Poblational and biochemical-structural analyses on CTX-M beta-lactamases harboring the D240G mutation reveal that E. coli populations with decreased susceptibility to ceftazidime preexist under a multifactorial resistance profile

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

Viena

Congreso; International Meeting on Emerging Diseases and Surveillance; 2016

International Society for Infectious Diseases

Purpose: CTX-M β-lactamases possess high catalytic efficiency towards cefotaxime (CTX) but spare ceftazidime (CAZ). Their molecular diversity has led to the emergence of variants harboring the replacement of D240 by a glycine (D240G substitution), and this mutation has been associated with a decreased susceptibility to CAZ. We analyzed the influence of D240G substitution in the overall behavior of CTX-M-producing Escherichia coli strains against CAZ.Methods: Parental blaCTX-M-genes and their corresponding D240G mutants from all 5 blaCTX-M clusters (2, 3, 8, 9, 25) were cloned in pK19 under regulation of the vector´s promoter and transformed in wild type and OmpF porin-deficient E. coli K12 strains. ESBL screening and susceptibility tests were performed according to CLSI recommendations. The selection of CAZ-resistant mutants upon challenging of cells with increasing concentrations of CAZ was performed. Steady-state kinetic parameters were determined for CTX and CAZ. Circular dichroism (CD) and fluorescence spectra were determined. Structural analysis and simulation models were obtained for D240G variants.Results: CTX-M producing clones remained susceptible to CAZ, while those lacking OmpF were resistant. Although kcat/Km toward CAZ for all CTX-M variants carrying Gly was between 5-10 times higher than that for CTX-M variants carrying Asp, it remained between 200-725 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. In vitro selection of CAZ-resistant E. coli strains yielded D240G CTX-M producers with MIC values of 32 and 64 µg/ml. Structural analyses indicated that both CTX and CAZ could be accommodated in the active site following the same pattern described in other CTX-M variants, regardless the presence of Asp or Gly at 240. Circular dichroism suggested that D240G mutation does not have negative impact in the overall structure. Thermal denaturation experiments showed slight differences between wild and mutant variants.Conclusions: D240G 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 G240 variants have only weak activity toward ceftazidime. The presence of this mutation may favor the selection of porin deficient variants.