National and local surveys have shown that during the last few years the incidence of resistance to trimethoprim/sulfamethoxazole has increased sharply among Streptococcus pneumoniae (SP). Although TMP resistance has been studied in several other microorganisms, the mechanism of resistance in SP has not been defined. Consequently, we cloned the TMP resistance determinant from two resistant (MIC = 256 μg/ml) independent clinical isolates by selection of Escherichia coli recombinants on TMP agar plates. Sequence analysis of these clones revealed that the pneumococcal DNA inserts encoded a 168 amino acid polypeptide which had high similarity to dihydrofolate reductases (DHFRs) from several other bacterial species. Based on the data obtained from these two isolates, oligonucleotide primers were designed to isolate the DHFR gene by the polymerase chain reaction from three other resistant and two susceptible SP clinical isolates, as well as the susceptible reference strain R6 (MIC ≤ 0.5 μg/ml). Comparison of the DHFR sequences from the five TMP-resistant and the three -susceptible clones showed remarkably few differences. In fact, the data suggest that high-level resistance to TMP may arise by four or fewer point mutations. The specific role of each of these mutations on the expression of TMP resistance is currently being examined by site-directed mutagenesis.

Analysis at the enzyme level showed that the susceptible and resistant pneumococci tested had similar specific DHFR activity. However, the concentration of TMP necessary to inhibit 50% of the enzyme activity was 10 to 100-fold higher in resistant strains.

We conclude that resistance to TMP in SP may originate from relatively few specific point mutations in the DHFR structural gene, which ultimately give rise to a much more drug-resistant enzyme.