title: Analysis of mutational resistance to trimethoprim in Staphylococcus aureus by genetic and structural modelling techniques.
abstract: OBJECTIVE
  This study sought to expand knowledge on the molecular mechanisms of mutational resistance to trimethoprim in Staphylococcus aureus, and the fitness costs associated with resistance.
  
  
  METHODS
  Spontaneous trimethoprim-resistant mutants of S. aureus SH1000 were recovered in vitro, resistance genotypes characterized by DNA sequencing of the gene encoding the drug target (dfrA) and the fitness of mutants determined by pair-wise growth competition assays with SH1000. Novel resistance genotypes were confirmed by ectopic expression of dfrA alleles in a trimethoprim-sensitive S. aureus strain. Molecular models of S. aureus dihydrofolate reductase (DHFR) were constructed to explore the structural basis of trimethoprim resistance, and to rationalize the observed in vitro fitness of trimethoprim-resistant mutants.
  
  
  RESULTS
  In addition to known amino acid substitutions in DHFR mediating trimethoprim resistance (F(99)Y and H(150)R), two novel resistance polymorphisms (L(41)F and F(99)S) were identified among the trimethoprim-resistant mutants selected in vitro. Molecular modelling of mutated DHFR enzymes provided insight into the structural basis of trimethoprim resistance. Calculated binding energies of the substrate (dihydrofolate) for the mutant and wild-type enzymes were similar, consistent with apparent lack of fitness costs for the resistance mutations in vitro.
  
  
  CONCLUSIONS
  Reduced susceptibility to trimethoprim of DHFR enzymes carrying substitutions L(41)F, F(99)S, F(99)Y and H(150)R appears to result from structural changes that reduce trimethoprim binding to the enzyme. However, the mutations conferring trimethoprim resistance are not associated with fitness costs in vitro, suggesting that the survival of trimethoprim-resistant strains emerging in the clinic may not be subject to a fitness disadvantage.
authors.affiliation: Antimicrobial Research Centre and Institute of Molecular and Cellular Biology, University of Leeds, UK.
authors.forename: Anna A
authors.initials: AA
authors.lastname: Vickers
authors.forename: Nicola J
authors.initials: NJ
authors.lastname: Potter
authors.forename: Colin W G
authors.initials: CW
authors.lastname: Fishwick
authors.forename: Ian
authors.initials: I
authors.lastname: Chopra
authors.forename: Alex J
authors.initials: AJ
authors.lastname: O'Neill
mesh_terms: D019943:Amino Acid Substitution / Q000235:genetics; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics; D004252:DNA Mutational Analysis; D004269:DNA, Bacterial / Q000235:genetics; D024881:Drug Resistance, Bacterial; D008958:Models, Molecular; D020125:Mutation, Missense; D011485:Protein Binding; D013211:Staphylococcus aureus / Q000187:drug effects* / Q000235:genetics* / Q000254:growth & development; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry / Q000235:genetics; D014295:Trimethoprim / Q000494:pharmacology*
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D004269:DNA, Bacterial; D014295:Trimethoprim; D013762:Tetrahydrofolate Dehydrogenase