Potential involvement of several nitroreductases in metronidazole resistance in Helicobacter pylori.

Susceptibility of Helicobacter pylori to the antibiotic metronidazole has been attributed to the activity of an oxygen-insensitive NADPH-dependent nitroreductase (RdxA), with resistance to this antimicrobial arising from null mutations in rdxA. To obtain a better understanding of the factors involved in resistance, nitroreductase and metronidazole reduction activities were investigated in matched pairs of clinical and laboratory-derived sensitive and resistant H. pylori strains. Significant differences in enzyme activities were observed between sensitive and resistant strains, suggesting that metronidazole susceptibility in H. pylori was associated with more than one enzyme activity. To establish the mutations occurring in rdxA, the genes from seventeen bacterial strains, including matched pairs were sequenced. To assess whether metronidazole was responsible for inducing random mutations in this gene, the complete nucleotide sequence of gene hp0630, encoding an NAD(P)H-quinone reductase which also has NADPH-dependent nitroreductase activity, was determined in the same strains. All resistant strains showed nonsense, missense, or frameshift mutations randomly throughout rdxA. In contrast, no mutations were observed in hp0630. The results confirmed the presence of rdxA null mutations in resistant strains and suggested that other factors involved in the metabolism of metronidazole contributed to the resistant phenotype.

Association between the IgG subclass response, inflammation and disease status in Helicobacter pylori infection.

BACKGROUND: In many viral, bacterial and parasitic infections the Immunoglobulin G (IgG) subclass response has been shown to correlate with severity of inflammation and disease outcome. The aim of the present study was to investigate the association between the IgG subclass response to Helicobacter pylori infection and disease and inflammation. METHODS: Eighty-three symptomatic patients undergoing endoscopic examination were included in the study. Upon endoscopic examination, the presence of ulceration was noted and biopsy specimens were collected from the gastric antrum, body and transitional zone. Blood was also collected from each patient. Gastric biopsy sections were graded using the Sydney system. H. pylori specific IgG, IgG1, IgG2, IgG3 and IgG4 were measured by ELISA. The IgG subclass was also examined retrospectively in sera collected from 20 patients previously proven to have duodenal ulcer (DU). RESULTS: The results of histological examination and IgG serology showed 35 subjects to be H. pylori negative and 48 to be H. pylori positive. Of the 48 H. pylori positive subjects, 25 were diagnosed with functional dyspepsia (FD), 14 with current DU and 9 with evidence of past DU. Significantly higher levels of IgG2 antibodies were found in patients with DU as compared with patients with FD (P < 0.01). In addition, significantly higher IgG3 subclass antibody levels were associated with chronic inflammatory cells in the body (P < 0.05) and active inflammatory cells in the transitional zone (P < 0.01). A significantly increased level of IgG1 antibodies was associated with lower levels of colonization in the gastric antrum. CONCLUSION: The results of this study suggest that the IgG subclass response in subjects infected with H. pylori may be a marker of DU disease as well as increased levels of inflammation.

Oxidases and reductases are involved in metronidazole sensitivity in Helicobacter pylori.

Helicobacter pylori is a contributing factor to the development of gastric and duodenal ulcers and some gastric cancers. Some therapeutic regimes comprise of a number of components, one of which is the antimicrobial metronidazole. A problem with these therapies is the increasing prevalence of metronidazole-resistant (MtrR) H. pylori strains. Several resistance mechanisms have been proposed, and this study addresses the 'scavenging of oxygen' hypothesis. Spectrophotometric assays of cytosolic fractions indicated that metronidazole-sensitive (MtrS) H. pylori isolates had 2.6-fold greater nicotinamide adenine dinucleotide (NADH) oxidase activity, 34-fold greater NADH nitroreductase activity, and eightfold greater nicotinamide adenine dinucleotide phosphate (NADPH) nitroreductase activity than cytosolic fractions from matched MtrR strains. Electrophoresis of cytosolic fractions in non-denaturing gels showed up to 10 protein bands when stained with Coomassie blue. Activity staining of non-denaturing, non-reducing polyacrylamide gels detected NAD(P)H oxidase, disulphide reductase, tetrazolium reductase and nitroreductase activities in the protein bands. Oxidase and reductase activities observed in a band from MtrS strains were absent in the corresponding band from MtrR strains. This band comprised at least 13 proteins, and the major constituent was identified as an alkyl hydroperoxide reductase AhpC subunit. The absence of oxidase and reductase activities in the band from MtrR strains indicated a correlation between the activity of the proteins in this band and the metronidazole-sensitive phenotype.

The Helicobacter pylori pyrB gene encoding aspartate carbamoyltransferase is essential for bacterial survival.

The production of defined isogenic Helicobacter pylori pyrB mutants was undertaken to investigate the role of aspartate carbamoyltransferase (encoded by pyrB) in the survival of the bacterium. The complete structural gene for aspartate carbamoyltransferase from H. pylori strain RU1 was cloned into Escherichia coli by complementation of a pyrB auxotrophic mutant to facilitate the construction of a pyrB-disrupted copy in E. coli. The H. pylori pyrB gene had high similarity to other bacterial pyrB genes, and the phylogenetic clustering with different species was consistent with functional characteristics of the ACTase. The transcription initiation site for H. pylori pyrB-mRNA was mapped 25 bp upstream of the ATG start codon, and potential promoter regions were identified. In order to construct an isogenic pyrB H. pylori mutant by natural transformation and allelic exchange, the plasmid insert containing pyrB was disrupted by insertional mutagenesis of a chloramphenicol transferase gene cassette. In multiple transformations of H. pylori cells, no chloramphenicol-resistant pyrB mutants were isolated. Successful mutagenesis of other H. pylori genes and PCR amplification of the recombined gene demonstrated that the ACTase-negative mutants had been constructed by allelic exchange involving simultaneous replacement of the pyrB gene with the chloramphenicol-pyrB-disrupted copy. These findings suggested that the ACTase enzyme is essential for the survival of H. pylori.

Use of digoxigenin-labelled ampicillin in the identification of penicillin-binding proteins in Helicobacter pylori.

Amoxycillin is used in current therapeutic regimens to treat the infection caused by the human gastric pathogen, Helicobacter pylori. The penicillin-binding proteins (PBPs) are the primary targets for the beta-lactam antibiotics, such as amoxycillin, and are involved in the terminal stages of peptidoglycan synthesis. They also play active roles in the determination and maintenance of cellular morphology. It was believed that an organism with a complex morphology, such as H. pylori, would have more than the three PBPs previously suggested. Using digoxigenin-labelled ampicillin (DIG-ampicillin), we report the identification of eight PBPs in H. pylori with masses of 72, 62, 54, 50, 44, 33.5, 30.5 and 28 kDa. A smaller (21 kDa) ninth band was also detected, which may represent another PBP. However, the relatively small size of this apparent PBP raises questions as to whether this is a true PBP. In an attempt to identify the PBPs to which amoxycillin preferentially binds, amoxycillin was used in competition assays with DIG-ampicillin. It appeared that amoxycillin inhibited the binding of DIG-ampicillin to only the 72 kDa PBP. The experimental data were also compared with the seven putative PBPs identified in the two published H. pylori genomes, most of which correlate with the experimental data. To investigate further the properties of these PBPs, the seven putative PBP genes identified in the H. pylori genomes were examined. The derived amino acid sequences of the putative PBPs were examined for the three characteristic motifs found in all conventional PBPs, SXXK, SXN and KTG. We were able to determine that all of the putative PBPs had at least one of these motifs, but none possessed all three motifs with the characteristics of conventional PBPs. These findings suggest that the PBPs of H. pylori are unique.

Fumarate metabolism and the microaerophily of Campylobacter species.

(1) The role of fumarate metabolism in the microaerophily of the Campylobacter genus and the effects of therapeutic agents against it were investigated. (2) NMR spectroscopy was employed to determine the properties of Campylobacter fumarase (Fum) and fumarate reductase (Frd). Radiotracer analysis was used to determine the production of carbon dioxide by Campylobacter cells. Standard microbiological techniques were used to measure the effects of environmental conditions and inhibitors on bacterial growth. (3) All Campylobacter species tested showed both Fum and Frd activities. Frd activity was observed with or without the addition of an exogenous electron donor in the particulate fractions obtained from lysates. Fumarate was oxidized to carbon dioxide via the acetyl-CoA cleavage pathway. The genes encoding proteins involved in fumarate metabolism were identified in the Campylobacter jejuni genome. Cells grew better in atmospheres with 5 and 10% oxygen levels. Fum activity was the same in cultures grown under different oxygen tensions and did not vary with the age of cultures. Frd activity was higher in cultures which grew at faster rates and decreased with the age of cultures. Four Frd inhibitors showed bactericidal effects against Campylobacter spp. with different potencies. The relative strengths of inhibition of the compounds followed the same order as the bactericidal effects. (4) The results suggested that Frd and Fum are constitutive and play a fundamental role in these microaerophiles which show characteristics of anaerobic metabolism, and that the Frd inhibitors tested would not be of therapeutic use.

Metabolism and genetics of Helicobacter pylori: the genome era.

The publication of the complete sequence of Helicobacter pylori 26695 in 1997 and more recently that of strain J99 has provided new insight into the biology of this organism. In this review, we attempt to analyze and interpret the information provided by sequence annotations and to compare these data with those provided by experimental analyses. After a brief description of the general features of the genomes of the two sequenced strains, the principal metabolic pathways are analyzed. In particular, the enzymes encoded by H. pylori involved in fermentative and oxidative metabolism, lipopolysaccharide biosynthesis, nucleotide biosynthesis, aerobic and anaerobic respiration, and iron and nitrogen assimilation are described, and the areas of controversy between the experimental data and those provided by the sequence annotation are discussed. The role of urease, particularly in pH homeostasis, and other specialized mechanisms developed by the bacterium to maintain its internal pH are also considered. The replicational, transcriptional, and translational apparatuses are reviewed, as is the regulatory network. The numerous findings on the metabolism of the bacteria and the paucity of gene expression regulation systems are indicative of the high level of adaptation to the human gastric environment. Arguments in favor of the diversity of H. pylori and molecular data reflecting possible mechanisms involved in this diversity are presented. Finally, we compare the numerous experimental data on the colonization factors and those provided from the genome sequence annotation, in particular for genes involved in motility and adherence of the bacterium to the gastric tissue.

The tricarboxylic acid cycle of Helicobacter pylori.

The composition and properties of the tricarboxylic acid cycle of the microaerophilic human pathogen Helicobacter pylori were investigated in situ and in cell extracts using [1H]- and [13C]-NMR spectroscopy and spectrophotometry. NMR spectroscopy assays enabled highly specific measurements of some enzyme activities, previously not possible using spectrophotometry, in in situ studies with H. pylori, thus providing the first accurate picture of the complete tricarboxylic acid cycle of the bacterium. The presence, cellular location and kinetic parameters of citrate synthase, aconitase, isocitrate dehydrogenase, alpha-ketoglutarate oxidase, fumarate reductase, fumarase, malate dehydrogenase, and malate synthase activities in H. pylori are described. The absence of other enzyme activities of the cycle, including alpha-ketoglutarate dehydrogenase, succinyl-CoA synthetase, and succinate dehydrogenase also are shown. The H. pylori tricarboxylic acid cycle appears to be a noncyclic, branched pathway, characteristic of anaerobic metabolism, directed towards the production of succinate in the reductive dicarboxylic acid branch and alpha-ketoglutarate in the oxidative tricarboxylic acid branch. Both branches were metabolically linked by the presence of alpha-ketoglutarate oxidase activity. Under the growth conditions employed, H. pylori did not possess an operational glyoxylate bypass, owing to the absence of isocitrate lyase activity; nor a gamma-aminobutyrate shunt, owing to the absence of both gamma-aminobutyrate transaminase and succinic semialdehyde dehydrogenase activities. The catalytic and regulatory properties of the H. pylori tricarboxylic acid cycle enzymes are discussed by comparing their amino acid sequences with those of other, more extensively studied enzymes.

The prevalence of antibody to CagA in children is not a marker for specific disease.

BACKGROUND: In adults, a high prevalence of antibody to the cytotoxin-associated antigen (CagA) of Helicobacter pylori has been linked to the development of more serious gastroduodenal disease. Few investigators have examined this association in children. The purpose of this study was to investigate the seroprevalence of antibody to the CagA antigen as well as other specific H. pylori antigens in children. METHODS: By use of an immunoblot analysis kit, the immune response to specific H. pylori antigens in serum collected from 21 H. pylori-positive symptomatic Australian children, 5 with peptic ulcer disease and 16 with nonulcer dyspepsia, and 33 H. pylori-positive asymptomatic Chinese children. Sera from 20 H. pylori-negative symptomatic Australian children were used as control subjects. RESULTS: Antibody responses to the 26.5-kDa, 30-kDa, and 116-kDa (CagA) antigens were found to be the most prevalent, with 81.5%, 79.6%, and 76% of children, respectively, mounting a response. In contrast, antibody responses to the 19.5-kDa, 35-kDa, 45-kDa, 60-kDa, 89 kDa (VacA), and 180-kDa antigens occurred in 55.5%, 24%, 16.7%, 63%, 37%, and 7.4% of children, respectively. A higher prevalence of antibody response to CagA was found in the symptomatic Australian children with peptic ulcer disease (100%) compared with prevalence in those with nonulcer dyspepsia (56.3%), but the difference did not reach statistical significance. No significant difference was found between the prevalence of antibody to CagA in the Australian peptic ulcer disease group (100%) and that in the asymptomatic Chinese children (81.8%). CONCLUSION: These results suggest that in children CagA is not a marker of specific disease development.

A novel mechanism for resistance to the antimetabolite N-phosphonoacetyl-L-aspartate by Helicobacter pylori.

The mechanism of resistance to N-phosphonoacetyl-L-aspartate (PALA), a potent inhibitor of aspartate carbamoyltransferase (which catalyzes the first committed step of de novo pyrimidine biosynthesis), in Helicobacter pylori was investigated. At a 1 mM concentration, PALA had no effects on the growth and viability of H. pylori. The inhibitor was taken up by H. pylori cells and the transport was saturable, with a Km of 14.8 mM and a Vmax of 19.1 nmol min-1 microliters of cell water-1. By 31P nuclear magnetic resonance (NMR) spectroscopy, both PALA and phosphonoacetate were shown to have been metabolized in all isolates of H. pylori studied. A main metabolic end product was identified as inorganic phosphate, suggesting the presence of an enzyme activity which cleaved the carbon-phosphorus (C-P) bonds. The kinetics of phosphonate group cleavage was saturable, and there was no evidence for substrate inhibition at higher concentrations of either compound. C-P bond cleavage activity was temperature dependent, and the activity was lost in the presence of the metal chelator EDTA. Other cleavages of PALA were observed by 1H NMR spectroscopy, with succinate and malate released as main products. These metabolic products were also formed when N-acetyl-L-aspartate was incubated with H. pylori lysates, suggesting the action of an aspartase. Studies of the cellular location of these enzymes revealed that the C-P bond cleavage activity was localized in the soluble fraction and that the aspartase activity appeared in the membrane-associated fraction. The results suggested that the two H. pylori enzymes transformed the inhibitor into noncytotoxic products, thus providing the bacterium with a mechanism of resistance to PALA toxicity which appears to be unique.

Metronidazole resistance and microaerophily in Campylobacter species.

Metronidazole is active against most anaerobic organisms and is also used in the treatment of the microaerophilic bacterium Helicobacter pylori. Resistance to metronidazole is uncommon in most anaerobic organisms, but it is increasingly prevalent in H. pylori. Previously we have suggested that metronidazole resistance in H. pylori is inherent in the microaerophilic nature of the organism and therefore would be present in other microaerophiles such as Campylobacter. Short periods of anaerobiosis caused metronidazole-resistant (MtrR) strains of Campylobacter spp. to become sensitive to metronidazole. Under microaerophilic conditions, cultures of the MtrR mutant Campylobacter coli R1 at bacterial cell densities of greater than 10(8) cfu/ml lost viability, whereas no loss in viability was observed in cultures at cell densities of less than 10(8). The MtrS C. coli strain lost viability at all cell densities. Comparisons of NAD(P)H oxidase activity between MtrS and MtrR strains indicated that the MtrS C. coli strain contained fourfold higher NADH oxidase activity and twofold higher NADPH oxidase activity than did the MtrR Campylobacter strains. These results show that MtrR Campylobacter spp. display resistance characteristics similar to those of H. pylori, suggesting that the resistance mechanism is a phenomenon of the microaerophilic nature of these bacteria.

Characterization of fumarate transport in Helicobacter pylori.

The fumarate transport system of the bacterium Helicobacter pylori was investigated employing radioactive tracer analysis. The transport of fumarate at micromolar concentrations was saturable with a KM of 220 +/- 21 micron and Vmax of 54 +/- 2 nmole/min/mg protein at 20 degrees C, depended on temperature between 4 and 40 degrees C, and was susceptible to inhibitors, suggesting the presence of one or more fumarate carriers. The release of fumarate from cells was also saturable with a KM of 464 +/- 71 micron and Vmax of 22 +/- 2 nmol/min/mg protein at 20 degrees C. The rates of fumarate influx at millomolar concentrations increased linearly with permeant concentration, and depended on the age of the cells. The transport system was specific for dicarboxylic acids suggesting that fumarate is taken up via dicarboxylate transporters. Succinate and fumarate appeared to form an antiport system. The properties of fumarate transport were elucidated by investigating the effects of amino acids, monovalent cations, pH and potential inhibitors. The results provided evidence that influx and efflux of fumarate at low concentrations from H. pylori cells was a carrier-mediated secondary transport with the driving force supplied by the chemical gradient of the anion. The anaerobic C4-dicarboxylate transport protein identified in the genome of the bacterium appeared to be a good candidate for the fumarate transporter.

An investigation of the molecular basis of the spontaneous occurrence of a catalase-negative phenotype in Helicobacter pylori.

BACKGROUND: The discovery of a highly active catalase in Helicobacter pylori that in some strains may lose its activity has generated strong scientific interest. We have characterized a spontaneous catalase-negative isolate of H. pylori (UNSW-RU1) and sequenced katA in the parent strain and the promoters of both phenotypes as a prelude to understanding the genetic processes leading to the failure to express catalase. MATERIALS AND METHODS: Protein extracts from both phenotypes were examined for catalase on 2D-PAGE and analyzed by Western blot-based immuno-analysis. Presence of catalase mRNA was detected by Northern blot. Hi-Fidelity PCR was used to sequence the katA promoter while katA was sequenced using cycle-sequencing. The transcription start site was located by primer extension. RESULTS: Catalase protein was absent in UNSW-RU1 (KatA-) by 2D-PAGE and Western blot, as was catalase mRNA by Northern blot, indicating that the cause of the KatA- phenotype was at the level of transcription. No mutations were found in the promoter region of the KatA- isolate. The transcription start site was identified 55 bp upstream of the ATG site and putative RNA polymerase binding sites were mapped at "-10" and "-35". A Fur box was identified 181 bp upstream of the transcription start site. The sequences of an 876 bp ORF and a 366 bp Escherichia coli phnA homologue were identified. CONCLUSIONS: The UNSW-RU1 (KatA-) phenotype does not express KatA or transcribe katA. The absence of defects in its promoter and a large part of its ORF indicates that loss of activity may be due to a mutation in an accessory gene essential for catalase expression, or to the binding of a repressor preventing katA transcription.

The mode of action of metronidazole in Helicobacter pylori: futile cycling or reduction?

The effects of metronidazole on catalase-positive and spontaneous catalase-negative mutants of Helicobacter pylori were studied to investigate whether the action of metronidazole on this microaerophilic organism occurs by reactive oxygen species generated by futile cycling or by the reduction of metronidazole to its active form. Increased sensitivity would be expected to occur in catalase-negative mutants if the mode of action of metronidazole was mediated through reactive oxygen species that may result from futile cycling of metronidazole. Two strains, RU1 and N6, were found to mutate spontaneously to a catalase-negative phenotype. The catalase-positive strain RU1(KatA+) and its catalase-negative counterpart RU1(KatA-) were sensitive to metronidazole, with MICs of 0.5 mg/L. The metronidazole-sensitive strain RU1(KatA-) lost viability at a rate similar to the parent RU1(KatA+) strain in the presence of 10 mg/L of metronidazole. Stable resistance to metronidazole was induced in RU1(KatA+) and RU1(KatA-) by passaging these strains in the presence of metronidazole. The catalase-positive and catalase-negative strains, N6(KatA+) and N6(KatA-), were resistant to metronidazole, with MICs of 96 mg/L. These observations indicated that the presence or absence of catalase activity did not affect the susceptibility of strains to metronidazole. The metabolism of metronidazole by H. pylori was investigated by 14N-NMR spectroscopy. Metronidazole was reduced in sensitive, catalase-positive and catalase-negative strains. Metronidazole-resistant cells reduced the 5-nitroimidazole more slowly, suggesting that resistance is achieved through the prevention or inhibition of metronidazole reduction.

A low rate of reinfection following effective therapy against Helicobacter pylori in a developing nation (China).

BACKGROUND & AIMS: In developed countries, reinfection after successful eradication of Helicobacter pylori appears unusual. High prevalences of H. pylori in developing countries may result in high reinfection rates. The aim of this study was to determine the rate of reinfection and ulcer recurrence in Chinese patients cured of H. pylori and duodenal ulcer disease. METHODS: One hundred eighty-four patients with duodenal ulcer disease shown by endoscopic examination (1 month) and 14C-urea breath test (3 months) after termination of treatment to have cleared their H. pylori were investigated. Patients were followed up by endoscopy (12 and 24 months) and breath test (6, 9, 12, 18, and 24 months). H. pylori status at endoscopic examination was determined by rapid urease, histology, and culture. In reinfected patients, random amplification of polymorphic DNA fingerprinting was used to compare isolates before and after therapy. RESULTS: Four patients were reinfected with H. pylori over 24 months (3 within 6 months and 1 at 24 months; average annual recurrence rate, 1.08%). Fingerprinting of isolates from 3 patients showed 1 patient (6 months) to have identical strains and the remainder to have nonidentical strains before and after treatment. Ulcer relapse occurred in 6 patients (4 H. pylori positive). CONCLUSIONS: Reinfection with H. pylori is rare in developing countries where treatment is effective.

Catalase, a novel antigen for Helicobacter pylori vaccination.

The efficacy of an orogastric vaccine comprised of purified Helicobacter pylori catalase plus the mucosal adjuvant cholera toxin (CT) was examined with both the Helicobacter felis and H. pylori mouse models with BALB/c mice. Native H. pylori catalase (200 microg) plus CT was initially used as a vaccine antigen in the H. felis mouse model and protected 80% (8 of 10) of the challenged animals, while all control animals were infected (20 of 20). In a follow-up experiment, recombinant H. pylori catalase plus CT was used for immunization, and groups of mice were challenged with the Sydney strain of H. pylori. Immunization with recombinant catalase protected a significant proportion (9 of 10) of the mice from H. pylori challenge, indicating that this enzyme should be considered as a candidate for a future vaccine. This study provides the first available data on the efficacy of protective immunization with the new Sydney strain of H. pylori in a mouse model. These data also provide indirect evidence that proteins which are normally intracellular, such as catalase, may be present on the surface of H. pylori and thus may provide targets for immunization.

In situ properties of Helicobacter pylori aspartate carbamoyltransferase.

The kinetic and regulatory properties of aspartate carbamoyltransferase (ACTase) of the human pathogen Helicobacter pylori were studied in situ in cell-free extracts. The presence of enzyme activity was established by identifying the end product as carbamoylaspartate using nuclear magnetic resonance spectroscopy. Activity was measured in all strains studied, including recent clinical isolates. Substrate saturation curves determined employing radioactive tracer analysis or a microtiter colorimetric assay were hyperbolic for both carbamoyl phosphate and aspartate, and there was no evidence for substrate inhibition at higher concentrations of either substrate. The apparent Km were 0.6 and 11.6 mm for carbamoyl phosphate and aspartate, respectively. Optimal pH and temperature were determined as 8.0 and 45 degrees C. Activity was observed with the l- but not the d-isomer of aspartate. Succinate and maleate inhibited enzyme activity competitively with respect to aspartate. The carbamoyl phosphate analogues acetyl phosphate and phosphonoacetic acid inhibited activity in a competitive manner with respect to carbamoyl phosphate. With limiting carbamoyl phosphate purine and pyrimidine nucleotides, tripolyphosphate, pyrophosphate, and orthophosphate inhibited competitively at millimolar concentrations. Ribose and ribose 5-phosphate at 10 mm concentration showed 20 and 35% inhibition of enzyme activity, respectively. N-Phosphonoacetyl-l-aspartate (PALA) was the most potent inhibitor studied, with 50% inhibition of enzyme activity observed at 0.1 microM concentration. Inhibition by PALA was competitive with carbamoyl phosphate (Ki = 0.245 microM) and noncompetitive with aspartate. The kinetic and regulatory data on the activity of the H. pylori enzyme suggest it is a Class A ACTase, but with some interesting characteristics distinct from this class.

Influence of a proton pump inhibitor-based therapy on Helicobacter pylori strain selection.

The influence of the proton pump inhibitor lansoprazole on strain diversity in Helicobacter pylori infected patients was investigated. Multiple isolates of Helicobacter pylori obtained pre- and post-therapy from gastric antral and body biopsies in 22 patients were compared using the random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) for analysis. Post-therapy strains exhibiting novel RAPD-profiles were found in 5 of 22 patients (4 of 11 patients treated with lansoprazole alone and 1 of 11 patients treated with lansoprazole plus amoxicillin). Proton pump inhibition may affect the microecology of the stomach by influencing the colonisation patterns of specific strains.

Genetic relationship among isolates of Helicobacter pylori: evidence for the existence of a Helicobacter pylori species-complex.

We investigated the population genetics of 23 isolates of H. pylori by allozyme electrophoresis using 16 enzyme loci. Isolates were obtained from adult patients of whom 48% were of Greek extraction. Eight patients (35%) had an active duodenal ulcer. Allelic variation per loci ranged from 2 to 11 alleles. Four major genetic clusters were apparent, having > 75% fixed genetic differences. There was no distinct clustering (clonal structure) on the basis of the geographical origin of the persons from whom isolates were obtained, indicating that this bacterium has not recently jumped a species barrier into humans. Isolates associated with ulcer disease were not monophyletic, with isolates from ulcer patients being found in phylogenetically diverse branches of the dendogram derived from the data. Based on the genetic diversity of H. pylori isolates, we propose that isolates should be classified as belonging not to a single species but to a 'Helicobacter pylori species-complex'.