title: Analysis of mupirocin resistance and fitness in Staphylococcus aureus by molecular genetic and structural modeling techniques.
abstract: Chromosomal resistance to mupirocin in clinical isolates of Staphylococcus aureus arises from V(588)F or V(631)F mutations in isoleucyl-tRNA synthetase (IRS). Whether these are the only IRS mutations that confer mupirocin resistance or simply those that survive in the clinic is unknown. Mupirocin-resistant mutants of S. aureus 8325-4 were therefore generated to examine their ileS genotypes and the in vitro and in vivo fitness costs associated with them before and after compensatory evolution. Most spontaneous first-step mupirocin-resistant mutants carried V(588)F or V(631)F mutations in IRS, but a new mutation (G(593)V) was also identified. Second-step mutants carried combinations of previously identified IRS mutations (e.g., V(588)F/V(631)F and G(593)V/V(631)F), but additional combinations also occurred involving novel mutations (R(816)C, H(67)Q, and F(563)L). First-step mupirocin-resistant mutants were not associated with substantial fitness costs, a finding that is consistent with the occurrence of V(588)F or V(631)F mutations in the IRS of clinical strains. Second-step mutants were unfit, but fitness could be restored by subculture in the absence of mupirocin. In most cases, this was the result of compensatory mutations that also suppressed mupirocin resistance (e.g., A(196)V, E(190)K, and E(195)K), despite retention of the original mutations conferring resistance. Structural explanations for mupirocin resistance and loss of fitness were obtained by molecular modeling of mutated IRS enzymes, which provided data on mupirocin binding and interaction with the isoleucyl-AMP reactive intermediate.
authors.affiliation: Antimicrobial Research Centre and School of Biochemistry and Microbiology, University of Leeds, Leeds LS2 9JT, United Kingdom.
authors.forename: Julian Gregston
authors.initials: JG
authors.lastname: Hurdle
authors.forename: Alexander John
authors.initials: AJ
authors.lastname: O'Neill
authors.forename: Eileen
authors.initials: E
authors.lastname: Ingham
authors.forename: Colin
authors.initials: C
authors.lastname: Fishwick
authors.forename: Ian
authors.initials: I
authors.lastname: Chopra
mesh_terms: D000249:Adenosine Monophosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D004269:DNA, Bacterial / Q000235:genetics; D024881:Drug Resistance, Bacterial; D004734:Energy Metabolism; D007532:Isoleucine / Q000378:metabolism; D007533:Isoleucine-tRNA Ligase / Q000235:genetics; D008826:Microbial Sensitivity Tests; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016712:Mupirocin / Q000494:pharmacology*; D009154:Mutation / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013211:Staphylococcus aureus / Q000187:drug effects* / Q000235:genetics*
chemical_list: D000900:Anti-Bacterial Agents; D004269:DNA, Bacterial; D007532:Isoleucine; D000249:Adenosine Monophosphate; D016712:Mupirocin; D007533:Isoleucine-tRNA Ligase