Genomic characteristics and hospital-associated clonal transmission analysis of ST11-K64 hypervirulent carbapenem-resistant Klebsiella pneumoniae co-harboring blaKPC-2 and blaNDM-13.

OBJECTIVES: The aim of this study was to characterize the co-occurrence of blaKPC-2 and blaNDM-13 genes in ST11-K64 carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKP), with particular focus on a hospital-associated clonal transmission caused by this clonal strain. METHODS: PCR and Sanger sequencing were employed to determine the MLST types, serotypes, and carbapenemase genes of carbapenem-resistant Klebsiella pneumoniae (CRKP) isolates. Clinical data, antimicrobial susceptibility, plasmid conjugation, and growth curves were evaluated. Virulence was assessed via serum killing assays, biofilm formation, and a Galleria mellonella infection model. Representative strains underwent whole-genome sequencing, and genomic analysis was performed using bioinformatic tools. RESULTS: Between February 2023 and May 2024, we consecutively collected 77 extensively drug-resistant CRKP isolates from a tertiary hospital in Heilongjiang Province, China, and identified six CRKP strains co-harboring KPC-2 and NDM-13, all belonging to the ST11-K64 type. Further clinical data analysis combined with core genome SNP analysis indicated that these ST11-K64 strains were responsible for a nosocomial outbreak. Whole-genome sequencing revealed that this clonal lineage carried an IncFII/IncR plasmid harboring blaKPC-2 and an IncI1 plasmid carrying blaNDM-13. Consistent with previous studies, the blaKPC-2 gene was located on a non-Tn4401 element, while the genetic environment of blaNDM-13 displayed the common features of the blaNDM genetic background: △ISAba125-blaNDM-bleMBL. This clonal strain also contained a pLVPK-like virulence plasmid, on which the virulence genes are localized in a combination of iucA-rmpA-rmpA2-peg-344. According to widely adopted genomic criteria, CRKP isolates carrying iuc and/or rmpA/rmpA2 are defined as CR-hvKP. Therefore, this clonal strain was classified as CR-hvKP. CONCLUSIONS: NDM-13 in Klebsiella pneumoniae, co-expressed with KPC-2, is extremely rare, with only one prior report. This particular strain also exhibits the potential for transmission both within and across regions. The emergence of this multidrug-resistant strain with hypervirulent potential presents a significant global health threat, demanding urgent action.