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

Abstract

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 β-lactamases containing single amino acid substitutions in the Ω-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 β-lactamase variants that possessed elevated MICs of ceftazidime/avibactam were selected for further biochemical analyses.

Results: Avibactam restored susceptibility to ceftazidime for all Ω-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. β-Lactamase kinetics showed that the Asp179Asn variant of KPC-2 demonstrated enhanced kinetic properties against ceftazidime. The Ki app, k2/K and koff of the Arg164Ala and Asp179Asn variant KPC-2 β-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 Ω-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 β-lactamases that prevent avibactam from binding to and inhibiting the β-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.

Keywords: ESBLs; antibiotic resistance; extended-spectrum β-lactamases; β-lactamase inhibitors.

Figure 1.

(a) Amino acid sequence alignment of the Ω-loop in four class A β-lactamases. The black arrows mark the sites of focus in this paper (Arg164, Leu/Pro167, Leu169 and Asp179). (b) Overlay of the X-ray crystallographic protein structure of SHV-1 (PDB ID: 1SHV, pink/magenta) and KPC-2 (PDB ID: 2OV5, cyan/purple) showing the Ω-loop in deeper colours with the catalytic Ser70, Arg164, Leu/Thr167, Leu169 and Asp179 labelled and the Arg164–Asp179 salt bridge drawn as a broken black line. (c) Chemical structures of the compounds tested in this paper. This figure appears in colour in the online version of JAC and in black and white in the print version of JAC.

Figure 2.

(a) Inhibition of nitrocefin (100 μM) hydrolysis by KPC-2 with increasing concentrations of avibactam (2–50 μM). (b) Inhibition of nitrocefin (100 μM) hydrolysis by KPC Arg164Ala with increasing concentrations of avibactam (1–20 μM). (c) Inhibition of nitrocefin (100 μM) hydrolysis by KPC Asp179Asn with increasing concentrations of avibactam (1–50 μM).

Figure 3.

(a) Hydrolysis of ceftazidime (50 μM) by periplasmic extracts of KPC-2 (dark grey) and Asp179Asn (light grey); ceftazidime alone (black) during a 100 s time course. (b) Hydrolysis of ceftazidime (50 μM) by periplasmic extracts of KPC-2 (dark grey) and Asp179Asn (light grey); ceftazidime alone (black) during a 1.5 ms time course.