title: 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: OBJECTIVE
  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.
authors.affiliation: Department of Molecular Biology and Microbiology, Case Western Reserve University, Cleveland, OH, USA Research Service, Louis Stokes Veteran Affairs Medical Center, Cleveland, OH, USA.
authors.forename: Marisa L
authors.initials: ML
authors.lastname: Winkler
authors.affiliation: Research Service, Louis Stokes Veteran Affairs Medical Center, Cleveland, OH, USA Department of Medicine, Case Western Reserve University, Cleveland, OH, USA.
authors.forename: Krisztina M
authors.initials: KM
authors.lastname: Papp-Wallace
authors.affiliation: Department of Molecular Biology and Microbiology, Case Western Reserve University, Cleveland, OH, USA Research Service, Louis Stokes Veteran Affairs Medical Center, Cleveland, OH, USA Department of Medicine, Case Western Reserve University, Cleveland, OH, USA Department of Pharmacology, Case Western Reserve University, Cleveland, OH, USA robert.bonomo@va.gov.
authors.forename: Robert A
authors.initials: RA
authors.lastname: Bonomo
mesh_terms: D019943:Amino Acid Substitution; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D053961:Azabicyclo Compounds / Q000494:pharmacology*; D002442:Ceftazidime / Q000494:pharmacology*; D004338:Drug Combinations; D024881:Drug Resistance, Bacterial; D004926:Escherichia coli / Q000187:drug effects* / Q000201:enzymology*; D008826:Microbial Sensitivity Tests; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism*; D001618:beta-Lactamases / Q000235:genetics / Q000378:metabolism*
chemical_list: D000900:Anti-Bacterial Agents; D053961:Azabicyclo Compounds; D004338:Drug Combinations; D050505:Mutant Proteins; C000595613:avibactam, ceftazidime drug combination; D002442:Ceftazidime; D001618:beta-Lactamases
keywords: ESBLs; antibiotic resistance; extended-spectrum β-lactamases; β-lactamase inhibitors