Benzoate- and Salicylate-Tolerant Strains of Escherichia coli K-12 Lose Antibiotic Resistance during Laboratory Evolution

Abstract

Escherichia coli K-12 W3110 grows in the presence of membrane-permeant organic acids that can depress cytoplasmic pH and accumulate in the cytoplasm. We conducted experimental evolution by daily diluting cultures in increasing concentrations of benzoic acid (up to 20 mM) buffered at external pH 6.5, a pH at which permeant acids concentrate in the cytoplasm. By 2,000 generations, clones isolated from evolving populations showed increasing tolerance to benzoate but were sensitive to chloramphenicol and tetracycline. Sixteen clones grew to stationary phase in 20 mM benzoate, whereas the ancestral strain W3110 peaked and declined. Similar growth occurred in 10 mM salicylate. Benzoate-evolved strains grew like W3110 in the absence of benzoate, in media buffered at pH 4.8, pH 7.0, or pH 9.0, or in 20 mM acetate or sorbate at pH 6.5. Genomes of 16 strains revealed over 100 mutations, including single-nucleotide polymorphisms (SNPs), large deletions, and insertion knockouts. Most strains acquired deletions in the benzoate-induced multiple antibiotic resistance (Mar) regulon or in associated regulators such as rob and cpxA, as well as the multidrug resistance (MDR) efflux pumps emrA, emrY, and mdtA. Strains also lost or downregulated the Gad acid fitness regulon. In 5 mM benzoate or in 2 mM salicylate (2-hydroxybenzoate), most strains showed increased sensitivity to the antibiotics chloramphenicol and tetracycline; some strains were more sensitive than a marA knockout strain. Thus, our benzoate-evolved strains may reveal additional unknown drug resistance components. Benzoate or salicylate selection pressure may cause general loss of MDR genes and regulators. IMPORTANCE Benzoate is a common food preservative, and salicylate is the primary active metabolite of aspirin. In the gut microbiome, genetic adaptation to salicylate may involve loss or downregulation of inducible multidrug resistance systems. This discovery implies that aspirin therapy may modulate the human gut microbiome to favor salicylate tolerance at the expense of drug resistance. Similar aspirin-associated loss of drug resistance might occur in bacterial pathogens found in arterial plaques.

Inverted Regulation of Multidrug Efflux Pumps, Acid Resistance, and Porins in Benzoate-Evolved Escherichia coli K-12

“…In fact, more than half the isolates sequenced had one of seven different mutations within the slp-gad island (Table S1). Large deletions were likely mediated by upstream insertion sequences, a common finding in stress evolution experiments (18,23). Thus, our early-generation sequences confirmed that the loss of Gad acid resistance was strongly selected by benzoate.…”

“…The MdtEF-TolC complex exports antibiotics from the cytoplasm, driven by PMF. Our benzoate-evolved isolates (18) have acquired point mutations or deletions throughout the slp-gad island, as well as knockout or point mutations in emrA, encoding the membrane fusion protein component of the EmrAB-TolC MDR pump (37,38); mdtA (39,40); and emrY (41). Other mutations affect genes for MDR regulators, such as cpxA (42), ariR (43), arcA (44), and rob (45).…”

“…MarA downregulates the large porin OmpF, which admits nutrients and antibiotics (48). Many targets of MarA are also subject to the MarA homolog Rob (45,46), which had a mutation in one benzoate-evolved isolate (18). Additionally, one strain acquired a mutation in the alpha subunit of RNA polymerase (RNAP) which could affect the regulation of a wide array of genes.…”

“…S1). At this benzoate concentration, the ancestral strain W3110 stopped growing and entered death phase by 12 h, whereas the 2,000-generation evolved strains grew more rapidly and reached stationary phase at an optical density at 600 nm (OD 600 ) of about 0.25 (18). All the A1 population isolates from generation 900 or 1,400 grew in 20 mM benzoate to a stable stationary phase.…”

“…MdtEF-TolC exports a variety of antibiotics and toxic metabolites, including chloramphenicol (44,51,52). It is one of a number of MDR pumps and regulators reported to be lost or mutated in genomes evolved with benzoate (18) or with CCCP (20). Note that while mdtEF mutations did appear early, sensitivity to chloramphenicol was not detected before generation 1,400 or 2,000 ( Table 2).…”