Teixobactin is a new antimicrobial of significant interest. It is active against a number of multidrug-resistant pathogens, including Staphylococcus aureus and Enterococcus faecalis, with no reported mechanisms of teixobactin resistance. However, historically, mechanisms of resistance always exist and arise upon introduction of a new antimicrobial into a clinical setting. Therefore, for teixobactin to remain effective long term, we need to understand how mechanisms of resistance could develop. Here we demonstrate that E. faecalis shows a remarkable intrinsic tolerance to high concentrations of teixobactin. This is of critical importance, as antimicrobial tolerance has been shown to precede the development of antimicrobial resistance. To identify potential pathways responsible for this tolerance, we determined the genomewide expression profile of E. faecalis strain JH2-2 in response to teixobactin using RNA sequencing. A total of 573 genes were differentially expressed (2.0-fold log2 change in expression) in response to teixobactin, with genes involved in cell wall biogenesis and division and transport/binding being among those that were the most upregulated. Comparative analyses of E. faecalis cell wall-targeting antimicrobial transcriptomes identified CroRS, LiaRS, and YclRK to be important two-component regulators of antimicrobial-mediated stress. Further investigation of CroRS demonstrated that deletion of croRS abolished tolerance to teixobactin and to other cell wall-targeting antimicrobials. This highlights the crucial role of CroRS in controlling the molecular response to teixobactin. IMPORTANCE Teixobactin is a new antimicrobial with no known mechanisms of resistance. Understanding how resistance could develop will be crucial to the success and longevity of teixobactin as a new potent antimicrobial. Antimicrobial tolerance has been shown to facilitate the development of resistance, and we show that E. faecalis is intrinsically tolerant to teixobactin at high concentrations. We subsequently chose E. faecalis as a model to elucidate the molecular mechanism underpinning teixobactin tolerance and how this may contribute to the development of teixobactin resistance.

Antimicrobial tolerance is the ability of a microbial population to survive, but not proliferate, during antimicrobial exposure. Significantly, it has been shown to precede the development of bona fide antimicrobial resistance. We have previously identified the two-component system CroRS as a critical regulator of tolerance to antimicrobials like teixobactin in the bacterial pathogenEnterococcus faecalis.To understand the molecular mechanism of this tolerance, we carried out RNA-seq analyses in theE. faecaliswild-type and isogeniccroRSmutant to determine the teixobactin-induced CroRS regulon. We identified a 132 gene CroRS regulon and show CroRS upregulates expression of all major components of the enterococcal cell envelope in response to teixobactin challenge. To gain further insight into the function of this regulon we isolated and characterized croRS mutants recovered for wild-type growth and tolerance. We show introduction of a single stop codon in a heptaprenyl diphosphate synthase (hppS), a key enzyme in the synthesis of the quinone electron carrier demethylmenaquinone (DMK), is sufficient to rescue loss of cell envelope integrity in thecroRSdeletion strain. Based on these findings, we propose a model where CroRS acts as a gate-keeper of isoprenoid biosynthesis, mediating flux of isoprenoids needed for cell wall synthesis (undecaprenyl pyrophosphate; UPP) and respiration (DMK) to maintain cell wall homeostasis upon antimicrobial challenge. Dysregulation of this flux in the absence ofcroRSleads to a loss of tolerance, which is rescued by loss of function mutations in HppS, allowing an increase in isoprenoid flow to UPP and subsequently cell wall synthesis.

Enterococcus faecalis and Enterococcus faecium are human and animal gut commensals. Vancomycin-resistant enterococci (VRE) are important opportunistic pathogens with limited treatment options. Historically, the glycopeptide antibiotics vancomycin and avoparcin selected for the emergence of vancomycin resistance in human and animal isolates, respectively, resulting in global cessation of avoparcin use between 1997 and 2000. To better understand human- and animal-associated VRE strains in the postavoparcin era, we sequenced the genomes of 231 VRE isolates from New Zealand (NZ; 75 human clinical, 156 poultry) cultured between 1998 and 2009. E. faecium lineages and their antibiotic resistance carriage patterns strictly delineated between agricultural and human reservoirs, with bacitracin resistance ubiquitous in poultry but absent in clinical E. faecium strains. In contrast, one E. faecalis lineage (ST108) predominated in both poultry and human isolates in the 3 years following avoparcin discontinuation. Both phylogenetic and antimicrobial susceptibility (i.e., ubiquitous bacitracin resistance in both poultry and clinical ST108 isolates) analyses suggest an agricultural origin for the ST108 lineage. VRE isolate resistomes were carried on multiple, heterogeneous plasmids. In some isolate genomes, bacitracin, erythromycin, and vancomycin resistance elements were colocalized, indicating multiple potentially linked selection mechanisms. IMPORTANCE Historical antimicrobial use in NZ agriculture has driven the evolution of ST108, a VRE lineage carrying a range of clinically relevant antimicrobial resistances. The persistence of this lineage in NZ for over a decade indicates that coselection may be an important stabilizing mechanism for its persistence.