Genetic and biochemical characterization of OXA-1186, a novel OXA-198-type carbapenemase hydrolysing cephalosporins in Citrobacter freundii.

Abstract

This study described OXA-1186, a novel carbapenemase related to OXA-198 carbapenemase and produced by a clinical isolate of Citrobacter freundii. WGS was used to characterize the resistome, virulome and plasmid types of the C. freundii 315C8 isolate and to reconstruct the blaOXA-1186-carrying plasmid. Disc diffusion and broth microdilution assays were used to determine MICs. The blaOXA-1186 gene was cloned into plasmid pTOPO and then transformed into Escherichia coli TOP10 or HB4. It was also cloned in pET41b and transformed into E. coli BL21 DE3 for protein purification. Steady-state kinetic parameters were determined on purified OXA-1186. C. freundii 315C8, belonging to ST8, was resistant to penicillins including temocillin and broad-spectrum cephalosporins and displayed reduced susceptibility to carbapenems. It was negative for one of the five main carbapenemases. WGS revealed that the blaOXA-1186 gene encoded a novel carbapenemase that shared 83% amino acid identity with OXA-198. The blaOXA-1186 gene was carried on an IncP6-type plasmid and was embedded within a class 1 integron. Cloning and expression in E. coli revealed that expression of the blaOXA-1186 gene conferred resistance to penicillins, cephalosporins and carbapenems, where it was associated with impaired outer membrane permeability. Kinetic parameters confirmed the hydrolysis of ceftazidime, cefepime and aztreonam, in addition to imipenem and meropenem. Here, we described a novel carbapenemase, OXA-1186, identified in C. freundii. Unlike OXA-198, OXA-1186 is able to hydrolyse broad-spectrum cephalosporins. This carbapenemase was carried on a broad-spectrum IncP6 plasmid identified in other Citrobacter species and non-fermenters.