Nikolay V. Volozhantsev

Isolation and characterization of wide host range lytic bacteriophage AP22 infecting Acinetobacter baumannii

Acinetobacter baumannii plays a significant role in infecting patients admitted to hospitals. Many A. baumannii infections, including ventilation-associated pneumonia, wound, and bloodstream infections, are common for intensive care and burn units. The ability of the microorganism to acquire resistance to many antibiotics, disinfectants, and dehydration assures its long-term survival in hospital settings. The application of bacteriophages is a potential tool to control A. baumannii infections. Bacteriophage AP22 lytic for A. baumannii was isolated from clinical materials and classified as a member of the Myoviridae family. The phage had an icosahedral head of 64 nm in diameter and a contractile tail of 85-90 nm in length. According to restriction analysis, AP22 had 46-kb double-stranded DNA genome. The phage AP22 exhibited rapid adsorption (> 99% adsorbed in 5 min), a large burst size (240 PFU per cell), and stability to the wide range of pH. The bacteriophage was shown to specifically infect and lyse 68% (89 of 130) genotype-varying multidrug-resistant clinical A. baumannii strains by forming clear zones. Thus, it could be used as a candidate for making up phage cocktails to control A. baumannii-associated nosocomial infections.

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 ORFs 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.

Comparative genomics of four closely related Clostridium perfringens bacteriophages reveals variable evolution among core genes with therapeutic potential.

BackgroundBecause biotechnological uses of bacteriophage gene products as alternatives to conventional antibiotics will require a thorough understanding of their genomic context, we sequenced and analyzed the genomes of four closely related phages isolated from Clostridium perfringens, an important agricultural and human pathogen.ResultsPhage whole-genome tetra-nucleotide signatures and proteomic tree topologies correlated closely with host phylogeny. Comparisons of our phage genomes to 26 others revealed three shared COGs; of particular interest within this core genome was an endolysin (PF01520, an N-acetylmuramoyl-L-alanine amidase) and a holin (PF04531). Comparative analyses of the evolutionary history and genomic context of these common phage proteins revealed two important results: 1) strongly significant host-specific sequence variation within the endolysin, and 2) a protein domain architecture apparently unique to our phage genomes in which the endolysin is located upstream of its associated holin. Endolysin sequences from our phages were one of two very distinct genotypes distinguished by variability within the putative enzymatically-active domain. The shared or core genome was comprised of genes with multiple sequence types belonging to five pfam families, and genes belonging to 12 pfam families, including the holin genes, which were nearly identical.ConclusionsSignificant genomic diversity exists even among closely-related bacteriophages. Holins and endolysins represent conserved functions across divergent phage genomes and, as we demonstrate here, endolysins can have significant variability and host-specificity even among closely-related genomes. Endolysins in our phage genomes may be subject to different selective pressures than the rest of the genome. These findings may have important implications for potential biotechnological applications of phage gene products.

The spread of bla OXA-48 and bla OXA-244 carbapenemase genes among Klebsiella pneumoniae, Proteus mirabilis and Enterobacter spp. isolated in Moscow, Russia.

BackgroundThe spread of carbapenemase-producing Enterobacteriaceae (CPE) is a great problem of healthcare worldwide. Study of the spread for blaOXA-48-like genes coding epidemically significant carbapenemases among hospital pathogens is important for the regional and global epidemiology of antimicrobial resistance.MethodsAntibacterial resistant isolates of Klebsiella pneumoniae (nxa0=xa095) from 54 patients, P.mirabilis (nxa0=xa032) from 20 patients, Enterobacter aerogenes (nxa0=xa06) from four patients, and Enterobacter cloacae (nxa0=xa04) from four patients were collected from January, 2013 to October, 2014 in neurosurgical intensive care unit (ICU) of the Burdenko Neurosurgery Institute, Moscow. Characteristics of the isolates were done using susceptibility tests, PCR detection of the resistance genes, genotyping, conjugation, DNA sequencing, and bioinformatic analysis.ResultsMajor strains under study were multi drug resistant (MDR), resistant to three or more functional classes of drugs simultaneously—98.9xa0% K. pneumoniae, 100xa0% P.mirabilis, one E.aerogenes isolate, and one E.cloacae isolate. Molecular-genetic mechanism of MDR in K.pneumoniae and P.mirabilis isolates were based on carrying of epidemic extended-spectrum beta-lactamase blaCTX-M-15 gene (87.2 and 90.6xa0% accordingly), carbapenemase blaOXA-48-like gene (55.3 and 23.3xa0% accordingly), and classxa01 (54.8 and 31.3xa0% accordingly) and class 2 (90.6xa0% P.xa0mirabilis) integrons. The blaOXA-48-like-positive K.xa0pneumoniae were collected during whole two-year surveillance period, while P.xa0mirabilis and Enterobacter spp. carrying blaOXA-48-like genes were detected only after four and 18xa0months after the research start, respectively. The blaOXA-48-like gene acquisition was shown for P.xa0mirabilis isolates collected from five patients and for E.xa0cloacae isolate collected from one patient during their stay in the ICU, presumably from blaOXA-48-like-positive K.xa0pneumoniae. The source of the blaOXA-244 gene acquired by E.xa0aerogenes isolates and the time of this event were not recognized.ConclusionsThe expanding of CPE in the surveyed ICU was associated with the spread of blaOXA-48 and blaOXA-244 carbapenemase genes documented not only among K.pneumoniae, well-known bacterial host for such genes, but among P.mirabilis, E.aerogenes, and E.xa0cloacae.

Atomic Force Microscopy Analysis of the Acinetobacter baumannii Bacteriophage AP22 Lytic Cycle

Background Acinetobacter baumannii is known for its ability to develop resistance to the major groups of antibiotics, form biofilms, and survive for long periods in hospital environments. The prevalence of infections caused by multidrug-resistant A. baumannii is a significant problem for the modern health care system, and application of lytic bacteriophages for controlling this pathogen may become a solution. Methodology/Principal Findings In this study, using atomic force microscopy (AFM) and microbiological assessment we have investigated A. baumannii bacteriophage AP22, which has been recently described. AFM has revealed the morphology of bacteriophage AP22, adsorbed on the surfaces of mica, graphite and host bacterial cells. Besides, morphological changes of bacteriophage AP22-infected A. baumannii cells were characterized at different stages of the lytic cycle, from phage adsorption to the cell lysis. The phage latent period, estimated from AFM was in good agreement with that obtained by microbiological methods (40 min). Bacteriophage AP22, whose head diameter is 62±1 nm and tail length is 88±9 nm, was shown to disperse A. baumannii aggregates and adsorb to the bacterial surface right from the first minute of their mutual incubation at 37°C. Conclusions/Significance High rate of bacteriophage AP22 specific adsorption and its ability to disperse bacterial aggregates make this phage very promising for biomedical antimicrobial applications. Complementing microbiological results with AFM data, we demonstrate an effective approach, which allows not only comparing independently obtained characteristics of the lytic cycle but also visualizing the infection process.

Complete genome sequence of novel T7-like virus vB_KpnP_KpV289 with lytic activity against Klebsiella pneumoniae

A novel bacteriophage, vB_KpnP_KpV289, lytic for hypermucoviscous strains of Klebsiella pneumoniae, was attributed to the family Podoviridae, subfamily Autographivirinae, genus T7likevirus based on transmission electron microscopy and genome analysis. The complete genome of the bacteriophage vB_KpnP_KpV289 consists of a linear double-stranded DNA of 41,054xa0bp including 179-bp direct-repeat sequences at the ends and 51 open reading frames (ORFs). The G+C content is 52.56xa0%. The phage was shown to lyse 15 out of 140 (10.7xa0%) K. pneumoniae strains belonged to the capsular types K-1, K-2, and K-57 and strains without a determined capsular type, including a hypermucoviscous strain of the novel sequence type ST-1554.

Genome sequencing and comparative analysis of three hypermucoviscous Klebsiella pneumoniae strains isolated in Russia

The prevalence and characteristics of hypermucoviscous (HV) strains among Klebsiella pneumoniae isolated in Russian hospitals were investigated. The HV strains accounted for 11% of the K. pneumoniae isolates collected in the period from 2011 to 2016, and were characterized as belonging to the K1, K2, K20 and K57 serotypes. Whole genome sequences (WGSs) of K. pneumoniae HV clinical strains KPi261 (SCPM-O-B-7850) and KPB4010 (SCPM-O-B-7846) belonging to the K1 and K2 capsular types, as well as WGSs of K. pneumoniae strain KPM9 (SCPM-O-B-7749) of the K20 capsular type isolated from freshwater, were completed. The final draft genome sequences of KPi261, KPB4010 and KPM9 strains consisted of 5 719 189, 5 431 785 and 5 427 926 bp with 57.0, 57.1 and 57.4% GC content, respectively. The chromosomal and plasmid genes associated with K. pneumoniae virulence including the capsular polysaccharide synthesis gene cluster, mucoid phenotype regulator rmpA and transcriptional activator rmpA2, the all operon associated with allantoin metabolism, the kfu operon involved in iron uptake, the aerobactin-producing system iucABCDiutA, and the iron-transport systems iroBCDN and fecIRA were detected.

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 180-246u2009bp. The Gu2009+u2009C 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.

Comparative analysis of Klebsiella pneumoniae strains isolated in 2012-2016 that differ by antibiotic resistance genes and virulence genes profiles.

Abstract The antibacterial resistance and virulence genotypes and phenotypes of 148 non-duplicate Klebsiella pneumoniae strains collected from 112 patients in Moscow hospitals in 2012–2016 including isolates from the respiratory system (57%), urine (30%), wounds (5%), cerebrospinal fluid (4%), blood (3%), and rectal swab (1%) were determined. The majority (98%) were multidrug resistant (MDR) strains carrying blaSHV (91%), blaCTX-M (74%), blaTEM (51%), blaOXA (38%), and blaNDM (1%) beta-lactamase genes, class 1 integrons (38%), and the porin protein gene ompK36 (96%). The beta-lactamase genes blaTEM-1, blaSHV-1, blaSHV-11, blaSHV-110, blaSHV-190, blaCTX-M-15, blaCTX-M-3, blaCTX-M-55, blaOXA-48, blaOXA-244, and blaNDM-1 were detected; class 1 integron gene cassette arrays (aadA1), (dfrA7), (dfrA1-orfC), (aadB-aadA1), (dfrA17-aadA5), and (dfrA12-orfF-aadA2) were identified. Twenty-two (15%) of clinical K. pneumoniae strains had hypermucoviscous (HV) phenotype defined as string test positive. The rmpA gene associated with HV phenotype was detected in 24% of strains. The intrapersonal mutation of rmpA gene (deletion of one nucleotide at the polyG tract) was a reason for negative hypermucoviscosity phenotype and low virulence of rmpA-positive K. pneumoniae strain KPB584. Eighteen virulent for mice strains with LD50 ≤ 104 CFU were attributed to sequence types ST23, ST86, ST218, ST65, ST2174, and ST2280 and to capsular types K1, K2, and K57. This study is the first report about hypervirulent K. pneumoniae strain KPB2580-14 of ST23K1 harboring extended-spectrum beta-lactamase CTX-M-15 and carbapenemase OXA-48 genes located on pCTX-M-15-like and pOXA-48-like plasmids correspondingly.