Biochemical characterization of OXA-244, an emerging OXA-48 variant with reduced β-lactam hydrolytic activity. (16th May 2021)

• Record Type: Journal Article
• Authors: Rima, Mariam; Emeraud, Cecile; Bonnin, Rémy A; Gonzalez, Camille; Dortet, Laurent; Iorga, Bogdan I; Oueslati, Saoussen; Naas, Thierry
• Is Part Of: Journal of antimicrobial chemotherapy. Volume 76:Number 8(2021)
• Page Start: 2024
• Page End: 2028
• Publication Date: 2021-05-16
• Subjects: Anti-infective agents -- Periodicals; Chemotherapy -- Periodicals; 615.58
• DOI: 10.1093/jac/dkab142
• Languages: English
• ISSNs: 0305-7453

Abstract

Abstract: Background: OXA-48-producing Enterobacterales have widely disseminated globally with an increasing number of variants identified. Among them, OXA-244 is increasingly reported, despite detection difficulties. Objectives: To determine the steady-state kinetic parameters of OXA-244. Methods: The bla OXA-244 gene was amplified, cloned into plasmids p-TOPO and pET41b+, and transformed into Escherichia coli TOP10 for MIC determination and E. coli BL21 DE3 for purification. Steady-state kinetic parameters and IC50 s of clavulanic acid, tazobactam and NaCl were determined using purified OXA-244. Molecular modelling was also performed. Results: A reduction in MICs of temocillin and carbapenems was observed in E. coli expressing OXA-244 as compared with OXA-48. The kinetic parameters revealed a reduced carbapenemase activity of OXA-244 as compared with OXA-48, especially for imipenem, which was 10-fold lower. Similarly, catalytic efficiency ( k cat / K m ) was reduced by 4-fold and 20-fold for ampicillin and temocillin, respectively. Kinetic parameters for cephalosporins were, however, similar. Molecular modelling studies evidenced the key role of R214 in OXA-48, establishing salt bridges with D159 and with the carboxylate group of the R1 substituent of temocillin. These interactions are not possible with G214 in OXA-244, explaining the reduced affinity of temocillin for this enzyme. The R214G mutation in OXA-244 is also likely to induce changes in the active site's water network that would explain the decrease in the hydrolysis rate of carbapenems. Conclusions: Our data confirm that the R214G mutation (present in OXA-244) results in reduced carbapenem- and temocillin-hydrolysing activity, confirming the crucial role of residue 214 in the hydrolysis of these substrates by OXA-48-like β-lactamases.