Friunavirus Phage-Encoded Depolymerases Specific to Different Capsular Types of Acinetobacter baumannii.

Acinetobacter baumannii is a critical priority nosocomial pathogen that produces a variety of capsular polysaccharides (CPSs), the primary receptors for specific depolymerase-carrying phages. In this study, the tailspike depolymerases (TSDs) encoded in genomes of six novel Friunaviruses, APK09, APK14, APK16, APK86, APK127v, APK128, and one previously described Friunavirus phage, APK37.1, were characterized. For all TSDs, the mechanism of specific cleavage of corresponding A. baumannii capsular polysaccharides (CPSs) was established. The structures of oligosaccharide fragments derived from K9, K14, K16, K37/K3-v1, K86, K127, and K128 CPSs degradation by the recombinant depolymerases have been determined. The crystal structures of three of the studied TSDs were obtained. A significant reduction in mortality of Galleria mellonella larvae infected with A. baumannii of K9 capsular type was shown in the example of recombinant TSD APK09_gp48. The data obtained will provide a better understanding of the interaction of phage-bacterial host systems and will contribute to the formation of principles of rational usage of lytic phages and phage-derived enzymes as antibacterial agents.

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.

Capsule-Targeting Depolymerases Derived from Acinetobacter baumannii Prophage Regions.

In this study, several different depolymerases encoded in the prophage regions of Acinetobacter baumannii genomes have been bioinformatically predicted and recombinantly produced. The identified depolymerases possessed multi-domain structures and were identical or closely homologous to various proteins encoded in other A. baumannii genomes. This means that prophage-derived depolymerases are widespread, and different bacterial genomes can be the source of proteins with polysaccharide-degrading activities. For two depolymerases, the specificity to capsular polysaccharides (CPSs) of A. baumannii belonging to K1 and K92 capsular types (K types) was determined. The data obtained showed that the prophage-derived depolymerases were glycosidases that cleaved the A. baumannii CPSs by the hydrolytic mechanism to yield monomers and oligomers of the K units. The recombinant proteins with established enzymatic activity significantly reduced the mortality of Galleria mellonella larvae infected with A. baumannii of K1 and K92 capsular types. Therefore, these enzymes can be considered as suitable candidates for the development of new antibacterials against corresponding A. baumannii K types.

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.

Emergence of Five Genetic Lines ST395NDM-1, ST13OXA-48, ST3346OXA-48, ST39CTX-M-14, and Novel ST3551OXA-48 of Multidrug-Resistant Clinical Klebsiella pneumoniae in Russia.

Aims: The objective of this study was phenotypic and genotypic characterization of antibacterial-resistant Klebsiella pneumoniae clinical strains isolated in Moscow Transplantology Intensive Care Unit in 2017-2019. Results: Major strains among K. pneumoniae (n = 63) isolated from 30 patients were recognized as extensive drug-resistant (n = 55) pathogens, and remaining strains were recognized as multidrug-resistant (n = 8) pathogens. The beta-lactamase genes blaSHV-1,-2a,-11,-27,-67,-187 (n = 63), blaCTX-M-14,-15 (n = 61), blaTEM-1 (n = 54), blaOXA-48 (n = 52), and blaNDM-1 (n = 2), as well as class 1 integrons (n = 19) carried gene cassette arrays aacA4 (n = 2), dfrA1-orfC (n = 6), aadB-aadA1 (n = 9), dfrA15-aadA1 (n = 3), and dfrA12-orfF-aadA2 (n = 1) were identified in the strains. All strains carried four virulence genes: wabG, fimH, uge, and allS, but two strains had additionally kfu gene. Six known sequence types (STs) of K. pneumoniae ST395 (n = 44), ST377 (n = 3), ST307 (n = 4), ST13 (n = 2), ST39 (n = 2), ST3346 (n = 1), and a novel sequence-type ST3551 (n = 7) were identified. Phylogenetic analysis showed that ST3551 belonged to the cluster of clonal group CG147, and the remaining six STs to the another cluster consisting of four subgroups. The emergence of K. pneumoniae genetic lines carrying epidemiologically significant beta-lactamase genes ST395NDM-1, ST13OXA-48, ST3346OXA-48/CTX-M-14, ST3551OXA-48, and ST39CTX-M-14 was the first case of detection in Russia. Conclusion: The emergence of novel carbapenemase-producing K. pneumoniae genetic lines in Russia highlights the global negative tendency of multidrug-resistant pathogens spread in high-technological medical centers.

Draft Genome Sequences of Clinical K1-Type Klebsiella pneumoniae Strains Isolated in Russia.

Klebsiella pneumoniae of capsular type K1 is the most common causative agent of both health care-associated and community-acquired infections. Here, we report the draft genome sequences of 10 K1-type K. pneumoniae strains isolated from patients in an infectious disease hospital and neurosurgical intensive care unit in Russia.

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.

Characterization of myophage AM24 infecting Acinetobacter baumannii of the K9 capsular type.

In the present study, we investigate the biological properties and genomic organization of virulent bacteriophage AM24, which specifically infects multidrug-resistant clinical Acinetobacter baumannii strains with a K9 capsular polysaccharide structure. The phage was identified as a member of the family Myoviridae by transmission electron microscopy. The AM24 linear double-stranded DNA genome of 97,177 bp contains 167 open reading frames. Putative functions were assigned for products of 40 predicted genes, including proteins involved in nucleotide metabolism and DNA replication, packaging of DNA into the capsid, phage assembly and structural proteins, and bacterial cell lysis. The gene encoding the tailspike, which possesses depolymerase activity towards the corresponding capsular polysaccharides, is situated in the phage genome outside of the structural module, upstream of the genes responsible for packaging of DNA into the capsid. The data on characterization of depolymerase-carrying phage AM24 contributes to our knowledge of the diversity of viruses infecting different capsular types of A. baumannii.

Draft Genome Sequences of 10 Clinical K2-Type Klebsiella pneumoniae Strains Isolated in Russia.

We report here the genome sequences of 10 Klebsiella pneumoniae strains of capsular type K2 isolated in Russia from patients in an infectious clinical hospital and neurosurgical intensive care unit. The draft genome sizes range from 5.34 to 5.87 Mb and include 5,448 to 6,137 protein-coding sequences.

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

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.

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.

Genome Sequences of Two NDM-1 Metallo-ß-Lactamase-Producing Multidrug-Resistant Strains of Klebsiella pneumoniae with a High Degree of Similarity, One of Which Contains Prophage.

We report genome sequences of two NDM-1 metallo-ß-lactamase-producing multidrug-resistant Klebsiella pneumoniae isolates of sequence type 147 (ST147) from one hospital. The genomes are highly similar and differ in prophage located in the chromosome of K. pneumoniae KPB-1470/16 and in the additional plasmid-carrying blaOXA-48 gene in K. pneumoniae KPB-417/16.

Complete Nucleotide Sequence of Klebsiella pneumoniae Bacteriophage vB_KpnM_KpV477.

The double-stranded DNA (dsDNA) bacteriophage vB_KpnM_KpV477, with a broad spectrum of lytic activity against Klebsiella pneumoniae, including strains of capsular serotypes K1, K2, and K57, was isolated from a clinical sample. The phage genome comprises 168,272 bp, with a G+C content of 39.3%, and it contains 275 putative coding sequences (CDSs) and 17 tRNAs.

Identification of IS1R and IS10R elements inserted into ompk36 porin gene of two multidrug-resistant Klebsiella pneumoniae hospital strains.

Hospital Klebsiella pneumoniae strains (n = 196) were collected in 2012-16 from the patients of a Moscow neurosurgical intensive care unit. Klebsiella pneumoniae strains were multidrug-resistant and carried beta-lactamase genes blaSHV (97.4% of strains), blaCTX-M (84.7%), blaTEM (56.1%), blaOXA-48-like (49.0%) and blaNDM-1 (one strain), class 1 integrons (43.4% of strains) and porin protein ompK36 gene (100% of strains). The ompK36 porin protein gene disruption by insertion sequence (IS) elements and OmpK36 production loss in two strains were detected in this study. Outer membrane proteins were isolated according to Carlone et al. (Rapid microprocedure for isolating detergent-insoluble outer membrane proteins from Haemophilus species. J Clin Microbiol 1986;24:330-2). The IS10R element belonging to the IS4 family, IS10 group was detected at the position of the 41st nucleotide of the ompK36 gene in K. pneumoniae strain KPB-2304K/15 (the first report for a certain IS element in K. pneumoniae). The IS1R element belonging to the IS1 family was identified at the position of the 86th nucleotide of the ompK36 gene in the K. pneumoniae strain KPB-367K/15 (novel insertion site for IS1 element into ompK36 gene). DNA transfer of the intact ompK36 gene into the strain KPB-367K/15 by vector plasmid restored OmpK36 porin protein production and resulted in a decrease of imipenem minimal inhibitory concentration. Such data confirm the importance of IS elements in ongoing multidrug-resistant evolution in hospital Klebsiella.

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.

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 41,054 bp including 179-bp direct-repeat sequences at the ends and 51 open reading frames (ORFs). The G+C content is 52.56 %. The phage was shown to lyse 15 out of 140 (10.7 %) 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.

Acinetobacter baumannii K27 and K44 capsular polysaccharides have the same K unit but different structures due to the presence of distinct wzy genes in otherwise closely related K gene clusters.

Capsular polysaccharides (CPSs), from Acinetobacter baumannii isolates 1432, 4190 and NIPH 70, which have related gene content at the K locus, were examined, and the chemical structures established using 2D(1)H and(13)C NMR spectroscopy. The three isolates produce the same pentasaccharide repeat unit, which consists of 5-N-acetyl-7-N-[(S)-3-hydroxybutanoyl] (major) or 5,7-di-N-acetyl (minor) derivatives of 5,7-diamino-3,5,7,9-tetradeoxy-D-glycero-D-galacto-non-2-ulosonic (legionaminic) acid (Leg5Ac7R), D-galactose, N-acetyl-D-galactosamine and N-acetyl-D-glucosamine. However, the linkage between repeat units in NIPH 70 was different to that in 1432 and 4190, and this significantly alters the CPS structure. The KL27 gene cluster in 4190 and KL44 gene cluster in NIPH 70 are organized identically and contain lga genes for Leg5Ac7R synthesis, genes for the synthesis of the common sugars, as well as anitrA2 initiating transferase and four glycosyltransferases genes. They share high-level nucleotide sequence identity for corresponding genes, but differ in the wzy gene encoding the Wzy polymerase. The Wzy proteins, which have different lengths and share no similarity, would form the unrelated linkages in the K27 and K44 structures. The linkages formed by the four shared glycosyltransferases were predicted by comparison with gene clusters that synthesize related structures. These findings unambiguously identify the linkages formed by WzyK27 and WzyK44, and show that the presence of different wzy genes in otherwise closely related K gene clusters changes the structure of the CPS. This may affect its capacity as a protective barrier for A. baumannii.

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

BACKGROUND: The spread of carbapenemase-producing Enterobacteriaceae (CPE) is a great problem of healthcare worldwide. Study of the spread for bla OXA-48-like genes coding epidemically significant carbapenemases among hospital pathogens is important for the regional and global epidemiology of antimicrobial resistance. METHODS: Antibacterial resistant isolates of Klebsiella pneumoniae (n = 95) from 54 patients, P. mirabilis (n = 32) from 20 patients, Enterobacter aerogenes (n = 6) from four patients, and Enterobacter cloacae (n = 4) 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. RESULTS: Major strains under study were multi drug resistant (MDR), resistant to three or more functional classes of drugs simultaneously-98.9 % K. pneumoniae, 100 % 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 bla CTX-M-15 gene (87.2 and 90.6 % accordingly), carbapenemase bla OXA-48-like gene (55.3 and 23.3 % accordingly), and class 1 (54.8 and 31.3 % accordingly) and class 2 (90.6 % P. mirabilis) integrons. The bla OXA-48-like-positive K. pneumoniae were collected during whole two-year surveillance period, while P. mirabilis and Enterobacter spp. carrying bla OXA-48-like genes were detected only after four and 18 months after the research start, respectively. The bla OXA-48-like gene acquisition was shown for P. mirabilis isolates collected from five patients and for E. cloacae isolate collected from one patient during their stay in the ICU, presumably from bla OXA-48-like-positive K. pneumoniae. The source of the bla OXA-244 gene acquired by E. aerogenes isolates and the time of this event were not recognized. CONCLUSIONS: The expanding of CPE in the surveyed ICU was associated with the spread of bla OXA-48 and bla OXA-244 carbapenemase genes documented not only among K. pneumoniae, well-known bacterial host for such genes, but among P. mirabilis, E. aerogenes, and E. cloacae.