METABOLIC-ACTIVITIES OF METRONIDAZOLE-SENSITIVE AND METRONIDAZOLE-RESISTANT STRAINS OF HELICOBACTER-PYLORI - REPRESSION OF PYRUVATE OXIDOREDUCTASE AND EXPRESSION OF ISOCITRATE LYASE ACTIVITY CORRELATE WITH RESISTANCE

In this study, we compared metronidazole (Mtz)-sensitive and -resistan t strains of Helicobacter pylori for metabolic differences that might correlate with drug resistance. Included in this study was an isogenic Mtz(r) strain, HP1107, that was constructed by transforming genomic D NA from Mtz(r) strain HP439 into Mtz(s) strain HP500. Enzyme activitie s were also measured for Mtz(r) strains grown in the presence or absen ce of 18 mu g of metronidazole per ml (ca. one-half of the MIC). These studies confirmed the presence of the Embden-Meyerhof-Parnas, Entner- Doudoroff, and pentose pathways, H. pylori strains expressed enzymatic activities indicative of a complete and active Krebs cycle. All strai ns expressed pyruvate oxidoreductase (POR) and alpha-keto-glutarate ox idoreductase (KOR) as measured with the redox-active dye benzyl violog en (30 to 96 nmol/min/mg of protein for POR and 30 nmol/min/mg of prot ein for KOR). When grown in the presence of Mtz at greater than or equ al to 3.5 mu g/ml, Mtz(r) strains expressed no detectable POR or MOR a ctivity. The apparent repression of POR and KOR activities by Mtz affe cted bacterial growth as manifest by extended lag periods and growth y ield reductions of >30%. A dose-dependent relationship was demonstrate d between the metronidazole concentration in the growth medium and the specific activity of POR measured in bacterial cell extracts. The obs erved repression was not due to inactivation of POR by Mtz. In additio n to repression of POR and KOR activities, growth in the presence of M tz also led to decreases in the activities of various Krebs cycle enzy mes, including aconitase, isocitrate dehydrogenase, and succinate dehy dragenase. All of the Mtz(r) strains examined expressed isocitrate lya se and malate synthase activities indicative of the glyoxylate bypass. No isocitrate lyase activity was detected ire Mtz(s) strain NP500. Is ocitrate lyase activity was expressed by HP500 following transformatio n to Mtz resistance (Mtz(r) strain HP1107) with DNA from an Mtz(r) str ain. The results of this study suggest that Mtz resistance may be a re cessive trait, possibly involving inactivation of a regulatory gene, t hat results in constitutive expression of isocitrate lyase. Repression of POR and KOR activities in response to low levels of Mtz may be a g eneral response of H. pylori strains to Mtz, but only resistant strain s manage to survive via activation of compensatory metabolic pathways.