In vitro induced anaerobic resistance to metronidazole in Trichomonas vaginalis.

Resistance to metronidazole detectable under anaerobic conditions was induced in two Trichomonas vaginalis strains (TV 10-02 and MRP-2) by cultivation at gradually increasing pressure of the drug (1-100 micrograms/ml) for 12 to 21 months. The resistant derivatives reproduced in anaerobic trypticase-yeast-extract-maltose medium at 100 micrograms/ml metronidazole and showed very high values of minimal lethal concentration for metronidazole in anaerobic in vitro assays (556-1,600 micrograms/ml at 48-h exposure to the drug). Stepwise selection was necessary to develop the resistance in either strain. Attempts to induce resistance by prolonged maintenance of trichomonads with constant, low or moderate drug concentrations (3-10 micrograms/ml) were unsuccessful. Freshly developed resistance to high concentrations of metronidazole was unstable in absence of drug pressure as well as after cryopreservation. Development of stable resistance required further cultivation at 100 micrograms/ml metronidazole. Unstable substrains did not revert to original susceptibility. They retained a moderate level of resistance, being able to grow at 10 micrograms/ml metronidazole. The strains with fully developed resistance had no activity of the hydrogenosomal enzymes pyruvate: ferredoxin oxidoreductase and hydrogenase and ceased uptake of [14C]-metronidazole. These findings indicate that the pyruvate oxidizing pathway responsible for metronidazole activation was inactivated and metabolism of the drug stopped.

Metabolic differences between metronidazole resistant and susceptible strains of Tritrichomonas foetus.

Tritrichomonas foetus mutants resistant to metronidazole lack the hydrogenosomal enzymes pyruvate: ferredoxin oxidoreductase and hydrogenase. Hydrogenosomes of these organisms did not oxidize pyruvate or produce ATP in its presence. Elimination of hydrogenosomal metabolism of pyruvate was compensated by an increased rate of glycolysis. The resistant mutants excreted no organic acids and H2 as metabolic end products. Glycolysis of the resistant T. foetus KV1-1MR-100 can be summarized as 1 mol glucose----2 mol ethanol + 2 mol CO2. The parent strain KV1, excreting H2, CO2 and acidic end products, converted about 10% of glucose to ethanol. Both strains produced ethanol from pyruvate through the action of two cytoplasmic enzymes: pyruvate decarboxylase and alcohol dehydrogenase. The specific activity of the former enzyme, catalyzing nonoxidative decarboxylation of pyruvate to acetaldehyde, was nearly seven times higher in the resistant than in the parent strain. Alcohol dehydrogenase reducing acetaldehyde to ethanol was specific to NADPH; it catalyzed the reverse reaction only slowly, and displayed similar activities in both resistant and sensitive trichomonads. Development of anaerobic metronidazole resistance in T. foetus depended on the loss of pyruvate:ferredoxin oxidoreductase as well as on the ability to increase alcoholic fermentation.

Tritrichomonas foetus: stable anaerobic resistance to metronidazole in vitro.

Stable anaerobic resistance of Tritrichomonas foetus to metronidazole was induced in vitro by cultivation of trichomonads in the Diamond's TYM medium with metronidazole in concentrations sublethal to the parasites. Nine metronidazole-resistant strains were derived from four drug-susceptible clones of the T. foetus strain KV-1. Subculturing the parasites at both increasing and constant pressure of the drug resulted in development of resistance if the medium contained at least 3 micrograms ml-1 of metronidazole and the organisms were exposed to the drug for 3 to 8 months. The development of resistance was gradual and in all clones investigated proceeded through similar sequence of stages: (1) Survival without growth and subsequent reproduction at low metronidazole concentrations (1 to 5 micrograms ml-1). (2) Survival and reproduction at moderate concentrations of the drug (10 to 15 micrograms ml-1). (3) Resistance to 100 micrograms ml-1 metronidazole, unstable in absence of selective pressure of the drug. (4) Resistance to high concentrations of metronidazole, stable when the organisms were maintained under nonselective conditions. The trichomonads with fully developed resistance were able to grow in anaerobic culture at 100 micrograms ml-1 metronidazole and could be maintained indefinitely under these conditions. The minimal lethal concentrations for metronidazole obtained with these strains in an anaerobic in vitro assay were, at 48 h, 500 to 1000 micrograms ml-1. This is 100 to 400 times higher than those obtained with the parent clones. The fully developed resistance was stable in organisms maintained in the absence of the drug over 2 years. The substrains with unstable resistance regained the susceptibility to high concentrations of metronidazole after 80 to 100 transfers in drug-free media. These strains, however, retained their resistance to moderate doses of metronidazole and full resistance could be restored by subculture in the presence of 10 micrograms ml-1 metronidazole.

Circular DNA and cardiolipin in hydrogenosomes, microbody-like organelles of trichomonads.

Circular molecules of DNA approximately 3 mum in length were revealed by electron microscopy in deproteinized extracts prepared from purified hydrogenosomal fraction of a protozoan Tritrichomonae foetus. This fraction contained also cardiolipin amounting to approximately 14.4% of its total phospholipids, as detected by thin-layer chromatography and quantitative phosphorus measurement. These characteristics extend a number of biochemical properties of hydrogenosome shared also by mitochondria and by prokaryotic cells.