Interferon gamma-signature transcript profiling and IL-23 upregulation in response to Helicobacter pylori infection.

Helicobacter pylori infection is the major cause of gastroduodenal pathologies including gastric cancer. The long persistence of bacteria and the type of immune and inflammatory response determine the clinical issue. In this study, the global gene expression profile after 6 and 12 months of H. pylori infection was investigated in the mouse stomach, using the Affymetrix GeneChip Mouse Expression Array A430. Genes related to the inflammatory and immune responses were focused. Levels of selected transcripts were confirmed by reverse transcription polymerase chain reaction. Twenty- five and nineteen percent of the differentially expressed genes observed at 6 and 12 months post-infection respectively, were related to immune response. They are characterized by an interferon (IFN)gamma-dependent expression associated to a T helper 1 (Th1) polarised response. In-depth analysis revealed that an up-regulation of IL-23p19, took place in the stomach of H. pylori infected-mice. Strong IL-23p19 levels were also confirmed in gastric biopsies from H. pylori-infected patients with chronic gastritis, as compared to healthy subjects. Our microarray analysis revealed also, a high decrease of H+K+-ATPase transcripts in the presence of the H. pylori infection. Association of gastric Th1 immune response with hypochlorhydria through the down-regulation of H+K+-ATPase contributes to the genesis of lesions upon the H. pylori infection. Our data highlight that the up-regulation of IL-23 and of many IFNgamma signature transcripts occur early on during the host response to H. pylori, and suggest that this type of immune response may promote the severity of the induced gastric lesions.

Second-line treatment for failure to eradicate Helicobacter pylori: a randomized trial comparing four treatment strategies.

AIM: To compare the efficacy of different regimens in patients in whom previous Helicobacter pylori eradication therapy has failed. METHODS: In this study named StratHegy patients (n=287) were randomized to receive one of three empirical triple therapy regimens or a strategy based on antibiotic susceptibility. The empirical regimens were omeprazole, 20 mg b.d., plus amoxicillin, 1000 mg b.d., and clarithromycin, 500 mg b.d., for 7 days (OAC7), clarithromycin, 500 mg b.d., for 14 days (OAC14) or metronidazole, 500 mg b.d., for 14 days (OAM14). In the susceptibility-based strategy, patients with clarithromycin-susceptible strains received OAC14, whilst the others received OAM14. The 13C-urea breath test was performed before randomization and 4-5 weeks after eradication therapy. RESULTS: In the intention-to-treat analysis, the eradication rates for empirical therapies were as follows: OAC7, 47.4% (27/57); OAC14, 34.5% (20/58); OAM14, 63.2% (36/57); it was 74.3% (84/113) for the susceptibility-based treatment (P<0.01 when compared with OAC7 and OAC14). In patients receiving clarithromycin, the eradication rates were 80% for clarithromycin-susceptible strains and 16% for clarithromycin-resistant strains; in patients receiving OAM14, the eradication rates were 81% for metronidazole-susceptible strains and 59% for metronidazole-resistant strains. CONCLUSIONS: Eradication rates of approximately 75% can be achieved with second-line triple therapy based on antibiotic susceptibility testing. If susceptibility testing is not available, OAM14 is an appropriate alternative.

The Helicobacter pylori UreI protein: role in adaptation to acidity and identification of residues essential for its activity and for acid activation.

Helicobacter pylori is a human gastric pathogen that survives the strong acidity of the stomach by virtue of its urease activity. This activity produces ammonia, which neutralizes the bacterial microenvironment. UreI, an inner membrane protein, is essential for resistance to low pH and for the gastric colonization of mice by H. pylori. In the heterologous Xenopus oocytes expression system, UreI behaves like an H+-gated urea channel, and His-123 was found to be important for low pH activation. We investigated the role of UreI directly in H. pylori and showed that, in the presence of urea, strains expressing wild-type UreI displayed very rapid stimulation of extracellular ammonia production upon exposure to pH

Identification of nonessential Helicobacter pylori genes using random mutagenesis and loop amplification.

Analysis of the published genome sequences of Helicobacter pylori revealed that approximately 40% of the predicted open reading frames (ORFs) were of unknown function. We have developed the random mutagenesis and loop amplification (RMLA) strategy, and used this approach both to characterize individual virulence factors and to collectively screen comparatively large numbers of H. pylori mutants to identify genes that are not essential for viability in vitro. The mini-Tn3-Km transposon was used to generate a random mutant library in H. pylori strain G27. By screening the library of mutants we were able to demonstrate that the transposon integrated randomly into the chromosome of H. pylori and that RMLA was able to identify mutants in known virulence genes (urease and catalase). To test whether this strategy could be used as a high-throughput approach for the simultaneous identification of a series of nonessential genes of H. pylori, the transposon-chromosomal junctions of a pool of mutants were amplified by inverse PCR using circular fragments of genomic DNA obtained after chromosomal DNA extracted from the pool of mutants had been digested with HindIII and self-ligated. The amplification products were radioactively labelled and hybridized to a high density macroarray membrane containing a duplicated target sequence for every gene of H. pylori strain 26695. For the positive ORFs the precise site of transposon insertion was confirmed by PCR mapping. In total 78 H. pylori genes were unambiguously identified as nonessential for viability in vitro, including 20 with orthologues of unknown function in other species and 21 which were H. pylori-specific.

[UreI: a Helicobacter pylori protein essential for resistance to acidity and for the early steps of murine gastric mucosa infection]. / UreI: une protéine de Helicobacter pylori essentielle à la résistance à l'acidité et aux étapes précoces de l'infection de la muqueuse gastrique murine.

UNLABELLED: Helicobacter pylori (H. pylori) is a Gram negative microaerophilic bacteria whose only known niche is the human gastric mucosa. The presence of H. pylori is associated with various pathologies ranging from peptic ulcer disease to gastric carcinoma. H. pylori virulence is dependent on its exceptional ability to resist to the stomach acidity by hydrolyzing urea into ammonia. Survival of H. pylori to acidity in the presence of urea relies on the activity of a membrane protein, UreI. AIMS: We decided to better characterize the role of UreI (i) in vitro in ammonia production through the action of urease, and (ii) in vivo in the colonization of the gastric mucosa. METHODS: Ammonia production by a wild type strain of H. pylori or by a UreI-deficient strain was measured as a function of extracellular pH. In addition, the kinetics of elimination of a UreI-deficient mutant in vivo were realized in the mouse model for colonization. RESULTS: UreI was associated with an increase of ammonia production in acidic conditions in vitro and was necessary for the initial steps of the mouse stomach colonization. CONCLUSION: UreI thus behaves as a sensor of extracellular pH. This protein activates urease at acidic pH; thereby, it probably allows H. pylori to resist to acidity in vivo during the first steps of infection.

Differentiation of metronidazole-sensitive and -resistant clinical isolates of Helicobacter pylori by immunoblotting with antisera to the RdxA protein.

Antimicrobial resistance in Helicobacter pylori is a serious and increasing problem, and the development of rapid, reliable methods for detecting resistance would greatly improve the selection of antibiotics used to treat gastric infection with this organism. We assessed whether detection of the RdxA protein could provide the basis for determining the susceptibility of H. pylori to metronidazole. In order to raise polyclonal antisera to RdxA, we cloned the rdxA gene from H. pylori strain 26695 into the commercial expression vector pMAL-c2, purified the resultant fusion protein by affinity chromatography, and used this recombinant RdxA preparation to immunize rabbits. We then used this specific anti-RdxA antibody to perform immunoblotting on whole bacterial cell lysates of 17 metronidazole-sensitive and 27 metronidazole-resistant clinical isolates of H. pylori. While a 24-kDa immunoreactive band corresponding to the RdxA protein was observed in all metronidazole-sensitive strains, this band was absent in 25 of 27 resistant isolates. Our results indicate that testing for the absence of the RdxA protein would identify the majority of clinical isolates that will respond poorly to metronidazole-containing eradication regimens and have implications for the development of assays capable of detecting metronidazole resistance in H. pylori.

Identification of the Helicobacter pylori anti-sigma28 factor.

Flagellar motility is essential for colonization of the human gastric mucosa by Helicobacter pylori. The flagellar filament is composed of two subunits, FlaA and FlaB. Transcription of the genes encoding these proteins is controlled by the sigma28 and sigma54 factors of RNA polymerase respectively. The expression of flagellar genes is regulated, but no sigma28-specific effector was identified. It was also unclear whether H. pylori possessed a checkpoint for flagellar synthesis, and no gene encoding an anti-sigma28 factor, FlgM, could be identified by sequence similarity searches. To investigate the sigma28-dependent regulation, a new approach based on genomic data was used. Two-hybrid screening with the H. pylori proteins identified a protein of unknown function (HP1122) interacting with the sigma28 factor and defined the C-terminal part of HP1122 (residues 48-76) as the interaction domain. HP1122 interacts with region 4 of sigma28 and prevents its association with the beta-region of H. pylori RNA polymerase. Thus, HP1122 presented the characteristics of an anti-sigma28 factor. This was confirmed in H. pylori by RNA dot-blot hybridization and electron microscopy. The level of sigma28-dependent flaA transcription was higher in a HP1122-deficient strain and was decreased by the overproduction of HP1122. The overproduction of HP1122 also resulted in H. pylori cells with highly truncated flagella. These results demonstrate that HP1122 is the H. pylori anti-sigma28 factor, FlgM, a major regulator of flagellum assembly. Potential anti-sigma28 factors were identified in Campylobacter jejuni, Pseudomonas aeruginosa and Thermotoga maritima by sequence homology with the C-terminal region of HP1122.

The AmiE aliphatic amidase and AmiF formamidase of Helicobacter pylori: natural evolution of two enzyme paralogues.

Aliphatic amidases (EC 3.5.1.4) are enzymes catalysing the hydrolysis of short-chain amides to produce ammonia and the corresponding organic acid. Such an amidase, AmiE, has been detected previously in Helicobacter pylori. Analysis of the complete H. pylori genome sequence revealed the existence of a duplicated amidase gene that we named amiF. The corresponding AmiF protein is 34% identical to its AmiE paralogue. Because gene duplication is widely considered to be a fundamental process in the acquisition of novel enzymatic functions, we decided to study and compare the functions of the paralogous amidases of H. pylori. AmiE and AmiF proteins were overproduced in Escherichia coli and purified by a two-step chromatographic procedure. The two H. pylori amidases could be distinguished by different biochemical characteristics such as optimum pH or temperature. AmiE hydrolysed propionamide, acetamide and acrylamide and had no activity with formamide. AmiF presented an unexpected substrate specificity: it only hydrolysed formamide. AmiF is thus the first formamidase (EC 3.5.1.49) related to aliphatic amidases to be described. Cys-165 in AmiE and Cys-166 in AmiF were identified as residues essential for catalysis of the corresponding enzymes. H. pylori strains carrying single and double mutations of amiE and amiF were constructed. The substrate specificities of these enzymes were confirmed in H. pylori. Production of AmiE and AmiF proteins is dependent on the activity of other enzymes involved in the nitrogen metabolism of H. pylori (urease and arginase respectively). Our results strongly suggest that (i) the H. pylori paralogous amidases have evolved to achieve enzymatic specialization after ancestral gene duplication; and (ii) the production of these enzymes is regulated to maintain intracellular nitrogen balance in H. pylori.

The protein-protein interaction map of Helicobacter pylori.

With the availability of complete DNA sequences for many prokaryotic and eukaryotic genomes, and soon for the human genome itself, it is important to develop reliable proteome-wide approaches for a better understanding of protein function. As elementary constituents of cellular protein complexes and pathways, protein-protein interactions are key determinants of protein function. Here we have built a large-scale protein-protein interaction map of the human gastric pathogen Helicobacter pylori. We have used a high-throughput strategy of the yeast two-hybrid assay to screen 261 H. pylori proteins against a highly complex library of genome-encoded polypeptides. Over 1,200 interactions were identified between H. pylori proteins, connecting 46.6% of the proteome. The determination of a reliability score for every single protein-protein interaction and the identification of the actual interacting domains permitted the assignment of unannotated proteins to biological pathways.

Short-term infection with Helicobacter pylori and 1 week exposure to metronidazole does not enhance gastric mutation frequency in transgenic mice.

The aim of this study was to determine whether exposure of Helicobacter pylori-infected mice to metronidazole resulted in the delivery of mutagenic compounds to the gastric epithelium via the oxygen-insensitive NADPH nitroreductase (RdxA) of H. pylori. C57BL/6 transgenic mice containing the lambda/lacI transgene were inoculated with peptone trypsin broth, H. pylori SS1 or SS1-rdxA(-), an SS1-derived mutant in rdxA. Twelve weeks after inoculation, the mice were treated for 7 days with a control solution or with the mouse equivalent of a human dose of metronidazole 1 g od. Three weeks after completion of treatment, the animals were killed and mutations in the target lacI gene assessed by a transgenic mutagenesis assay system. There was no increase in lacI mutations in cells harvested from mice infected with H. pylori and/or exposed to metronidazole. These data suggest that short-term infection with H. pylori and exposure to metronidazole does not enhance the mutation frequency in the gastric cells of mice. Whether chronic infection and/or repeated exposure to metronidazole or other nitroaromatic compounds causes genetic damage to gastric epithelial cells remains to be determined.

[Bacteriology and pathogenicity of Helicobacter pylori]. / Bactériologie et pathogénicité d'Helicobacter pylori.

Helicobacter pylori is the prototype of bacteria belonging to a new genus, the Helicobacter genus. It is a gram-negative, highly motile and microaerophilic bacterium, with a spiral shape, that colonizes the human gastric mucosa and causes several gastroduodenal diseases. Pathogenicity of H. pylori relies upon its capacity to adapt to a hostile environment and to escape the host response. Resistance to acidity, motility, adhesion, molecular mimicry, resistance to phagocytosis, synthesis of a cytotoxin, induction of an inflammatory response are the major strategies developed by H. pylori to colonize persistently and damage gastric tissue.

Evaluation of nitrofurantoin combination therapy of metronidazole-sensitive and -resistant Helicobacter pylori infections in mice.

The main objectives of this study were to determine whether the nitroreductase enzyme encoded by the rdxA gene of Helicobacter pylori was responsible for reductive activation of nitrofurantoin and whether a triple-therapy regimen with nitrofurantoin was able to eradicate metronidazole-sensitive and -resistant H. pylori infections from mice. The susceptibilities to nitrofurantoin of parent and isogenic rdxA mutant strains (three pairs), as well as a series of matched metronidazole-sensitive and -resistant strains isolated from mice (30) and patients (20), were assessed by agar dilution determination of the MIC. Groups of mice colonized with the metronidazole-sensitive H. pylori SS1 strain or a metronidazole-resistant rdxA SS1 mutant were treated with either metronidazole or nitrofurantoin as part of a triple-therapy regimen. One month after the completion of treatment the mice were sacrificed and their stomachs were cultured for H. pylori. The nitrofurantoin MICs for all strains tested were between 0.5 and 4.0 microg/ml. There was no significant difference between the susceptibility to nitrofurantoin of the parental strains and those of respective rdxA mutants or between those of matched metronidazole-sensitive and -resistant H. pylori isolates. The regimen with metronidazole eradicated infection from all eight SS1-infected mice and from one of eight mice inoculated with the rdxA mutant (P < or =0.001). The regimen with nitrofurantoin failed to eradicate infection from any of the six SS1-infected mice (P < or =0.001) and cleared infection from one of seven mice inoculated with the rdxA mutant. These results demonstrate that, despite the good in vitro activity of nitrofurantoin against H. pylori and the lack of cross-resistance between metronidazole and nitrofurantoin, eradication regimens involving nitrofurantoin are unable to eradicate either metronidazole-sensitive or -resistant H. pylori infections from mice.

Outbred mice with long-term Helicobacter felis infection develop both gastric lymphoid tissue and glandular hyperplastic lesions.

Experimental infection of mice with Helicobacter felis reproduces many aspects of the gastritis observed in Helicobacter pylori-infected humans. The development of gastric inflammatory lesions in chronically infected inbred mice is host-dependent; in BALB/c mice, gastric B-cell MALT lymphomas were observed, whilst other murine hosts (e.g. C57BL/6) developed severe glandular hyperplasia. The aims of this investigation were to characterize and immunophenotype Helicobacter-induced inflammatory lesions in mice with an outbred genetic background. Swiss mice (n=10 per group) were either inoculated with a suspension of H. felis or left untreated. H. felis-inoculated mice and age-matched control animals were killed 13 months later. The severity of gastric inflammatory lesions in the animals was graded and the number and distribution of B (CD45R(+)) and T (CD3(+)) lymphocytes in lymphoid tissues was determined by immunohistochemistry. Compared with control mice, animals with long-term H. felis infection developed severe hyperplastic gastritis (0.80+/-0.63 vs. 2.7+/-0.68), with epithelial dedifferentiation (0. 40+/-0.52 vs. 2.3+/-0.82) and lengthening of the pits and glands (0. 46+/-0.05 vs. 0.8+/-0.19). Gastric CD45R(+) and CD3(+) lymphocyte scores were significantly elevated (r=0.803) in infected animals, while lymphoepithelial lesions and polymorphonuclear leucocyte infiltrates were absent. Although prominent lymphoid follicles were present in the tissues of all infected animals, and in one control animal, only a proportion (55%) of the mucosal follicles had a dominant B-cell phenotype (defined as > or =75% CD45R(+) labelling), and all were poorly labelled with anti-mouse immunoglobulin antibodies. It was concluded that the lesions in outbred Swiss mice differed from B-cell MALT lymphomas. In contrast to inbred mice, outbred animals developed both glandular and lymphoid tissue lesions to chronic H. felis infection. It is suggested that the default T-helper phenotype of the host influences glandular lesion formation or B-cell lymphomagenesis in this model of infection.

A novel 3-methyladenine DNA glycosylase from Helicobacter pylori defines a new class within the endonuclease III family of base excision repair glycosylases.

The cloning, purification, and characterization of MagIII, a 3-methyladenine DNA glycosylase from Helicobacter pylori, is presented in this paper. Sequence analysis of the genome of this pathogen failed to identify open reading frames potentially coding for proteins with a 3-methyladenine DNA glycosylase activity. The putative product of the HP602 open reading frame, reported as an endonuclease III, shares extensive amino acid sequence homology with some bacterial members of this family and has the canonic active site helix-hairpin-helix-GPD motif. Surprisingly, this predicted H. pylori endonuclease III encodes a 25,220-Da protein able to release 3-methyladenine, but not oxidized bases, from modified DNA. MagIII has no abasic site lyase activity and displays the substrate specificity of the 3-methyladenine-DNA glycosylase type I of Escherichia coli (Tag) because it is not able to recognize 7-methylguanine or hypoxanthine as substrates. The expression of the magIII open reading frame in null 3-methyladenine glycosylase E. coli (tag alkA) restores to this mutant partial resistance to alkylating agents. MagIII-deficient H. pylori cells show an alkylation-sensitive phenotype. H. pylori wild type cells exposed to alkylating agents present an adaptive response by inducing the expression of magIII. MagIII is thus a novel bacterial member of the endonuclease III family, which displays biochemical properties not described for any of the members of this group until now.

Inferring regulatory elements from a whole genome. An analysis of Helicobacter pylori sigma(80) family of promoter signals.

Helicobacter pylori is adapted to life in a unique niche, the gastric epithelium of primates. Its promoters may therefore be different from those of other bacteria. Here, we determine motifs possibly involved in the recognition of such promoter sequences by the RNA polymerase using a new motif identification method. An important feature of this method is that the motifs are sought with the least possible assumptions about what they may look like. The method starts by considering the whole genome of H. pylori and attempts to infer directly from it a description for a family of promoters. Thus, this approach differs from searching for such promoters with a previously established description. The two algorithms are based on the idea of inferring motifs by flexibly comparing words in the sequences with an external object, instead of between themselves. The first algorithm infers single motifs, the second a combination of two motifs separated from one another by strictly defined, sterically constrained distances. Besides independently finding motifs known to be present in other bacteria, such as the Shine-Dalgarno sequence and the TATA-box, this approach suggests the existence in H. pylori of a new, combined motif, TTAAGC, followed optimally 21 bp downstream by TATAAT. Between these two motifs, there is in some cases another, TTTTAA or, less frequently, a repetition of TTAAGC separated optimally from the TATA-box by 12 bp. The combined motif TTAAGCx(21+/-2)TATAAT is present with no errors immediately upstream from the only two copies of the ribosomal 23 S-5 S RNA genes in H. pylori, and with one error upstream from the only two copies of the ribosomal 16 S RNA genes. The operons of both ribosomal RNA molecules are strongly expressed, representing an encouraging sign of the pertinence of the motifs found by the algorithms. In 25 cases out of a possible 30, the combined motif is found with no more than three substitutions immediately upstream from ribosomal proteins, or operons containing a ribosomal protein. This is roughly the same frequency of occurrence as for TTGACAx(15-19)TATAAT (with the same maximum number of substitutions allowed) described as being the sigma(70 )promoter sequence consensus in Bacillus subtilis and Escherichia coli. The frequency of occurrence of the new motif obtained, TTAAGCx(19-23)TATAAT, remains high when all protein genes in H. pylori are considered, as is the case for the TTGACAx(15-19)TATAAT motif in B. subtilis but not in E. coli.

Frequent association between alteration of the rdxA gene and metronidazole resistance in French and North African isolates of Helicobacter pylori.

Mutations in the rdxA gene have been associated with the acquisition of resistance to metronidazole in Helicobacter pylori. This gene encodes an NADPH nitroreductase whose expression is necessary for intracellular activation of the drug. We wished to examine whether mutations in rdxA were present in resistant H. pylori isolates infecting either French or North African patients. We determined the complete nucleotide sequences of the rdxA genes from seven French and six North African patients infected with paired resistant and sensitive strains. Genotyping by random amplified polymorphic DNA analysis confirmed the close genetic relatedness of the susceptible and resistant isolates from individual biopsies. Eight French and five North African individual resistant strains were also studied. For the French strains, an alteration in rdxA most probably implicated in resistance was found in 10 cases (seven frameshift mutations, two missense mutations, and one deletion of 211 bp). One to three putative missense mutations were identified in four cases, and a missense mutation possibly not implicated in resistance was discovered in the last case. For the North African strains, an alteration in rdxA was found in eight cases (three frameshift mutations, three missense mutations, one deletion of 6 bp, and one insertion of a variant of IS605). Two strains contained putative missense mutations, and no change was observed in rdxA of the last strain. Thus, inactivation of the rdxA gene is frequently, but not always, associated with resistance to metronidazole in French and North African clinical isolates of H. pylori. In addition, a variety of alterations of rdxA are associated with the resistant phenotype.

Gastric helicobacters in cats.

The types of helicobacter which are found in the stomachs of carnivorous pets, especially cats, have been traditionally referred to as 'gastric helicobacter-like organisms' (GHLOs). These are microaerophilic, Gram-negative, spiral bacteria with multiple terminal flagellae and are endowed with high-level urease activity which allows them to survive in an acidic environment. Certain species have one or more periplasmic fibrils. The two GHLOs most commonly found in cats are Helicobacter felis and a species related to H heilmannii which was recently cultured from dogs. All phenotypic and genotypic (16S RNA gene sequences) evidence suggests that both of these bacteria belong in the genus Helicobacter. Whether or not helicobacters can be transmitted to humans from carnivorous pets is controversial but the recent discovery of H pylori -infected cats may be evidence of an animal reservoir for this pathogen. Although the role of H pylori in inducing antral gastritis and perpetuating pyloric ulcers in humans is well established, whether or not Helicobacter spp are causally involved in any feline gastric inflammatory conditions is unknown.

Performance of native and recombinant antigens for diagnosis of Helicobacter pylori infection.

The aim of this study was to evaluate the performance of three antigenic preparations for serological diagnosis of Helicobacter pylori infection: (i) native antigens from Helicobacter pylori strain N6 or its aflagellated isogenic mutant N6flbA-, or an acellular extract (antigen AgFA) from a pool of six clinical strains; (ii) recombinant antigens consisting of CagA fused to MS2 polymerase and HspA or recombinant UreA and UreB fused to the maltose-binding protein, and (iii) the preparations provided with two commercial kits, the Cobas Core (Roche Diagnostic Systems, France) and the Pylori Stat (BioWhittaker, Belgium). All preparations were used in an enzyme immunoassay to test 92 sera from dyspeptic patients for whom the status of Helicobacter infection was established. Sensitivities were higher (90 to 100%) for the native antigens and the commercial kits than for the recombinant antigens. Specificities were higher than 90%, except with UreA + UreB (42%). The most useful antigens were those extracted from strains N6 and N6flbA-.

The role of the rdxA gene in the evolution of metronidazole resistance in Helicobacter pylori.

It was recently demonstrated that inactivation of the rdxA gene, which encodes an oxygen-insensitive NADPH nitroreductase, is associated with the development of resistance to metronidazole by Helicobacter pylori. In order to further evaluate the contribution of rdxA to metronidazole resistance, the sequence of the rdxA gene was determined for a series of metronidazole-sensitive and -resistant isolates derived from a single, metronidazole-sensitive strain using an H. pylori mouse model. These strains were cultured from the stomachs of mice experimentally infected with H. pylori strain SS1 and then treated orally with metronidazole. The sequence of the rdxA gene of all 10 sequenced metronidazole-sensitive and two (7%) of the 27 metronidazole-resistant isolates was identical to that of the parental strain. In contrast, the rdxA gene of the other 25 metronidazole-resistant isolates contained between one and three frameshift or missense mutations. This suggests that while the development of metronidazole resistance in H. pylori is frequently associated with mutational inactivation of the rdxA gene, other mechanisms of resistance are likely to exist in this bacterium.