High-Level Antibiotic Tolerance of a Clinically Isolated Enterococcus faecalis Strain

Bacteria can survive antibiotic treatment both by acquiring antibiotic resistance genes and through mechanisms of tolerance that are based on phenotypic changes and the formation of metabolically inactive cells. Here, we report an Enterococcus faecalis strain (E. faecalis UM001B) that was isolated from a cystic fibrosis patient and had no increase in resistance but extremely high-level tolerance to ampicillin, vancomycin, and tetracycline. Specifically, the percentages of cells that survived 3.5-h antibiotic treatment (at 100 mu g . ml(-1)) were 25.4% +/- 4.3% and 51.9% +/- 4.0% for ampicillin and tetracycline, respectively; vancomycin did not exhibit any significant killing. Consistent with the changes in antibiotic susceptibility, UM001B was found to have reduced penetration of ampicillin and vancomycin and accumulation of tetracycline compared to the reference strain ATCC 29212. Based on whole-genome sequencing, four amino acid substitutions were identified in one of the tetracycline efflux pump repressors (TetRs), compared to ATCC 29212. Results of molecular simulations and experimental assays revealed that these mutations could lead to higher levels of tetracycline efflux activity. Consistently, replicating these mutations in Escherichia coli MG1655 increased its tolerance to tetracycline. Overall, these findings provide new insights into the development of multidrug tolerance in E. faecalis, which can facilitate future studies to better control enterococcal infections. IMPORTANCE Enterococcus faecalis represents a major group of pathogens causing nosocomial infections that are resistant to multiple classes of antibiotics. An important challenge associated with E. faecalis infection is the emergence of multidrug-tolerant strains, which have normal MICs but do not respond to antibiotic treatment. Here, we report a strain of E. faecalis that was isolated from a cystic fibrosis patient and demonstrated high-level tolerance to ampicillin, vancomycin, and tetracycline. Whole-genome sequencing revealed critical substitutions in one of the tetracycline efflux pump repressors that are consistent with the increased tolerance of E. faecalis UM001B to tetracycline. These findings provide new information about bacterial antibiotic tolerance and may help develop more effective therapeutics.