Dataset Information

Characterization and Genomic Analysis of Bacteriophage vB_KpnM_IME346 Targeting Clinical Klebsiella pneumoniae Strain of the K63 Capsular Type.

ABSTRACT: A Klebsiella pneumoniae bacteriophage (vB_KpnM_IME346) was isolated from a hospital sewage sample. This bacteriophage specifically infects a clinical K. pneumoniae strain with a K63 capsular polysaccharide structure. The phage genome was evaluated by next-generation sequencing, which revealed a linear double-stranded DNA genome consisting of 49,482 base pairs with a G+C content of 49.1%. The latent period of vB_KpnM_IME346 was shown to be 20 min, and the burst size was 25-30 pfu (plaque-forming units)/infected cell. Transmission electron microscopy and phylogenetic analysis showed that the JD001-like phage belongs to the genus Jedunavirus of the family Myoviridae. The newly isolated vB_KpnM_IME346 shows infectivity in the clinical host K. pneumoniae KP576 strain, indicating that it is a promising alternative to antibacterial agents for removing K. pneumoniae from patients.

SUBMITTER: Gao M

PROVIDER: S-EPMC9007800 | biostudies-literature | 2022 Apr

REPOSITORIES: biostudies-literature

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Characterization and Genomic Analysis of Bacteriophage vB_KpnM_IME346 Targeting Clinical Klebsiella pneumoniae Strain of the K63 Capsular Type.

Gao Mingming M Yi Lingxian L Wang Yuan Y Gao Jie J Liu Huiying H Zhang Xianglilan X Pei Guangqian G Tong Yigang Y Bai Changqing C

Current microbiology 20220413 6

A Klebsiella pneumoniae bacteriophage (vB_KpnM_IME346) was isolated from a hospital sewage sample. This bacteriophage specifically infects a clinical K. pneumoniae strain with a K63 capsular polysaccharide structure. The phage genome was evaluated by next-generation sequencing, which revealed a linear double-stranded DNA genome consisting of 49,482 base pairs with a G+C content of 49.1%. The latent period of vB_KpnM_IME346 was shown to be 20 min, and the burst size was 25-30 pfu (plaque-forming ...[more]

PMID: 35416546

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Project description:BACKGROUND: Klebsiella pneumoniae is one of the major pathogens causing hospital-acquired multidrug-resistant infections. The capsular polysaccharide (CPS) is an important virulence factor of K. pneumoniae. With 78 capsular types discovered thus far, an association between capsular type and the pathogenicity of K. pneumoniae has been observed. METHODOLOGY/PRINCIPAL FINDINGS: To investigate an initially non-typeable K. pneumoniae UTI isolate NTUH-K1790N, the cps gene region was sequenced. By NTUH-K1790N cps-PCR genotyping, serotyping and determination using a newly isolated capsular type-specific bacteriophage, we found that NTUH-K1790N and three other isolates Ca0507, Ca0421 and C1975 possessed a new capsular type, which we named KN2. Analysis of a KN2 CPS(-) mutant confirmed the role of capsule as the target recognized by the antiserum and the phage. A newly described lytic phage specific for KN2 K. pneumoniae, named 0507-KN2-1, was isolated and characterized using transmission electron microscopy. Whole-genome sequencing of 0507-KN2-1 revealed a 159 991 bp double-stranded DNA genome with a G+C content of 46.7% and at least 154 open reading frames. Based on its morphological and genomic characteristics, 0507-KN2-1 was classified as a member of the Myoviridae phage family. Further analysis of this phage revealed a 3738-bp gene encoding a putative polysaccharide depolymerase. A recombinant form of this protein was produced and assayed to confirm its enzymatic activity and specificity to KN2 capsular polysaccharides. KN2 K. pneumoniae strains exhibited greater sensitivity to this depolymerase than these did to the cognate phage, as determined by spot analysis. CONCLUSIONS/SIGNIFICANCE: Here we report that a group of clinical strains possess a novel Klebsiella capsular type. We identified a KN2-specific phage and its polysaccharide depolymerase, which could be used for efficient capsular typing. The lytic phage and depolymerase also have potential as alternative therapeutic agents to antibiotics for treating K. pneumoniae infections, especially against antibiotic-resistant strains.

Project description:Capsular polysaccharides enable clinically important clones of Klebsiella pneumoniae to cause severe systemic infections in susceptible hosts. Phage-encoded capsule depolymerases have the potential to provide an alternative treatment paradigm in patients when multiple drug resistance has eroded the efficacy of conventional antibiotic chemotherapy. An investigation of 164 K. pneumoniae from intensive care patients in Thailand revealed a large number of distinct K types in low abundance but four (K2, K51, K1, K10) with a frequency of at least 5%. To identify depolymerases with the capacity to degrade capsules associated with these common K-types, 62 lytic phage were isolated from Thai hospital sewage water using K1, K2 and K51 isolates as hosts; phage plaques, without exception, displayed halos indicative of the presence of capsule-degrading enzymes. Phage genomes ranged in size from 41-348 kb with between 50 and 535 predicted coding sequences (CDSs). Using a custom phage protein database we were successful in applying annotation to 30 - 70% (mean = 58%) of these CDSs. The largest genomes, of so-called jumbo phage, carried multiple tRNAs as well as CRISPR repeat and spacer sequences. One of the smaller phage genomes was found to contain a putative Cas type 1E gene, indicating a history of host DNA acquisition in these obligate lytic phage. Whole-genome sequencing (WGS) indicated that some phage displayed an extended host range due to the presence of multiple depolymerase genes; in total, 42 candidate depolymerase genes were identified with up to eight in a single genome. Seven distinct virions were selected for further investigation on the basis of host range, phage morphology and WGS. Candidate genes for K1, K2 and K51 depolymerases were expressed and purified as his6-tagged soluble protein and enzymatic activity demonstrated against K. pneumoniae capsular polysaccharides by gel electrophoresis and Anton-Paar rolling ball viscometry. Depolymerases completely removed the capsule in K-type-specific fashion from K. pneumoniae cells. We conclude that broad-host range phage carry multiple enzymes, each with the capacity to degrade a single K-type, and any future use of these enzymes as therapeutic agents will require enzyme cocktails for utility against a range of K. pneumoniae infections.

Project description:The genomic organization of the chromosomal cps region that is responsible for capsular polysaccharide synthesis in Klebsiella pneumoniae Chedid (O1:K2) was investigated. Deletion analyses and Southern hybridization studies suggested that the central region of the cloned 29-kb BamHI fragment is indispensable for K2 capsular polysaccharide synthesis. The 24,329-bp nucleotide sequence of the Klebsiella cps region was determined and deposited in the EMBL and GenBank databases through DDBJ and assigned accession number D21242. Nineteen possible open reading frames (ORFs) were identified in the sequenced area. Among them, 13 ORFs are very close to each other. Six of the 19 ORFs show considerable nucleotide sequence similarities to Salmonella typhimurium cpsG, cpsB, rfbP, and orf2.8, Escherichia coli gnd, and Haemophilus influenzae bexD, respectively. Moreover, the deduced amino acid sequence of the ORF10 product demonstrated a highly hydrophobic profile and showed putative membrane topology similarity to Rickettsia prowazekii ATP/ADP translocase. Nucleotide sequence similar to the sigma 54-dependent promoter, as well as the usual -35 and -10 sequences, were identified just upstream of ORF3, which is the first ORF in the polycistronic structure. Furthermore, a sequence (GGGCGGTAGCGT) found just downstream of the sigma 54-dependent promoter-like sequence was generally conserved among gene clusters implicated in cell surface polysaccharide synthesis, such as Salmonella rfb and viaB and E. coli kpsMT and rfaQPG. A possible transcriptional terminator with a hairpin loop structure found just downstream of ORF15 that is a homolog of E. coli gnd. K2 capsular polsaccharide biosynthesis in E. coli K-12 depends on cpsB (mannose-1-phosphate guanyltransferase gene), and Klebsiella cpsB, found in the downstream region of the polycistronic structure, was able to complement cpsB of E. coli. Results of transposon insertion and promoter-cloning analyses were consistent with the results of nucleotide sequence analysis.

Dataset Information

Characterization and Genomic Analysis of Bacteriophage vB_KpnM_IME346 Targeting Clinical Klebsiella pneumoniae Strain of the K63 Capsular Type.

Publications

Characterization and Genomic Analysis of Bacteriophage vB_KpnM_IME346 Targeting Clinical Klebsiella pneumoniae Strain of the K63 Capsular Type.

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