Emergence of Ceftazidime-Avibactam Resistance Mediated by KPC Variants KPC-71 and KPC-78 in ST463 Pseudomonas aeruginosa

Pseudomonas aeruginosa has long been a model organism in microbiological research and a prevalent opportunistic pathogen in clinical settings. The limitations of current antimicrobial regimens have exacerbated the global threat of carbapenem-resistant P. aeruginosa (CRPA). Here, we elucidate the mechanisms of ceftazidime-avibactam (CZA) resistance mediated by two novel KPC variants, KPC-71 and KPC-78, identified during the treatment of CRPA infections. Two CZA-resistant P. aeruginosa strains, SY-206885 and HZ-231016032, were isolated from critically ill male patients with severe pneumonia. Whole-genome sequencing assigned both isolates to the high-risk sequence type 463 (ST463). Isolate SY-206885 harbors the blaKPC-71 gene, while HZ-231016032 carries blaKPC-78. Cloning and expression of these genes in P. aeruginosa PAO1 conferred a 16-fold increase in the minimum inhibitory concentration (MIC) of CZA. Structural analysis and kinetic profiling revealed that, compared to wild-type KPC-2, both KPC-71 and KPC-78 exhibit reduced hydrolytic efficiency (kcat) but higher affinity (lower Km) for ceftazidime, alongside significantly weakened binding affinity for the inhibitor avibactam. These findings underscore the clinical risk of ST463 CRPA strains evolving CZA resistance via KPC mutations under therapeutic pressure.