Kinetic characterization of novel CTX-M-96 harboring D240G substitution and its role in the phenotypic resistance to oxyimino-cephalosporins

GHIGLIONE BÁRBARA; RODRIGUEZ MARGARITA; ADAM KAT; BLATEZKY; FERRARI; DI CONZA JOSÉ; MARCELA RADICE; POWER PABLO; GUTKIND GABRIEL

Chicago

Conferencia; 51th ICAAC Interscience Conference on Antimicrobial Agents and Chemotherapy Chicago; 2011

American Society of Microbiology

Background: Compared to other ESBL, CTX-M hydrolyze ceftazidime less effectively than cefotaxime. Variants responsible for decreased susceptibility to ceftazidime are associated with mutations at several amino acid positions, being D240G the most prevalent. We evaluated the role of this mutation in the resistance profile of CTX-M-96, a D240G natural variant of CTX-M-12, and compared to other hypothetical “ceftazidimases”. Methods: blaCTX-M-3, -15, -12, -96, and -37 were PCR-amplified from native strains, and CTX-M-37 D240G mutant generated by PCR site-directed mutagenesis. Genes were cloned in pK19 under regulation of vector’s promoter. ESBL screening and susceptibility tests were performed according to CLSI. blaCTX-M-96 was cloned in pET28a, the enzyme purified by ion-exchange chromatography and the main kinetic parameters determined. Results: Positive synergy with clavulanic acid was observed in all CTX-M recombinant clones. Mutant-CTX-M producing clones displayed smaller inhibition zones for ceftazidime compared to their parental ones. CTX-M-96 and -15 clones showed MIC values of ceftazidime 32-fold higher than CTX-M-12 and -3, respectively; and for CTX-M-37 D240G/CTX-M-37WT was 4-fold higher. No difference was observed in MIC values of cefotaxime. CTX-M-96 shows high catalytic efficiencies towards benzyl-penicillin, cephalothin and cefuroxime. Even when kcat/Km of ceftazidime was 3-fold higher than that to CTX-M-12, it remains 100-fold lower than the one for cefotaxime (0.012 vs. 1 uM-1.sec-1, respectively). The IC50 value for clavulanate was 760 nM. Conclusions: Even when D240G mutation led to an increase in the MIC of ceftazidime in the producing clones, the overall increase in the catalytic activity of CTX-M-96 towards this antibiotic seems to be insufficient to yield phenotypic resistance. As MICs of cefotaxime remain at least 8-fold higher than the corresponding values to ceftazidime, the term “ceftazidimases” currently applied for these CTX-M should be used carefully.