Genome-facilitated discovery of RND efflux pump-mediated resistance to cephalosporins in Vibrio spp. isolated from the mummichog fish gut

Abstract Objectives This study examines the role of RND effluxpumps in resistance to first- and third-generation cephalosporinsas well as the potential contribution to increased virulence in two Vibrioisolates from the gut microbiota of a forage- feeder fish. Methods We performed phenotypic MIC testing in the presence and absence of an RND effluxpump inhibitor, phenylalanine-arginine-beta-napthylamide (PAβN), and further compared the genomes of the two Vibriospp. to characterized RND effluxpump gene homologs. Results We identified 13 and 12 RND operons, respectively, in Vibriospp. T21 and T9, with Vibriosp. T21 containing an additional RND operoncompared to other V. parahaemolyticus strains. Both the inner-membrane protein (IMP) and the membrane facilitator protein (MFP) sequences of this operonare homologous to VexD and VexC, respectively, an RND operonin V. cholerae . More generally, the other RND proteins in our strains show homology to RND effluxpumps characterized in E. coli and V. cholerae . In Vibriosp. T21, we observed decreased resistance to cefoperazoneand cephradine, and in Vibriosp. T9 , decreased resistance to cefoperazoneand cefsulodinin the presence of PAβN. RND pumps may also mediate transportof kanamycin. Conclusions By analyzing the genomes of two Vibriospp. isolated from the mummichogfish gut we discovered RND effluxpump-mediated resistance to first- and third-generation cephalosporinsin these strains. This work highlights the need for further research into this unique Vibriosp. operon, and more generally, RND effluxpumps in Vibriospecies, as Vibriooften cause seafood-borne illness.