Salicylate, Bile Acids and Extreme Acid Cause Fitness Tradeoffs for Multidrug Pumps in Escherichia coli K-12

ABSTRACT The aspirin derivative salicylate selects against bacterial multidrug efflux pumps of Escherichia coli K-12 such as MdtEF-TolC and EmrAB-TolC, and acid stress regulators such as GadE. Salicylate uptake is driven by the transmembrane pH gradient (ΔpH) and the proton motive force (PMF) which drives many efflux pumps. We used flow cytometry to measure the fitness tradeoffs of salicylate, bile acids, and extreme low pH for E. coli cultured with pump deletants. The AcrAB-TolC efflux pump conferred a fitness advantage in the presence of bile acids, an efflux substrate. Without bile acids, AcrA incurred a small fitness cost. The fitness advantage with bile acids was eliminated by the PMF uncoupler CCCP. The Gad acid fitness island encodes components of MdtEF-TolC (an acid-adapted efflux pump) as well as acid regulator GadE. The fitness advantage of E. coli cocultured with a Gad deletant (Δ slp-gadX ) was lost in the presence of salicylate. Salicylate caused an even larger fitness cost for GadE. MdtE incurred negative or neutral fitness under all media conditions, as did EmrA. But when the competition cycle included two hours at pH 2, MdtE conferred a fitness advantage. The MdtE advantage required the presence of bile acids. Thus, the MdtEF-TolC pump is useful to E. coli for transient extreme acid exposure comparable to passage through the acidic stomach. Salicylate selects against some multidrug efflux pumps, whereas bile acids selects for them; and these fitness tradeoffs are amplified by extreme acid. IMPORTANCE Control of drug resistance in gut microbial communities is a compelling problem for human health. Growth of gut bacteria is limited by host-produced acids such as bile acids, and may be modulated by plant-derived acids such as salicylic acid. Membrane-soluble organic acids can control bacterial growth by disrupting membranes, decreasing cell pH, and depleting PMF. Our flow cytometry assay measures the fitness effects of exposure to membrane-soluble organic acids, with growth cycles that may include a period of extreme acid. We find that extreme-acid exposure leads to a fitness advantage for a multidrug pump, MdtEF-TolC, which otherwise incurs a large fitness cost. Thus, organic acids and stomach acid may play important roles in controlling multidrug resistance in the gut microbiome. Therapeutic acids might be developed to limit the prevalence of multidrug resistance pumps in environmental and host-associated communities.