Pandemic β-lactamases: CTX-M and the risk of emergence of ceftazidime resistant variants.

GHIGLIONE, B; RODRIGUEZ, MM; CURTO L.M.; BRUNETTI, F; POWER, P; GUTKIND, G

Congreso; III International Congress in Translational Medicine; 2016

Background: CTX-M β-lactamases display high catalytic efficiency towards cefotaxime, while they poorly hydrolyze ceftazidime. Diversification of CTX-M enzymes led to the emergence of Asp240Gly variants responsible for decreased susceptibility to ceftazidime that is not always associated with an evident improvement in kinetic behavior towards ceftazidime. Methods: Parental blaCTX-M-genes and their corresponding mutants from CTX-M clusters 3, 2, 8, 9 and 25 were cloned in pK19 under regulation of the vector´s promoter and transformed in wild type and OmpF deficient E. coli K12 strains. ESBL screening and susceptibility tests were performed according to CLSI recommendations. The emergence of resistant mutants to CAZ was evaluated in E. coli K12 clones expressing CTX-M from cluster 3 and susceptibility of two selected mutants was assessed. Steady-state kinetic parameters were determined for cephalothin, cefotaxime and ceftazidime. Circular dichroism, fluorescence spectra and thermal denaturation experiments were performed. Structural analysis and simulation models were obtained for Asp240Gly variants with YASARA software. Results: CTX-M producing clones remained susceptible to ceftazidime, while those lacking OmpF were resistant. Although kcat/Km toward ceftazidime for all CTX-M variants carrying Gly was between 5 and 10 times higher than that for CTX-M variants carrying Asp, it remained between 200 and 725 times lower than the corresponding value for cefotaxime. This behavior is only due to a slight increase in kcat values, while Km values remain at the millimolar range. The in vitro assay to detect the emergence of E. coli K12 strains resistant to CAZ, selected CTXM Asp240Gly producers with MIC values of 32 and 64 µg/ml. The overall fold of CTX-M-96 was similar to previously reported CTX-M enzymes and simulated models indicated that both cefotaxime and ceftazidime could be accommodated in the active site following the same pattern described in other CTX-M variants, either harboring Asp or Gly at position 240. Circular dichroism suggested that Asp240Gly mutation does not have negative impact in the overall structure. Thermal denaturation experiments showed slight differences between wild and mutant variants.Conclusions: Asp240Gly mutation leads to a remarkable increase in ceftazidime resistance in CTX-M producing clones only when expressed in an OmpF deficient background, suggesting the importance of this porin in the entrance of ceftazidime. Kinetic analysis showed that Gly240 variants have only weak activity toward ceftazidime. The presence of this mutation may favor the selection of porin deficient variants.