In Vitro Selection of Ceftazidime-Avibactam Resistance in Enterobacteriaceae with KPC-3 Carbapenemase

Ceftazidime-avibactam is active against most Enterobacteriaceae isolates with KPC carbapenemases. We investigated whether this activity could be compromised by mutation. Single-step and multistep selections were attempted using ceftazidime-avibactam (avibactam fixed at 1 or 4 mu g/ml) versus two strains each of Enterobacter cloacae and Klebsiella pneumoniae, all with the KPC-3 enzyme. Mutant bla(KPC) alleles were sequenced, and their parentage was confirmed by typing. Ceftazidime-avibactam selected mutants at up to 16 x MIC, with frequencies of ca. 10(-9). This contrasted with previous experience for ceftaroline-avibactam, where mutant frequencies under similar conditions were <10(-9). The MICs of ceftazidime with 1 mu g/ml avibactam for the ceftazidime-avibactam-selected mutants rose from 1 to 8 mu g/ml to 16 to >256 mu g/ml and those of ceftazidime with 4 mu g/ml avibactam from 0.25 to 1 mu g/ml to 4 to 128 mu g/ml; ceftaroline-avibactam MICs rose less, typically from 0.5 to 1 mu g/ml to 1 to 8 mu g/ml. The MICs of carbapenems and cephalosporins except ceftazidime and piperacillin-tazobactam were reduced for many mutants. Sequencing of bla(KPC) revealed point and insertion changes in 12/13 mutants investigated, representing all four parents; one mutant lacked bla(KPC) changes and possibly had reduced permeability. Amino acid changes commonly involved Omega loop alterations or 1 to 6 amino acid insertions immediately C-terminal to this loop. The most frequent change, seen in four mutants from three strains, was Asp179Tyr, replacing a residue that ordinarily forms a salt bridge to stabilize the Omega loop. Since ceftaroline-avibactam was less affected than ceftazidime-avibactam, we postulate that these mutations increase ceftazidimase specificity rather than conferring avibactam resistance. The clinical relevance remains uncertain.