Characterization of an AcrE-AcrF-TolC-like Putative Efflux Pump of Bacteroides spp.

Abstract

The Bacteroides spp. are the major component of microbial flora in the human GI tract and have the highest antibiotic resistance rates compared to other opportunistic anaerobic pathogens when they are found away from the GI tract. Hence, mechanisms of resistance to antibiotics within the Bacteroides spp. are important to study. In our study, we assess the ability of an operon in B. thetaiotaomicron to functionally replace two homologues, the AcrA-AcrB-TolC and AcrE-AcrF-TolC pumps of E. coli. These operons encode for Resistance-Nodulation-Division (RND) family efflux pumps with a tripartite structure allowing for efflux of a substrate (e.g. antibiotics, dyes, etc.) through the periplasmic space. To assess the functionality of the B. thetaiotaomicron derived pump in its ability to replace the E.coli homologs, Minimum Inhibitory Concentration (MIC) assays and isogenic E.coli strains were employed. These microdilution assays included antibiotics and dyes which have previously been identified as bactericidal or bacteriostatic. We found that the B. thetaiotaomicron AcrE-like portion of the efflux pump strongly recognizes crystal violet and, to a lesser extent, other antibiotics and dyes resulting in altered MIC values. The crystal violet MIC differed by approximately 10-fold in E. coli when the AcrE-like gene was introduced into the ∆AcrE::kan strain. These results suggest that, while similar, E.coli and B. thetaiotaomicron AcrE-AcrF-TolC efflux pumps may have slightly different substrate ranges. In the future, the same functional replacement study could be applied for the replacement of the efflux pump under study in B. thetaiotaomicron with the AcrE-AcrF-TolC system of E. coli.