Background: Class D carbapenem-hydrolyzing β-lactamases (CHDLs), such as OXA-23, OXA-24/40 and OXA-48 commonly share very poor catalytic activity on expanded-spectrum cephalosporins, especially ceftazidime. OXA-146, an OXA-23 clinical variant differing by a single alanine (A220) duplication exhibits significant activity on ceftazidime. Interestingly, the duplicated amino acid residue is located in the β5-β6 loop, a structural element whose role in enzyme functional properties was previously recognized. In order to understand the impact of such residue duplication in other CHDLs, similar variants of OXA-24/40 and OXA-48 were generated and characterized. Methods: OXA-24/40 and OXA-48 alanine insertion variants at the structurally-equivalent position to OXA-23 220 (222 and 213 in OXA-24/40 and OXA-48, respectively) were obtained by mutagenesis. Escherichia coli laboratory strains producing the two β-lactamase variants (OXA-24ins222 and OXA-48ins213) were obtained and their in vitro susceptibility profile determined and compared to that of the wild-type CHDL-producing strains. The functional characterization of the variants was carried out using enzyme assays and the carbapenemase plate assay. Results: As compared to WT OXA-24/40, the OXA-24ins222 insertion variant confers, in an E. coli background, a susceptibility profile similar to that of OXA-146, with a significant increase of the MIC values of ceftriaxone, ceftazidime, cefepime (16-fold) and aztreonam (128-fold). In addition, the Ala222 insertion in OXA-24/40 also determined an apparent decrease of activity towards imipenem. Furthermore, the OXA-24ins222 variant also showed a delayed accumulation in E. coli, as compared to the WT OXA-24/40, which suggests that this modification might also impact on enzyme stability. Strikingly, the insertion of an Ala residue at position 213 of OXA-48 did not confer any of the properties observed with OXA-146 and OXA-24ins222 variants. Conclusions: These data further highlight the functional and structural importance of the β5-β6 loop of CHDLs and the mechanistic heterogeneity observed between OXA-23/24 and OXA-48 CHDLs.
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|Titolo:||Mimicking the Ala220 Duplication of OXA-146 (an OXA-23 variant) in OXA-40 and OXA-48 Highlights Mechanistic Differences among Class D Carbapenemases.|
|Appare nelle tipologie:||4.2 Abstract in Atti di convegno|
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