(347c) High-Level Antibiotic Tolerance of a Clinically-Isolated Enterococcus Faecalis strain

¹Department of Biomedical and Chemical Engineering, ²Syracuse Biomaterials Institute, ⁴Department of Civil and Environmental Engineering, ⁵Department of Biology, Syracuse University, Syracuse, NY 13244, USA

³Pediatric Pulmonary Center, Upstate University Hospital, Syracuse, NY, 13210 USA

Multidrug-resistant infections present a major threat to affected individuals and public health. Most of the studies on this topic have been focused on antibiotic-resistant strains that can grow in the presence of an antibiotic. However, bacteria can also survive antibiotic treatment by forming dormant phenotypic variants, known as persister cells, which have greatly contributed to persistent infections. Here, we report an Enterococcus faecalis strain (E. faecalis UM001B) isolated from a local hospital. E. faecalis cells commonly inhabit the gastrointestinal (GI) tract of mammalians; they can also cause infections when entering wounds, bloodstream, or urinary tract. This clinical isolate demonstrated low minimal inhibitory concentrations (MICs) but remarkable high-level tolerance to three clinically used antibiotics, e.g., ampicillin, vancomycin, and tetracycline. Specifically, the percentage of persister cells based on 3.5 h antibiotic treatment at a concentration of 100 µg/mL (in Lysogenic Broth at 37°C) was 25.4 ± 4.3% and 51.9 ± 4.0% for ampicillin and tetracycline, respectively. Vancomycin did not show any killing under the same treatment condition. The high tolerance of E. faecalis UM001B to vancomycin was further enhanced by its robust biofilm formation in Brian and Heart Infusion (BHI) medium. Compared to the genome of a wild-type E. faecalis ATCC 29212 that is sensitive to these three antibiotics, whole genome sequencing of E. faecalis UM001B identified 326 variants in 100 membrane-associated proteins and transporters. These changes are consistent with the reduced penetration of ampicillin and vancomycin into E. faecalis UM001B. Sequencing results also revealed variations in four nucleotides in a gene tetR that encodes a tetracycline efflux pump repressor, leading to a higher level of tetracycline efflux activities in E. faecalis UM001B during tetracycline treatment. These results were confirmed by both molecular simulation and experiments with the variants and complemented strains, which explain the high-level tolerance of E. faecalis UM001B against tetracycline. Overall, these findings provide new insights into the development of antibiotic tolerance in E. faecalis, which can help future studies to control Enterococcal infections.