Characterization of the first OXA-10 natural variant with increased carbapenemase activity [Mechanisms of Resistance]

While carbapenem resistance in Gram-negatives is mainly due to production of efficient carbapenemases, β-lactamases with narrower spectrum may also contribute to resistance when combined with additional mechanisms. OXA-10 type class D β-lactamases, previously shown to be weak carbapenemases, could represent such a case. In this study two novel OXA-10 variants were identified as the sole carbapenem hydrolyzing enzymes in meropenem resistant Enterobacteria isolated from hospital waste water and found by NGS to express additional β-lactam resistance mechanisms. The new variants, OXA-655 and OXA-656, were carried by two related IncQ1 broad-host plasmids. Compared to OXA-10 they both harbor a Thr26Met substitution with OXA-655 also baring leucine instead of valine in position 117 of the SAV catalytic motif. Susceptibility profiling of laboratory strains replicating the natural bla_OXA plasmids and of recombinant clones expressing OXA-10 and the novel variants in isogenic background indicated that OXA-655 is a more efficient carbapenemase. The carbapenemase activity of OXA-655 is due to the Val117Leu substitution as shown by steady state kinetic experiments where the k_cat of meropenem hydrolysis was increased 4-fold. In contrast, OXA-655 has no activity towards oxyimino β-lactams while its catalytic efficiency against oxacillin is significantly reduced. Moreover, the Val117Leu variant is more efficient against temocillin and cefoxitin. Molecular dynamics indicated that Val117Leu affects the 117-Leu155 interaction leading to structural shifts in the active site that may alter carbapenem alignment. The evolutionary potential of OXA-10 enzymes towards carbapenem hydrolysis combined with their spread by promiscuous plasmids indicates that they may pose a future clinical threat.

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