Prolonged exposure to teixobactin generates cross-tolerance to other cell wall-targeting antimicrobials in Enterococcus faecalis

Antimicrobial tolerance, i.e., the ability to survive despite growth inhibition, is an important precursor to the development of antimicrobial resistance. However, very little is known about the evolution of drug-induced antimicrobial tolerance. Teixobactin (TXB) is an antimicrobial peptide that targets the cell envelope precursors lipid II and lipid III in Gram-positive bacteria. We have previously shown that Enterococcus faecalis displays high intrinsic tolerance to killing by TXB, and this may aid in the development of TXB resistance in this species. Here, we demonstrate that prolonged exposure to TXB led to the emergence of enhanced tolerance, but not TXB resistance. Whole-genome sequencing of these TXB-tolerant mutants identified mutations in the mevalonate and Epa (enterococcal polysaccharide antigen) biosynthesis pathways, hprK, a key regulator of carbon metabolism, and liaF, a negative regulator of the LiaSR cell envelope stress response. Increased susceptibility to TXB in single gene deletion mutants of the sensor kinase liaS and cognate response regulator liaR provides further support for a novel role of the LiaFSR cell envelope stress response system in TXB tolerance. Finally, we demonstrate that constitutive hyper-signaling of the CroRS and LiaSR cell envelope stress responses is consistent with broad tolerance to TXB and the clinically relevant antimicrobials daptomycin and ampicillin, suggesting expression profiling of the cell envelope stress response may serve as a key indicator of antimicrobial tolerance in E. faecalis.