Genetic and kinetic characterization of the novel AmpC beta-lactamases DHA-6 and DHA-7.

During a Spanish surveillance study, two natural variants of DHA beta-lactamases, DHA-6 and DHA-7 were found, with the replacements Ala226Thr and Phe322Ser respect to DHA-1, respectively. The enzymes were isolated from Escherichia coli and Enterobacter cloacae clinical isolates, respectively. The aim of the study was the genetic, microbiological and biochemical characterization of the DHA-6 and DHA-7 beta-lactamases. The blaDHA-6 andblaDHA-7 genes were located in I1 and HI2 incompatibility group plasmids of 87.3 and 310.4 kb, respectively. The gene context of both blaDHA-6 andblaDHA-7 was similar to that already described for blaDHA-1 gene and included the qnrB4 and aadA genes. The MICs for cephalothin, aztreonam, cefotaxime and ceftazidime were 8 to 30 fold lower for the DHA-6 than for DHA-1 and DHA-7 expressed in the same isogenic E.coli TG1 strain. Interestingly the MIC for cefoxitin was higher in DHA-6 expressing transformant compared to DHA-1 and DHA-7. Biochemical studies with pure beta-lactamases revealed a slightly lower catalytic efficiency of DHA-6 against cephalothin, ceftazidime and cefotaxime compared to DHA-1 and DHA-7. To understand this behavior, stability experiments were carried out and showed that the DHA-6 protein displayed a significantly higher stability than DHA-1 and DHA-7 enzymes. The proximity of Thr226 to the N-terminal in the tertiary protein structure in DHA-6 may promote this stabilization and consequently could induce a slight reduction of the dynamic of this enzyme primarily affecting the hydrolysis of some of the bulkiest antibiotics.