The mode of action of metronidazole in Helicobacter pylori: futile cycling or reduction?

The effects of metronidazole on catalase-positive and spontaneous catalase-negative mutants of Helicobacter pylori were studied to investigate whether the action of metronidazole on this microaerophilic organism occurs by reactive oxygen species generated by futile cycling or by the reduction of metronidazole to its active form. Increased sensitivity would be expected to occur in catalase-negative mutants if the mode of action of metronidazole was mediated through reactive oxygen species that may result from futile cycling of metronidazole. Two strains, RU1 and N6, were found to mutate spontaneously to a catalase-negative phenotype. The catalase-positive strain RU1(KatA+) and its catalase-negative counterpart RU1(KatA-) were sensitive to metronidazole, with MICs of 0.5 mg/L. The metronidazole-sensitive strain RU1(KatA-) lost viability at a rate similar to the parent RU1(KatA+) strain in the presence of 10 mg/L of metronidazole. Stable resistance to metronidazole was induced in RU1(KatA+) and RU1(KatA-) by passaging these strains in the presence of metronidazole. The catalase-positive and catalase-negative strains, N6(KatA+) and N6(KatA-), were resistant to metronidazole, with MICs of 96 mg/L. These observations indicated that the presence or absence of catalase activity did not affect the susceptibility of strains to metronidazole. The metabolism of metronidazole by H. pylori was investigated by 14N-NMR spectroscopy. Metronidazole was reduced in sensitive, catalase-positive and catalase-negative strains. Metronidazole-resistant cells reduced the 5-nitroimidazole more slowly, suggesting that resistance is achieved through the prevention or inhibition of metronidazole reduction.