Antimicrob Agents Chemother. 2021 Jul 6:AAC0089021. doi: 10.1128/AAC.00890-21. Online ahead of print.
Carbapenem-resistant Enterobacterales, such as KPC-producing Klebsiella pneumoniae, represent a major threat to public health due to their rapid spread. Novel drug combinations such as ceftazidime-avibactam (CZA), combining a broad-spectrum cephalosporin along with a broad-spectrum ß-lactamase inhibitor, have recently been introduced and have been shown to exhibit excellent activity towards multidrug-resistant KPC-producing Enterobacterale strains. However, CZA-resistantK. pneumoniae isolates are now being increasingly reported, mostly corresponding to producers of KPC variants. In this study, we evaluated in vitro the nature of the mutations in the KPC-2 and KPC-3 ß-lactamase sequences (the most frequent KPC-type enzymes) that lead to CZA resistance, and the subsequent effects of these mutations on susceptibility to other ß-lactam antibiotics. Single-step in vitro selection assays were conducted resulting in the identification of a series of mutations in the KPC sequence which conferred the ability to those mutated enzymes to confer resistance to CZA. Hence, 16 KPC-2 variants and 10 KPC-3 variants were obtained. Production of the KPC variants in an Escherichia coli recombinant strain resulted in a concomitant increased susceptibility to broad-spectrum cephalosporins and carbapenems, with the exceptions of ceftazidime and piperacillin-tazobactam, compared to wild-type KPC enzymes. Enzymatic assays showed that all of the KPC variants identified exhibited an increased affinity toward ceftazidime and a slightly decreased sensitivity to avibactam, sustaining their impact on CZA resistance. However their respective carbapenemase activities were concurrently negatively impacted.