title: 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.
  
  
  
  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.
authors: Department of Biology, Kenyon College, Gambier, Ohio, USA.
authors: Kaitlin E
authors: KE
authors: Creamer
authors: Department of Biology, Kenyon College, Gambier, Ohio, USA.
authors: Frederick S
authors: FS
authors: Ditmars
authors: Department of Biology, Kenyon College, Gambier, Ohio, USA.
authors: Preston J
authors: PJ
authors: Basting
authors: Department of Biology, Kenyon College, Gambier, Ohio, USA.
authors: Karina S
authors: KS
authors: Kunka
authors: Department of Biology, Kenyon College, Gambier, Ohio, USA.
authors: Issam N
authors: IN
authors: Hamdallah
authors: Department of Biology, Kenyon College, Gambier, Ohio, USA.
authors: Sean P
authors: SP
authors: Bush
authors: Department of Biology, Kenyon College, Gambier, Ohio, USA.
authors: Zachary
authors: Z
authors: Scott
authors: Department of Biology, Kenyon College, Gambier, Ohio, USA.
authors: Amanda
authors: A
authors: He
authors: Department of Biology, Kenyon College, Gambier, Ohio, USA.
authors: Stephanie R
authors: SR
authors: Penix
authors: Department of Biology, Kenyon College, Gambier, Ohio, USA.
authors: Alexandra S
authors: AS
authors: Gonzales
authors: Department of Biology, Kenyon College, Gambier, Ohio, USA.
authors: Elizabeth K
authors: EK
authors: Eder
authors: Department of Biology, Kenyon College, Gambier, Ohio, USA.
authors: Dominic W
authors: DW
authors: Camperchioli
authors: Department of Biology, Kenyon College, Gambier, Ohio, USA.
authors: Adama
authors: A
authors: Berndt
authors: Department of Biology, Kenyon College, Gambier, Ohio, USA.
authors: Michelle W
authors: MW
authors: Clark
authors: Department of Chemistry, Kenyon College, Gambier, Ohio, USA.
authors: Kerry A
authors: KA
authors: Rouhier
authors: Department of Biology, Kenyon College, Gambier, Ohio, USA slonczewski@kenyon.edu.
authors: Joan L
authors: JL
authors: Slonczewski
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology; D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000378:metabolism; D001565:Benzoates / Q000378:metabolism*; D005075:Biological Evolution; D004305:Dose-Response Relationship, Drug; D004352:Drug Resistance, Microbial / Q000235:genetics*; D048168:Escherichia coli K12 / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D005520:Food Preservatives / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D012459:Salicylates / Q000378:metabolism*
chemical_list: D000900:Anti-Bacterial Agents; D000894:Anti-Inflammatory Agents, Non-Steroidal; D001565:Benzoates; D005520:Food Preservatives; D012459:Salicylates
keywords: Escherichia coli; acid; antibiotic resistance; aspirin; benzoate; chloramphenicol; experimental evolution; low pH; salicylate