Reply to Frère: Covalent Trapping and Bacterial Resistance to Ceftazidime

Abstract

This is a response to a letter by Frere (1). Dr. Frere's letter and insightful comments are appreciated. We investigated the role of Arg-164 in the KPC-2 carbapenemase using biochemical and microbiological assays (2). Our variants were expressed in a laboratory strain, Escherichia coli DH10B, and the gene was encoded in a high copy vector (pBC SK). The R164S variant demonstrated increased ceftazidime minimum inhibitory concentrations (MICs); our argument for covalent trapping was based upon the detection of the acyl-enzyme complex of the R164S variant and ceftazidime. Our results suggested the hypothesis that rapid turnover of ceftazidime did not appear to be the only cause of the high ceftazidime MIC of the R164S variant. From our observations (mass spectrometry), the R164S variant instead appeared to “trap ceftazidime,” thus preventing it from reaching its PBP targets. We also ran Western blots using a polyclonal antibody that measured KPC β-lactamase. Our Western blots showed that steady-state expression levels paralleled the resistance to ampicillin and cephalothin. As Fig. 3 shows, the R164S variant expressed in this laboratory strain of E. coli produced a significant amount of R164S KPC β-lactamase. We agree that accurately measuring the amount of β-lactamase produced in these experimental strains may give us better insights. Efforts will be undertaken to develop a specific ELISA for KPC-β-lactamase as we have done in other studies to help approximate the true amount of enzyme (3). However, we wonder if, in strains of E. coli or Klebsiella pneumoniae recovered from the clinic, calculations may not be entirely precise as many factors impact the MIC (porin loss, other β-lactamase production, permeability, etc.).