding twenty-six to twenty-eight ORF's with inverted terminal repeats and an average GC content of 34.6%. Structural proteins identified by mass spectrometry in the purified ΦCP7R virion included a pre-neck/appendage with putative lyase activity, major head, tail, connector/upper collar, lower collar and a structural protein with putative lysozyme-peptidase activity. All three podoviral bacteriophage genomes encoded a predicted N-acetylmuramoyl-L-alanine amidase and a putative stage V sporulation protein. Each putative amidase contained a predicted bacterial SH3 domain at the C-terminal end of the protein, presumably involved with binding the C. perfringens cell wall. The predicted DNA polymerase type B protein sequences were closely related to other members of the Podoviridae including Bacillus phage Φ29. Whole-genome comparisons supported this relationship, but also indicated that the Russian and USA viruses may be unique members of the sub-family Picovirinae.

The genome sequence and proteome of bacteriophage ΦCPV1 virulent for Clostridium perfringens.

Application of bacteriophages and their lytic enzymes to control Clostridium perfringens is one potential approach to reduce the pathogen on poultry farms and in poultry-processing facilities. Bacteriophages lytic for C. perfringens were isolated from sewage, feces and broiler intestinal contents and ΦCPV1, a virulent bacteriophage, was classified in the family Podoviridae. The purified virus had an icosahedral head and collar of approximately 42nm and 23nm in diameter, respectively, with a structurally complex tail of 37nm lengthwise and a basal plate of 30nm. The ΦCPV1 double-stranded DNA genome was 16,747 base pairs with a GC composition of 30.5%. Twenty-two open reading frames (ORFs) coding for putative peptides containing 30 or more amino acid residues were identified and analyzed in the genome. Amino acid sequences of the predicted proteins from the ΦCPV1 genome ORFs were compared with those from the NCBI database and potential functions of 12 proteins were predicted by sequence homology. Three putative proteins were similar to hypothetical proteins with unknown functions, whereas seven proteins did not have similarity with any known bacteriophage or bacterial proteins. Identified ORFs formed at least four genomic clusters that accounted for predicted proteins involved with replication of the viral DNA, its folding, production of structural components and lytic properties. One bacteriophage genome encoded lysin was predicted to share homology with N-acetylmuramoyl-l-alanine amidases and a second structural lysin was predicted to be a lysozyme-endopeptidase. These enzymes digest peptidoglycan of the bacterial cell wall and could be considered potential therapeutics to control C. perfringens.