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

• Published in: Journal of antimicrobial chemotherapy Vol. 63; no. 6; pp. 1112 - 1117
• Main Authors: Vickers, Anna A., Potter, Nicola J., Fishwick, Colin W. G., Chopra, Ian, O’Neill, Alex J.
• Format: Journal Article
• Language: English
• Published: Oxford Oxford University Press 01.06.2009; Oxford Publishing Limited (England)
• Subjects: Amino Acid Substitution - genetics; Anti-Bacterial Agents - pharmacology; Antibiotics; Antibiotics. Antiinfectious agents. Antiparasitic agents; Antimicrobial agents; antimicrobial drug resistance; Bacterial diseases; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Biological and medical sciences; DNA Mutational Analysis; DNA, Bacterial - genetics; drug binding; Drug resistance; Drug Resistance, Bacterial; fitness costs; Genotype & phenotype; Human bacterial diseases; Infectious diseases; Medical sciences; Models, Molecular; Mutation; Mutation, Missense; Pharmacology. Drug treatments; Protein Binding; Staphylococcal infections, streptococcal infections, pneumococcal infections; Staphylococcus aureus; Staphylococcus aureus - drug effects; Staphylococcus aureus - genetics; Staphylococcus aureus - growth & development; Staphylococcus infections; substrate binding; Tetrahydrofolate Dehydrogenase - chemistry; Tetrahydrofolate Dehydrogenase - genetics; Trimethoprim - pharmacology; fitness costs; antimicrobial drug resistance; drug binding; substrate binding; Costs; Drug susceptibility test; Trimethoprim; Modeling; Antimicrobial agent; Antifolate; Bacteria; Micrococcales; Genetics; Micrococcaceae; Antiinfectious; Staphylococcus aureus; Drug; Treatment resistance; Infection; Substrate; Analysis; Health economy; Bacteriosis; Pyrimidine derivatives; Economic aspect; Mutation; Technique; Antibacterial agent; Staphylococcal infection; Fitness
• ISSN: 0305-7453; 1460-2091; 1460-2091
• DOI: 10.1093/jac/dkp090

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.