Mutation-based mechanism and evolution of the potent multidrug efflux pump RE-CmeABC in .

The resistance-nodulation-cell division (RND) superfamily of multidrug efflux systems are important players in mediating antibiotic resistance in gram-negative pathogens. , a major enteric pathogen, utilizes an RND-type transporter system, CmeABC, as the primary mechanism for extrusion of various antibiotics. Recently, a functionally potent variant of CmeABC (named RE-CmeABC) emerged in clinical isolates, conferring enhanced resistance to multiple antibiotic classes. Despite the clinical importance of RE-CmeABC, the molecular mechanisms for its functional gain and its evolutionary trajectory remain unknown. Here, we demonstrated that amino acid substitutions in RE-CmeB (inner membrane transporter), but not in RE-CmeA (periplasmic protein) and RE-CmeC (outer membrane protein), in conjunction with a nucleotide mutation in the promoter region of the efflux operon, are responsible for the functional gain of the multidrug efflux system. We also showed that RE- is emerging globally and distributed in genetically diverse strains, suggesting its possible spread by horizontal gene transfer. Notably, many of RE- harboring isolates were associated with the human host including strains from large disease outbreaks, indicating the clinical relevance and significance of RE-CmeABC. Evolutionary analysis indicated that RE- likely originated from , but its expansion mainly occurred in possibly driven by antibiotic selection pressure. Additionally, RE-, but not RE- and RE-, experienced a selective sweep and was progressing to be fixed during evolution. Together, these results identify a mutation-based mechanism for functional gain in RE-CmeABC and reveal the key role of RE-CmeB in facilitating adaptation to antibiotic selection.