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JAC Advance Access originally published online on April 21, 2009
Journal of Antimicrobial Chemotherapy 2009 63(6):1112-1117; doi:10.1093/jac/dkp090
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© The Author 2009. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Original research

Analysis of mutational resistance to trimethoprim in Staphylococcus aureus by genetic and structural modelling techniques

Anna A. Vickers1, Nicola J. Potter2, Colin W. G. Fishwick2, Ian Chopra1 and Alex J. O’Neill1,*

1 Antimicrobial Research Centre and Institute of Molecular and Cellular Biology, University of Leeds, Leeds LS2 9JT, UK 2 Antimicrobial Research Centre and School of Chemistry, University of Leeds, Leeds LS2 9JT, UK

Received 12 November 2008; returned 27 December 2008; revised 18 February 2009; accepted 23 February 2009

* Corresponding author. Tel: +44-113-233-5600; Fax: +44-113-233-5638; E-mail: a.j.oneill{at}leeds.ac.uk

Objectives: 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 (F99Y and H150R), two novel resistance polymorphisms (L41F and F99S) 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 L41F, F99S, F99Y and H150R 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.

Keywords: antimicrobial drug resistance , drug binding , substrate binding , fitness costs


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