Aspirin inhibits the growth of Helicobacter pylori and enhances its susceptibility to antimicrobial agents.

BACKGROUND AND AIM: The role of Helicobacter pylori and aspirin in peptic ulcer formation and recurrence remains an important clinical topic. The interaction between aspirin and H pylori in vitro is also not clear. We investigated the effect of aspirin on the growth of H pylori and on the susceptibility of H pylori to antimicrobials. METHODS: Time killing studies of H pylori were performed with different concentrations of aspirin and salicylate. Growth of bacteria was assessed spectrophotometrically and by viable colony count. The effects of aspirin on the efficiency of colony formation and on metronidazole induced mutation to rifampicin resistance in H pylori were determined. Minimal inhibitory concentrations (MICs) of aspirin and metronidazole were tested by the standard agar dilution method. MICs of amoxycillin and clarithromycin were determined by the E test method. RESULTS: Aspirin and salicylate inhibited the growth of H pylori in a dose dependent manner and bactericidal activity was due to cell lysis. Aspirin 400 micro g/ml caused a 2 logs decrease in colony forming units/ml at 48 hours, and suppressed the normal ability of metronidazole to induce new mutations to rifampicin. The IC(90) of aspirin was 512 micro g/ml. Increased susceptibility of amoxycillin, clarithromycin, and metronidazole to H pylori was observed at 1 mM (180 micro g/ml) aspirin. CONCLUSIONS: Aspirin inhibited the growth of H pylori, suppressed the mutagenic effect of metronidazole, and enhanced the susceptibility of H pylori to antimicrobial agents. This mechanism is important in future drug development for effective clearing and overcoming resistance.

Roles of FrxA and RdxA nitroreductases of Helicobacter pylori in susceptibility and resistance to metronidazole.

The relative importance of the frxA and rdxA nitroreductase genes of Helicobacter pylori in metronidazole (MTZ) susceptibility and resistance has been controversial. Jeong et al. (J. Bacteriol. 182:5082--5090, 2000) had interpreted that Mtz(s) H. pylori were of two types: type I, requiring only inactivation of rdxA to became resistant, and type II, requiring inactivation of both rdxA and frxA to become resistant; frxA inactivation by itself was not sufficient to confer resistance. In contrast, Kwon et al. (Antimicrob. Agents Chemother. 44:2133--2142, 2000) had interpreted that resistance resulted from inactivation either of frxA or rdxA. These two interpretations were tested here. Resistance was defined as efficient colony formation by single cells from diluted cultures rather than as growth responses of more dense inocula on MTZ-containing medium. Tests of three of Kwon's Mtz(s) strains showed that each was type II, requiring inactivation of both rdxA and frxA to become resistant. In additional tests, derivatives of frxA mutant strains recovered from MTZ-containing medium were found to contain new mutations in rdxA, and frxA inactivation slowed MTZ-induced killing of Mtz(s) strains. Northern blot analyses indicated that frxA mRNA, and perhaps also rdxA mRNA, were more abundant in type II than in type I strains. We conclude that development of MTZ resistance in H. pylori requires inactivation of rdxA alone or of both rdxA and frxA, depending on bacterial genotype, but rarely, if ever, inactivation of frxA alone, and that H. pylori strains differ in regulation of nitroreductase gene expression. We suggest that such regulatory differences may be significant functionally during human infection.

Sequential inactivation of rdxA (HP0954) and frxA (HP0642) nitroreductase genes causes moderate and high-level metronidazole resistance in Helicobacter pylori.

Helicobacter pylori is a human-pathogenic bacterial species that is subdivided geographically, with different genotypes predominating in different parts of the world. Here we test and extend an earlier conclusion that metronidazole (Mtz) resistance is due to mutation in rdxA (HP0954), which encodes a nitroreductase that converts Mtz from prodrug to bactericidal agent. We found that (i) rdxA genes PCR amplified from 50 representative Mtz(r) strains from previously unstudied populations in Asia, South Africa, Europe, and the Americas could, in each case, transform Mtz(s) H. pylori to Mtz(r); (ii) Mtz(r) mutant derivatives of a cultured Mtz(s) strain resulted from mutation in rdxA; and (iii) transformation of Mtz(s) strains with rdxA-null alleles usually resulted in moderate level Mtz resistance (16 microg/ml). However, resistance to higher Mtz levels was common among clinical isolates, a result that implicates at least one additional gene. Expression in Escherichia coli of frxA (HP0642; flavin oxidoreductase), an rdxA paralog, made this normally resistant species Mtz(s), and frxA inactivation enhanced Mtz resistance in rdxA-deficient cells but had little effect on the Mtz susceptibility of rdxA(+) cells. Strains carrying frxA-null and rdxA-null alleles could mutate to even higher resistance, a result implicating one or more additional genes in residual Mtz susceptibility and hyperresistance. We conclude that most Mtz resistance in H. pylori depends on rdxA inactivation, that mutations in frxA can enhance resistance, and that genes that confer Mtz resistance without rdxA inactivation are rare or nonexistent in H. pylori populations.