Trimethoprim resistance in <em>Escherichia coli</em> exhibits an allele-specific growth advantage

© 2025 The Authors.Introduction. The antibiotic trimethoprim has been used to treat urinary tract infection (UTI) since ~1962. Alongside the nitrofurantoin, there are still justified reasons for trimethoprim use, especially in non-pregnant women. Trimethoprim resistance is commonly the result of acquiring the trimethoprim-insensitive dihydrofolate reductase gene: dfrA. Assessment of clinical Escherichia coli isolates from two clinical trials, AnTIC and ALTAR, identified carriage of two copies of dfrA. Hypothesis. The hypothesis tested here was that dual dfrA carriage provided E. coli with a growth advantage. Methodology. Two hundred and seventy-eight clinical isolates from AnTIC/ALTAR were assessed for dfrA carriage. Microplate-based growth assays assessed growth behaviour with and without 64 mg l−1 trimethoprim. Allelic replacement of dfrA5 with five other alleles was also performed. Results. One hundred and four isolates (37%) were identified to carry a total of 112 dfrA genes. Eight isolates (2.9%) carried two copies of dfrA. Comparison of dfrA+ to dual dfrA carriage could be differentiated by their growth behaviour when exposed to trimethoprim but had comparable MIC (&gt;512 mg l−1). Analysis of all dfrA+ isolates determined that the growth behaviour exhibited an allelic bias. Allelic replacement of dfrA5 with dfrA1, dfrA7, dfrA8, dfrA14 and dfrA17 demonstrated that the growth behaviour was dfrA specific. Conclusion. This analysis determined that the dual carriage of two dfrA alleles generated a growth advantage to E. coli. However, the growth behaviour was dictated by allele carriage and not specifically dual carriage, as single carriage isolates also possessed the identified phenotype. This data suggests that there is a potential clinical impact dictated by dfrA allele carriage that could improve clinical decisions on management strategies of UTI.