Mutations in the dihydrofolate reductase gene of trimethoprim-resistant isolates of Streptococcus pneumoniae

Streptococcus pneumoniae isolates resistant to several antimicrobial agent classes including trimethoprim-sulfamethoxazole have been reported with increasing frequency throughout the world. The MICs of trimethoprim, sulfamethoxazole, and trimethoprim-sulfamethoxazole (1:19) for 259 clinical isolates from South Africa were determined, and 166 of these 259 (64%) isolates were resistant to trimethoprim-sulfamethoxazole (MICs greater than or equal to 20 mg/liter). Trimethoprim resistance was found to be more strongly correlated with trimethoprim-sulfamethoxazole resistance (correlation coefficient, 0.744) than was sulfamethoxazole resistance (correlation coefficient, 0.441). The dihydrofolate reductase genes from 11 trimethoprim-resistant (MICs, 64 to 512 mu g/ml) clinical isolates of Streptococcus pneumoniae were amplified by PCR, and the nucleotide sequences were determined. Two main groups of mutations to the dihydrofolate reductase gene were found. Both groups shared six amino acid changes (Glu20-Asp, Pro70-Ser, Gln81-His, Asp92-Ala, Ile100-Leu, and Leu135-Phe). The first group included two extra changes (Lys60-Gln and Pro111-Ser), and the second group was characterized by six additional amino acid changes (Glu14-Asp, Ile74-Leu, Gln91-His, Glu94-Asp, Phe147-Ser, and Ala149-Thr). Chromosomal DNA from resistant isolates and cloned PCR products of the genes encoding resistant dihydrofolate reductases were capable of transforming a susceptible strain of S. pneumoniae to trimethoprim resistance. The inhibitor profiles of recombinant dihydrofolate reductase from resistant and susceptible isolates revealed that the dihydrofolate reductase from trimethoprim-resistant isolates was 50-fold more resistant (50% inhibitory doses [ID(50)s], 3.9 to 7.3 mu M) than that from susceptible strains (ID(50)s, 0.15 mu M). Site-directed mutagenesis experiments revealed that one mutation, Ile100-Leu, resulted in a 50-fold increase in the ID50 of trimethoprim. The resistant dihydrofolate reductases were characterized by highly conserved redundant changes in the nucleotide sequence, suggesting that the genes encoding resistant dihydrofolate reductases may have evolved as a result of inter- or intraspecies recombination by transformation.