Novel KPC-2 variants and epidemic ST463 clones underlie ceftazidime/avibactam resistance in carbapenem-resistant <i>Pseudomonas aeruginosa</i>

Carbapenem-resistant <i>Pseudomonas aeruginosa</i> (CRPA) presents a serious clinical challenge due to limited treatment options. Ceftazidime-avibactam (CZA) is a critical last-resort agent against multidrug-resistant Gram-negative pathogens; however, resistance is emerging, particularly mediated by <i>bla</i>_KPC variants. In this study, we investigated the prevalence and mechanisms of CZA resistance among CRPA isolates collected from two tertiary hospitals in Hangzhou between 2021 and 2022. A total of 273 non-duplicate CRPA isolates were screened, among which 13.9% (38/273) exhibited CZA resistance (MIC ≥ 16 µg/mL). Whole-genome sequencing revealed that resistant strains were highly resistant to carbapenems, cephalosporins, and aztreonam but retained partial susceptibility to aminoglycosides and polymyxins. ST463 was the predominant sequence type (76.3%), strongly associated with <i>bla</i>_KPC-2 carriage and virulence genes, such as <i>exoU</i> and <i>pldA</i>. Notably, three <i>bla</i>_KPC variants-KPC-14, KPC-33, and KPC-86-were identified, all conferring high-level resistance to CZA (MIC > 32 µg/mL). KPC-86 was discovered for the first time in <i>P. aeruginosa</i>, embedded in an IS<i>26</i>-flanked transposon, suggesting potential for horizontal transfer. Sequential recovery of KPC-86 and KPC-33 from the same patient supports <i>in vivo</i> evolution under antibiotic pressure. These findings highlight the evolving threat of CZA resistance in CRPA, driven by <i>bla</i>_KPC diversification within high-risk clones. Continued genomic surveillance and antimicrobial stewardship are urgently needed to preserve the efficacy of CZA.IMPORTANCECeftazidime-avibactam (CZA) is one of the few remaining options to treat serious infections caused by drug-resistant <i>Pseudomonas aeruginosa</i>. However, its effectiveness is now being threatened by emerging genetic changes that reduce susceptibility. In this study, we investigated CZA-resistant strains and uncovered three variants of the resistance gene <i>bla</i>_KPC, including one-KPC-86-never before seen in <i>P. aeruginosa</i>. This variant was located on a mobile genetic element, raising concerns about its potential to spread between bacteria. We also documented the evolution of resistance within a single patient, showing how treatment pressure can drive genetic change. By combining clinical, microbiological, and genomic data, our findings highlight an urgent need for ongoing surveillance and careful antibiotic use to preserve the usefulness of last-resort drugs like CZA. This work informs both clinicians and public health experts on emerging resistance threats.