title: The TCA cycle is not required for selection or survival of multidrug-resistant Salmonella.
abstract: OBJECTIVE
  The initial aim of this study was to use a systems biology approach to analyse a ciprofloxacin-selected multidrug-resistant (MDR) Salmonella enterica serotype Typhimurium, L664.
  
  
  METHODS
  The whole genome sequence and transcriptome of L664 were analysed. Site-directed mutagenesis to recreate each mutation was carried out, followed by phenotypic characterization and mutation frequency analysis. As a mutation in the TCA cycle was detected we tested the controversial hypothesis regarding the bacterial response to bactericidal antibiotics, put forward by Kohanski et al. (Cell 2007; 130: 797-810 and Mol Cell 2010; 37: 311-20), that exposure of bacteria to agents such as ciprofloxacin produces reactive oxygen species (ROS), which transiently increase the mutation rate giving rise to MDR bacteria.
  
  
  RESULTS
  L664 contained a mutation in ramR that conferred MDR. A mutation in tctA affected the TCA cycle and conferred the inability to grow on minimal agar. The virulence of L664 was not attenuated. Ciprofloxacin exposure produced ROS in L664 and SL1344 (tctA::aph), but it was reduced and occurred later. There were no significant differences in the rates of killing or mutations per generation to antibiotic resistance between the strains.
  
  
  CONCLUSIONS
  Whilst we confirm production of ROS in response to ciprofloxacin, we have no data to support the hypothesis that this leads to selection of MDR strains. Our results indicate that the mutations in tctA and glgA were random as they did not pre-exist in the parental strain, and that the mutation in tctA did not provide a survival advantage or disadvantage in the presence of antibiotic.
authors.affiliation: Antimicrobial Agents Research Group, School of Immunity and Infection, University of Birmingham, Edgbaston, Birmingham, UK.
authors.forename: Vito
authors.initials: V
authors.lastname: Ricci
authors.forename: Nick
authors.initials: N
authors.lastname: Loman
authors.forename: Mark
authors.initials: M
authors.lastname: Pallen
authors.forename: Alasdair
authors.initials: A
authors.lastname: Ivens
authors.forename: Maria
authors.initials: M
authors.lastname: Fookes
authors.forename: Gemma C
authors.initials: GC
authors.lastname: Langridge
authors.forename: John
authors.initials: J
authors.lastname: Wain
authors.forename: Laura J V
authors.initials: LJ
authors.lastname: Piddock
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology; D002939:Ciprofloxacin / Q000494:pharmacology*; D002952:Citric Acid Cycle; D024901:Drug Resistance, Multiple, Bacterial; D006801:Humans; D050296:Microbial Viability / Q000187:drug effects*; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000187:drug effects; D017382:Reactive Oxygen Species / Q000378:metabolism; D012486:Salmonella typhimurium / Q000187:drug effects* / Q000378:metabolism / Q000502:physiology*; D012641:Selection, Genetic; D049490:Systems Biology / Q000379:methods; D059467:Transcriptome
chemical_list: D000900:Anti-Bacterial Agents; D017382:Reactive Oxygen Species; D002939:Ciprofloxacin