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

J Antimicrob Chemother. 2015 Aug;70(8):2279-86. doi: 10.1093/jac/dkv094. Epub 2015 May 8.


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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Amino Acid Substitution*
  • Anti-Bacterial Agents / pharmacology*
  • Azabicyclo Compounds / pharmacology*
  • Ceftazidime / pharmacology*
  • Drug Combinations
  • Drug Resistance, Bacterial
  • Escherichia coli / drug effects*
  • Escherichia coli / enzymology*
  • Microbial Sensitivity Tests
  • Mutant Proteins / genetics
  • Mutant Proteins / metabolism*
  • beta-Lactamases / genetics
  • beta-Lactamases / metabolism*


  • Anti-Bacterial Agents
  • Azabicyclo Compounds
  • Drug Combinations
  • Mutant Proteins
  • avibactam, ceftazidime drug combination
  • Ceftazidime
  • beta-Lactamases