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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 isogenic ΔcroRSmutant 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 ΔcroRSmutants 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.ImportanceAntimicrobial tolerance is the ability of a microorganism to survive, but not grow upon antimicrobial challenge, and is an important precursor to the development of antimicrobial resistance (the ability to profilerate). Understanding the molecular mechanisms that underpin tolerance will therefore aid in hampering the development of resistance to novel antimicrobials such as teixobactin. CroRS is an essential two-component regulator of antimicrobial tolerance in the bacterial pathogenEnterococcus faecalis. We have determined the antimicrobial-induced CroRS regulon and identified key mutations in a heptaprenyl diphosphate synthase to uncover a novel mechanism of antimicrobial tolerance.

Bacteria have developed resistance against every antimicrobial in clinical use at an alarming rate. There is a critical need for more effective use of antimicrobials to both extend their shelf life and prevent resistance from arising. Significantly, antimicrobial tolerance, i.e., the ability to survive but not proliferate during antimicrobial exposure, has been shown to precede the development of bona fide antimicrobial resistance (AMR), sparking a renewed and rapidly increasing interest in this field. As a consequence, problematic infections for the first time are now being investigated for antimicrobial tolerance, with increasing reports demonstrating in-host evolution of antimicrobial tolerance. Tolerance has been identified in a wide array of bacterial species to all bactericidal antimicrobials. Of particular interest are enterococci, which contain the opportunistic bacterial pathogens Enterococcus faecalis and Enterococcus faecium. Enterococci are one of the leading causes of hospital-acquired infection and possess intrinsic tolerance to a number of antimicrobial classes. Persistence of these infections in the clinic is of growing concern, particularly for the immunocompromised. Here, we review current known mechanisms of antimicrobial tolerance, and include an in-depth analysis of those identified in enterococci with implications for both the development and prevention of AMR.