Mutant prevention concentrations, resistance evolution dynamics, and mechanisms of resistance to imipenem and imipenem/relebactam in carbapenem-susceptible isolates showing ceftazidime/avibactam resistance.

carbapenemase (KPC) variants selected during ceftazidime/avibactam treatment usually develop susceptibility to carbapenems and carbapenem/β-lactamase inhibitors, such as imipenem and imipenem/relebactam. We analyzed imipenem and imipenem/relebactam single-step mutant frequencies, resistance development trajectories and differentially selected resistance mechanisms using two representative isolates that had developed ceftazidime/avibactam resistance during therapy (ST512/KPC-31 and ST258/KPC-35). Mutant frequencies and mutant prevention concentrations were measured in Mueller-Hinton agar plates containing incremental concentrations of imipenem or imipenem/relebactam. Resistance dynamics were determined after incubation for 7 days in 10 mL MH tubes containing incremental concentrations of each antibiotic or combination, up to 64 times their baseline MIC. Two colonies per strain from each experiment were characterized by antimicrobial susceptibility testing and whole genome sequencing. The impact of KPC variants identified in resistant mutants on β-lactam resistance was investigated by cloning experiments. Imipenem/relebactam suppressed the emergence of resistant mutants at lower concentrations than imipenem, slowed down resistance development for both strains, and the resulting mutants yielded lower MICs of carbapenems and carbapenem/β-lactamase inhibitors than those selected with imipenem alone. Characterization of resistant mutants revealed that imipenem resistance was mainly caused by inactivation of OmpK36 and mutations in the KPC β-lactamase. Imipenem/relebactam-resistant mutants also maintained OmpK36 alterations, but mutations in KPC were much less frequent compared with those selected with imipenem alone. Genetic and biochemical characterization of the KPC derivatives identified in the resistant mutants confirmed their role in carbapenem resistance. Our data positions imipenem/relebactam as an attractive therapeutic option for combating ceftazidime/avibactam-resistant KPC-producing infections.