Characterization of KPC-176, an S171P variant derived from KPC-2, associated with ceftazidime-avibactam resistance in ST11-KL64 Klebsiella pneumoniae.

BACKGROUND

The adaptive evolution of Klebsiella pneumoniae carbapenemase (KPC) is the primary mechanism mediating the development of ceftazidime-avibactam (CZA) resistance, and the continuous emergence of novel KPC variants has posed a critical challenge to contemporary CZA-based therapeutic strategies.

OBJECTIVES

This study aimed to investigate the mechanisms underlying the development of CZA resistance in an ST11-KL64 K. pneumoniae strain Kpn_54H carrying the blaKPC-176 gene.

METHODS

Whole-genomic sequencing (WGS), gene cloning, antimicrobial susceptibility testing (AST), agar well diffusion assay, molecular docking, and molecular dynamics simulations were conducted to explore CZA resistance mechanisms. Conjugation experiments, plasmid stability testing, and growth assays were performed to assess fitness costs.

RESULTS

The carbapenem-resistant Klebsiella pneumoniae (CRKP) strain Kpn_54H (ST11-KL64) harbours KPC-176, a variant derived from a T508C mutation in blaKPC-2, resulting in an S171P substitution within the its Omega loop (residues 164-179). CZA resistance emerged post-drug exposure, and the variant reverted to wild-type KPC-2 after drug withdrawal. Molecular dynamics simulations indicated this mutation may enhance hydrogen bonds and residue rigidity during ceftazidime (CAZ) binding, increasing enzyme affinity for CAZ while reducing avibactam (AVI) affinity. Consequently, KPC-176 exhibits markedly elevated CAZ hydrolysis. blaKPC-176 is embedded in a core △ISKpn6-blaKPC-176-ISKpn27 structure, flanked by IS26 elements, and resides on a horizontally transmissible IncFII (pHN7A8) plasmid. KPC-176 conferred lower competitive fitness, but enhanced tolerance to sub-inhibitory CZA.

CONCLUSIONS

KPC-176 mediates K. pneumoniae resistance to CZA by enhancing ceftazidime affinity while impairing avibactam inhibition. It poses a significant transmission threat due to the conjugative pKpn_54H-KPC plasmid and IS26 flanked genetic environment.