Stepwise KPC mutational accumulation confers high-level ceftazidime-avibactam resistance in ST11 carbapenem-resistant Klebsiella pneumoniae.

Ceftazidime-avibactam (CZA) has become an effective therapeutic option against carbapenem-resistant Klebsiella pneumoniae (CRKP). The emergence of Klebsiella pneumoniae carbapenemase (KPC) variants in CRKP is a major cause of CZA resistance. However, high-level CZA resistance conferred by accumulated KPC mutations was seldom reported. In this study, we observed that high-level ceftazidime-avibactam resistance was attributed to stepwise KPC mutational accumulation. A total of 21 KPC-producing K. pneumoniae isolates were collected from a single patient over one year, encompassing 17 harboring blaKPC-2, 2 harboring blaKPC-33, and 2 harboring blaKPC-275. The patient's treatment course indicated that prolonged CZA pressure likely drove the accumulation of mutations, leading to the sequential evolution from blaKPC-2 to blaKPC-33 and ultimately to blaKPC-275. Molecular docking and steady-state kinetics revealed that the newly identified variant KPC-275 confers high-level CZA resistance, mediated by the cumulative effects of D179Y and T243P mutations, which enhance ceftazidime affinity and diminish avibactam inhibitory activity. Antimicrobial susceptibility testing demonstrated that compared to blaKPC-2, expression of blaKPC-33 and blaKPC-275 in E. coli DH5α elevated the MIC of CZA by 128-fold and 1024-fold, respectively. Moreover, unlike the individual D179Y or T243P mutations, the combination of both conferred high-level resistance to CZA but imposed a fitness cost. Whole-genome sequencing results demonstrated that these highly homologous ST11 CRKP strains carried blaKPC within Tn6296 on IncFII/IncR plasmids. Additionally, the plasmid carrying blaKPC-275 integrated virulence-associated fragments via IS5075, forming a hybrid virulence-resistance plasmid. Furthermore, global phylogeographic analysis of 928 KPC variant-producing K. pneumoniae isolates revealed pronounced geographical clustering and clone-driven dissemination, with Asia, particularly China, representing the largest reservoir and ST11 identified as the predominant lineage. In view of the widespread prevalence of KPC-KP strains, clinicians should be aware of the risk of treatment failure due to blaKPC mutation-mediated CZA resistance during therapy.