Emergence of an IncX3 Plasmid Co-Harbouring the Carbapenemase Genes Blandm-5 and Blaoxa-181.

Background: The spread of transmissible plasmids with carbapenemase genes has contributed to a global increase in carbapenemase-producing Enterobacterales over the past two decades, with bla(NDM) and bla(OXA) among the most prevalent carbapenemase genes. Objectives: To characterize an Escherichia coli isolate co-carrying bla(NDM-5) and bla(OXA-181) (JBEHAAB-19-0176) that was isolated in the Japan Antimicrobial Resistant Bacterial Surveillance in 2019-20, and to evaluate the functional advantage of carrying both genes as opposed to only one. Methods: The whole-genome sequence of the isolate was determined using long- and short-read sequencing. Growth assay and co-culture experiments were performed for phenotypic characterization in the presence of different beta-lactam antibiotics. Results: WGS analysis showed that bla(NDM-5) and bla(OXA-181) were carried by the same IncX3 plasmid, pJBEHAAB-19-0176_NDM-OXA. Genetic characterization of the plasmid suggested that the plasmid emerged through the formation of a co-integrate and resolution of two typical IncX3 plasmids harbouring bla(NDM-5) and bla(OXA-181), which involved two recombination events at the IS3000 and IS26 sequences. When cultured in the presence of piperacillin or cefpodoxime, the growth rate of the transformant co-harbouring bla(NDM-5) and bla(OXA-181) was significantly higher than the transformant with only bla(NDM-5). Furthermore, in co-culture where the two bla(NDM-5)-harbouring transformants were allowed to compete directly, the strain additionally harbouring bla(OXA-181) showed a marked growth advantage. Conclusions: The additional carriage of bla(OXA-181) confers a selective advantage to bacteria in the presence of piperacillin and cefpodoxime. These findings may explain the current epidemiology of carbapenemase-producing Enterobacterales, in which bacteria carrying both bla(NDM-5) and bla(OXA-48)-like genes have emerged independently worldwide.