[Genetic heterogeneity of helicobacter pylori putative virulence genes and association with clinical outcomes, recent studies and future perspectives]

Helicobacter pylori [H. pylori] plays an important role in the development of peptic and duodenal ulcerations as well as gastric cancer. Several studies have shown a strong association between specific genotypes of H. pylori and gastrointestinal tract diseases. The vacA and cagA genes, which are the putative virulence factors of the bacterium, are important determinants of H. pylori-related diseases. Polymorphisms of the signal peptide [s], middle [m], and intermediate [i] regions of the vacA gene, presence of the cagA, and genetic heterogeneity of the C-terminal motifs of CagA might result in varying clinical outcomes in H. pylori-infected patients. In this review article, the intracellular activities of VacA and CagA proteins, the genetic diversity of the coding sequences of these proteins, their association with clinical outcomes with regard to the status of H. pylori infection in Iran, and future perspectives are discussed

Evolutionary dynamics of Helicobacter pylori cagA and vacA genes in Iran and their association with clinical outcomes

There is a relationship between specific genotypes of Helicobacter pylori cagA and vacA genes and the increased risk of peptic ulcer diseases and gastric cancer. These genes also possess strong patterns of geographical differentiation. The present study aims to determine the patterns of variation of the virulence genes in Iran and their association with clinical status. Sequence fragments for cagAand vacA were obtained from a total of 147 H. pylori isolates from diverse geographical and ethnic sources within Iran. We used phylogenetic methods to determine the patterns of allelic diversity, and the relationship between evolutionary lineages and clinical status. Phylogenetic analyses of Iranian cagA gene disclosed four lineages, whereas the vacA gene had two distinct lineages. The cagA lineage II showed extensive genetic diversity compared with lineage I. cagA lineages III and IV disclosed mixed ancestries with recombinant nucleotides that originated from lineages I and H Iranian strains with vac A lineage II genotype were significantly cagA+ [> 90%, p = 0.0] and correlated with a higher rate of peptic ulcers in infected individuals [p =0.003]. Most strains in the cagA lineage I showed a vacA lineage II genotype [p = 0.003] and significantly correlated with an increased risk of peptic ulcers in infected individuals [p = 0.022]. Strains with cagA lineage III genotype significantly correlated with gastritis [p = 0.0]. The increased level of allelic diversity in the virulence genes shows strong evolutionary dynamics, resulting in the emergence of new clonal genealogies of the cagA gene within Iran. Particular lineages of the Iranian cagA and vac A genes correlate with peptic ulcer diseases

Preparative sds-page electroelution for rapid purification of alkyl hydroperoxide reductase from helicobacterpylori

Alkyl hydroperoxide reductase [AhpC] of Helicobacter pylori is considered as a diagnostic antigen. Therefore, this antigen can be used to detect H, pylori infection by stool immunoassays such as ELISA. The aim of this study was to simplify the AhpC protein purification procedures. For whole cell protein extraction, the bacterial cells were ruptured by octly-beta-D glucopyranoside. The isolation and purification of AhpC protein were attempted by various techniques including ammonium sulfate precipitation, dialysis, preparative sodium dodecyl sulfate polyacrylamide gel electrophoresis [SDS-PAGE] and electroelution. A simple method was used for protein purification AhpC protein. One-dimensional preparative gel electrophoresis allows a single and short purification step; the high resolution capacity of this technique leads to a high level of purity of the protein. Moreover, it avoids contamination by other non-specific proteins which often appear during protein purification by column chromatography. The present method is simple, rapid and makes it possible to preparate AhpC from H. pylori

simple and cost-effective method for rapid purification of alkyl hydroperoxide reductase [AhpC] from helicobacterpylori and its antibody production

Helicobacter pylori express abundant amounts of AhpC enzyme that functions to reduce organic hydroperoxides [ROOH] into the corresponding non-toxic alcohols [ROH]. This conserved antigen has been earlier described as specific and unique for H. pylori and therefore, both H. pylori AhpC and Anti-AhpC could be useful in the development of serologic and stool antigen tests, to detecting and monitoring H. pylori infection. AhpC may also serves as a potential target for an antimicrobial agent or for vaccine development. The aim of this study was to simplify isolation and purification of the AhpC and production of a highly specific polyclonal antibody against it. In this paper a simple method was used for protein purification and antibody production which avoids both the long term AhpC protein purification procedure and the addition of Freund's adjuvant. One-dimensional preparative gel electrophoresis allows a single and short purification step and the high resolution capacity of this technique leads to a high level of purity of the protein and consequently to a very high specificity of the antibody. Moreover, it avoids contamination by other non-specific proteins which often appear during protein purification by column chromatographic techniques. The present method is simple, rapid and cost-effective and makes it possible to produce antibody for stool antigen enzyme immunoassay in short time and at low cost

[Evaluation of anti-Helicobacter pylori activity of methanol extracts of some species of Stachys and Melia]

The gram-negative bacterium Helicobacter pylori [H. pylori], identified in 1982, is now recognized as the primary etiological factor associated with the development of gastritis and peptic ulcer disease. The growing problem of antibiotic resistance by the organism demands the search for novel compounds from plant based sources. The present study is aimed at evaluating the anti-Helicobacter pylori activity of 10 Iranian plant extracts on clinical isolates of H. pylori. Gastric biopsy samples were obtained from patients presenting with gastroduodenal complications. H. pylori was isolated from the specimens following standard microbiology procedures. The disk diffusion method was used to determine the susceptibility of 12 isolates to methanol plant extracts [Fruit and leaves of Melia azedarach, Melia indica and aerial parts of Stachys setifera, Stachys turcomanica, Stachys trinervis, Stachys subaphylla, Stachys byzanthina, Stachys persica, Stachys inflata, Stachys laxa]. The plants tested at 8 mg/disc concentration demonstrated anti-Helicobacter pylori activity with zone diameters of inhibition ranging from 12-38 mm. Of these, Stachys setifera [aerial parts], Melia indica [Fruit] and Melia azedarach [leaves] showed the most potent anti -H. pylori activity on the isolates. Due to the rise in antibiotic resistance, new sources of anti-H. pylori drugs are needed. The use of medicinal plants may have potential benefit in eradicating such problems. According to the results of this study, further studies will be necessary to investigate the effects of other plants of Iran against H. pylori infectio

Assessment of helicobacter pylori viability by flow cytometry

Flow cytometry is a rapid, sensitive, and reliable method for determination of bacterial viability. Here we assayed the capability of flow cytometry to detect Helicobacter pylori viable cells in both forms of spiral and coccoid. Viable bacteria stained with Rhodamin 123 and fluoresced with laser beam of 488nm. The rate of Rh123 absorption was determined in both forms of bacteria. In positive control that consisted of live bacteria, the rate of rh123 absorption was at highest, but negative control that consisted of dead bacteria, the rate of Rh 123 absorption was at lowest absorption. This method showed that non-culturable coccoid forms of H. pylori, which could resist environmental stresses, were alive and might be responsible for bacterial transmission and failure in disease treatment. Due to simplicity, reliability, and sensitivity of flow cytometry, this method is preferred to other expensive and no reliable methods such as autoradiography, PCR and Electron microscopy used for assessment viability

Antimicrobial effectiveness of furazolidone against metronidazole-resistant strains of Helicobacter pylori

The occurrence of strains resistant to metronidazole is causing failure of the 4-drug regimen for eradication of Helicobacter pylori in the Islamic Republic of Iran. This study compared the in vitro efficacy of furazolidone with metronidazole, clarithromycin, amoxicillin and tetracycline in 70 H. pylori isolates from dyspeptic patients. Of the isolates, 33% were resistant to metronidazole but all were susceptible to furazolidone. Furazolidone could be considered as an appropriate substitute for metronidazole for H. pylori infections

[DNA fingerprinting of h. pylori isolates from Iranian patients by REP-PCR]

H. pylori has been implicated in peptic diseases, some with detrimental consequences such as ulcer or cancer. Since considerable genetic heterogeneity has been observed within H. pylori population worldwide, it appears an ideal achievement to recruit PCR-based methods and design genetic markers which recognize isolates from normal and symptomatic individuals. In this study 61 H. pylori isolates from dyspeptic patients were fingerprinted by REP-PCR. REP-PCR was performed on extracted DNAs of 61 H. pylori isolates from 39 normal, 18 ulcer and 4 cancer patients. Synthetic 18-nt primers, specific for interspersed repetitive elements in the bacterial genome, were recruited. PCR conditions were optimized and reproducibility of the reactions were confirmed. The size and number of PCR products were determined and DNA fingerprints of all isolates were analyzed by NTSYSpc programme, and dendrograms were generated. Results: Among 39 H. pylori isolates from normal patients 28 comprised a distinct cluster and 5 clustered along with isolates from ulcer patients. The remaining 6 isolates comprised a separate cluster distinct from other groups. Among 18 isolates from ulcer patients, 17 classified in a specific cluster, only one isolate was clustered along with isolates from normal patients. Isolates from cancer patients consisted a quite distinct cluster. In this study REP-PCR was used to show that majority of isolates from normal, ulcer, and cancer patients have distinct fingerprints which can be recruited for predicting the outcome of the infection with certain H. pylori isolates. It is concluded that REP-PCR is an effective and reproducible technique for fingerprinting H. pylori isolates from different human origins

[Detection of helicobacter pylori-specific genes in the oral yeast]

Until today human stomach is the only recognized habitat of H. pylori. However, recruitment of DNA-based methods has made possible the detection of H. pylori in water and oral cavity, thus suggesting faecal-oral and oral-oral routes for transmission of H. pylori, respectively. In this study yeast has been proposed as a common vector for transmission of H. pylori. Thus designed primers were recruited to target 16S rDNA and cagA genes in the oral yeasts by PCR. Eighteen yeasts were examined microscopically for the presence of bacterial-like bodies. DNAs were extracted from oral yeasts using phenol-chloroform method. Amplification conditions were optimized as 33 cycles and annealing temperatures of 63°C for 16S rDNA and 51°C and 52°C for cagA gene which was targetted in two steps. DNAs of H. pylori and saccharomyces cerevisiae were used as controls. PCR products of two genes from one yeast and from H. pylori were cloned in pCAP and subsequently subcloned in pSK+ and sequenced. Bacterial-like bodies were observed in all oral yeasts. The amplified products of 16S rDNA from all oral yeasts were homologous in size with those of H. pylori. 15/18 [83%] yeasts contained cagA gene, homologous to H. pylori. CagA was not amplified from three yeasts and S. cerevisiae. Analysis of sequenced products of 16S rDNA and cagA from one oral yeast showed 98% homology with those of H. pylori. The presence of H. pylori inside the yeast was indicated by light microscopy and PCR. It appears that yeasts which are abundant in nature and thrive the mucosal surfaces of human might serve as reservoirs and vehicles of H. pylori

[Neutralization of helicobacter pylori cytotoxicity on vero cells by omeprazole]

Omeprazole is a gastric parietal cells proton pump inhibitor that is also active against H. pylori in vitro. This study was designed to examine the neutralization of H. pylori cytotoxicity on Vero cells by omeprazole micronized in strains isolated from gastritis, ulcer, cancer and Barrett's ulcer, to determine whether omeprazole can inhibit vacuolation of the Vero cells induced by cytotoxin of H. pylori or by urease. The effect of omeprazole on motility of H. pylori was assessed using concentrations lower than MIC. The antimicrobial activity of omeprazole micronized was studied by determining the MICs for 15 H. pylori strains. Water extract of the bacteria [concentrated culture supernatant] and different concentrations of omeprazole were added to Vero cells in culture. Also extracted urease from H. pylori strains with urea [10 mM] and omeprazole were added to Vero cells in culture. The inhibitory effect of omeprazole on motility of H. pylori was tested in semi-solid medium. MIC of omeprazole micronized was 20 micro g/ml. Omeprazole could inhibit induced vacuolation by the water extract of H. pylori strains in Vero cells. It could also inhibit vacuolation induced by urease. Inhibition of vacuolation strains was assessed microscopically and by the neutral red method. It was also found that omeprazole inhibits the motility of H. pylori strains at concentrations lower than MIC. The results of this study suggest that omeprazole micronized could neutralize the vacuolation effect of H. pylori cytotoxin on Vero cells probably by targeting v-type ATPase. The bacterial motility was also inhibited by low concentrations of omeprazole. The results of this study considers omeprazole micronized as an effective drug which targets important virulence factors of H. pylori including vacuolating cytotoxin, urease, and motility

[Genotype variation in H. pylori isolates from Iranian patients by RAPD-PCR]

Do H. pylori isolates from normal and dyspeptic patients have similar genetic profiles? Since genotype variation occurs within H. pylori population with high frequency, it is tempting to exploit techniques such as RAPD-PCR to examine the possible correlation between specific H. pylori genotypes and different peptic diseases. In this study, H. pylori isolates from different dyspeptic patients were genotyped by RAPD-PCR. H. pylori isolates from 66 patients, 41 normal, 21 with ulcer, and 4 with cancer were cultured, DNAs were extracted by phenol-chloroform. RAPD-PCR was optimized, using 10-nt primers of arbitrary sequences [1281, 1254, 1247] and isolate-specific fingerprints were generated. Analysis of PCR products on agarose gel was performed using NTSYSpc program. Dendrograms were calculated according to Jaccard and Nei. According to differences in genetic profiles, H. pylori isolates were clustered into 4 distinct groups: 2 groups consisted of isolates from normal patients, 2 groups of isolates from patients with ulcer, and isolates from patients with cancer were clustered along with isolates from normal and ulcer patients. Furthermore, isolates from ulcer patients appeared in the cluster related to isolates from normal patients. Genetic variation is quite frequent within H. pylori populations; thus RAPD-PCR is an effective technique to reveal genetic diversity of isolates from different dyspeptic patients. In this study, H. pylori strains were clustered into 4 groups: 2 groups from normal patients, and 2 from patients with ulcer. Further studies in larger populations will help to correlate a certain peptic disease to specific strain of H. pylori