Sulfa Resistance in Mouse‐Derived Pneumocystis carinii

Sulfa drugs, such as sulfamethoxazole and dapsone, are pivotal for the prophylaxis and therapy of P. curinii pneumonia. Sulfa drugs act by inhibiting dihydropteroate synthetase (DHPS), an enzyme involved in de novo folate biosynthesis. In P. carinii, DHPS is part of a trifunctional protein, Fas, which has previously been cloned and sequenced from rat-derived organisms ( I ) . Sulfa resistance has developed in a variety of bacterial and protozoan pathogens. In most cases, resistance has been shown to be due to point mutations in the gene coding for DHPS. In order to investigate whether sulfa resistance mutations could occur in P . carinii, the DHPS coding sequence was PCR-amplified and sequenced from infected mice before, during and after exposure to several rounds of subtherapeutic doses of sulfa. MATERIALS AND METHODS. Latently infected SCID mice were immunosuppressed as described (2). For each treatment cycle, mice were given trimethoprim/sulfamethoxazole (251125 mg/kg) p.0. 5 times per week for 3 weeks. There were 4 treatment cycles. In between any two treatment cycles was a 3 week period without treatment. Mice were sacrificed before and after the experiment and the lungs were frozen. Lung homogenates were then injected intratracheally into immunosuppressed germ-free mice (3). These mice were given sulfamethoxazole (0.3 mg/kg/d) in their drinking water for 6 weeks and sacrificed. Lung homogenates from these mice were passaged one more time into immunosuppressed mice, this time administered sulfamethoxazole at 0.4 mg/kg/d. As a control for the latter passage, mice infected with the IU strain of P. curinii were also treated with sulfamethoxazole at 0.4 mg/kg/d. At the end of 6 weeks, both sets of mice were sacrificed and the degree of infection scored. DNA was extracted from frozen lungs and PCR-amplified as previously described (4). Amplifed products were gel-purified and sequenced by dye-terminator automated sequencing. RESULTS AND DISCUSSION. In order to ascertain whether the drug-treated organisms were resistant to sulfa, lungs were removed from treated and untreated mice and assessed for degree of infection (Table 1) (5). While mice infected with the IU strain demonstrated marked decreases in organism burden after treatment with sulfamethoxazole (0.8 vs 4.1), mice infected with the P. carinii strainexposed to sulfa showed very poor responses to sulfa treatment (2.2 vs 3.5). These data suggest that the treated organisms had developed resistance to sulfa. Table 1. Effects of sulfamethoxazole on degree of infection in