Identification of a CTX-M-255 β-lactamase containing a G239S substitution selectively conferring resistance to penicillin/β-lactamase inhibitor combinations

Objectives: An Escherichia coli isolate, WGS1363, showed resistance to piperacillin/tazobactam but susceptibility to cephalosporins and contained a previously unrecognized β-lactamase, CTX-M-255, as the only acquired β-lactamase. CTX-M-255 was identical to CTX-M-27 except for a G239S substitution. Here, we characterize the hydrolytic spectrum of CTX-M-255 and a previously reported β-lactamase, CTX-M-178, also containing a G239S substitution and compare it to their respective parental enzymes, CTX-M-27 and CTX-M-15. Methods: All β-lactamase genes were expressed in E. coli TOP10 and MICs to representative β-lactam-antibiotics were determined. Furthermore, bla bla, bla and bla with C-terminal His-tag fusions were affinity purified for enzyme kinetic assays determining Michaelis–Menten kinetic parameters against representative β-lactam-antibiotics and ICs of clavulanate, sulbactam, tazobactam and avibactam. Results: TOP10-transformants expressing bla and bla showed resistance to penicillin/β-lactamase combinations and susceptibility to cephalothin and cefotaxime in contrast to transformants expressing bla and bla. Determination of enzyme kinetic parameters showed that CTX-M-178 and CTX-M-255 both lacked hydrolytic activity against cephalosporins and showed impaired hydrolytic efficiency against penicillin antibiotics compared to their parental enzymes. Both enzymes appeared more active against piperacillin compared to benzylpenicillin and ampicillin. Compared to their parental enzymes, ICs of β-lactamaseinhibitors were increased more than 1000-fold for CTX-M-178 and CTX-M-255. Conclusions: CTX-M-178 and CTX-M-255, both containing a G239S substitution, conferred resistance to piperacillin/tazobactam and may be characterized as inhibitor-resistant CTX-M β-lactamases. Inhibitor resistance was accompanied by loss of activity against cephalosporins and monobactams. These findings add to the necessary knowledge base for predicting antibiotic susceptibility from genotypic data.