Development of an internal control for evaluation and standardization of a quantitative PCR assay for detection of Helicobacter pylori in drinking water

Due to metabolic and morphological changes that can prevent Helicobacter pylori cells in water from growing on conventional media, an H. pylori-specific TaqMan quantitative PCR (qPCR) assay was developed that uses a 6-carboxyfluorescein-labeled probe (A. E. McDaniels, L. Wymer, C. Rankin, and R. Haugland, Water Res. 39:4808-4816, 2005). However, proper internal controls are needed to provide an accurate estimate of low numbers of H. pylori in drinking water. In this study, the 135-bp amplicon described by McDaniels et al. was modified at the probe binding region, using PCR mutagenesis. The fragment was incorporated into a single-copy plasmid to serve as a PCR-positive control and cloned into Escherichia coli to serve as a matrix spike. It was shown to have a detection limit of five copies, using a VIC dye-labeled probe. A DNA extraction kit was optimized that allowed sampling of an entire liter of water. Water samples spiked with the recombinant E. coli cells were shown to behave like H. pylori cells in the qPCR assay. The recombinant E. coli cells were optimized to be used at 10 cells/liter of water, where they were shown not to compete with 5 to 3,000 cells of H. pylori in a duplex qPCR assay. Four treated drinking water samples spiked with H. pylori (100 cells) demonstrated similar cycle threshold values if the chlorine disinfectant was first neutralized by sodium thiosulfate.     

Helicobacter pylori DNA in drinking water in Japan

Helicobacter pylori has been detected in drinking water in Peru and Sweden, suggesting the possibility of water-borne transmission. To date there have been few reports of H. pylori being detected in water; one was of the ureA gene of H. pylori in wells and springs in rural Japan. We examined water sampled in or near urban areas of Japan for H. pylori DNA by three assays using the polymerase chain reaction (PCR). Near Tokyo, samples were obtained: 10 of tap water, 6 of well water, 10 of river water, and 10 of sea water. Samples were filtered with membranes with 0.05- or 0.22-microm pores, which bacterial cells are caught by. Bacterial nucleic acids were extracted and purified and the PCR was done to amplify adhesin specific for H. pylori and the ureA gene, if present. Real-time PCR that measured the yield in terms of fluorescence was done with primers for 16S rRNA. None of the samples of tap, river, or sea water contained adhesin, ureA or 16S rRNA. None of the 6 samples of well water contained adhesin or ureA, but 2 of the 6 samples contained 16S rRNA. Some of the users of the well had had H. pylori infection in the past. H. pylori DNA was detected in well water and the users had been infected, so water-borne transmission via well water may occur even in towns in Japan.     

Occurrence of Helicobacter pylori DNA in the coastal environment of southern Italy (Straits of Messina)

AIMS: The occurrence of Helicobacter pylori in the coastal zone of the Straits of Messina (Italy) as free-living and associated with plankton was studied. METHODS AND RESULTS: Monthly sampling of seawater and plankton was carried out from April 2002 to March, 2003. All environmental samples analysed by cultural method, did not show the presence of H. pylori. The DNA extracted from all environmental samples was tested by PCR by using primers for H. pylori 16S rRNA, ureA and cagA. 16S rRNA PCR yielded amplified products of 522-bp in 15 of 36 (41.7%) of the environmental samples. By using the ureA primers to amplify the urea signal sequences, the predicted PCR products of 491-bp were obtained from eight (22.2%) of 36 environmental samples. PCR with cagA primers yielded amplified products of 349-bp in DNA extracted of seven of 36 (19.4%) of the environmental samples. When 16S rRNA, ureA and cagA amplified gene sequences were aligned with H. pylori 26695 and J99 genome sequences, we obtained a percentage of alignment over 90%. CONCLUSIONS: The detection of H. pylori genes in marine samples allows us to consider the marine environment a possible reservoir for this pathogenic bacterium. SIGNIFICANCE AND IMPACT OF THE STUDY: The direct detection of H. pylori genes may be relevant in order to consider the marine environment as significant reservoir for this bacterium.     

Detection of Helicobacter pylori by PCR but not culture in water and biofilm samples from drinking water distribution systems in England

AIMS: To investigate treated water distribution systems in England as a source of Helicobacter pylori. METHODS AND RESULTS: Water and biofilms were obtained from 11 domestic and seven educational properties and from hydrants, reservoirs and water meters supplied by three water utilities. Samples were cultured on nonselective and antibiotic containing media combined with immunomagnetic separation concentration. Viable helicobacters were not detected in any of the 151 samples but Helicobacter-specific PCR assays detected DNA in 26% of samples from domestic properties, schools and hydrants with the highest frequency in biofilms (42%). Direct sequencing of six selected amplicons confirmed >95% sequence homology to H. pylori. CONCLUSIONS: While viable helicobacters were not isolated, evidence was obtained for the presence of Helicobacter DNA, including that of H. pylori. Biofilms on surfaces within water distribution systems may act either as sites for the passive accumulation of helicobacters or as potentially important reservoirs of infection. SIGNIFICANCE AND IMPACT OF THE STUDY: Our findings strengthen evidence that H. pylori may be transmitted through drinking water. However, there is currently no evidence that viable cells can survive the disinfection levels used in UK mains supplies and the health risk from this source remains unclear.     

Detection of Helicobacter pylori in water by direct PCR

AIMS: This paper demonstrates a rapid, simple method for the detection of Helicobacter pylori in water that eliminates the need for recovery of cells or DNA extraction prior to PCR. METHODS AND RESULTS: Direct polymerase chain reaction (DPCR) with primers specific for H. pylori ureA (urease, subunit A) were used to detect H. pylori added to groundwater. DPCR also detected H. pylori in a naturally contaminated water sample. CONCLUSIONS: DPCR should provide an improved method to assess contamination of water by H. pylori. SIGNIFICANCE AND IMPACT OF THE STUDY: This simple, rapid method for detection of H. pylori in water will provide an improved means to investigate the possible role of water as a disease vector.     

Construction of a Helicobacter pylori-Escherichia coli shuttle vector for gene transfer in Helicobacter pylori.

In this study, a Helicobacter pylori-Escherichia coli shuttle vector was constructed for transferring DNA into H. pylori. The smallest cryptic plasmid (1.2 kb), pHP489, among those harbored by 77 H. pylori isolates was selected as a base replicon for constructing vectors. HindIII-digested pHP489 was ligated with a kanamycin resistance gene [aph(3')-III], which originated from Campylobacter jejuni, to produce the recombinant plasmid pHP489K. pHP489K was efficiently transformed into and stably maintained in H. pylori strains. The shuttle vector pBHP489K (3.6 kb) was constructed by the recombination of pHP489, ColE1, and aph(3')-III sequences. pBHP489K was reciprocally transformed into and maintained in both H. pylori and E. coli. Introduction of the shuttle vector clone DNA (pBHP489K/AB; 6.7 kb), containing the ureA and ureB genes of H. pylori, into urease-negative mutants of H. pylori led to the restoration of their urease activity. The transformants were confirmed to contain the incoming plasmid DNA. pBHP489K satisfied the requirements for an H. pylori-E. coli shuttle vector, implying that it might be a useful vector for investigating pathogenicity and restriction-modification systems of H. pylori.     

Helicobacter pylori cagA Gene Polymorphism Affects the Total Antioxidant Capacity of Human Saliva

Background:  We aimed to evaluate the total antioxidant capacity (TAC) of saliva in healthy Helicobacter pylori -positive and negative saliva individuals.
Materials and Methods:  A total of 102 human saliva samples were checked for the presence of H. pylori DNA ( ureA and cagA gene fragments).
TAC of saliva was estimated by ABTS radical cation (ABTS^{• +} ) decolorization assay.
Results:  PCR analysis revealed that 36 subjects were ureA-/cagA- , 24 were ureA+/cagA- and 42 were ureA+/cagA+ . Smoking habits had no evident effect on H. pylori infection.
We found that TAC of the ureA-/cagA- material, after 10 seconds reaction reflecting fast-reacting antioxidants, was significantly higher than of ureA+/cagA- and ureA+/cagA+ samples (p < .01 and p < .001, respectively). Similar results were obtained for reaction time of 3 minutes measuring slow-reacting antioxidants (p < .001). We also estimated ureA+/cagA- and ureA+/cagA+ samples alone and reported a statistically significant decrease in the TAC_3min value of ureA+/cagA+ compared with ureA+/cagA- samples (p < .05).
Conclusions:  Our data demonstrated that altered redox equilibrium may be associated with more frequent occurrence of H. pylori in the saliva samples.     

Activation of Helicobacter pylori ureA promoter by a hybrid Escherichia coli-H. pylori rpoD gene in E. coli.

We constructed and analyzed hybrid Escherichia coli-Helicobacter pylori rpoD genes in an E. coli rpoD mutant. It turned out that a hybrid consisting of E. coli rpoD with subdomain 4.2 of H. pylori rpoD (for -35 recognition) was functional. On the other hand, hybrids consisting of E. coli rpoD with domain 2 and the adjacent sequence of H. pylori rpoD (for core enzyme binding and -10 recognition) were non-functional. Intriguingly, a hybrid rpoD containing H. pylori subdomain 4.2 conferred higher activity for the H. pylori PureA as determined by xylE expression of PureA-xylE fusions, although the activity of the hybrid rpoD for the tac promoter was comparable to that of E. coli rpoD. The tsp of ureA in E. coli with the hybrid rpoD and E. coli rpoD were 15 and 17bp upstream from that in H. pylori, respectively. The comparison of PureA sequences in both E. coli and H. pylori indicated the existence of a -10 consensus sequence but little conservation of -35 sequences. Instead, the PureA in both H. pylori and E. coli contained an identical heptamer, GTTAATA, in the extended -35 region.     

Cutting edge: urease release by Helicobacter pylori stimulates macrophage inducible nitric oxide synthase

Inducible NO synthase (iNOS) expression and production of NO are both up-regulated with Helicobacter pylori infection in vivo and in vitro. We determined whether major pathogenicity proteins released by H. pylori activate iNOS by coculturing macrophages with wild-type or mutant strains deficient in VacA, CagA, picB product, or urease (ureA(-)). When filters were used to separate H. pylori from macrophages, there was a selective and significant decrease in stimulated iNOS mRNA, protein, and NO(2)(-) production with the ureA(-) strain compared with wild-type and other mutants. Similarly, macrophage NO(2)(-) generation was increased by H. pylori protein water extracts of all strains except ureA(-). Recombinant urease stimulated significant increases in macrophage iNOS expression and NO(2)(-) production. Taken together, these findings indicate a new role for the essential H. pylori survival factor, urease, implicating it in NO-dependent mucosal damage and carcinogenesis.     

Detection of Helicobacter pylori DNA in human faeces and water with different levels of faecal pollution in the north-east of Spain

AIMS: To assess the role of water in the faecal transmission of Helicobacter pylori by detecting the DNA of this pathogen in human faecal samples and environmental water samples with a range of faecal pollution from the north-east of Spain. METHODS AND RESULTS: Semi-nested PCR was used to detect H. pylori in stools and water, both matrices with a complex biota. DNA was detected using highly specific primers of an ureA gene fragment. In addition, antigens were used to detect the bacteria in stools. Helicobacter pylori was detected in 33% of 36 human faecal samples and in 66% of wastewater samples, and 11% of river samples, but in none of the spring waters samples. Faecal pollution of the aquatic environment was tested analysing the presence of microbial indicators. CONCLUSIONS: We report the presence of H. pylori DNA in stools and in aquatic environments with different levels of faecal pollution, from the north-east of Spain. In this study a higher number of positive results were obtained in the more faecally polluted waters. SIGNIFICANCE AND IMPACT OF THE STUDY: These data indicate that water may be a vector of H. pylori in its faecal-oral route.     

Development of an Internal Control for Evaluation and Standardization of a Quantitative PCR Assay for Detection of Helicobacter pylori in Drinking Water

Due to metabolic and morphological changes that can prevent Helicobacter pylori cells in water from growing on conventional media, an H. pylori-specific TaqMan quantitative PCR (qPCR) assay was developed that uses a 6-carboxyfluorescein-labeled probe (A. E. McDaniels, L. Wymer, C. Rankin, and R. Haugland, Water Res. 39:4808-4816, 2005). However, proper internal controls are needed to provide an accurate estimate of low numbers of H. pylori in drinking water. In this study, the 135-bp amplicon described by McDaniels et al. was modified at the probe binding region, using PCR mutagenesis. The fragment was incorporated into a single-copy plasmid to serve as a PCR-positive control and cloned into Escherichia coli to serve as a matrix spike. It was shown to have a detection limit of five copies, using a VIC dye-labeled probe. A DNA extraction kit was optimized that allowed sampling of an entire liter of water. Water samples spiked with the recombinant E. coli cells were shown to behave like H. pylori cells in the qPCR assay. The recombinant E. coli cells were optimized to be used at 10 cells/liter of water, where they were shown not to compete with 5 to 3,000 cells of H. pylori in a duplex qPCR assay. Four treated drinking water samples spiked with H. pylori (100 cells) demonstrated similar cycle threshold values if the chlorine disinfectant was first neutralized by sodium thiosulfate.     

兰州地区幽门螺杆菌分离株主要毒力基因的研究

本文首次报道了兰州地区胃病患者幽门螺杆菌分离株主要毒力基因ｕｒｅＡ ｖａｃＡ 和ｃａｇＡ的 ＰＣＲ 检测情况。共获 ４１ 株Ｈｐ 分离株,分别来自于慢性胃炎病人（３２ 株）、胃－十二指肠溃疡病人（７株）和胃癌病人（２ 株）。检测结果表明,４１ 株Ｈｐ 分离株的ｕｒｅＡ,ｖａｃＡ 及ｃａｇＡ 的阳性率分别为１００％ ,１００％ 和９７．６％ ;含有ｕｒｅＡ,ｖａｃＡ 和ｃａｇＡ 基因的Ｈｐ 与人类胃部疾患密切相关,而ｃａｇＡ 基因的存在可能与更加严重的胃部疾病有关。Ｈｐ 毒力基因的检测结果与其它地区Ｈｐ 分离株的检测结果相似。作者建议,对ｕｒｅＡ 基因的ＰＣＲ 检测可以作为鉴定Ｈｐ 的一个指标。     

Construction of isogenic urease-negative mutants of Helicobacter pylori by allelic exchange.

Isogenic urease-negative mutants of Helicobacter pylori were constructed by allelic replacement. A region of cloned H. pylori DNA containing the structural urease genes (ureA and ureB) was disrupted by insertion of a mini-Tn3-Km transposon. Electrotransformation of H. pylori cells with kanamycin-ureB-disrupted derivative plasmids resulted in isolation of kanamycin-resistant H. pylori transformants. Competence for electrotransformation appeared to be restricted to certain wild-type H. pylori isolates; only 1 isolate (of 10 tested) was consistently transformed. Two of the kanamycin-resistant H. pylori transformants were further studied and shown to be urease negative. Southern hybridization analyses demonstrated that the urease-negative mutants had been constructed by allelic exchange involving simultaneous replacement of the ureB gene with the kanamycin-ureB-disrupted copy and loss of the vector. Immunoblot studies of whole-cell extracts of the isogenic ureB mutants with anti-H. pylori sera indicated the absence of a polypeptide with an apparent molecular mass of 61 kDa; thus, the mutants no longer synthesized the UreB product. Generation of stable, genetically engineered urease mutants of H. pylori will be useful for addressing the role of urease in the pathogenesis of H. pylori infection.     

Adhesion of water stressed Helicobacter pylori to abiotic surfaces

AIM: The main aim of this work was to study and compare the adhesion of water exposed Helicobacter pylori to six different substrata and correlate any changes in morphology, physiology, ability to form aggregates and cultivability when in the planktonic or in the sessile phase. METHODS AND RESULTS: The number of total cells adhered for different water exposure times and modifications in the cell shape were evaluated using epifluorescence and scanning electron microscopy, and physiology assessed using Syto9 and propidium iodide (PI) cellular uptake. All abiotic surfaces were rapidly colonized by H. pylori, and colonization appeared to reach a steady state after 96 h with levels ranging from 2.3 x 10(6) to 3.6 x 10(6) total cells cm(-2). Cell morphology was largely dependent on the support material, with spiral bacteria, associated with the infectious form of H. pylori, subsisting in a higher percentage on nonpolymeric substrata. Also, sessile bacteria were generally able to retain the spiral shape for longer when compared with planktonic bacteria, which became coccoid more quickly. The formation of large aggregates, which may act as a protection mechanism against the negative impact of the stressful external environmental conditions, was mostly observed on the surface of copper coupons. However, Syto9 and PI staining indicates that most of H. pylori attached to copper or SS304 have a compromised cell membrane after only 48 h. Cultivability methods were only able to detect the bacteria up to the 2 h exposure-time and at very low levels (up to 500 CFU cm(-2)). CONCLUSIONS: The fact that the pathogen is able to adhere, retain the spiral morphology for longer and form large aggregates when attached to different plumbing materials appeared to point to pipe materials in general, and copper plumbing in particular, as a possible reservoir of virulent H. pylori in water distribution systems. However, the Syto9/PI staining results and cultivability methods indicate that the attached H. pylori cells quickly enter in a nonviable physiological state. SIGNIFICANCE AND IMPACT OF THE STUDY: This represents the first study of H. pylori behaviour in water-exposed abiotic surfaces. It suggests that co-aggregation with the autochthonous heterotrophic consortia present in water is necessary for a longer survival of the pathogen in biofilms associated to drinking water systems.     

Helicobacter pylori NikR protein exhibits distinct conformations when bound to different promoters

Helicobacter pylori NikR (HpNikR) is a ribbon-helix-helix (RHH) DNA-binding protein that binds to several different promoter regions. The binding site sequences are not absolutely conserved. The ability of HpNikR to discriminate specific DNA sites resides partly in its nine-amino acid N-terminal arm. Previously, indirect evidence indicated that the arm exists in different conformations when HpNikR is bound to the nixA and ureA promoters. Here, we directly examined HpNikR conformation when it was bound to nixA and ureA DNA fragments by tethering (S)-1{[bis(carboxymethyl)amino]methyl}-2-{4-[(2-bromoacetyl)amino]phenylethyl}(carboxymethyl)amino]acetic acid, iron(III) to different positions in the N-terminal arm and RHH DNA binding domain. Different cleavage patterns at each promoter directly demonstrated that both the RHH domain and the arm adopt different conformations on the nixA and ureA promoters. Additionally, the two RHH domain dimers of the HpNikR tetramer are in distinct conformations at ureA. Site-directed mutagenesis identified an interchain salt bridge (Lys(48)-Glu(47')) in the RHH domain remote from the DNA binding interface that is required for high affinity binding to ureA but not nixA. Finally, DNA affinity measurements of wild-type HpNikR and a salt bridge mutant (K48A) to hybrid nixA-ureA promoters demonstrated that inverted repeat half-sites, spacers, and flanking DNA are all required for sequence-specific DNA binding by HpNikR. Notably, the spacer region made the largest contribution to DNA affinity. HpNikR exhibits a substantially expanded regulon compared with other NikR proteins. The results presented here provide a molecular basis for understanding regulatory network expansion by NikR as well as other prokaryotic regulatory proteins.     

Effect of oxidizing disinfectants (chlorine, monochloramine, and ozone) on Helicobacter pylori

The susceptibility of Helicobacter pylori to disinfectants was compared to that of Escherichia coli. H. pylori is more resistant than E. coli to chlorine and ozone but not monochloramine. H. pylori may be able to tolerate disinfectants in distribution systems and, therefore, may be transmitted by a waterborne route.     

Role of mast cells as a trigger of inflammation in Helicobacter pylori infection.

Helicobacter pylori (H. pylori) induces severe inflammation and plays a key role in gastric mucosal diseases. In general, mast cells have been believed to play an important role in inflammation. Although mast cells were detected in the gastric mucosa, the role of mast cells in the gastric mucosal inflammation caused by H. pylori is still unclear. Therefore, we examined the effects of H. pylori water extract on the degranulation of mast cells to clarify the role of these cells in gastric mucosal inflammation induced by H. pylori. Mast cells prepared from rat abdominal cavity were incubated with H. pylori for 30 min. The protein concentrations of H. pylori water extract used in this study were 0.5-3 mg/ml. The degranulation of mast cells were monitored morphologically by phase contrast microscopy equipped with time-lapse video recording system and biochemically by measuring histamine and beta-hexosaminidase. H. pylori water extract induced the degranulation of mast cells dose-dependently. The identical experiment was performed without extracellular calcium, and no significant degranulation was found. The data indicates that the degranulation of mast cells by H. pylori water extract depend on extracellular calcium. The present results indicate that H. pylori might be involved in the gastric mucosal inflammation as a trigger of mast cell degranulation for releasing chemical mediators.     

Detection of Helicobacter pylori in cow's milk

AIMS: To investigate the existence of Helicobacter pylori in cow's milk as one of the foods which most Japanese children eat. METHODS AND RESULTS: Detection of H. pylori was demonstrated by the semi-nested polymerase chain reaction (PCR), a culture method and electron microscopy. Semi-nested PCR demonstrated the ureA gene of H. pylori in 13 of 18 (72.2%) raw milk samples and in 11 of 20 (55%) commercial pasteurized milk samples. Helicobacter pylori binding immunomagnetic beads with H. pylori-specific goat anti-H. pylori antibody was shown by electron microscopy in both raw and pasteurized milk positive for the ureA gene. Helicobacter pylori was cultured in one raw milk sample, whereas it was not cultured in pasteurized milk samples. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: There is a possibility that cow's milk is a transmission vehicle in childhood H. pylori infection, although we failed to confirm the survival of H. pylori in pasteurized milk.     

Biofilm formation by Escherichia coli O157:H7 on stainless steel: effect of exopolysaccharide and Curli production on its resistance to chlorine

The resistance of Escherichia coli O157:H7 strains ATCC 43895-, 43895-EPS (an exopolysaccharide [EPS]-overproducing mutant), and ATCC 43895+ (a curli-producing mutant) to chlorine, a sanitizer commonly used in the food industry, was studied. Planktonic cells of strains 43895-EPS and/or ATCC 43895+ grown under conditions supporting EPS and curli production, respectively, showed the highest resistance to chlorine, indicating that EPS and curli afford protection. Planktonic cells (ca. 9 log(10) CFU/ml) of all strains, however, were killed within 10 min by treatment with 50 microg of chlorine/ml. Significantly lower numbers of strain 43895-EPS, compared to those of strain ATCC 43895-, attached to stainless steel coupons, but the growth rate of strain 43895-EPS on coupons was not significantly different from that of strain ATCC 43895-, indicating that EPS production did not affect cell growth during biofilm formation. Curli production did not affect the initial attachment of cells to coupons but did enhance biofilm production. The resistance of E. coli O157:H7 to chlorine increased significantly as cells formed biofilm on coupons; strain ATCC 43895+ was the most resistant. Population sizes of strains ATCC 43895+ and ATCC 43895- in biofilm formed at 12 degrees C were not significantly different, but cells of strain ATCC 43895+ showed significantly higher resistance than did cells of strain ATCC 43895-. These observations support the hypothesis that the production of EPS and curli increase the resistance of E. coli O157:H7 to chlorine.     

Detection of Lactobacillus gasseri OLL2716 strain administered with yogurt drink in gastric mucus layer in humans

In animal models and human trials, Lactobacillus gasseri OLL2716 (LG21) strain suppressed Helicobacter pylori colonization in the stomach. The aim of the present study was to clarify whether orally administered LG21 strain can enter the gastric mucus layer. Biopsy samples were taken from the gastric antrum and corpus of two healthy volunteers (H. pylori infected and non-infected) who drank yogurt supplemented with LG21 strains. DNA of LG21 and H. pylori in the mucus layer was detected using the laser-assisted microdissection and non-contact pressure catapulting (LMPC) method and the semi-nested PCR method with primer sets of RNA helicases of superfamily II gene-Insertion sequence for LG21 strain and those of ureA gene for H. pylori. In the volunteer with H. pylori infection, DNA fragments of LG21- and H. pylori-specific regions from both antrum and corpus were amplified, whereas in a non-infected volunteer, only the LG21 DNA from the antrum was amplified. The present study demonstrated that LG21 strains administered through a yogurt drink can enter into the gastric mucus layer. Our novel method may be useful in studying gastric probiotics for H. pylori infection.