The emergence of carbapenemase-producing Enterobacteriaceae (CPE) has become a major public health concern. Among these CPE, OXA-48-producing Enterobacteriaceae have now widely disseminated throughout European countries. OXA-48 confers high-level resistance to penicillins, including temocillin, and hydrolyzes carbapenems at a low level, but spares extended spectrum cephalosporins (3GC). Since the first identification of OXA-48, different variants have been reported, differing by a few amino acid substitutions or deletions mostly in the region of the β5-β6 loop made of 7 amino acids (YSTRIEP). Whereas some OXA-48-variants with single amino acid substitutions in this loop have similar hydrolytic activities as OXA-48, others, such as OXA-163 or OXA-405, have a four amino acid deletion that result in loss of carbapenem hydrolysis and in gain of expanded-spectrum cephalosporin hydrolysis. Despite the availability of structural information of OXA-48, the factors responsible for the heterogeneity observed in the behavior of these variants toward carbapenems have not been yet fully understood. In order to gain further insights into the different hydrolytic profiles of OXA-48 variants, we performed site-directed mutagenesis in the β5-β6 loop, in order to delete one to six amino acids in both directions. The phenotypical changes induced by deletions were assessed by susceptibility testing and minimal inhibitory concentration (MIC) determination. The enzymes corresponding to the different mutants were then purified and the kinetic parameters for β-lactams were determined and compared with those of OXA-48. Progressive deletions in the β5-β6 loop starting either from the left side or from the right side identified key amino acids involved in carbapenem and 3GC hydrolysis. Comparative modeling, covalent docking and molecular dynamics simulations evidenced the conformational changes induced by mutations or deletions within the β5-β6 loop. The direct correlations between the structural determinants of this loop and different hydrolytic profiles were also highlighted. These results clearly demonstrate the key role of the β5-β6 loop conformation in the substrate specificity of OXA-48. The plasticity of this loop provides OXA-48 with a tremendous evolutionary potential, and the present work identifies a number of variants that may be selected in future clinical isolates.

Key role of the β5-β6 loop conformation in the substrate specificity of OXA-48–like enzymes: Implications for the β-lactamase-mediated antibiotic resistance

253rd National Meeting (2017)

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