The bactericidal activity of tea against Helicobacter pylori

Extracts of black and green tea inhibited in-vitro growth of six clinical isolates of Helicobacter pylori in an agar diffusion assay. Tea extracts killed H. pylori (10⁶ cfu ml^-1) within 5 h. Heat treatment of extracts did not affect the inhibitory or bactericidal activity.     

Lipid profile of Helicobacter spp.: presence of cholesteryl glucoside as a characteristic feature.

The lipid and fatty acid profiles of eight Helicobacter spp. (H. nemestrinae, H. acinonyx, H. canis, Helicobacter sp. strain CLO-3, "H. rappini" [Flexispira rappini], H. pametensis, Helicobacter sp. strain Bird-B, and Helicobacter sp. strain Bird-C) and the fatty acid profiles of five additional species (H. pylori, H. felis, H. muridarum, H. mustelae, and H. fennelliae) were analyzed and compared. A heterologous fatty acid profile was observed among the Helicobacter spp., and on that basis the species could be divided into two groups. Group A had 19-carbon cyclopropane fatty acid (19:0cyc) and tetradecanoic acid (14:0) as the major fatty acids, and group B characteristically lacked the 19:0cyc and had hexadecanoic acid (16:0) and octadecenoic (18:1) acids as the major fatty acids. The species of group A are primarily gastric colonizers, and those of group B are primarily intestinal colonizers. Seven of the eight species studied showed the unusual and characteristic presence of cholesteryl glucosides (CGs), and most of these seven showed a very large amount (9.7 to 27.4% of the weight of total extractable lipid). The types of CGs and their distribution in different species were as follows: cholesteryl-6-O-acyl-alpha-D-glucopyranoside (cholesteryl-6-O-tetradecanoyl-alpha-D-glucopyranoside in H. nemestrinae and mainly cholesteryl-6-O-dodecanoyl-alpha-D-glucopyranoside in "H. rappini"), cholesteryl-alpha-D-glucopyranoside (H. nemestrinae, H. acinonyx, H. canis, Helicobacter sp. strain CLO-3, and "H. rappini"), and cholesteryl-6-O-phosphatidyl-alpha-D-glucopyranoside (H. nemestrinae, H. acinonyx, H. canis, and Helicobacter sp. strain CLO-3). Besides this, we could also detect cholesteryl acyl glucoside in H. acinonyx, cholesteryl glucoside in Helicobacter sp. strains Bird-B and -C, and cholesteryl phosphatidyl glucoside in "H. rappini" and Helicobacter sp. strain Bird-C. A selective accumulation of free cholesterol was observed in the neutral lipid fractions. On the basis of the detection of CGs in 11 of the 13 species studied so far, the presence of CGs appears to be a characteristic feature of the genus Helicobacter. In view of this and also because of a simple and rapid detection method described herein, the CGs can be used as a valuable chemotaxonomic marker.     

幽门螺杆菌分离培养的研究

采用细菌培养法及快速尿素酶法分别检测了１６９例消化道疾病患者幽门螺杆菌（Ｈｅｌｉ－ｃｏｂａｃｔｅｒｐｙｌｏｒｉ,ＨＰ）感染情况。标本经２０％葡萄糖运送培养基运送后,分别涂布于选择性培养基和本室改良的选择性培养基,３７℃微氧环境（５％Ｏ２,１０％ＣＯ２,８５％Ｎ２）培养８天。标本同时采用快速尿毒酶法进行检测。结果表明,细菌培养法与尿素酶法检测阳性率接近,分别为４６％和５５％;细菌培养第６天与第８天其阳性检出率相同;选择性培养基与本室改良选择性培养基对幽门螺杆菌的检出率差别明显,分别为３４％（１７／４９）和５１％（６１／１２０）,同时其杂菌生长率分别为５４％（２９／４９）和２５％（３０／１２０）。     

The role of Helicobacter spp. in the pathogenesis of primary biliary cirrhosis and primary sclerosing cholangitis

Helicobacter species DNA has been detected in liver tissue of patients affected by primary biliary cirrhosis (PBC) and primary sclerosing cholangitis (PSC). To investigate a potential causative relation between Helicobacter species and PBC/PSC, we compared the presence of Helicobacter species-specific DNA in liver tissue of patients with PBC/PSC (n=18/n=13) with those of a control group of patients with various liver diseases with known cause (n=29). A PCR with Helicobacter genus-specific 16S rRNA primers was performed on DNA isolated from paraffin embedded liver tissue. Control patients had hepatitis-B (n=9), alcoholic cirrhosis (n=14), or non-cirrhotic metabolic liver disease (n=6). There was no significant difference between the incidence of Helicobacter spp.-specific DNA in PBC/PSC (9/31; 29%) and the control group (10/29; 34%). Sequence analysis confirmed Helicobacter spp. DNA. Because Helicobacter spp. DNA can be found in approximately one-third of all samples tested, it is unlikely that PSC and PBC are caused by Helicobacter infection.     

Helicobacter infection in the surfactant protein D-deficient mouse

BACKGROUND: Surfactant protein D (SP-D), a component of innate immunity, is expressed in the gastric mucosa and is up-regulated in the presence of Helicobacter infection. SP-D binds to Helicobacter in vitro, suggesting the involvement of SP-D in Helicobacter-induced immune responses. The aim of this study was to determine the role of SP-D in gastric epithelial defense in vivo. METHODS: Specific pathogen-free SP-D-deficient mice (SP-D(-/-)) and C57BL/6 wild-type controls were challenged by gavage with different doses of Helicobacter felis, a mouse-adapted Helicobacter strain. Mice were assessed for colonization rates and density of infection. Inflammatory responses were measured by neutrophil counting and T-cell responses by proliferation assays on spleen cells stimulated with H. felis sonicate. The in vitro effect of SP-D on Helicobacter uptake by monocyte-derived dendritic cells was assessed by confocal microscopy and FACS analyses. RESULTS: SP-D(-/-) mice were more susceptible to low-dose infectious challenge than C57BL/6 controls (p = .02). The density of colonization was higher in the SP-D(-/-) infected mice. Neutrophil infiltrates were lower in the SP-D(-/-) mice, particularly in the acid-secreting regions of the stomach. T-cell proliferative responses to Helicobacter antigen were reduced in SP-D(-/-) mice (p = .001) after 12 weeks infection. In vitro uptake of Helicobacter by dendritic cells was significantly enhanced in the presence of SP-D (p = .001). CONCLUSION: In the absence of SP-D, Helicobacter uptake by dendritic cells is impaired. This provides an explanation for the diminished inflammation and immune responses in the SP-D(-/-) mice.     

The respiratory chain of Helicobacter pylori: identification of cytochromes and the effects of oxygen on cytochrome and menaquinone levels

Abstract The quinone and cytochrome components of the respiratory chain of the microaerophilic bacterium Helicobacter pylori have been investigated. The major isoprenoid quinone was menaquinone-6, with traces of menaquinone-4; no methyl-substituted or unusual menaquinone species were found. Cell yield was highest after growth at 10% (v/v) oxygen and menaquinone levels (per dry cell mass) were maximal at 5–10% (v/v) oxygen. Helicobacter pylori cells and membranes contained b -and c -type cytochromes, but not terminal oxidases of the a -or d -types, as judged by reduced minus oxidised difference spectra. Spectra consistent with the presence of a CO-binding terminal oxidase of the cytochrome b -or o -type were obtained. The soluble fraction from disrupted cells also contained cytochrome c . There were no significant qualitative differences in the cytochrome complements of cells grown at oxygen concentrations in the range 2–15% (v/v) but putative oxidases were highest in cells grown at 5–10% (v/v) oxygen.     

Development of a plating medium for selection of Helicobacter pylori from water samples

The goal of this study was to develop a simple plating medium to allow large-scale screening of water samples for the presence of Helicobacter pylori. Five conventional plating media (brain heart infusion, brucella agar, Columbia blood agar base, campylobacter agar kit Skirrow, and HPSPA medium), each containing a commercial antibiotic supplement, were initially evaluated. Eight strains selected as common waterborne organisms (Acinetobacter, Aeromonas, Bacillus, Escherichia coli, Enterobacter, Enterococcus, Helicobacter pylori, and Pseudomonas strains) were individually plated onto each of these media. Three organisms (Acinetobacter, E. coli, and H. pylori) were able to grow on all five media. This growth was unacceptable since Helicobacter grows very slowly and competing organisms must be inhibited for up to 7 days. Therefore, a more selective medium (HP agar) containing a novel mixture of growth supplements plus amphotericin B and polymyxin B was developed. This medium also included a phenol red color indicator for urease production. Aliquots of nonsterile well water that contained native flora (Flavobacterium, Serratia, Citrobacter, Pasteurella, Ochrobactrum, Rahnella, and unidentified molds) and were further adulterated with the eight strains listed above (10(6) CFU of each strain per 100 ml) were spiked with H. pylori and were plated. In spite of the heavy mixed microbial load, only H. pylori colonies grew during 7 days of incubation at 37 degrees C. The color indicator system allowed presumptive identification of H. pylori colonies sooner (12 to 20 h) than the conventional media tested allowed. The HP formulation developed in this study provides a medium with superior selectivity for H. pylori from mixed microbial populations in water and reduces the time required to complete the assay.     

An evaluation of a nutrient medium for isolating and cultivating Helicobacter pylori

Selective Helicobacter agar containing the selective supplement and blood, adding ex tempore, for the isolation and cultivation of H. pylori was developed. The Helicobacter agar was studied with the use of 5 newly isolated H. pylori strains, 13 bacterial associated cultures, as well as 21 inoculated biopsy specimens of the gastric and duodenal mucosa of patients with peptic ulcer. The study revealed that Helicobacter agar ensured the growth of H. pylori and their isolation from clinical material. The positive results after the inoculation of the specimens of biopsy material on Helicobacter agar and control media was 85%. In addition, the study of Helicobacter agar showed that it also exhibited pronounced selective properties with respect to bacterial associations, not inhibiting the growth of Helicobacter organisms and retaining their main biological properties. It is possible to recommend Helicobacter agar for use in laboratory practice in diagnosing Helicobacter-associated diseases.     

Urea, fluorofamide, and omeprazole treatments alter helicobacter colonization in the mouse gastric mucosa

BACKGROUND: Helicobacter pylori is a causative agent of gastric and duodenal ulcers and gastric cancer. Its urease enzyme allows survival in acid conditions and drives bacterial intracellular metabolism. We aimed to investigate the role of urease in determining the intragastric distribution of Helicobacter species in vivo. MATERIALS AND METHODS: The C57BL/6 mouse model of gastritis was used for infection with Helicobacter felis (CS1) or H. pylori (SS1). Urease-modulating compounds urea and/or fluorofamide (urease inhibitor) were administered to mice over 7 days. Concurrent gastric acid inhibition by omeprazole was also examined. Bacterial distribution in the antrum, body, antrum/body, and body/cardia transitional zones was graded "blindly" by histologic evaluation. Bacterial colony counts on corresponding tissue were also conducted. RESULTS: Urease inhibition by fluorofamide decreased H. pylori survival in most gastric regions (p < .05); however, there were no marked changes to H. felis colonization after this treatment. There was a consistent trend for decreased antral colonization, and an increase in antrum/body transitional zone and body colonization with excess 5% or 6% (w/v) urea treatment. Significant reductions of both Helicobacter species were observed with the co-treatment of urea and fluorofamide (p < .05). Collateral treatment with omeprazole did not alter H. pylori colonization patterns caused by urea/fluorofamide. CONCLUSIONS: Urease perturbations affect colonization patterns of Helicobacter species. Combined urea and fluorofamide treatment reduced the density of both Helicobacter species in our infection model.     

Characteristics of Helicobacter pylori natural transformation

For Helicobacter pylori, which exhibits substantial genetic diversity, many strains are naturally competent for transformation by exogenous DNA. To better understand the mechanism of natural transformation and its role in the generation of diversity, we sought to systematically identify factors important for natural transformation in H. pylori. We now show that the highest frequency of H. pylori transformation occurs when DNA is introduced prior to exponential phase growth, and that it is a saturable phenomenon. That transformation can be inhibited by DNA from Helicobacter (H. pylori and Helicobacter bilis) but not Escherichia coli suggests specificity based on DNA source. Finally, the cag island was determined to be unnecessary for high-frequency transformation.     

Expression of Helicobacter pylori ggt gene in baculovirus expression system and activity analysis of its products

The gamma-glutamyltranspeptidase (GGT) of Helicobacter pylori (HpGT) is a newly found virulence factor. In an approach to gain insight into the gene function, the four domains of the HpGT were cloned and expressed in baculovirus expression system. The results of a functional assay showed that the HpGT products acted as GGT, even when the N-terminal 380 amino acids were deleted. However, only the full length open reading frame (ORF) of the HpGT gene was apparently effective on cell growth. This result indicated that the products of the full length ORF might have an important role in gastric carcinogenesis. In this paper, we are the first to report that changes of mitochondrial membrane potential can be detected using 5, 5', 6, 6'-tetrachloro-1, 1', 3, 3'-tetraethylbenzimidazole carbocyanine iodide (JC-1) staining in insect cells.     

Natural colonization with Helicobacter species and the development of inflammatory bowel disease in interleukin-10-deficient mice

BACKGROUND: The interleukin-10-deficient (IL-10-/-) mice maintained in specific-pathogen-free (SPF) conditions develop typhlocolitis when experimentally infected with Helicobacter species. However, there is limited information regarding the role of Helicobacter species that naturally colonize IL-10-/- mice in typhlocolitis development. The aim of this study was to examine in SPF IL-10-/- mice the association between natural colonization specific Helicobacter species and typhlocolitis development. MATERIAL AND METHODS: Cecum and proximal colon from 72 C57BL/6 x 129/Ola IL-10-/- mice (8-20 weeks old) were removed for DNA extraction and histologic evaluation. Genus-specific polymerase chain reaction- denaturing gradient gel electrophoresis (PCR-DGGE) and species-specific PCR were used to detect Helicobacter species. Mice were grouped by age, sex, and Helicobacter colonization status, and their histologic scores were compared. The development of clinical typhlocolitis was observed in a further 12 mice. RESULTS: Species-specific PCR showed that mice were colonized with Helicobacter ganmani and/or Helicobacter hepaticus. The PCR-DGGE detected H. ganmani, H. hepaticus and an H. ganmani-like organism. The histologic scores in mice colonized with H. hepaticus were significantly higher than that in mice colonized with H. ganmani. Male mice showed significantly higher histologic scores than female mice. Four of the 12 mice developed clinical typhlocolitis in 38 weeks. CONCLUSION: Natural colonization with different Helicobacter species was found in IL-10-/- mice within the same breeding colony. The severity of typhlocolitis differed according to the colonizing Helicobacter species. Furthermore, the rate of typhlocolitis development in IL-10-/- mice naturally colonized with Helicobacter species was significantly slower than that reported in experimentally infected mice.     

The morphological transition of Helicobacter pylori cells from spiral to coccoid is preceded by a substantial modification of the cell wall.

The peptidoglycan (murein) of Helicobacter pylori has been investigated by high-performance liquid chromatography and mass spectrometric techniques. Murein from H. pylori corresponded to the A1gamma chemotype, but the muropeptide elution patterns were substantially different from the one for Escherichia coli in that the former produced high proportions of muropeptides with a pentapeptide side chain (about 60 mol%), with Gly residues as the C-terminal amino acid (5 to 10 mol%), and with (1-->6)anhydro-N-acetylmuramic acid (13 to 18 mol%). H. pylori murein also lacks murein-bound lipoprotein, trimeric muropeptides, and (L-D) cross-linked muropeptides. Cessation of growth and transition to coccoid shape triggered an increase in N-acetylglucosaminyl-N-acetylmuramyl-L-Ala-D-Glu (approximately 20 mol%), apparently at the expense of monomeric muropeptides with tri- and tetrapeptide side chains. Muropeptides with (1-->6)anhydro-muramic acid and with Gly were also more abundant in resting cells.     

Helicobacter typhlonius was detected in the sex organs of three mouse strains but did not transmit vertically

Helicobacter typhlonius, one of the most recently identified members of this rapidly expanding genus of bacteria, was detected by polymerase chain reaction (PCR) in an animal facility and studied for tissue distribution in sentinel mice (Helicobacter-free, barrier maintained). Immunodeficient athymic nude-nu (nu/nu), Helicobacter-sensitive C3H/HeJ and Helicobacter-resistant C57BL/6J mouse strains were used to study whether Helicobacter species could spread to the sex organs and be transmitted vertically. Sentinel mice of these three strains became infected at different times after first exposure and Helicobacter was detected by PCR for a brief period in the sex organs. The potential for PCR-positive tissues from the ovary, uterus, testis and epididymis to transmit infection was investigated via in vitro fertilization (IVF), embryo transfer (ET) or ovary transplantation in immunodeficient athymic nude-nu mice and did not result in contamination of the recipient females.     

Role of chronic infection and inflammation in the gastrointestinal tract in the etiology and pathogenesis of idiopathic parkinsonism. Part 1: eradication of Helicobacter in the cachexia of idiopathic parkinsonism

BACKGROUND: Neuronal damage in idiopathic parkinsonism may be in response to ubiquitous occult infection. Since peptic ulceration is prodromal, Helicobacter is a prime candidate. AIM: To consider the candidature of Helicobacter in parkinsonism with cachexia. METHODS: We explore the relationship between being underweight and inflammatory products in 124 subjects with idiopathic parkinsonism and 195 controls, and present the first case-series evidence of efficacy of Helicobacter eradication, in parkinsonism advanced to the stage of cachexia. RESULTS: Association of a low body mass index with circulating interleukin-6 was specific to parkinsonism (p = .002), unlike that with antibodies against Helicobacter vacuolating-toxin and cytotoxicity-associated gene product (p < .04). Marked reversibility in both cachexia and disability of idiopathic parkinsonism followed Helicobacter heilmannii eradication in one case, Helicobacter pylori eradication in another, follow-up being > or = 3.5 years. The latter presented with postprandial bloating, and persistent nausea: following eradication, radioisotope gastric-emptying returned towards normal, and upper abdominal symptoms regressed. Reversibility of their cachexia/disability contrasts with the outcome of anti-Helicobacter therapy where eradication repeatedly failed (one case), and in non-Helicobacter gastritis (three cases). Anti-parkinsonian medication remained constant. Intestinal absorption and barrier function were normal in all. CONCLUSION: Categorization, according to presence or absence of Helicobacter infection, was a useful therapeutic tool in late idiopathic parkinsonism.     

Is Helicobacter pylori a True Microaerophile?

BACKGROUND: There is no general consensus about the specific oxygen and carbon dioxide requirements of the human pathogen Helicobacter pylori. This bacterium is considered a microaerophile and consequently, it is grown under atmospheres at oxygen tensions 5-19% and carbon dioxide tensions 5-10%, both for clinical and basic and applied research purposes. The current study compared the growth of H. pylori in vitro, under various gas atmospheres, and determined some specific changes in the physiology of bacteria grown under different oxygen partial pressures. METHODS: Measurements of bacterial growth under various conditions were carried out employing classical solid and liquid culture techniques. Enzymatic activities were measured using spectrophotometric assays. RESULTS: H. pylori and all the other Helicobacter spp. tested had an absolute requirement for elevated carbon dioxide concentrations in the growth atmosphere. In contrast with other Helicobacter spp., H. pylori can tolerate elevated oxygen tensions when grown at high bacterial concentrations. Under 5% CO(2), the bacterium showed similar growth in liquid cultures under oxygen tensions from microaerobic (< 5%) to fully aerobic (21%) at cell densities higher than 5 x 10(5) cfu/ml for media supplemented with horse serum and 5 x 10(7) cfu/ml for media supplemented with beta-cyclodextrin. Evidence that changes occurred in the physiology of H. pylori was obtained by comparing the activities of ferredoxin:NADH (nicotinamide adenine dinucleotide) oxidoreductases of bacteria grown under microaerobic and aerobic atmospheres. CONCLUSIONS: H. pylori is a capnophile able to grow equally well in vitro under microaerobic or aerobic conditions at high bacterial concentrations, and behaved like oxygen-sensitive microaerophiles at low cell densities. Some characteristics of H. pylori cells grown in vitro under microaerobic conditions appeared to mimic better the physiology of organisms grown in their natural niche in the human stomach.     

In vitro inhibitory activity of boropinic acid against Helicobacter pylori

In this study, we assessed in vitro minimum inhibitory concentration (MIC) values of some natural geranyloxycoumarins, geranyloxy- and isopentenyloxy acids against growth of Helicobacter pylori. Boropinic acid, active principle isolated from Boronia pinnata (Fam. Rutaceae), was seen to be the most effective compound with a MIC value of 1.62 microg/mL.     

Growth inhibition of Helicobacter pylori by apolyamine synthesis inhibitor, methylglyoxal bis(cyclopentylamidinohydrazone)

The anti-proliferative effect of methylglyoxal bis(cyclopentyl-amidino-hydrazone) (MGBCP), a multi-enzyme inhibitor of polyamine biosynthesis, on the growth of Helicobacter pylori was investigated. MGBCP inhibited the cell growth of H. pylori in a dose-dependent manner. The inhibition was partially reversed by the addition of spermidine. Synthesis of macromolecules, DNA, RNA and protein, was inhibited in the spermidine-depleted H. pylori cells. These findings suggest that MGBCP exhibits an anti-proliferative effect on H. pylori by suppression of macromolecule synthesis.     

Steryl glycosides: a characteristic feature of the Helicobacter spp.?

The lipids of different species of Helicobacter (H.felis, H. muridarum, H. mustelae, H. fennelliae, and H. cinaedi) were studied. Different types of cholesteryl glucosides were found in all of the species studied except H. cinaedi. The total amount of cholesteryl glucosides varied from 14.8% of total lipids in H. mustelae to 33.1% of total lipids in H. felis. The different types of cholesteryl glucosides and their species distribution are cholesteryl-6-O-acyl-alpha-D-glucopyranoside (cholesteryl-6-O-tetradecanoyl-alpha-D-glucopyranoside in H. felis and cholesteryl-6-O-dodecanoyl-alpha-D-glucopyranoside in H. muridarum), cholesteryl-alpha-D-glucopyranoside (H. felis, H. muridarum, H. mustelae, and H. fennelliae), and cholesteryl-6-O-phosphatidyl-alpha-D-glucopyranoside (H. fennelliae). The neutral lipid fractions showed a high percentage of cholesterol, with selective accumulation of free cholesterol. The study thus shows that the characteristic presence of steryl glycosides in Helicobacter spp. may be an important chemotaxonomic marker for many of the species, and the helicobacters show a selective accumulation of free cholesterol from the media.     

Immunogenic proteins of Helicobacter pullorum,Helicobacter bilis and Helicobacter hepaticus identified by two-dimensional gel electrophoresis and immunoblotting

The ecological niches occupied by various species of Helicobacter are not yet known and the full spectrum of diseases associated with Helicobacter infections are not yet defined. Since these fastidious microaerofilic bacteria require special growth conditions new and improved molecular and serologic diagnostic methods have been developed to increase our understanding of their pathogenesis and virulence characteristics. Immunogenic cell surface proteins of Helicobacter pullorum, Helicobacter bilis, and Helicobacter hepaticus were characterised by proteomic techniques using two-dimensional electrophoresis and immunoblotting with antisera from immunised rabbits. Cross-reactivity between the three Helicobacter species were analysed after a four-step cross-absorption experiment. For H. pullorum, H. bilis and H. hepaticus 21, 13 and 27 specific immunogenic proteins, respectively, were identified. These proteins could be of important sero-diagnostic value for analyses of sera from humans, laboratory animals and for the veterinarian field.