Characterization of dihydrofolate reductase genes from trimethoprim-susceptible and trimethoprim-resistant strains of Enterococcus faecalis

Enterococci are usually susceptible in vitro to trimethoprim; however, high -level resistance (HLR) (MICs, >1,024 mu g/ml) has been reported. We studie d Enterococcus faecalis DEL, for which the trimethoprim MIC was >1,024 mu g /ml. No transfer of resistance was achieved by broth or filter matings, Two different genes that conferred trimethoprim resistance when they were clon ed in Escherichia coli (MICs, 128 and >1,024 mu g/ml) were studied. One gen e that coded for a polypeptide of 165 amino acids (MIC, 128 mu g/ml for E. coli) was identical to dfr homologs that we cloned from a trimethoprim-susc eptible E. faecalis strain, and it is presumed to be the intrinsic E. faeca lis dfr gene (which causes resistance in E. coli when cloned in multiple co pies); this gene was designated dfrE, The nucleotide sequence 5' to this df r gene showed similarity to thymidylate synthetase genes, suggesting that t he dfr and thy genes from E. faecalis are located in tandem. The E. faecali s gene that conferred HLR to trimethoprim in E. coli, designated dfrF, code s for a predicted polypeptide of 165 amino acids with 38 to 64% similarity with other dihydrofolate reductases from gram-positive and gramnegative org anisms. The nucleotide sequence 5' to dfrF did not show similarity to the t hy sequences, A DNA probe for dfrF hybridized under high-stringency conditi ons only to colony lysates of enterococci for which the trimethoprim MIC wa s > 1,024 mu g/ml; there was no hybridization to plasmid DNA from the strai n of origin. To confirm that this gene causes trimethoprim resistance in en terococci, we cloned it into the integrative vector pAT113 and electroporat ed it into RH110 (E. faecalis OG1RF::Tn916 Delta Em) (trimethoprim MIC, 0.5 mu g/ml), which resulted in RH110 derivatives for which the trimethoprim M IC was >1,024 mu g/ml. These results indicate that dfrF is an acquired but probably chromosomally located gene,which is responsible for in vitro HLR t o trimethoprim in E. faecalis.