Resistance development in KPC-producing <i>Klebsiella pneumoniae</i> under ceftazidime/avibactam-meropenem pressure: KPC-2 Ω-loop mutations attenuate avibactam efficacy

ABSTRACT The ceftazidime/avibactam (CAZ-AVI, CZA) combination is a potent agent against KPC-producing Klebsiella pneumoniae (KPC-Kp) infections, but resistance readily emerges via KPC mutations that restore carbapenem susceptibility. Previous studies have shown that the CZA-meropenem (MEM) combination exhibits favorable therapeutic efficacy by exploiting this collateral sensitivity. Here, we investigated in vitro how this adjunctive combination impacts resistance evolution in KPC-Kp and delineated the underlying molecular mechanisms. Resistance evolution in 26 KPC-Kp isolates was selected by multipassaging under dual CZA-MEM pressure. The underlying resistant mechanisms were systematically dissected by integrating MICs, enzyme kinetics, molecular docking, and molecular dynamics simulations. In contrast to CZA alone, the CZA-MEM combination significantly suppressed resistance development, but five strains still adapted. Under dual-drug selection, mutations arose solely within KPC but were restricted to the Ω loop. Kinetic analysis showed that these mutations drive CZA resistance (R164S variants chiefly by increasing ceftazidime hydrolysis, R164L variants by weakening AVI inhibition, and D179Y by both mechanisms) while simultaneously restoring MEM susceptibility (all variants reduced MEM hydrolysis). Importantly, every mutant elevated the AVI IC 50 , suggesting this is the optimal evolutionary path for KPC-Kp under CZA-MEM pressure. Mechanistic studies reveal that R164L/T243S mutations destabilize the Ω loop to reposition Ser70 at the active site and confer AVI resistance by impairing acylation, destabilizing the enzyme-inhibitor complex, and potentially accelerating deacylation. Our study provides actionable clues for positioning and deploying different β-lactam agents in the treatment of KPC-Kp infections. IMPORTANCE The global rise of KPC-producing Klebsiella pneumoniae (KPC-Kp) severely limits available therapeutic options, underscoring the urgent need for strategies that curb resistance evolution. The ceftazidime/avibactam-meropenem combination exploits collateral sensitivity to suppress resistance development, yet adaptive mutations still emerge. However, the mechanisms underlying resistance development under dual-drug pressure remain unclear. This study focused on Ω-loop mutations in KPC under dual-drug pressure, revealing that these mutations concurrently enhance avibactam resistance and restore carbapenem susceptibility, although the potential contribution of additional genetic alterations cannot be fully excluded. These findings offer critical insights for optimizing combination therapies and monitoring resistance mechanisms during treatment of KPC-Kp infections.