In vivo expression of the 25-kDa laminin-binding protein of Helicobacter pylori

The gastroduodenal pathogen Helicobacter pylori has been shown to inhibit the interaction between the extracellular matrix protein laminin and its receptor on gastric epithelial cells, potentially contributing to a loss of mucosal integrity. As a 25-kDa outer membrane protein of H. pylori in association with the bacterial lipopolysaccharides (LPS) mediates attachment to laminin, the aim of this study was to determine whether the 25-kDa protein is produced by H. pylori in infected hosts. We examined the immune response to the 25-kDa laminin binding protein in 12 paediatric patients; samples from a H. pylori-negative healthy adult were used as controls. In immunoblotting, antibodies to a 25-kDa protein were found in the serum and saliva of H. pylori-positive individuals only, and using the positive sera and saliva, laminin binding to the 25-kDa protein was inhibited. Thus, the 25-kDa laminin-binding protein is produced by H. pylori in infected hosts.     

In vitro and in vivo effects of the Mongolian drug Amu‐ru 7 on Helicobacter pylori growth and viability

Amu‐ru 7, a Mongolian folk medicine, is used to treat digestive diseases such as gastritis and gastric and duodenal ulcers. We examined the effect of Amu‐ru 7 on the growth and viability of Helicobacter pylori in vivo and in vitro. By the agar dilution method, the MIC of Amu‐ru 7 for H. pylori strains was shown to be 100–200 μg/mL with a MIC₉₀ of 200 μg/mL. Two hundred micrograms per milliliter of Amu‐ru 7 exhibited potent bactericidal activity against H. pylori in the stationary phase of growth 6 hr after treatment. Amu‐ru 7 inhibited the growth of both AMPC‐resistant and CAM‐resistant strains, and also had a combined effect with AMPC on AMPC‐resistant strain 403. The Amu‐ru 7 inhibited biofilm formation by H. pylori and induced morphological changes, such as bleb‐like formation and shortening of the cell. Although colonization of the stomach of the Mongolian gerbil by H. pylori was not cured by treatment with Amu‐ru 7, both the mean number of H. pylori colonized and the colonization rate were decreased in Amu‐ru 7 treated gerbils. These results suggest the effectiveness Amu‐ru 7 as an adjunct therapy for eradication therapies consisting of a PPI combined with antibiotics.     

Susceptibility in vitro of Helicobacter pylori to cetylpyridinium chloride

The antimicrobial agent cetylpyridinium chloride (CPC) which is used in therapy of oro-pharyngeal infections and for antiseptic treatment of the oral cavity is active against different bacterial species. Determination of the minimal inhibitory concentration (MIC) using the agar dilution technique revealed that the gastric pathogen Helicobacter pylori in vitro is highly susceptible to CPC as indicated by an MIC of 10 microM (3.4 microg ml(-1)) which was significantly lower than the MIC of CPC against other bacterial species, which were analyzed in comparison to H. pylori. Bacteria of the genus Campylobacter, various Streptococcus spp., Staphylococcus aureus and Escherichia coli showed higher MICs ranging from 100 microM to 2 mM. In summary, this finding renders CPC-containing drugs candidates possibly useful for eradication or for the prevention of transmission of the gastric pathogen.     

Functional complementation of E. coli secD and secG mutants by Helicobacter pylori homologues

The Sec machinery is one mechanism used by bacteria to translocate proteins across their cytoplasmic membrane. Most of the Sec components have been identified within the important gastric pathogen, Helicobacter pylori, however their functionality has not yet been demonstrated. Here we report the existence of putative homologues to the Sec components yajC (HP1450) and yidC (HP1551), and demonstrate the ability of the H. pylori secD (HP1550) and secG (HP1255) homologues to facilitate inner membrane translocation of the maltose-binding protein MalE, by complementation of the respective secretion-deficient Escherichia coli mutants, thus providing evidence of their functionality.     

抗重组VacA IgY体内抗幽门螺杆菌感染的实验研究

目的在构建H.pylori的基因工程菌pQE30-v-DH5a的基础上,诱导表达VacA重组蛋白,以此为抗原,制备抗VacA的蛋黄抗体（VacA IgY）。通过小鼠口服试验,证实VacA IgY治疗H.pylori感染的作用,为进一步制备抗H．pylori感染的IgY制剂提供实验依据。方法用重组H.pylori VacA蛋白免疫母鸡,水稀释结合氯仿有机沉淀法提取IgY,ELISA法测定其针对VacA的效价。建立H.pylori感染的Balb／c小鼠动物模型,治疗组在小鼠灌喂菌液后灌喂不同剂量的VacA IgY。以H.pylori培养和病理切片观察胃黏膜H.pylori定植和炎症反应程度。结果制备了高效价的IgY（1：12800）。动物实验阳性对照组H.pylori的总感染率为70．4％,12周后的感染率为88．9％。治疗组的感染率与同期阳性对照组相似,胃黏膜的炎症反应程度比阳性对照组弱,随IgY剂量的增加,炎症减弱明显,IgY剂量为4mg／ml时,能达到较理想的治疗效果。结论成功制备了高效价的特异性VacA IgV,小鼠体内实验证实了口服VacA IgY具有治疗H.pylori感染的作用,可用于制备口服制剂。     

Genetic complementation of the urease-negative Helicobacter pylori mutant N6ureB::TnKm

Helicobacter pylori produces urease composed of the structural subunits UreA and UreB. Isogenic mutants produced by shuttle mutagenesis from the wild-type strain N6 are widely used in the literature. We describe the genetic complementation of the mutant N6ureB::TnKm by stable transformation with the vector pHel2 containing the cloned genes ureA and ureB and their specific promoter sequence. The orientation of the cloned insert was found to be crucial for urease expression. The majority of complemented clones functionally expressed urease at higher levels than did N6. Homologous recombination between chromosomal and cloned genes occurred at a frequency of 5%.     

幽门螺杆菌随机扩增多态性DNA指纹图谱分析

应用随机扩增多态性DNA指纹法(RandomamplifiedpolymorphicDNA,RAPD)对来自29例消化性溃疡和19例单纯性胃炎病人的幽门螺杆菌(Helicobacterpylori,Hp)菌株基因组DNA进行PCR反应,建立Hp的DNA指纹图谱,运用统计分析软件(Statisticanalysissoftware,SAS)对HpDNA指纹图的相似性以及与疾病的相关性进行分析。结果显示,这些菌株按HpDNA指纹图可分为两大类,两种来源的菌株在两大类中的比例有明显差异(P<0.05)。提示可能存在与疾病相关的特异基因型。     

A novel in vitro infection model of Helicobacter pylori using mucin-producing murine gastric surface mucous cells

BACKGROUND: Helicobacter pylori is found within the gastric surface mucous gel layer and in the epithelial surface. Gastric cancer cells have been used in experimental H. pylori infection in vitro, although cancer cells have some abnormalities in cellular properties. The aim of this study was to develop an in vitro H. pylori infection model using normal gastric surface cells that produce gastric mucin. MATERIALS AND METHODS: Normal murine gastric surface mucous cells (GSM06) were cultured by the liquid interface method using a serum-free medium and a collagen gel containing a fibroblast cell line (L929) and infected with H. pylori. Infection by H. pylori was assessed by enumerating the colony-forming units (CFU) of H. pylori adhered to GSM06 cells and by transmission electron microscopy. The production of mucin was determined by a lectin binding assay, sugar analysis, and MUC5AC gene expression. RESULTS: GSM06 cells cultured under these conditions produced mucin containing N-acetylgalactosamine and MUC5AC as the core protein. Significantly higher numbers of H. pylori adhered to GSM06 cells under mucin-producing conditions than under nonproducing conditions. Microscopic observation showed a filamentous structure resembling a type IV secretion system apparatus formed between the surface of GSM06 cells and H. pylori. CONCLUSIONS: This study demonstrates a novel in vitro H. pylori infection model using mucin-producing murine GSM06 cells for early stages of infection.     

High Level of Antimicrobial Resistance in French Helicobacter pylori Isolates

Background:  Helicobacter pylori is a human pathogen responsible for serious diseases including peptic ulcer disease and gastric cancer. The recommended triple therapy included clarithromycin but increasing resistance has undermined its effectiveness. It is therefore important to be aware of the local prevalence of antimicrobial resistance to adjust treatment strategy.
Materials and Methods:  Overall, 530 biopsies were collected between 2004 and 2007. The antimicrobial susceptibility of H. pylori was determined by E-test and molecular methods.
Results:  Among these, 138/530 (26%) strains were resistant to clarithromycin, 324/530 (61%) to metronidazole and 70/530 (13.2%) to ciprofloxacin. Whereas no resistance against amoxicillin and tetracycline was observed, only one strain was resistant to rifampicin. Compared to the patients never treated for H. pylori infection, the prevalence of resistance was significantly higher in patients previously treated (19.1% vs 68% for clarithromycin; 13.2% vs 53.3% for both clarithromycin and metronidazole). The trend analysis revealed an increase of primary resistance to ciprofloxacin between 2004 and 2005 (7.3%) vs 2006–2007 (14.1%) ( p  = .04) and the secondary resistance reached 22.7% in 2007. Interestingly, 27 biopsies (19.6%) contained a double population of clarithromycin-susceptible and -resistant strains.
Conclusions:  The reported high prevalence of clarithromycin and multiple resistances of H. pylori suggest that the empiric therapy with clarithromycin should be abandoned as no longer pretreatment susceptibility testing has assessed the susceptibility of the strain. As culture and antibiogram are not routinely performable in most clinical laboratories, the use of molecular test should be developed to allow a wide availability of pretreatment susceptibility testing.     

Helicobacter pylori mutagenesis by mariner in vitro transposition

We have developed a method for generating transposon insertion mutants using mariner in vitro mutagenesis. The gene of interest was PCR-amplified and cloned. A kanamycin-marked mariner transposon was randomly inserted into the purified plasmid in an in vitro transposition reaction. After repair and propagation in Escherichia coli, purified mutagenized plasmid was introduced into Helicobacter pylori by natural transformation. Transformants were selected by plating on kanamycin. Mutants were predominantly the result of double homologous recombination, and multiple mutants (with insertions in distinct positions) were often obtained. The site of insertion was determined by PCR or sequencing. We have made mutations in known or potential virulence genes, including ureA, hopZ, and vacA, using kanamycin- and kanamycin/lacZ-marked transposons. Colonies carrying a kanamycin/lacZ transposon appeared blue on medium containing the chromogenic agent X-gal, allowing discrimination of mutant and wild-type H. pylori in mixed competition experiments.     

幽门螺杆菌空泡毒素研究进展

幽门螺杆菌空泡毒素是该菌产生的已知其它细菌毒素无明显源性的唯一蛋白毒素。该毒素是幽门螺杆菌重要的毒力致病因子,它的产生与感染胃肠上皮损伤和溃疡形成密切相关。本就幽门螺杆菌空泡毒素的结构与功能研究进展以及在未来免疫预防与免疫治疗中的作用进行了阐述。     

益生菌在幽门螺杆菌根除治疗中的应用

幽门螺杆菌(Helicobacter pylori,H.pylori)是导致活动性胃炎、消化性溃疡、胃癌、胃黏膜相关淋巴组织淋巴瘤等消化系统疾病的重要病因之一,已被世界卫生组织确认为Ⅰ类致癌因子,根除H.pylori对防治上述疾病有重要意义。目前临床上主要采用含抗生素的三联或四联药物进行H.pylori的根除,虽然取得一定的疗效,但随着抗生素耐药率逐年增加,根除率持续下降,限制了其广泛应用。此外,初次或多次治疗失败后再治疗可选择的药物很少。近年来人们开始尝试将益生菌应用在H.pylori根除治疗中,并取得一定疗效。本文就益生菌在辅助根除幽门螺杆菌方面的研究进展作一简单综述。     

In vitro inhibition of Helicobacter pylori by micromycetes

The anti-Helicobacter pylori effect of 22 micromycetes were studied against one standard strain and 11 clinical isolates of H. pylori. Penicillium ochlochloron and Penicillium funiculosum have been proven the most active fungi against this microorganism. Further bio-guided chemical analysis of P. funiculosum afforded an active component identified as (-) 2,3,4-trihydroxybutanamide.     

Evaluation of the homologous recombination in Helicobacter pylori

BACKGROUND: Most strategies for direct mutagenesis of Helicobacter pylori primarily involve genomic DNA cloning which is a time-consuming and expensive technique. METHODS: To make a gene replacement, we propose a strategy using polymerase chain reaction (PCR) amplicons to allow a double homologous recombination in the genome of H. pylori. Different strains were used to validate this strategy and we describe how the amplicon insertion was made with accuracy. Moreover, we looked for the shortest homologous sequence needed to allow a specific gene replacement in H. pylori without any deletion, insertion or mutation at the recombination site. All of the experiments were performed at the flaA locus, whose gene encodes the major flagellin. RESULTS: Amplicons bearing 500 or 150 bp flanking regions of flaA on each side (depending on the strain) were sufficient to allow the specific insertion of a 1173 bp chloramphenicol cassette into the genome of H. pylori. The insertion was accurate with no substitutions at the insertion locus. CONCLUSIONS: This information opens the door to other strategies for mutagenesis used for the identification of virulence factors without deleting genes, which would not be based on a negative screening system. For example, they could be useful in performing protein fusion for a better understanding of the virulence factor's mechanism.     

Factors related to Helicobacter pylori prevalence in an adult population in Brazil

Background: The prevalence of Helicobacter pylori is higher in developing countries. Sanitary facilities, crowding and ethnic group are some of the factors related to H. pylori infection. The aim of this study was to investigate in blood donors, free of dyspeptic symptoms, the prevalence and factors influencing H. pylori infection. Materials and Methods: This study was conducted in São Paulo, a city known to have a mixed population coming from all over the country. A total of 1008 blood donors were initially included in the study. After a final revision of all the questionnaires, 993 were included in the final analysis (746 males). H. pylori status was checked by an ELISA test. The following associations to infection were analyzed: sex, age, ethnic group, previous upper gastrointestinal (GI) endoscopy, smoking, alcoholism, drug addiction, type of drinking water, crowding, sanitary facilities, and family income. Results: Infection was observed in 496 of 746 male (66.5%) and in 156 of 247 female (63.2%) blood donors. Infection prevalence increased according to age group, regardless of sex. Prevalence was lower in White population than in non‐White. No relationship was observed between infection and smoking, drug addiction, and alcohol. A positive relation was observed between infection and previous upper GI endoscopy, and type of drinking water, regardless if currently or during childhood. Crowding and lack of toilet in the house during childhood resulted in a higher infection rate. Lower familial income and educational level showed a positive association to infection. Conclusions: Prevalence of H. pylori is higher in non‐White population, independent of gender. A positive association was observed in aging, previous upper GI endoscopy, crowding, type of drinking water, lack of toilet during childhood, lower family income, and lower educational level.     

Relationship between gastric ulcer and Helicobacter pylori VacA detected in gastric juice using bead-ELISA method

Background. VacA is an important pathogenetic factor produced by Helicobacter pylori. VacA has often been detected in supernatants of liquid cultures or lysates of whole bacterial cells. However, no studies have ever tried to assay VacA produced in the human stomach. We applied a very sensitive and simple method, bead‐ELISA, to detect VacA in gastric juice. Materials and Methods. Forty‐eight H. pylori‐positive patients (16 nonulcer dyspepsia, 16 gastric ulcer, and 16 duodenal ulcer) and four H. pylori‐negative nonulcer dyspepsia patients had endoscopy performed and gastric juice were aspirated. Polystyrene beads coated with the antibody to VacA, were used in this bead‐ELISA method. The nucleotide sequences of vacA in the signal and middle regions were investigated. Results. Of the 48 samples that were positive for H. pylori, 21 [43.8%] were found to be VacA positive in gastric juice. The average and maximum concentrations of detected VacA in gastric juice were 143.2 ± 216.5 and 840 pg/ml, respectively. The average density of VacA from gastric ulcer patients (227.5 ± 276.7 pg/ml) was higher than that found in nonulcer dyspepsia (51.8 ± 39.8 pg/ml) and duodenal ulcer (49.2 ± 21.5 pg/ml) patients. There was no relationship between VacA in gastric juice and vacA genotype. Conclusions. VacA in gastric juice could be directly detected by bead‐ELISA. In this study, the diversity of disease outcome was associated with not the quality but the quantity of VacA. Therefore, not only the quality but also the quantity of VacA is important etiological factors in the pathogenesis of mucosal damage.     

Carbohydrate-dependent inhibition of Helicobacter pylori colonization using porcine milk

Breast milk has a well-known anti-microbial effect, which is in part due to the many different carbohydrate structures expressed. This renders it a position as a potential therapeutic for treatment of infection by different pathogens, thus avoiding the drawbacks of many antibiotics. In a previous study, we showed that pigs express the Helicobacter pylori receptors, sialyl Lewis x (Le x) and Le b, on various milk proteins. Here, we investigate the pig breed- and individual-specific expression of these epitopes, as well as the inhibitory capacity of porcine milk on H. pylori binding and colonization. Milk proteins from three different pig breeds were analysed by western blotting using antibodies with known carbohydrate specificity. An adhesion assay was used to investigate the capacity of pig milk to inhibit H. pylori binding to neoglycoproteins carrying Le b and sialyl-di-Le x. alpha1,3/4-fucosyltransferase transgenic FVB/N mice, known to express Le b and sialyl Le x in their gastric epithelium, were colonized by H. pylori and were subsequently treated with Le b- and sialyl Le x-expressing or nonexpressing porcine milk, or water (control) only. The degree of H. pylori colonization in the different treatment groups was quantified. The expression of the Le b and sialyl Le x carbohydrate epitopes on pig milk proteins was breed- and individual specific and correlated to the ability of porcine milk to inhibit H. pylori adhesion in vitro and H. pylori colonization in vivo. Milk from certain pig breeds may have a therapeutic and/or prophylactic effect on H. pylori infection.     

Organ-specific autoantibodies in children with Helicobacter pylori infection

BACKGROUND: We compared the prevalence of organ-specific autoantibodies in a group of Helicobacter pylori infected children and a group of uninfected children and investigated the relationship between the presence of relevant autoantibodies and the status of the target organs. PATIENTS AND METHODS: One hundred and twenty-four children with dyspepsia (54 boys, 70 girls; mean age 10.5 years; range 4-19) underwent gastroscopy: 56 had H. pylori infection (31 girls, 25 boys), while 68 (37 girls and 31 boys), were H. pylori-negative. All sera were tested for the presence of: parietal cell autoantibodies (PCA), intrinsic factor autoantibodies (IFA), microsomial autoantibodies, thyroglobulin autoantibodies, islet cell autoantibodies, glutamic acid decarboxylase autoantibodies, adrenal cortex autoantibodies, steroid-producing cell autoantibodies; gastrin, pepsinogen A, pepsinogen C and anti-H. pylori antibodies. The histological features and the ureA and cagA genes were also considered. RESULTS: The frequency of organ-specific autoantibodies was higher in patients with H. pylori infection than in uninfected patients (chi2-test p < .0001). Specifically gastric autoantibodies were significantly higher: seven of the 56 H. pylori-positive children were PCA-positive and one was IFA-positive (chi2-test p = .0004). The presence of autoantibodies was not associated with any clinical or biohumoral signs of disease. CONCLUSIONS: Our study detected a relationship between H. pylori infection in childhood and the presence of organ-specific autoantibodies unassociated with any clinical or biohumoral signs of disease. Helicobacter pylori infection in childhood could trigger the onset of clinical autoimmune gastritis, and/or other clinical autoimmune diseases.