ABC and MFS efflux transporters mediate antibiotic resistance and bile salt tolerance in Enterococcus faecalis

Bacteria that reside in the gastrointestinal tract must adapt to the antibacterial properties of bile salts (BSs) and other compounds. We investigated five putative transporters of the enteric-colonizing Enterococcus faecalis for their roles in adaptation to BS and antibiotic resistance. Transcript levels of ef2593 and ef0575 increased over fivefold, and ef0420 increased 12-fold when exposed to 0.02% and 0.1% BS concentrations. No change in the expression of ef2592 or ef1814 was seen. E. faecalis wild-type MMH594 overexpressing efflux pumps by plasmid constructs (EF2592, EF2593, EF0575, EF0420) or by a pre-exposure to 0.02% BS (EF2593 or EF0420) showed a higher CFU/mL than wild type alone when exposed to 0.3% BS, indicating adaptation of E. faecalis to resist a higher BS level in the medium, and that E. faecalis BS response involved multiple transporters. In MIC assays, the following overexpressors also showed a twofold increase in antibiotic resistance: EF2592 and EF2593 (daptomycin, vancomycin); EF0420 (daptomycin, oritavancin); and EF1814 (daptomycin, levofloxacin, oritavancin, and vancomycin). While no change in EF0575-overexpressor, mutant 0575 showed a significant increase in susceptibility to daptomycin, linezolid, levofloxacin, and oritavancin. In addition, nisin-induced overexpression of several transporters from a plasmid in Lactococcus lactis also increased the MICs of multiple antibiotics, most notably for EF1814, with a 4- to 16-fold increase in the MICs of daptomycin, levofloxacin, linezolid, and vancomycin. These data indicate that expression of multiple E. faecalis transporters responds to bile salts and may contribute to survival and colonization in the gut, as well as causing low-level multidrug resistance.Bile salts are key antimicrobial components of the gastrointestinal tract, and bacterial adaptation to bile salt stress is critical for host colonization and persistence. In this study, we demonstrate that multiple E. faecalis transporters are induced by bile salts and contribute to adaptation at physiologically relevant bile salt concentrations. Importantly, several of these transporters also confer low-level resistance to clinically important antibiotics, including daptomycin, vancomycin, levofloxacin, linezolid, and oritavancin. These findings suggest that transport systems selected for survival in the intestinal environment may have the unintended consequence of increasing antibiotic tolerance and multidrug resistance. By linking host-associated environmental stress with antimicrobial resistance phenotypes, this work advances our understanding of how gut colonization factors can influence bacterial persistence and therapeutic outcomes. The identified transporters represent potential targets for strategies to limit both intestinal colonization and antibiotic resistance in E. faecalis.