Within-host co-evolution of KPC variants: plasmid-mediated dissemination of blaKpc-194 and blaKpc-33 in ST11-KL64 hypervirulent Klebsiella pneumoniae driving ceftazidime-avibactam resistance.

UNLABELLED: KPC variants are the primary cause of treatment failure in patients with Klebsiella pneumoniae infections. This study reports the molecular mechanism by which two novel KPC variants (KPC-194 and KPC-33), isolated from a single patient, mediate resistance to ceftazidime-avibactam in ST11-KL64 K. pneumoniae, as well as the evolutionary trajectory of these variants within the host. The broth microdilution method (BMD) was used to determine bacterial susceptibility to antimicrobial agents. Whole-genome sequencing (WGS) technology was employed to identify the drug-resistant genes, virulence genes, and genetic environment carried by the bacterial strains. Molecular cloning experiments and plasmid conjugation experiments were conducted to clarify the susceptibility of KPC-194 to ceftazidime-avibactam and carbapenem. The BMD showed that the KPC-194-producing K. pneumoniae strain was resistant to ceftazidime-avibactam and other antimicrobial agents but susceptible to imipenem (with a minimum inhibitory concentration [MIC] of 0.5 mg/L). Compared with KPC-2, KPC-194 had two amino acid changes, namely, D179Y and P183L. In comparison with Escherichia coli EC 600, the MIC of ceftazidime-avibactam against E. coli EC 600 carrying the blaKPC-194 plasmid increased by 256-fold. When compared with pHSG398-DH5α, the MIC of ceftazidime-avibactam against the cloned strain blaKPC-194-pHSG398-DH5α was elevated by 64-fold. WGS revealed that blaKPC-194 was located on both the IncFII(pHN7A8)-type plasmid and the IncR-type plasmid and that it was horizontally transferred from the IncR-type plasmid to the IncFII(pHN7A8)-type plasmid via an IS26-mediated replicative transposition mechanism. This study elucidates the key mechanism by which the novel KPC variant, KPC-194 (D179Y/P183L), mediates resistance to ceftazidime-avibactam.

IMPORTANCE

This study elucidates the critical molecular mechanism and evolutionary pathway of a novel KPC variant, KPC-194, that confers resistance to the last-resort antibiotic combination ceftazidime-avibactam in a high-risk Klebsiella pneumoniae strain. We identified that two amino acid substitutions (D179Y/P183L) in KPC-194 are responsible for ceftazidime-avibactam resistance. Crucially, our work reveals a dual-threat dynamic: the resistance phenotype is not only caused by the KPC mutation but also profoundly exacerbated by horizontal gene transfer. blaKPC-194 mobilized from a low-risk IncR plasmid to a highly transmissible IncFII plasmid via IS26-mediated replicative transposition. This event dramatically enhances the potential for widespread dissemination among clinical pathogens.