Global Phylogeography and Genomic Epidemiology of Carbapenem-Resistant blaOXA-232-Carrying Klebsiella pneumoniae Sequence Type 15 Lineage.

Prevalence of carbapenem-resistant Klebsiella pneumoniae (CRKP) has compromised antimicrobial efficacy against severe infections worldwide. To monitor global spread, we conducted a comprehensive genomic epidemiologic study comparing sequences from 21 blaOXA-232-carrying CRKP isolates from China with K. pneumoniae sequence type (ST) 15 strains from 68 countries available in GenBank. Phylogenetic and phylogeographic analyses revealed all blaOXA-232-carrying CRKP isolates belonged to ST15 lineage and exhibited multidrug resistance. Analysis grouped 330 global blaOXA-232-carrying ST15 CRKP strains into 5 clades, indicating clonal transmission with small genetic distances among multiple strains. The lineage originated in the United States, then spread to Europe, Asia, Oceania, and Africa. Most recent common ancestor was traced back to 2000; mutations averaged ≈1.7 per year per genome. Our research helps identify key forces driving global spread of blaOXA-232-carrying CRKP ST15 lineage and emphasizes the importance of ongoing surveillance of epidemic CRKP.

High-Resolution Genomic Profiling of Carbapenem-Resistant Klebsiella pneumoniae Isolates: A Multicentric Retrospective Indian Study.

BACKGROUND: Carbapenem-resistant Klebsiella pneumoniae (CRKP) is a threat to public health in India because of its high dissemination, mortality, and limited treatment options. Its genomic variability is reflected in the diversity of sequence types, virulence factors, and antimicrobial resistance (AMR) mechanisms. This study aims to characterize the clonal relationships and genetic mechanisms of resistance and virulence in CRKP isolates in India. MATERIALS AND METHODS: We characterized 344 retrospective K. pneumoniae clinical isolates collected from 8 centers across India collected in 2013-2019. Susceptibility to antibiotics was tested with VITEK 2. Capsular types, multilocus sequence type, virulence genes, AMR determinants, plasmid replicon types, and a single-nucleotide polymorphism phylogeny were inferred from their whole genome sequences. RESULTS: Phylogenetic analysis of the 325 Klebsiella isolates that passed quality control revealed 3 groups: K. pneumoniae sensu stricto (n = 307), K. quasipneumoniae (n = 17), and K. variicola (n = 1). Sequencing and capsular diversity analysis of the 307 K. pneumoniae sensu stricto isolates revealed 28 sequence types, 26 K-locus types, and 11 O-locus types, with ST231, KL51, and O1V2 being predominant. blaOXA-48-like and blaNDM-1/5 were present in 73.2% and 24.4% of isolates, respectively. The major plasmid replicon types associated with carbapenase genes were IncF (51.0%) and Col group (35.0%). CONCLUSION: Our study documents for the first time the genetic diversity of K and O antigens circulating in India. The results demonstrate the practical applicability of genomic surveillance and its utility in tracking the population dynamics of CRKP. It alerts us to the urgency for longitudinal surveillance of these transmissible lineages.

Characterization of ST15-KL112 Klebsiella pneumoniae Co-Harboring Bla oxa-232 and rmtF in China.

Introduction: This study aimed to investigate the emergence and characteristics of carbapenem-resistant Klebsiella pneumoniae (CRKP) strains that demonstrate resistance to multiple antibiotics, including aminoglycosides and tigecycline, in a Chinese hospital. Methods: A group of ten CRKP strains were collected from the nine patients in a Chinese hospital. Antimicrobial Susceptibility Testing (AST) and phenotypic inhibition assays precisely assess bacterial antibiotic resistance. Real-time quantitative PCR (RT-qPCR) was used to analyze the mRNA levels of efflux pump genes (acrA/acrB and oqxA/oqxB) and the regulatory gene (ramA). The core-genome tree and PFGE patterns were analyzed to assess the clonal and horizontal transfer expansion of the strains. Whole-genome sequencing was performed on a clinical isolate of K. pneumoniae named Kpn20 to identify key resistance genes and antimicrobial resistance islands (ARI). Results: The CRKP strains showed high resistance to carbapenems, aminoglycosides (CLSI, 2024), and tigecycline (EUCAST, 2024). The mRNA expression levels of efflux pump genes and regulatory genes were detected by RT-qPCR. All 10 isolates had significant differences compared to the control group of ATCC13883. The core-genome tree and PFGE patterns revealed five clusters, indicating clonal and horizontal transfer expansion. Three key resistance genes (blaoxa-232, blaCTX-M-15 , and rmtF) were observed in the K. pneumoniae clinical isolate Kpn20. Mobile antibiotic resistance islands were identified containing bla CTX-M-15 and rmtF, with multiple insertion sequences and transposons present. The coexistence of bla oxa-232 and rmtF in a high-risk K. pneumoniae strain was reported. Conjugation assay was utilized to investigate the transferability of bla oxa-232-encoding plasmids horizontally. Conclusion: The study highlights the emergence of ST15-KL112 high-risk CRKP strains with multidrug resistance, including to aminoglycosides and tigecycline. The presence of mobile ARI and clonal and horizontal transfer expansion of strains indicate the threat of transmission of these strains. Future research is needed to assess the prevalence of such isolates and develop effective control measures.

A Nationwide Genomic Study of Clinical Klebsiella pneumoniae Carrying blaOXA-232 and rmtF in China.

OXA-232 carbapenemase is becoming a threat in China due to its high prevalence, mortality, and limited treatment options. However, little information is available on the impact of OXA-232-producing Klebsiella pneumoniae in China. This study aims to characterize the clonal relationships, the genetic mechanisms of resistance, and the virulence of OXA-232-producing K. pneumoniae isolates in China. We collected 81 OXA-232-producing K. pneumoniae clinical isolates from 2017 to 2021. Antimicrobial susceptibility testing was performed using the broth microdilution method. Capsular types, multilocus sequence types, virulence genes, antimicrobial resistance (AMR) determinants, plasmid replicon types, and single-nucleotide polymorphism (SNP) phylogeny were inferred from whole-genome sequences. OXA-232-producing K. pneumoniae strains were resistant to most antimicrobial agents. These isolates showed partial differences in susceptibility to carbapenems: all strains were resistant to ertapenem, while the resistance rates to imipenem and meropenem were 67.9% and 97.5%, respectively. Sequencing and capsular diversity analysis of the 81 K. pneumoniae isolates revealed 3 sequence types (ST15, ST231, and one novel ST [ST-V]), 2 K-locus types (KL112 and KL51), and 2 O-locus types (O2V1 and O2V2). The predominant plasmid replicon types associated with the OXA-232 and rmtF genes were ColKP3 (100%) and IncFIB-like (100%). Our study summarized the genetic characteristics of OXA-232-producing K. pneumoniae circulating in China. The results demonstrate the practical applicability of genomic surveillance and its utility in providing methods to prevent transmission. It alerts us to the urgent need for longitudinal surveillance of these transmissible lineages. IMPORTANCE In recent years, the detection rate of carbapenem-resistant K. pneumoniae has increased and represents a major threat to clinical anti-infective therapy. Compared with KPC-type carbapenemases and NDM-type metallo-ß-lactamases, OXA-48 family carbapenemases are another important resistance mechanism mediating bacterial resistance to carbapenems. In this study, we investigated the molecular characteristics of OXA-232 carbapenemase-producing K. pneumoniae isolated from several hospitals to clarify the epidemiological dissemination characteristics of such drug-resistant strains in China.

Co-occurrence of OXA-232, RmtF-encoding plasmids, and pLVPK-like virulence plasmid contributed to the generation of ST15-KL112 hypervirulent multidrug-resistant Klebsiella pneumoniae.

The emergence of carbapenem-resistant Klebsiella pneumoniae (CRKP) strains and restricted therapeutic options pose a global threat to public health. Aminoglycosides are a wise choice, which can effectively reduce the mortality rate when combined with ß-lactam drugs. However, in this study, we identified a ST15-KL112 CRKP FK3006 which not only exhibited resistance to carbapenems, but also exhibited high level resistance to aminoglycosides. In addition to the multidrug resistant phenotype, FK3006 also owned typical pathogenic characteristic, including hypermucoviscosity and hypervirulence phenotypes. According to the whole-genome sequencing, one pLVPK-like virulence plasmid, and three key resistant plasmids (bla OXA-232, bla CTX-M-15, and rmtF) were observed in FK3006. Compared to other typical ST15 CRKP, the presence of pLVPK-like virulence plasmid (p3006-2) endowed the FK3006 with high virulence features. High siderophore production, more cell invasive and more resistant to serum killing was observed in FK3006. The Galleria mellonella infection model also further confirmed the hypervirulent phenotype of FK3006 in vivo. Moreover, according to the conjugation assay, p3006-2 virulence plasmid also could be induced transfer with the help of conjugative IncFIIK p3006-11 plasmid (bla CTX-M-15). In addition to the transmissible plasmid, several insertion sequences and transposons were found around bla CTX-M-15, and rmtF to generate the mobile antimicrobial resistance island (ARI), which also make a significant contribution to the dissemination of resistant determinants. Overall, we reported the uncommon co-existence of bla OXA-232, rmtF-encoding plasmids, and pLVPK-like virulence plasmid in ST15-KL112 K. pneumoniae. The dissemination threatens of these high-risk elements in K. pneumoniae indicated that future studies are necessary to evaluate the prevalence of such isolates.

Characterisation of OXA-48-like carbapenemases in Escherichia coli and Klebsiella pneumoniae from North India.

The oxacillinase-48 (OXA-48)-like carbapenemases are class D ß-lactamases and increasingly reported in Enterobacterial species. The detection of these carbapenemases is challenging and little information is available on the epidemiology and plasmid characteristics of OXA-48-like carbapenemase producers. We detected the presence of OXA-48-like carbapenemases in 500 clinical isolates of Escherichia coli and Klebsiella pneumoniae, followed by detection of other carbapenemases, extended spectrum ß-lactamases (ESBLs) and 16S rRNA methyltransferases in OXA-48 producers. Clonal relatedness was studied using pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST). Finally, plasmid characterisation was performed through conjugation experiment, S1-PFGE and Southern hybridisation. Around 40% of E. coli and K. pneumoniae isolates harboured OXA-48-like ß-lactamases. Two OXA-48 allele variants, OXA-232 and OXA-181 were detected in our study. OXA-48 producers co-harbored diverse drug-resistant genes belonging to other classes of carbapenemases, ESBLs and 16S rRNA methyltransferases. OXA-48-like carbapenemase producers exhibited high clonal diversity. Bla OXA-48 carrying plasmids were conjugative, untypable and their size was ~ 45 kb and ~ 104.5 kb in E. coli and K. pneumoniae respectively. In conclusion, OXA-48-like carbapenemases have emerged as major cause of carbapenem resistance in Enterobacteriaceae and probably still being under reported. Strict surveillance and adequate detection methods are needed to prevent the dissemination of OXA-48-like carbapenemases.

blaOXA-48-like genome architecture among carbapenemase-producing Escherichia coli and Klebsiella pneumoniae in the Netherlands.

Carbapenem-hydrolysing enzymes belonging to the OXA-48-like group are encoded by blaOXA-48-like alleles and are abundant among Enterobacterales in the Netherlands. Therefore, the objective here was to investigate the characteristics, gene content and diversity of the blaOXA-48-like carrying plasmids and chromosomes of Escherichia coli and Klebsiella pneumoniae collected in the Dutch national surveillance from 2014 to 2019 in comparison with genome sequences from 29 countries. A combination of short-read genome sequencing with long-read sequencing enabled the reconstruction of 47 and 132 complete blaOXA-48-like plasmids for E. coli and K. pneumoniae, respectively. Seven distinct plasmid groups designated as pOXA-48-1 to pOXA-48-5, pOXA-181 and pOXA-232 were identified in the Netherlands which were similar to internationally reported plasmids obtained from countries from North and South America, Europe, Asia and Oceania. The seven plasmid groups varied in size, G+C content, presence of antibiotic resistance genes, replicon family and gene content. The pOXA-48-1 to pOXA-48-5 plasmids were variable, and the pOXA-181 and pOXA-232 plasmids were conserved. The pOXA-48-1, pOXA-48-2, pOXA-48-3 and pOXA-48-5 groups contained a putative conjugation system, but this was absent in the pOXA-48-4, pOXA-181 and pOXA-232 plasmid groups. pOXA-48 plasmids contained the PemI antitoxin, while the pOXA-181 and pOXA-232 plasmids did not. Furthermore, the pOXA-181 plasmids carried a virB2-virB3-virB9-virB10-virB11 type IV secretion system, while the pOXA-48 plasmids and pOXA-232 lacked this system. A group of non-related pOXA-48 plasmids from the Netherlands contained different resistance genes, non-IncL-type replicons or no replicons. Whole genome multilocus sequence typing revealed that the blaOXA-48-like plasmids were found in a wide variety of genetic backgrounds in contrast to chromosomally encoded blaOXA-48-like alleles. Chromosomally localized blaOXA-48 and blaOXA-244 alleles were located on genetic elements of variable sizes and comprised regions of pOXA-48 plasmids. The blaOXA-48-like genetic element was flanked by a direct repeat upstream of IS1R, and was found at multiple locations in the chromosomes of E. coli. Lastly, K. pneumoniae isolates carrying blaOXA-48 or blaOXA-232 were mostly resistant for meropenem, whereas E. coli blaOXA-48, blaOXA-181 and chromosomal blaOXA-48 or blaOXA-244 isolates were mostly sensitive. In conclusion, the overall blaOXA-48-like plasmid population in the Netherlands is conserved and similar to that reported for other countries, confirming global dissemination of blaOXA-48-like plasmids. Variations in size, presence of antibiotic resistance genes and gene content impacted pOXA-48, pOXA-181 and pOXA-232 plasmid architecture.

Emergence of ST15 Klebsiella pneumoniae Clinical Isolates Producing Plasmids-Mediated RmtF and OXA-232 in China.

BACKGROUND: RmtF, as 16S rRNA methyltransferase, leads to high-level resistance to aminoglycoside and is now barely reported. METHODS AND RESULTS: Three rmtF-positive Klebsiella pneumoniae isolates, belonging to the pandemic clone sequence type 15, were isolated from children and coproduced bla OXA-232 and bla CTX-M-15. The rmtF gene was located on an IncFIB transformable plasmid of 128,536-bp and bla OXA-232 was on a 6141-bp ColKP3 plasmid, respectively. CONCLUSION: Plasmids with rmtF found worldwide, shared relatively low similarity, and merely matched partly in their multidrug resistance region. Notably, clinical isolates coproducing rmtF and bla OXA-232 are gradually increasing in China.