Diverse evolutionary trajectories of carbapenemase: unraveling the impact of amino acid substitutions on β-lactam susceptibility and the role of avibactam in driving resistance.

carbapenemases (KPCs) have evolved into over 245 distinct variants, with over one-third of variants exhibiting reduced susceptibility to ceftazidime-avibactam, while the underlying selection mechanisms remain elusive. To better elucidate these resistant phenotypes, we cloned 33 clinically described KPC variants (from KPC-2 to KPC-36) and 8 artificially created variants into a common plasmid vector and assessed their impact on β-lactam susceptibility. Strains expressing KPC-14, KPC-28, and KPC-31 exhibited increased resistance to ceftazidime and ceftazidime-avibactam but decreased resistance to carbapenems. We further studied the catalytic mechanism of β-lactam hydrolysis by KPC-4, KPC-14, KPC-15, KPC-16, KPC-21, KPC-25, KPC-28, KPC-31, and the ancestral KPC-2 and KPC-3 enzymes. Antimicrobial susceptibility test, enzyme kinetics, and molecular modeling revealed diverse selective pressures, including but not limited to aztreonam and ceftriaxone, driving KPC evolution, with ceftazidime playing a central role. Substitutions within the KPC hydrolytic active sites notably reduced the inhibitory effect of avibactam on KPC, demonstrated by isothermal titration calorimetry analysis, resulting in enhanced hydrolysis of ceftazidime by enzyme kinetics. This highlights that avibactam may serve as an additional driving force in KPC evolution.IMPORTANCEThe rapid evolution of KPC carbapenemases, including resistance to ceftazidime-avibactam, threatens the effectiveness of last-resort antibiotics against infections, necessitating understanding of of the underlying selection pressures. This study investigates the evolutionary mechanisms driving KPC diversification and resistance to ceftazidime-avibactam, providing crucial information for developing effective strategies to combat carbapenem-resistant (CRKP) infections and preserve antibiotic efficacy.