Enterococcusbacteria inhabit human and soil environments that show a wide range of pH. Strains include commensals as well as antibiotic-resistant pathogens. We investigated adaptation to pH stress inE. faecalisOG1RF by conducting experimental evolution in acid (pH 4.8), neutral pH (pH 7.0), and base (pH 9.0). Serial planktonic culture was performed for 500 generations, and in high-pH biofilm culture for four serial bead transfers. Nearly all mutations led to nonsynonomous codons, indicating adaptive selection. All acid-adapted clones from planktonic culture showed a mutation infusA(encoding elongation factor G). The acid-adaptedfusAmutants had a tradeoff of decreased resistance to fusidic acid (fusidate). All base-adapted clones from planktonic cultures, and some from biofilm-adapted cultures, showed mutations affecting the Pst phosphate ABC transporter (pstA, pstB, pstB2, pstC) andpyrR(pyrimidine biosynthesis regulator/uracil phosphoribosyltransferase). Biofilm culture produced small-size colonies on brain-heart infusion agar; these variants each contained a single mutation inpstB2, pstC, orpyrR. ThepstandpyrRmutants outgrew the ancestral strain at pH 9.2, with a tradeoff of lower growth at pH 4.8. Additional genes that had a mutation in multiple clones evolved at high pH (but not at low pH) includeoppBCDF(oligopeptide ABC transporter),ccpA(catabolite control protein A), andftsZ(septation protein). Overall, experimental evolution ofE.faecalisshowed strong pH dependence, favoring fusidate-sensitive elongation factor G modification at low pH and loss of phosphate transport genes at high pH.

E. faecalis bacteria are found in dental biofilms, where they experience low pH as a result of fermentative metabolism. Thus, the effect of pH on antibiotic resistance has clinical importance.