A novel ceftazidime-hydrolysing extended-spectrum β-lactamase, CTX-M-54, with a single amino acid substitution at position 167 in the omega loop

Abstract

Objectives: To characterize a novel ceftazidime-hydrolysing CTX-M mutant, designated CTX-M-54, produced by Klebsiella pneumoniae clinical isolate BDK0419 and to investigate its genetic environment. Methods: Antimicrobial susceptibilities were determined by disc diffusion and agar dilution methods, and the double-disc synergy test was carried out. Detection of genes encoding class A β-lactamases was performed by PCR amplification, and the genetic organization of the blaCTX-M-54 gene was investigated by PCR and sequencing of the regions surrounding this gene. Kinetic parameters were determined from purified CTX-M-54. Results: The strain BDK0419 contained a transferable plasmid with a molecular size of ∼21 kbp that carries both blaSHV-2a and blaCTX-M-54 β-lactamase genes, along with two other plasmids. The blaCTX-M-54 gene was flanked upstream by an IS Ecp1 insertion sequence and downstream by an IS 903-like element. CTX-M-54 had a P167Q substitution within the omega loop region of class A β-lactamases compared with the sequence of CTX-M-3. The MIC of ceftazidime for K. pneumoniae BDK0419 was 16-fold higher than that of cefotaxime; however, the kinetic parameter of CTX-M-54 against ceftazidime revealed a low catalytic efficiency. Conclusions: This work shows once again that novel CTX-M enzymes with an expanded activity towards ceftazidime through a single amino acid substitution can be identified from clinical isolates. Thus, detection of CTX-M enzymes can no longer be based solely on the resistance phenotypes of clinical isolates towards ceftazidime and cefotaxime. © 2006 Oxford University Press.