Stuart L. Hazell

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.

The urea cycle of Helicobacter pylori

The presence and activities of the enzymes of the urea cycle in the bacterium Helicobacter pylori were investigated employing one- and two-dimensional NMR spectroscopy and radioactive tracer analysis. Cell suspensions, lysates and membrane preparations generated L-ornithine and ammonium at high rates in incubations with L-arginine, indicating the presence of arginase activity. Anabolic ornithine transcarbamoylase (OTCase) activity was identified by the formation of heat-stable products in incubations of cell-free extracts with ornithine and radiolabelled carbamoyl phosphate. The heat-labile product that resulted from incubations of cell-free extracts with citrulline radiolabelled in the guanidino moiety revealed the presence of catabolic OTCase activity. Argininosuccinate formation and catalysis indicated the presence of argininosuccinate synthetase and argininosuccinase activities. The findings suggested that H. pylori has a urea cycle which acts as an effective mechanism to extrude excess nitrogen from cells.

Phospholipase activity of Helicobacter pylori and its inhibition by bismuth salts - Biochemical and biophysical studies

In this study we measured phospholipase A (PLA) and C (PLC) activity of media filtrates and French Press lysates of the gastritis-inducing bacteriaHelicobacter pylori. We report here that bothH. pylori lysates and filtrates contain PLA1, PLA2, and C enzymes, which readily hydrolyze a radiolabeled dipalmitoylphosphatidylcholine (DPPC) and phosphorylcholine substrates, respectively. The specific activity of both PLA and C enzymes were greatest in the 6.5–7.0 and 8.4–8.8 pH ranges, respectively. Colloidal bismuth subcitrate (CBS) induced a dose-dependent inhibition of PLA2 and C activity of bothH. pylori lysates and filtrates. This inhibitory effect of CBS on PLA2 was antagonized in a dose-dependent fashion by the addition of CaCl2 to the incubation mixture, suggesting that calcium and bismuth may be competing for the same site on the enzyme. In contrast, the ability of bismuth salts to inhibit PLC activity ofH. pylori lysates was not antagonized by CaCl2. Employing a biophysical assay system for surface wettability, it was determined thatH. pylori lysates had the capacity to remove a synthetic phospholipid monolayer off a glass in a dose-dependent fashion. This ability of the bacterial lysates to catalyze the transformation of a hydrophobic surface to a wettable state was significantly attenuated in the presence of bismuth salts. Our experimental results are, therefore, consistent with the possibility thatH. pylori colonization compromises the stomachs barrier to acid by eroding a phospholipid lining, possibly a monolayer, on the surface of the gastric mucus gel and that this process is blocked in response to bismuth therapy.In this study we measured phospholipase A (PLA) and C (PLC) activity of media filtrates and French Press lysates of the gastritis-inducing bacteria Helicobacter pylori. We report here that both H. pylori lysates and filtrates contain PLA1, PLA2, and C enzymes, which readily hydrolyze a radiolabeled dipalmitoylphosphatidylcholine (DPPC) and phosphorylcholine substrates, respectively. The specific activity of both PLA and C enzymes were greatest in the 6.5-7.0 and 8.4-8.8 pH ranges, respectively. Colloidal bismuth subcitrate (CBS) induced a dose-dependent inhibition of PLA2 and C activity of both H. pylori lysates and filtrates. This inhibitory effect of CBS on PLA2 was antagonized in a dose-dependent fashion by the addition of CaCl2 to the incubation mixture, suggesting that calcium and bismuth may be competing for the same site on the enzyme. In contrast, the ability of bismuth salts to inhibit PLC activity of H. pylori lysates was not antagonized by CaCl2. Employing a biophysical assay system for surface wettability, it was determined that H. pylori lysates had the capacity to remove a synthetic phospholipid monolayer off a glass in a dose-dependent fashion. This ability of the bacterial lysates to catalyze the transformation of a hydrophobic surface to a wettable state was significantly attenuated in the presence of bismuth salts. Our experimental results are, therefore, consistent with the possibility that H. pylori colonization compromises the stomachs barrier to acid by eroding a phospholipid lining, possibly a monolayer, on the surface of the gastric mucus gel and that this process is blocked in response to bismuth therapy.

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.

Aminoacid utilization by Helicobacter pylori.

Utilization of aminoacids during growth by laboratory adapted and wild type Helicobacter pylori strains was investigated employing nuclear magnetic resonance spectroscopy and aminoacid analysis. All H. pylori strains tested showed growth rates with doubling times of approx. 11.5 hr in liquid cultures with semi-defined media or with defined aminoacid broth without carbohydrates. Fast utilization of several aminoacids at rates between 80 and 250 microM/hr was observed in culture broths inoculated with approx. 10(7) cells/ml; and acetate, formate and succinate accumulated as catabolic products in the growth media. Suspensions of bacterial cells and lysates in isotonic solutions converted arginine, asparagine, aspartate, glutamine, and serine used as sole substrates at significant rates; and under these conditions the principal metabolic products observed were acetate, formate, succinate and lactate. The findings of the study indicated that H. pylori can survive employing aminoacids as the basic nutrients, and suggested some of these metabolites were utilized via fermentative pathways with common characteristics to those found in anaerobes.

Immunisation against gastric infection with Helicobacter species: First step in the prophylaxis of gastric cancer?

The discovery of the gastric bacterium, Helicobacter pylori and the demonstration of its role in the pathogenesis of gastroduodenal disease, has been one of the major microbiological advances in the last decade. Recent demonstration of long term infection with this bacterium as a risk factor in gastric carcinoma suggests that intervention in a disease of major morbidity and mortality is possible. Using a model of Helicobacter infection in mice it has been shown that oral immunisation with a sonicate of Helicobacter felis plus the adjuvant cholera toxin results in protection against an oral challenge with large numbers of viable bacteria. The success of the immunising regimen has been shown to correlate with the development of local immunity. Formulation of equivalent safe vaccines of H. pylori will make possible the immunisation of children in countries such as China, Japan and Columbia and so prevent the establishment of long term inflammation and thus significantly reduce the incidence of gastric cancer in those societies. This animal model is proposed as a major tool in the development of effective oral immunisation.

Pyruvate metabolism in Helicobacter pylori

The metabolism of pyruvate by Helicobacter pylori was investigated employing one- and two-dimensional 1H and 13C nuclear magnetic resonance spectroscopy. Generation of pyruvate from l-serine in incubations with whole cell lysates indicated the presence of serine dehydratase activity in the bacterium. Pyruvate was formed also in cell suspensions and lysates from phosphoenol pyruvate. Metabolically competent cells incubated aerobically with pyruvate yielded alanine, lactate, acetate, formate, and succinate. The production of alanine and lactate indicated the presence of alanine transaminase and lactate dehydrogenase activities, respectively. Accumulation of acetate and formate as metabolic products provided evidence for the existence of a mixed-acid fermentation pathway in the microorganism. Formation of succinate suggested the incorporation of the pyruvate carbon skeleton into the Krebs cycle. Addition of pyruvate to various liquid culture media did not affect bacterial growth or loss of viability. The variety of products formed using pyruvate as the sole substrate showed the important role of this metabolite in the energy metabolism of H. pylori.

Acetyl-CoA carboxylase activity in Helicobacter pylori and the requirement of increased CO2 for growth.

A biotinylated acetyl-CoA carboxylase from the microaerophilic bacterium Helicobacter pylori was partially purified and characterized. The approximate molecular mass of the native enzyme was estimated at 235 kDa by native PAGE. A single band corresponding to approximately 24 kDa was detected by SDS-PAGE, suggesting that the native enzyme is a multi-protein complex. The protein was isolated from the soluble fraction of the cell. Catalytic activity was acetyl-CoA-dependent and inhibited by avidin but unaffected by avidin pretreated with excess biotin. The end-product of the reaction was identified as malonyl-CoA and the reaction was shown to be reversible by NMR spectroscopy. The activity of the enzyme was 0.29 mumol min-1 (mg protein)-1. The Vmax for bicarbonate was calculated at 0.73 mumol min-1 (mg protein)-1, and the affinity of the enzyme for this substrate was relatively low, with an apparent Km of 16.6 mM. These data provide the first evidence of a possible physiological role for the requirement of high levels of CO2 for growth in vitro of this bacterium.

How Helicobacter pylori works: an overview of the metabolism of Helicobacter pylori.

elicobacter pylori is a microaerophilic bacterium. The organism is able to take up, phosphorylate, and use glucose in the synthesis of pentose sugars and in the generation of reducing power via the pentose phosphate pathway or is able to catabolize glucose by the Entner-Doudoroff pathway. There is also evidence for mixed acid fermentation pathways in H. pylori. The bacterium exhibits attributes common to many anaerobic bacteria, having a pyruvate, flavodoxin oxidoreductase, to generate acetyl-coenzyme A from pyruvate. In addition, there is evidence that fumarate may act as a terminal electron acceptor in oxidative phosphorylation. Further evidence demonstrates a branched Krebs cycle, with the tricarboxylic arm and the dicarboxylic arm operating in opposite directions, the latter in a reductive direction from oxaloacetate to succinate. Amino acids are an important source of carbon, nitrogen, and energy; however, the deamination of amino acids presents problems for the bacterium in the management of intracellular nitrogen balance. To address this problem, urease in concert with a urea cycle may act to “pump” nitrogen from the cytosol into the external environment. Degradation of lipids is a potential source of carbon, phosphate, and energy. The requirements for purine and pyrimidine nucleotides may be met by salvage synthesis of purines and by both salvage and de novo synthesis of pyrimidines. The microaerophily of the bacterium is related both to its oxygen requirements and to the need for an increased partial pressure of CO 2 . Catalase, superoxide dismutase, and the recombination repair protein (recA) are important to resist oxidative damage, particularly in inflamed gastric mucosae, and may represent important systems for survival in the host. In the study of H. pylori and associated disease, there have been numerous and substantive publications in the areas of clinical studies, treatment, pathogenesis, molecular biology, and epidemiology. One area that has advanced dramatically only in the last few years has been the understanding of the physiology and biochemistry of H. pylori . Advances in these areas have served to increase our knowledge both of how H. pylori works and of the phenomenon of microaerophilicity. In this article, we describe key findings about the basic biochemistry of this important pathogen and identify areas of controversy where appropriate. Because our understanding of how the bacterium works remains incomplete, this review may serve as a resource to facilitate further investigations.

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.