Apr. 17, 1998 St. Louis, April 13 -- Scientists in Halifax, Nova Scotia, and St. Louis, Mo., have discovered why the bacterium Helicobacter pylori, which causes peptic ulcer disease, is sensitive to metronidazole, a critical component of the leading H. pylori therapy. They also have determined how the bacterium becomes resistant to this drug. H. pylori infects more than half the world's people and is a major early risk factor for stomach cancer.
The researchers' findings also raise concern about a possible link between the drug and stomach cancer in people infected with H. pylori. "The real danger lurks when a person takes metronidazole without the complete complement of drugs that eradicate this bacterium," says Paul S. Hoffman, Ph.D., professor of microbiology and immunology and medicine at Dalhousie University Medical School in Halifax. "When metronidazole is taken alone, it can be activated by one of the bacterium's enzymes to produce hydroxylamine, a mutagen and cancer-causing chemical."
The collaborators describe their findings in the April 14 issue of Molecular Microbiology. Hoffman's graduate student, Avery Goodman, is lead author of the paper.
Metronidazole -- a generic drug sold as Flagyl, MetroGel and Protostat -- is prescribed for dental abscesses, certain vaginal infections and conditions where anaerobic bacteria or protozoan parasites are suspected. It also is the key component in combination therapies for peptic ulcer disease. But between 10 percent and 30 percent of H. pylori strains in the United States and Western Europe are metronidazole-resistant. In developing countries, the proportion may be as high as 80 percent. This resistance is the most common reason for treatment failure, renewal of infection and recurrence of peptic ulcers and other stomach lesions.
The researchers discovered that metronidazole resistance results from mutation in a gene called rdxA. This gene codes for one of the nitroreductase enzymes that allow H. pylori to break down organic nitrogen compounds. The enzyme also happens to convert metronidazole to hydroxylamine, which damages DNA, proteins and other macromolecules and kills the bacterium. So the bacterium changes a harmless chemical into a lethal drug. When the rdxA gene is inactivated by mutation, however, H. pylori can?t break down metronidazole and therefore becomes resistant.
After the Dalhousie scientists cloned and sequenced rdxA, they and their Washington University collaborators showed that this gene is responsible for resistance. First, the researchers found that the bacterium E. coli, which normally is metronidazole-resistant, became sensitive to the drug when they inserted rdxA from H. pylori into it. Second, they made resistant H. pylori sensitive again by adding extra copies of rdxA.
They also specifically inactivated rdxA in H. pylori simply by inserting another marker gene into it, thereby disrupting its DNA sequence. The H. pylori with the mutant rdxA gene became fully metronidazole-resistant. This critical experiment showed that rdxA alone confers metronidazole sensitivity and that its loss of function is sufficient to cause resistance. "It was very satisfying to see that the altered strains, whose only difference from the wild type was having this inactivated gene, showed metronidazole resistance," says Douglas E. Berg, Ph.D., the Alumni Professor in Molecular Microbiology and professor of genetics at Washington University School of Medicine in St. Louis.
Berg and research associate Dangeruta Kersulyte, Ph.D., also looked to see whether normally sensitive H. pylori strains become etronidazole-resistant by picking up mutant genes from already resistant strains -- many bacteria donate pieces of DNA that carry resistance genes to other strains or species. The researchers therefore examined pairs of H. pylori isolates from patients in Peru and Lithuania, where infection rates are very high. The two members of each pair came from the same patient and were chosen because one was metronidazole-sensitive while the other was resistant.
By analyzing DNA from the H. pylori chromosome, the researchers determined whether the members of each pair differed significantly from each other, which might suggest that resistance was due to an extra piece of DNA, or whether they differed at just one or two points, suggesting new mutation.
Because transferable drug resistance is so common in other bacterial species, they were intrigued to find that all the resistant strains they examined had new mutations in the rdxA gene that had made the parental strain metronidazole-sensitive.
"This indicates that mutation is the easiest way for resistance to arise in H. pylori," Berg says. "Our guess is that it occurs because the bacterium converts metronidazole to hydroxylamine, a powerful mutagen, and use of metronidazole again in later therapies selects for these newly resistant mutant variants."
Metronidazole-resistant strains often arise in people who have never before been treated for H. pylori infection but who may have taken metronidazole periodically for other reasons. In many countries, for example, the drug can be purchased very cheaply without a prescription, and it usually is used at doses that are insufficient to kill all the H. pylori cells a person might carry. That person therefore would accumulate resistant strains, selected by the drug, and sensitive strains, which would make hydroxylamine in the stomach.
"If I lived in a society where gastric cancer was a major problem, the last thing I would want would be to have H. pylori delivering mutagen to my gastric epithelial cells at the same time this bacterium was creating a long-term inflammation that also is known to contribute to gastric cancer," Berg says.
Goodwin A, Kersulyte D, Sisson G, Veldhuyzen van Zanten SJO, Berg DE, Hoffman PS. Metronidazole resistance in Helicobacter pylori is due to null mutations in a gene (rdxA) that encodes an oxygen-insensitive NADP nitroreductase. Molecular Microbiology, 28(2), April 14, 1998.
Grants from Astra Pharm, Canada, the Canadian Medical Research Council, the U.S. National Institutes of Health and the American Cancer Society supported this research.
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