A novel extended-spectrum ²-lactamase CTX-M-23 with a P167T substitution in the active-site omega loop associated with ceftazidime resistance

Objective: In recent years, cefotaximases of the CTX-M type have become a predominant cause of resistance to extended-spectrum cephalosporins in Gram-negative bacteria. Although most enzymes provide higher levels of resistance to cefotaxime than to ceftazidime, mutants with enhanced catalytic efficiency against ceftazidime have recently been described. This report identifies another ceftazidime-resistant mutant of the CTX-M class of enzymes. Methods: Two ceftazidime-resistant strains, Escherichia coli IFI-1 and Klebsiella pneumoniae IFI-2, were isolated from a 46-year-old man during treatment of postoperative peritonitis with ceftazidime. Susceptibility testing, mating-out assays, isoelectric focusing as well as PCR and sequencing techniques were carried out to investigate the underlying mechanism of resistance. Results: E. coli IFI-1 and K. pneumoniae IFI-2 exhibited a clavulanic acid-inhibited substrate profile that included extended-spectrum cephalosporins. Notably, both strains had up to a 32-fold higher level of resistance to ceftazidime than to cefotaxime. Further characterization revealed that a novel bla CTX-M gene encoding a ²-lactamase with a pI of 8.9 was implicated in this resistance: CTX-M-23. Along with the substitutions D114N and S140A, CTX-M-23 differed from CTX-M-1, the most closely related enzyme, by a P167T replacement in the active-site omega loop, which has not previously been observed in other CTX-M enzymes. By analogy with what was observed with certain TEM/PSE/BPS-type ²-lactamases, the amino acid substitution in the omega loop may explain ceftazidime resistance, which has only rarely been reported for other CTX-M enzymes. Conclusion: The emergence of a new ceftazidime-resistant CTX-M-type mutant provides evidence that these enzymes are able to broaden their substrate spectrum towards ceftazidime, probably due to substitutions in the active-site omega loop.