title: Spontaneous and evolutionary changes in the antibiotic resistance of Burkholderia cenocepacia observed by global gene expression analysis.
abstract: BACKGROUND
  Burkholderia cenocepacia is a member of the Burkholderia cepacia complex group of bacteria that cause infections in individuals with cystic fibrosis. B. cenocepacia isolate J2315 has been genome sequenced and is representative of a virulent, epidemic CF strain (ET12). Its genome encodes multiple antimicrobial resistance pathways and it is not known which of these is important for intrinsic or spontaneous resistance. To map these pathways, transcriptomic analysis was performed on: (i) strain J2315 exposed to sub-inhibitory concentrations of antibiotics and the antibiotic potentiator chlorpromazine, and (ii) on spontaneous mutants derived from J2315 and with increased resistance to the antibiotics amikacin, meropenem and trimethoprim-sulfamethoxazole. Two pan-resistant ET12 outbreak isolates recovered two decades after J2315 were also compared to identify naturally evolved gene expression changes.
  
  
  RESULTS
  Spontaneous resistance in B. cenocepacia involved more gene expression changes and different subsets of genes than those provoked by exposure to sub inhibitory concentrations of each antibiotic. The phenotype and altered gene expression in the resistant mutants was also stable irrespective of the presence of the priming antibiotic. Both known and novel genes involved in efflux, antibiotic degradation/modification, membrane function, regulation and unknown functions were mapped. A novel role for the phenylacetic acid (PA) degradation pathway genes was identified in relation to spontaneous resistance to meropenem and glucose was found to repress their expression. Subsequently, 20 mM glucose was found to produce greater that 2-fold reductions in the MIC of multiple antibiotics against B. cenocepacia J2315. Mutation of an RND multidrug efflux pump locus (BCAM0925-27) and squalene-hopene cyclase gene (BCAS0167), both upregulated after chlorpromazine exposure, confirmed their role in resistance. The recently isolated outbreak isolates had altered the expression of multiple genes which mirrored changes seen in the antibiotic resistant mutants, corroborating the strategy used to model resistance. Mutation of an ABC transporter gene (BCAS0081) upregulated in both outbreak strains, confirmed its role in B. cenocepacia resistance.
  
  
  CONCLUSIONS
  Global mapping of the genetic pathways which mediate antibiotic resistance in B. cenocepacia has revealed that they are multifactorial, identified potential therapeutic targets and also demonstrated that putative catabolite repression of genes by glucose can improve antibiotic efficacy.
authors.affiliation: Cardiff School of Biosciences, Cardiff University, Cardiff, Wales, CF10 3AT, UK.
authors.forename: Andrea
authors.initials: A
authors.lastname: Sass
authors.forename: Angela
authors.initials: A
authors.lastname: Marchbank
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Tullis
authors.forename: John J
authors.initials: JJ
authors.lastname: Lipuma
authors.forename: Eshwar
authors.initials: E
authors.lastname: Mahenthiralingam
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology* / Q000627:therapeutic use; D057508:Burkholderia cenocepacia / Q000187:drug effects* / Q000235:genetics*; D003550:Cystic Fibrosis / Q000188:drug therapy / Q000453:epidemiology / Q000382:microbiology; D004196:Disease Outbreaks; D024881:Drug Resistance, Bacterial / Q000235:genetics*; D019143:Evolution, Molecular; D020869:Gene Expression Profiling; D006801:Humans; D009154:Mutation / Q000235:genetics; D020411:Oligonucleotide Array Sequence Analysis
chemical_list: D000900:Anti-Bacterial Agents