- 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
- 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
- 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
- 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.