Genomic and functional characterization of ST11-KL64 hypervirulence-associated carbapenem-resistant Klebsiella pneumoniae co-harboring blaKPC-2 and blaNDM-13

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Background Hypervirulence-associated carbapenem-resistant Klebsiella pneumoniae (hv-CRKP) is a major clinical and public health threat. However, ST11-KL64 hv-CRKP co-harboring bla KPC-2 and bla NDM-13 remains poorly characterized, particularly regarding genomic relatedness, plasmid dynamics, and attenuated virulence-associated phenotypes. Methods We retrospectively investigated clinical K. pneumoniae isolates collected at a tertiary hospital in Chengdu, China, between January and December 2024. Hypervirulence-associated markers were screened by PCR, followed by antimicrobial susceptibility testing and carbapenemase inhibitor enhancement assay to identify genotype-defined hv-CRKP. All isolates were subjected to molecular typing. ST11-KL64 isolates co-harboring bla KPC-2 and bla NDM-13 were subjected to Illumina sequencing, with the representative isolate K3 undergoing hybrid whole-genome sequencing and functional characterization. Results Among the 46 hvKP isolates recovered from 43 patients, 35 were identified as hv-CRKP, predominantly ST11-KL64. Three ST11-KL64 hv-CRKP isolates co-harbored bla KPC-2 / bla NDM-13 , and Illumina sequencing coupled with core-genome SNP (cgSNP) typing revealed minimal genetic variation. The expanded cgSNP analysis supported close relatedness between K3 and Beijing isolate K56649. K3 carried a pLVPK-like virulence plasmid, a bla KPC-2 -bearing IncFII/IncR plasmid, and a bla NDM-13 -bearing IncI1 plasmid. Relative to pK2044, K3 exhibited an rmpA -proximal ISKpn26 -associated insertion and a complex alteration of the 5′-terminal coding region of rmpA . The bla NDM-13 plasmid was conjugatively transferred to Escherichia coli C600 with a mean conjugation frequency of 5.213 × 10 −3 transconjugants per recipient cell and bla NDM-13 maintained high stability following approximately 100 generations of antibiotic-free passage, whereas bla KPC-2 was not detected under the tested conditions. Phenotypically, K3 showed a negative string test, low mucoviscosity, and attenuated virulence-associated phenotypes. Conclusion Our results reveal that the three isolates formed a closely related local genomic cluster, among which K3 was closely related to the K56649 clone. In addition, K3 exhibited conjugative transfer capacity of the bla NDM-13 -bearing IncI1 plasmid, and alterations at the rmpA locus accompanied by reduced rmpA transcript abundance were associated with low mucoviscosity.