Oxygen scavenging, NADH oxidase and metronidazole resistance in helicobacter pylori

Abstract

Failure of triple-therapy regimes to eradicate Helicobacter pylori from the stomach is thought to be due to the occurrence of a metronidazole-resistant bacterial population. Exposure of metronidazole-resistant (Mtz(R)) strains of H. pylori to an anaerobic environment causes the activation of metronidazole and the loss of resistance. Using metronidazole-sensitive (Mtz(S)) clinical isolates, we selected mutants conferring resistance to metronidazole, which were used to investigate the effect of bacterial cell density upon the activation of metronidazole. The addition of metronidazole, at a final concentration of 10 mg/L, to Mtz(R) cultures of a bacterial cell density > 1 x 106 cfu/mL, caused a loss in viability. No loss in viability, however, occurred upon addition of metronidazole to Mtz(R) cultures of a cell density of < 1 x 106 cfu/mL. Mtz(S) cultures lost viability irrespective of the initial cell density, indicating that oxygen scavenging at the site of metronidazole reduction may occur in these cultures. The ability of Mtz(S) wild types, Mtz(R) isogenic mutants and Mtz(R) wild types to scavenge oxygen from the intracellular environment was investigated. H. pylori cultures contained NADH and NADPH oxidase activity. NADPH oxidase activity was always more than double the NADH oxidase activity. Mtz(R) mutants possessed approximately one-third the NADH oxidase activity found in their respective Mtz(S) parent wild types. Mtz(R) wild types possessed a low NADH oxidase level similar to that found in the Mtz(R) mutants. We propose that metronidazole resistance may be mediated through an inability of Mtz(R) strains to remove oxygen from the site of metronidazole reduction, thereby preventing metronidazole activation.