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ABSTRACT Enterococcus bacteria inhabit human and soil environments that show a wide range of pH values. Strains include commensals as well as antibiotic-resistant pathogens. We investigated the adaptation to pH stress in E. faecalis OG1RF by conducting experimental evolution under acidic (pH 4.8), neutral pH (pH 7.0), and basic (pH 9.0) conditions. A serial planktonic culture was performed for 500 generations and in a high-pH biofilm culture for 4 serial bead transfers. Nearly all of the mutations led to nonsynonomous codons, indicating adaptive selection. All of the acid-adapted clones from the planktonic culture showed a mutation in fusA (encoding elongation factor G). The acid-adapted fusA mutants had a trade-off of decreased resistance to fusidic acid (fusidate). All of the base-adapted clones from the planktonic cultures as well as some from the biofilm-adapted cultures showed mutations that affected the Pst phosphate ABC transporter ( pstA , pstB , pstB2 , pstC ) and pyrR (pyrimidine biosynthesis regulator/uracil phosphoribosyltransferase). The biofilm cultures produced small-size colonies on brain heart infusion agar. These variants each contained a single mutation in pstB2 , pstC , or pyrR . The pst and pyrR mutants outgrew the ancestral strain at pH 9.2, with a trade-off of lower growth at pH 4.8. Additional genes that had a mutation in multiple clones that evolved at high pH (but not at low pH) include opp1BCDF (oligopeptide ABC transporter), ccpA (catabolite control protein A), and ftsZ (septation protein). Overall, the experimental evolution of E. faecalis showed a strong pH dependence, favoring the fusidate-sensitive elongation factor G modification at low pH and the loss of phosphate transport genes at high pH. IMPORTANCE 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. The loss of fusidate resistance is notable for OG1RF strains in which fusidate resistance is assumed to be a stable genetic marker. In endodontal infections, enterococci can resist calcium hydroxide therapy that generates extremely high pH values. In other environments, such as the soil and plant rhizosphere, enterococci experience acidification that is associated with climate change. Thus, the pH modulation of natural selection in enterococci is important for human health as well as for understanding soil environments. 

Abstract The perennial ice-covered lakes of the Antarctic McMurdo Dry Valleys harbour oligotrophic microbial communities that are separated geographically from other aquatic systems. Their microbiomes include planktonic microbes as well as lift-off mat communities that emerge from the ice. We used the ShortBRED protein family profiler to quantify the antibiotic resistance genes (ARGs) from metagenomes of lift-off mats emerging from ice and from filtered water samples of Lake Fryxell and Lake Bonney. The overall proportion of ARG hits was similar to that found in temperate-zone rural ponds with moderate human inputs. Specific ARGs showed distinct distributions for the two lakes and for mat vs planktonic sources. Metagenomic taxa distributions showed that mat phototrophs consisted mainly of cyanobacteria or Betaproteobacteria, whereas the water column phototrophs were mainly protists. An enrichment culture of the Betaproteobacterium Rhodoferax antarcticus from a Lake Fryxell mat sample showed an unusual mat-forming phenotype not previously reported for this species. Its genome showed no ARGs associated with Betaproteobacteria but had ARGs consistent with a minor Pseudomonas component. The Antarctic lake mats and water showed specific ARGs distinctive to the mat and water sources, but overall ARG levels were similar to those of temperate water bodies with moderate human inputs. 

Bacterial genomes encode various multidrug efflux pumps (MDR) whose specific conditions for fitness advantage are unknown. We show that the efflux pump MdtEF-TolC, in Escherichia coli, confers a fitness advantage during exposure to extreme acid (pH 2). Our flow cytometry method revealed pH-dependent fitness tradeoffs between bile acids (a major pump substrate) and salicylic acid, a membrane-permeant aromatic acid that induces a drug-resistance regulon but depletes proton motive force (PMF). The PMF drives MdtEF-TolC and related pumps such as AcrAB-TolC. Deletion of mdtE (with loss of pump MdtEF-TolC) increased the strain’s relative fitness during growth with or without salicylate or bile acids. However, when the growth cycle included a 2-h incubation at pH 2 (below the pH growth range), MdtEF-TolC conferred a fitness advantage. The fitness advantage required bile salts but was decreased by the presence of salicylate, whose uptake is amplified by acid. For comparison, AcrAB-TolC, the primary efflux pump for bile acids, conferred a PMF-dependent fitness advantage with or without acid exposure in the growth cycle. A different MDR pump, EmrAB-TolC, confered no selective benefit during growth in the presence of bile acids. Without bile acids, all three MDR pumps incurred a large fitness cost with salicylate when exposed at pH 2. These results are consistent with the increased uptake of salicylate at low pH. Overall, we showed that MdtEF-TolC is an MDR pump adapted for transient extreme-acid exposure; and that low pH amplifies the salicylate-dependent fitness cost for drug pumps. IMPORTANCE Antibiotics and other drugs that reach the gut must pass through stomach acid. Yet little is known of how extreme acid modulates the effect of drugs on gut bacteria. We find that extreme-acid exposure leads to a fitness advantage for a multidrug pump that otherwise incurs a fitness cost. At the same time, extreme acid amplifies the effect of salicylate selection against multidrug pumps. Thus, organic acids and stomach acid could play important roles in regulating multidrug resistance in the gut microbiome. Our flow cytometry assay provides a way to measure the fitness effects of extreme-acid exposure to various membrane-soluble organic acids including plant-derived nutrients and pharmaceutical agents. Therapeutic acids might be devised to control the prevalence of multidrug pumps in environmental and host-associated habitats. 

Evolutionary innovations allow populations to colonize new ecological niches. We previously reported that aerobic growth on citrate (Cit+) evolved in an Escherichia coli population during adaptation to a minimal glucose medium containing citrate (DM25). Cit+ variants can also grow in citrate-only medium (DM0), a novel environment for E. coli. To study adaptation to this niche, we founded two sets of Cit+ populations and evolved them for 2500 generations in DM0 or DM25. The evolved lineages acquired numerous parallel mutations, many mediated by transposable elements. Several also evolved amplifications of regions containing the maeA gene. Unexpectedly, some evolved populations and clones show apparent declines in fitness. We also found evidence of substantial cell death in Cit+ clones. Our results thus demonstrate rapid trait refinement and adaptation to the new citrate niche, while also suggesting a recalcitrant mismatch between E. coli physiology and growth on citrate.