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Antimicrobial Agents and Chemotherapy, November 2004, p. 4366-4376, Vol. 48, No. 11
0066-4804/04/$08.00+0     DOI: 10.1128/AAC.48.11.4366-4376.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Analysis of Mupirocin Resistance and Fitness in Staphylococcus aureus by Molecular Genetic and Structural Modeling Techniques

Julian Gregston Hurdle,¹ Alexander John O'Neill,¹ Eileen Ingham,¹ Colin Fishwick,² and Ian Chopra^1*

Antimicrobial Research Centre and School of Biochemistry and Microbiology,¹ Antimicrobial Research Centre and Department of Chemistry, University of Leeds, Leeds, United Kingdom²

Received 6 February 2004/ Returned for modification 2 May 2004/ Accepted 17 July 2004

Chromosomal resistance to mupirocin in clinical isolates of Staphylococcus aureus arises from V₅₈₈F or V₆₃₁F 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 V₅₈₈F or V₆₃₁F mutations in IRS, but a new mutation (G₅₉₃V) was also identified. Second-step mutants carried combinations of previously identified IRS mutations (e.g., V₅₈₈F/V₆₃₁F and G₅₉₃V/V₆₃₁F), but additional combinations also occurred involving novel mutations (R₈₁₆C, H₆₇Q, and F₅₆₃L). First-step mupirocin-resistant mutants were not associated with substantial fitness costs, a finding that is consistent with the occurrence of V₅₈₈F or V₆₃₁F 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., A₁₉₆V, E₁₉₀K, and E₁₉₅K), 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.

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* Corresponding author. Mailing address: Antimicrobial Research Centre and School of Biochemistry and Microbiology, University of Leeds, Leeds LS2 9JT, United Kingdom. Phone: 44-113-343-5604. Fax: 44-113-343-5638. E-mail: i.chopra{at}leeds.ac.uk.

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Antimicrobial Agents and Chemotherapy, November 2004, p. 4366-4376, Vol. 48, No. 11
0066-4804/04/$08.00+0     DOI: 10.1128/AAC.48.11.4366-4376.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

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