Activity of ceftazidime/avibactam against isogenic strains of Escherichia coli containing KPC and SHV beta-lactamases with single amino acid substitutions in the Omega-loop

Objectives: The objective of this study was to explore the activity of ceftazidime and ceftazidime/avibactam against a collection of isogenic strains of Escherichia coli DH10B possessing SHV and KPC beta-lactamases containing single amino acid substitutions in the V-loop (residues 164-179). Methods: Ceftazidime and ceftazidime/avibactam MICs were determined by the agar dilution method for a panel of isogenic E. coli strains expressing SHV-1 and KPC-2 with amino acid substitutions at positions 164, 167, 169 or 179. Two KPC-2 beta-lactamase variants that possessed elevated MICs of ceftazidime/avibactam were selected for further biochemical analyses. Results: Avibactam restored susceptibility to ceftazidime for all V-loop variants of SHV-1 with MICs <8 mg/L. In contrast, several of the Arg164 and Asp179 variants of KPC-2 demonstrated MICs of ceftazidime/avibactam >8 mg/L. beta-Lactamase kinetics showed that the Asp179Asn variant of KPC-2 demonstrated enhanced kinetic properties against ceftazidime. The K-i app, k(2)/K and k(off) of the Arg164Ala and Asp179Asn variant KPC-2 beta-lactamases indicated that avibactam effectively inhibited these enzymes. Conclusions: Several KPC-2 variants demonstrating ceftazidime resistance as a result of single amino acid substitutions in the V-loop were not susceptible to ceftazidime/avibactam (MICs. 8 mg/L). We hypothesize that this observation is due to the stabilizing interactions (e.g. hydrogen bonds) of ceftazidime within the active site of variant beta-lactamases that prevent avibactam from binding to and inhibiting the beta-lactamase. As ceftazidime/avibactam is introduced into the clinic, monitoring for new KPC-2 variants that may exhibit increased ceftazidime kinetics as well as resistance to this novel antibiotic combination will be important.