ABSTRACT
During a Spanish surveillance study, a natural variant of a CMY-type ß-lactamase related to CMY-2 with a GluLeu217-218 insertion in the Ω-loop (designated CMY-54) was found to increase the minimum inhibitory concentractions to ß-lactams in a clinical strain of Escherichia coli. The aim of this study was to characterize CMY-54 by genetic, microbiological, and biochemical analysis. The blaCMY-54 gene is encoded by a plasmid of around 100 kb that hybridizes with K and FIB probes. The genetic context of blaCMY-54 and blaCMY-2 genes was found to be very similar. E. coli expressing CMY-54 under isogenic conditions showed a clear fourfold to eightfold increase in MICs to penicillins, cefotaxime, ceftazidime, and aztreonam compared with CMY-2. The catalytic efficiencies of pure CMY-2 and CMY-54 proteins correlated with their microbiological parameters. CMY-2 protein was more resistant to thermal denaturation than CMY-54, indicating that the Ω-loop of CMY-54 may be wider and more relaxed and probably enables better accommodation of these antimicrobials. Otherwise, the higher stabilization of CMY-2 may induce a slight reduction of the dynamics of this enzyme and primarily affect the hydrolysis of some of the bulkiest antibiotics. In summary, the GluLeu217-218 insertion observed in CMY-54 compared to CMY-2 produces a ß-lactamase with a distinctive catalytic efficacy for ß-lactam antimicrobials likely caused by an increased flexibility slightly affecting the active site shape, highlighting the relevance of single mutations on the hydrolytic spectrum in class C ß-lactamases.
