Antibiotic resistance mediated by site-specific mutations in the multidrug efflux transporter CmeB of zoonotic...

The resistance‒nodulation‒division (RND) family of multidrug efflux transporters is widely distributed in Gram-negative bacteria. Although their roles in mediating antibiotic resistance have been well known, our understanding of how they are altered to augment bacterial adaptation to antibiotic selection remains at an infancy stage. Here, we report the identification of a mutation-based mechanism that empowers the function of the CmeB efflux protein, an RND-type transporter in the zoonotic pathogen Campylobacter. During our surveillance study, we identified Campylobacter isolates that were phenotypically resistant to florfenicol but lacked known florfenicol resistance mechanisms. Using natural transformation and whole genome sequencing, we first linked the phenotype to sequence polymorphisms in the cmeB and subsequently demonstrated that both the T136A and M292I mutations in CmeB are required for the resistance phenotype. The mutations elevated Campylobacter resistance to florfenicol, ciprofloxacin, and other classes of antimicrobial agents. Structural modeling and molecular dynamics simulations revealed that the two residues were localized in the drug-binding pocket of CmeB, and the T136A and M292I substitutions enhanced hydrophobic interactions, stabilized CmeB-antibiotic binding, and lessened steric hindrance in the drug-binding pocket, thereby facilitating antibiotic extrusion by CmeB. Analysis of the Campylobacter genomic sequences deposited in the NCBI database revealed that T136A- and M292I-harboring isolates were found in 35 different countries and associated with various host species, indicating the widespread distribution and clinical relevance of the two mutations. Together, these results identified a new mechanism underlying CmeB-mediated multidrug resistance and provide a potential target for clinical surveillance of antibiotic-resistant Campylobacter.