Evaluation of Helicobacter pylori OipA protein as a vaccine candidate and propolis as an adjuvant in C57BL/6 mice.

OBJECTIVES: Outer inflammatory protein A (OipA) is an essential adhesin of Helicobacter pylori. We aimed to evaluate the effects of a recombinant OipA in the induction of crucial cytokines as a vaccine candidate and propolis as an adjuvant in C57BL/6 mice. MATERIALS AND METHODS: C57BL/6 mice were divided into nine groups according to the disposition of antigen and adjuvant and route of administration: subcutaneous (sc) or gavage. The administrated recombinant purified OipA and propolis concentrations were 10 µg/ml and 40 µg/ml, respectively. After vaccination, we measured expression levels of IFN-γ and IL-4 cytokine genes in the spleen cells of mice by real-time PCR. RESULTS: All results were contrasted with the negative sample. By sc injection, the expression of INF-γ was increased 3.5 and 2.9-fold for OipA and OipA plus propolis, respectively. By gavage 4.4 and 11-fold increase was found for OipA and OipA plus propolis, respectively. The administration of propolis by gavage showed more increase than Sc injection concerning the production of INF-γ. The 11-fold increase for injection of OipA plus propolis by gavage was comparable OipA plus Freund's adjuvant injected subcutaneously. This result suggested an excellent immunological response toward OipA concerning the production of INF-γ in mice. In all cases there were no notable IL-4 production increases. CONCLUSION: The results confirm the efficiency of OipA in induction of IFN-γ production, and thereby the cellular immune response. Propolis could be a suitable adjuvant.

The Role of Helicobacter pylori Proinflammatory Outer Membrane Protein and Propolis in Immunomodulation on U937 Macrophage Cell Model.

BACKGROUND: Regarding the important role of proinflammatory outer membrane protein (OipA) in the pathogenesis of Helicobacter pylori infection and immunomodulatory activity of propolis, we aimed to evaluate the immunogenicity effect of a purified recombinant OipA protein and propolis in the induction of two cytokines, interferon-gamma (IFN-γ) and interleukin-4 (IL-4), in a macrophage cell model. MATERIALS AND METHODS: The recombinant protein used in the present study corresponding to the oipA expressing a 34-35 kDa protein. OipA protein was purified by Ni-NTA affinity chromatography. The purified OipA protein (2.5- 40 µg /mL) and the propolis ethanolic extract (5-40 µg/mL) were incubated with phorbol 12-myristate 13-acetate-treated human myelomonocytic cell line U937 cells. IL-4 and IFN-γ levels were measured after 48 hours of incubation using enzyme-linked immunosorbent assay. RESULTS: The amounts of IL-4 and IFN-γ were significantly increased. The optimum concentration of OipA for the secretion of IL-4 was 5 µg/ml (P<0.0001). At higher concentrations, the amount of IL-4 diminished until suppression at 40 µg/mL. The optimum concentration of propolis, resulting in the most significant increased secretion of both IL-4 and IFN-γ was 40 µg/mL (P=0.0001 and P=0.0004). CONCLUSION: We found that an OipA concentration of 10 µg/mL was more effective for IFN-γ production; however, it was not effective for the high production of IL-4. Therefore, it is postulated that the OipA could mainly induce a Th1 response through the production of IFN-γ. We also observed propolis's capability to induce IFN-γ production; however, the effective concentration for this was the same as for IL-4. Therefore, as an adjuvant, proper concentration of propolis is required for OipA to give the optimum response.

Antagonistic and Immunomodulant Effects of Two Probiotic Strains of Lactobacillus on Clinical Strains of Helicobacter pylori.

BACKGROUND: The present study aimed to evaluate the in vitro and in situ antagonistic effects of Lactobacillus probiotic strains on clinical strains of Helicobacter pylori. Also to investigate their immunomodulation effects on a macrophage cell model. MATERIALS AND METHODS: Anti-microbial effects of probiotic lactobacilli against H. pylori was assessed using the well and disk diffusion methods. Effects of lactobacilli probiotics strains, as well as their cell-free supernatant on adhesion of H. pylori to MKN-45 gastric epithelial cells, were examined in their presence and absence. Immunomodulation effects of probiotic lactobacilli were performed using the U937 macrophage cell model. Incubation of host cells with probiotics and their cell-free supernatants with cultured host cells was performed in different optimized conditions. The supernatant of host cells cultured in their presence and absence was used for cytokines measurement. RESULTS: Two probiotics,Lactobacillus acidophilus ATCC4356, and Lactobacillus rhamnosus PTCC1607, could inhibit the growth of clinical H. pylori in vitro. They could also inhibit attachment of H. pylori to MKN-45 cells. Cell-free supernatant of L. acidophilus had a stimulating effect on the production of Interferon-gamma (IFN-γ) by U937 cells. CONCLUSION: The present study demonstrates that, L. acidophilus ATCC4356 and L. rhamnosus PTCC1607 probiotic strains can inhibit the growth of clinical H. pylori in vitro. Treatment of U937 with alive H. pylori plus cell-free supernatant of L. acidophilus, have a significantly higher capacity to stimulate IFN-γ production than H. pylori alone. So, the metabolite (s) of this probiotic may have an immunomodulatory effect in immune response versus H. pylori.

Antibacterial activity of lactobacilli probiotics on clinical strains of Helicobacter pylori.

OBJECTIVES: Treatment of Helicobacter pylori infection by common drugs may be associated with several problems such as antimicrobial resistance to commonly used antibiotics and side effects of employed drugs. Therefore, exploration of non-chemical compounds which are safer than chemical ones is becoming important as an alternative therapy. The purpose of this study was to evaluate the effects of lactic acid bacteria (LAB) against clinical strains of H. pylori. MATERIALS AND METHODS: Study of antibacterial effects of LAB against H. pylori strains included: evaluation of LAB effects as well as its cell-free supernatant (CFS) to reduce the number of H. pylori, and to examine the effects of CFS to inhibit the urease activity of H. pylori. The anti-adhesion effect of LAB on adherence of H. pylori to epithelial cell line was also evaluated. RESULTS: Evaluation of the anti H. pylori effect of LAB depended on the strain of H. pylori and Lactobacillus. However, CFS of LAB reduced significantly the growth of all H. pylori strains. Also, urease activity of H. pylori strains was inhibited by CFS of LAB demonstrating that their organic acid may have a role in this inhibition. The significant anti-adhesion effect of LAB on adherence of H. pylori was also observed. CONCLUSION: Presence of LAB and/or their CFS can reduce the count of H. pylori, inhibit the urease activity of H. pylori, and reduce adhesion of H. pylori to epithelial cell line. This may be important for the impact of H. pylori colonization in the host stomach.

Identification of Factors Associated with Biofilm Formation Ability in the Clinical Isolates of Helicobacter pylori.

BACKGROUND: A few reports confirm the ability of Helicobacter pylori to form biofilm. However, conclusive data do not exist concerning the factors that favor this ability. OBJECTIVES: Evaluation of the factors associated with the biofilm formation ability of H. pylori including bacterial, physical and chemical, and environmental factors was the research's aim. MATERIALS AND METHODS: H. pylori isolates from gastric biopsy specimens of patients infected chronically were screened for biofilm formation ability. Association of bacterial properties such as motility, auto-aggregation, cell hydrophobicity, and extracellular polymeric substances (EPS) with in vitro biofilm formation ability of H. pylori was evaluated. The effects of environmental factors such as growth-medium, temperature, oxygen-tension, pH, ß-cyclodextrin, gastric secreted mucins, and sub-inhibitory concentration of amoxicillin were also evaluated. RESULTS: Ability of clinical H. pylori isolates to form biofilm in was quantitatively compared. The coccoid shape H. pylori cells were observed by scanning electron microscopy, the images were illustrative of the attachment of cells to form microcolony. The levels of hydrophobicity, motility and auto aggregation of two isolates with highest and lowest biofilm formation ability were the same. However, the signifi cant role of mucins (P < 0.05) in elevating the biofilm formation was observed. Other factors influencing biofilm formation were: pH, atmosphere and sub-MIC of antibiotics. CONCLUSION: Mucins have a signifi cant role in elevating the biofilm formation, also pH, atmosphere and sub-MIC of antibiotics influence biofilm formation.

Relationship between histopathological status of the Helicobacter pylori infected patients and proteases of H. pylori in isolates carrying diverse virulence genotypes.

Helicobacter pylori is the main cause of several gastroduodenal diseases in Humans. Among various virulence factors of H. pylori, proteases may also be involved in its pathogenicity. In this study, relationship between proteolytic activity of H. pylori strains and histopathological changes of the stomach was investigated in the patients infected with strains carrying diverse virulence factors. H. pylori strains were isolated from the biopsies of 116 patients who referred to hospital for their gastroduodenal disorders, in Tehran, Iran. Biopsies were sent to microbiology and pathology laboratories for further analysis. All the suspected grown colonies were characterized by both biochemical tests and polymerase chain reaction (PCR). Presence of seven protease genes, htrA, clpP, hp0169, hp1012, hp0382, hp1350 and hp1435, and distinct allelic variants of H. pylori virulence factors, cagA, vacA, iceA, babA2 and sabA, were analyzed in each strain. Protease activity of the strains was assessed using spectrophotometric assay. Furthermore, association between diversity in protease genes and virulence genes, protease activity, as well as pathological changes was estimated statistically. Proteases genes, htrA, clpP, hp0169, hp1012, hp0382, hp1350, hp1435, were detected among 100%, 100%, 98%, 98%, 98%, 98%, and 8% of fifty H. pylori strains isolated from the patients, respectively. Status of cagA, vacA s1, vacA s2, vacA m1, vacA m2, iceA1, iceA2, babA2 and sabA genes in isolates were 64%, 68%, 30%, 26%, 74%, 48%, 52%, 100%, and 96%, respectively. Predominant (84%) combined status for protease genes was: htrA/clpP/hp0169/hp1012/hp0382/hP1350/hp1435, while the prevalent combined status (16%) for virulence genes was: cagA+/vacA s1m2/iceA1+/sabA+/babA2+. Although most of the strains (91.4%) presented moderate protease activity in vitro, lowest activity was measured in strains isolated from the patients with chronic gastritis (4.25%). Present study provide the new data on diversity of protease genes in H. pylori, as well as the proteolytic activity of these genes in H. pylori strains from the sick patients. Presence of significant association between lower protease activity of the strains and mildness of the pathological changes propose involvement of these proteases in the pathogenesis of H. pylori in vivo.

The role of outer inflammatory protein A (OipA) in vaccination of theC57BL/6 mouse model infected by Helicobacter pylori.

BACKGROUND/AIM: Outer inflammatory protein A (OipA) is an important adhesin of Helicobacter pylori. Our goal was to assess the role of OipA in protection of C57BL/6 mice against H. pylori. MATERIALS AND METHODS: C57BL/6 mice were mucosally immunized with recombinant OipA protein, OipA + propolis, propolis, and phosphate-buffered saline. After vaccination, anti-OipA IgA was measured. Mice were challenged three times with 5 × 107 CFU of the H. pylori B19 strain. Two weeks later, bacterial colonization and inflammation in the stomach was analyzed using standard methods. RESULTS: The CFU number in the OipA group was significantly (P < 0.05) lower than that of the control. The CFU number in the OipA + propolis group was higher than those of the OipA and propolis groups. IgA titers were significantly higher (P 6lt; 0.0001) in the OipA group compared to the control and OipA + propolis groups. Propolis did not play an adjuvant effect but it interfered with the efficient vaccine effect of OipA. CONCLUSION: Results show the effect of vaccination by OipA in protection of the mouse model and the importance of OipA in H. pylori pathogenesis. OipA may be proposed as a suitable oral vaccine candidate against H. pylori infection; however, further study is required to determine adjuvant or adverse effects of propolis toward OipA.

Effect of biofilm formation by clinical isolates of Helicobacter pylori on the efflux-mediated resistance to commonly used antibiotics.

AIM: To evaluate the role of biofilm formation on the resistance of Helicobacter pylori (H. pylori) to commonly prescribed antibiotics, the expression rates of resistance genes in biofilm-forming and planktonic cells were compared. METHODS: A collection of 33 H. pylori isolates from children and adult patients with chronic infection were taken for the present study. The isolates were screened for biofilm formation ability, as well as for polymerase chain reaction (PCR) reaction with HP1165 and hp1165 efflux pump genes. Susceptibilities of the selected strains to antibiotic and differences between susceptibilities of planktonic and biofilm-forming cell populations were determined. Quantitative real-time PCR (qPCR) analysis was performed using 16S rRNA gene as a H. pylori-specific primer, and two efflux pumps-specific primers, hp1165 and hefA. RESULTS: The strains were resistant to amoxicillin, metronidazole, and erythromycin, except for one strain, but they were all susceptible to tetracycline. Minimum bactericidal concentrations of antibiotics in the biofilm-forming cells were significantly higher than those of planktonic cells. qPCR demonstrated that the expression of efflux pump genes was significantly higher in the biofilm-forming cells as compared to the planktonic ones. CONCLUSION: The present work demonstrated an association between H. pylori biofilm formation and decreased susceptibility to all the antibiotics tested. This decreased susceptibility to antibiotics was associated with enhanced functional activity of two efflux pumps: hp1165 and hefA.

Association of hp1181 and hp1184 Genes With the Active Efflux Phenotype in Multidrug-Resistant Isolates of Helicobacter pylori.

BACKGROUND: During the last decades the rate of multidrug resistance among clinical Helicobacter pylori isolates has increased. Active pumping out of the drugs may be an important mechanism for multidrug resistance in H. pylori strains. OBJECTIVES: The aim of this study was to evaluate the association of two H. pylori efflux-genes, hp1181 and hp1184 with the active-efflux phenotype in MDR clinical-strains of H. pylori. MATERIALS AND METHODS: Minimal inhibitory concentration (MIC) and drug accumulation for ß-lactames, Tetracycline (TET), Erythromycin (ERY), Metronidazole (MTZ), Ciprofloxacin (CIP) and Ethidium Bromide (EtBr) was performed in the presence and absence of carbonyl cyanide M-Chlorophenyl Hydrazone (CCCP). Presence of hp1181 and hp1184 genes was detected by the polymerase chain reaction (PCR). RT-PCR was performed to compare expression of efflux genes by MDR strains, demonstrating active efflux with the strains without active efflux. RESULTS: Two- to four-fold decrease in minimum inhibitory concentration (MIC) and two-fold increase in accumulation were observed for EtBr in the presence of CCCP for 67% (8) of 12 MDR strains. With CCCP, two- to four-fold decrease in MIC and 1.4- to 1.8-fold increase in the accumulation of ß-lactames, TET, CIP and MTZ were obtained for 42% (5) of the MDR strains. Six, five and three of the 12 MDR strains amplified hp1184, hp1181, and both of them, respectively. The RT-PCR product for expression of hp1181 by MDR strains was approximately 100 bp shorter than that of the 26695 susceptible standard strain. CONCLUSIONS: Expression of the genes hp1184 and hp1181 are associated with the specific active efflux of EtBr and non-related antibiotics, respectively. For displaying these phenotypes, a post-transcriptional regulation step may be required.

Study of biofilm formation in C57Bl/6J mice by clinical isolates of Helicobacter pylori.

BACKGROUND/AIM: Despite the significant number of studies on H. pylori pathogenesis, not much data has been published concerning its ability to form biofilm in the host stomach. This study aims to evaluate the potential of clinical isolates of H. pylori to form biofilm in C57BL/6J mice model. MATERIALS AND METHODS: Two strains of H. pylori were selected from a collection of clinical isolates; one (19B), an efficient biofilm producer and the other (4B), with weak biofilm-forming ability. Mice infected through gastric avages were examined after one and two weeks. Colonization was determined by CFU and urease activity; the anti-H. pylori IgA was measured by ELISA, and chronic infections were evaluated by histopathology. Bacterial communities within mucosal sections were studied by immunofluorescence and scanning electron microscopy (SEM). RESULTS: Successful infection was obtained by both test strains. Strain 19B with higher ability to form biofilm in vitro also showed a higher colonization rate in the mice stomach one week after infection. Difference (P < 0.05) in IgA titers was observed between the infected mice and the controls as well as between 19B and 4B infected mice, two weeks after the last challenge. Immunofluorescence and SEM results showed tightly colonizing H. pylori in stomach mucosal sections and in squamous and glandular epithelium. CONCLUSION: H. pylori is able to form biofilm in the mouse stomach and induce IgA production, reflecting the same potential as in humans. Firm attachment of coccoid form bacteria to host cells suggests the importance of this state in biofilm formation by H. pylori. Occurrence of biofilm in squamous and glandular epithelium of the mouse stomach proposes that H. pylori can all parts of the upper gastrointestinal tract.

Analysis of vacA/cagA genotypes/status in Helicobacter pylori isolates from Iranian children and their association with clinical outcome.

BACKGROUND/AIM: More than 50% of Iranian children are infected with Helicobacterpylori; however, no data exist about the association of vacA/cagA genotype/status with disease outcomes in them. We analyzed association of vacA/cagA genotypes/status of children's isolates with gastric inflammation status as the first step in H. pylori pathogenesis. MATERIALS AND METHODS: Antral biopsies for culture and histopathology were taken from 328 children in 1997-2009. vacA (s, m) alleles and cagA statuses of the isolates were determined by PCR. Histopathology was performed according to the Sydney system; gastritis was scored as normal, mild, moderate, severe, and follicular. RESULTS: A total of 159 culture-positive cases, with no mixed infections, were enrolled in the study. Of them, 60% were cagA-positive; 21.4%, 37.1%, 16.3%, and 25.2% cases were slm1, slm2, s2m1, and s2m2, respectively. Histopathology showed normal (4.4%), mild- chronic (31.4%), moderate-chronic (38.4%), severe-chronic (10.7%), and follicular gastritis (15.1%) cases. Thirty-four (21.4%) of the children had ulcers. Correlation (P < 0.05) was observed between more severe (moderate, severe, follicular) status and both vacAs1 allele and cagA-positive status. No significant relation was observed between genotype/status of vacA/cagA and ulcers (P > 0.05). CONCLUSION: vacAs1 and cagA are associated with more severe gastric inflammation in Iranian children. Association ofvacAs1 and cagA with more severe pathology in Iran may be similar to that of other parts of the world.

Chemical composition and anti-Helicobacter pylori effect of Satureja bachtiarica Bunge essential oil.

Resistance of H. pylori strains to common antibiotics has been developed in different parts of the world and continues to increase. It is important to investigate the novel and efficient anti-H. pylori drugs, among which the plants would be suitable sources. Satureja bachtiarica Bunge is traditionally used as antimicrobial agent. In this study, we evaluated the antibacterial activity of S. bachtiarica Bunge essential oil against 10 clinical isolates of Helicobacter pylori by disc diffusion and agar dilution methods. The chemical composition of essential oil was analyzed by GC and GC-MS. Carvacrol (45.5%) and thymol (27.9%) were the primary constituents of oil, followed by p-cymene (4.4%), and γ-terpinene (4.0%). S. bachtiarica essential oil showed strong antibacterial activity against clinical isolates of H. pylori (17.6 ± 1.1 mm and 0.035 ± 0.13 µl/ml). Carvacrol, as the first main component, had a significant role in this effect, whereas in the presence of thymol, the antibacterial effect of carvacrol was reduced. Therefore, S. bachtiarica essential oil can be applied as an alternative agent for treatment of H. pylori infections. More studies would be required to better clarify its mechanism of action on H. pylori.

Isolation of Enteropathogenic Escherichia coli from lettuce samples in Tehran.

AIM: The purpose of this study was to find the isolation rate of enteropathogenic Escherichia coli (EPEC) from lettuce samples collected in Tehran. BACKGROUND: During the last decade, the prevalence of infectious diarrheal diseases due to consumption of contaminated food especially raw vegetable has been increasingly reported. Enteropathogenic Escherichia coli strains are an important group of diarrheagenic E. coli that can cause infant diarrhea especially in the developing world. MATERIAL AND METHODS: One hundred lettuce samples collected in Tehran were transported to the laboratory, homogenized by a stomacher in EC broth containing cefixime, and cultured on MacConkey agar plates. Bacterial DNA was extracted by boiling method and PCR was performed using three pairs of primers targeting stx 1, stx 2 and eaeA genes. RESULTS: Screening of 100 lettuce samples by PCR showed four samples were positive for the presence of EPEC. CONCLUSION: This study suggests contamination of the lettuce by the EPEC and its possible role as the source of infection in this region.

Evaluation of an Iranian Home-made Helicobacter pylori Stool Antigen ELISA Kit.

BACKGROUND: Current diagnosis of Helicobacter pylori infection by biopsy-based tests requires invasive sampling. Non-invasive methods such as the H. pylori stool-antigen (HpSA) test may be the best alternative for diagnosis of active infection. However, due to the presence of antigenic-diversity among the strains, various commercial tests have shown some discrepancies in different geographical-areas. OBJECTIVES: This study evaluates a homemade HpSA kit developed by using the H. pylori antigens from Iranian-isolates for detection of H. pylori in the stool of infected patients. PATIENTS AND METHODS: Based on the endoscopic features and/or a rapid-urease test (RUT), 30 child and 50 adult patients, were recruited. From these candidates, three biopsies for RUT, culture and histology, and a stool-sample, were obtained. Patients were considered as H. pylori-positive if culture alone or RUT plus histology were found to be positive. Presence of H. pylori antigens in their stools was detected by the homemade HpSA test and an imported HpSA kit (Immundiagnostik, Germany). RESULTS: Using the biopsy-based tests with RUT, histology and culture, 53% (16/30) of children were diagnosed as H. pylori-positive while using the imported kit 57% and the homemade kit 50% of the candidates showed positive results. Also by the biopsy-based tests, 54% of the adults were diagnosed as H. pylori-positive while by the homemade kit 56% showed positive results. Considering the biopsy-based tests as the gold standard, sensitivity and specificity for the imported kit was 94% and 86%, respectively, while the mean sensitivity and specificity for the homemade kit was 96% and 98%, respectively. CONCLUSIONS: The homemade kit, compared with the imported kit and biopsy-proven tests may be a valid and reliable method for determining the presence of H. pylori infection in Iran.

Analysis of Genomic Diversity among Helicobacter pylori Strains Isolated from Iranian Children by Pulsed Field Gel Electrophoresis.

OBJECTIVE: Presence of genomic diversity among Helicobacter pylori (H. pylori) strains have been suggested by numerous investigators. Little is known about diversity of H. pylori strains isolated from Iranian children and their association with virulence of the strains. Our purpose was to assess the degree of genomic diversity among H. pylori strains isolated from Iranian-children, on the basis of vacA genotype, cagA status of the strains, sex, age as well as the pathological status of the patients. METHODS: Genomic DNA from 44 unrelated H. pylori strains isolated during 1997-2009, was examined by pulse-field gel electrophoresis (PFGE). Pathological status of the patients was performed according to the modified Sydney-system and genotype/status of vacA/cagA genes was determined by PCR. PFGE was performed using XbaI restriction-endonuclease and the field inversion-gel electrophoresis system. FINDINGS: No significant relationship was observed between the patterns of PFGE and the cagA/vacA status/genotype. Also no relationship was observed between age, sex, and pathological status of the children and the PFGE patterns of their isolates. Similar conclusion was obtained by Total Lab software. However, more relationship was observed between the strains isolated in the close period (1997-2009, 2001-2003, 2005-2007, and 2007-2009) and more difference was observed among those obtained in the distant periods (1997 and 2009). CONCLUSION: H. pylori strains isolated from children in Iran are extremely diverse and this diversity is not related to their virulence characteristics. Occurrence of this extreme diversity may be related to adaptation of H. pylori strains to variable living conditions during transmission between various host individuals.

Immunological detection of 34 KDa outer membrane protein as a functional form of OipA in clinical isolates of Helicobacter pylori.

BACKGROUND AND OBJECTIVE: An outer membrane protein (OMP) of Helicobacter pylori namely OipA, is an important virulence factor associated with peptic ulcer and gastric cancer risks. The purpose of this study was to isolate the 34 KDa OMP of H. pylori and evaluate its immunogenicity in experimental animals for rapid detection of more virulent H. pylori isolates. MATERIAL AND METHODS: Sarcosine insoluble fraction of membrane proteins (OMPs) were prepared from 15 clinical isolates of H. pylori and their profiles were analyzed by SDS-PAGE. Two out of 15 isolates which demonstrated higher expression for apparent 34 KDa proteins were selected. Under optimal conditions, 34 KDa protein was recovered from 5% SDS-Agarose gel, purified and injected into the New Zealand white rabbits with Fruend's adjuvant in multiple stages with two weeks intervals. Collected antiserum was purified through affinity chromatography with Sepharose column and its titer was determined by ELISA. Specific immune response was demonstrated by Dot blot and western blotting methods. RESULTS: The titer of antibody was determined about 1/3000 and western blotting demonstrated a 34 KD-protein. Screening of various strains by Dot blot method for its presence showed that its expression was more frequent in strains isolated from the patients with more severe pathology. CONCLUSION: High titer obtained for pAbs antibody, suggested the high immunogenicity of this protein in experimental animals. Detection of 34 KDa OMP in strains isolated from the patients with more severe pathology proposes the possible application of this pAbs in detecting more virulent strains of H. pylori.

Construction of an expression plasmid (vector) encoding Brucella melitensis outer membrane protein, a candidate for DNA vaccine.

BACKGROUND: DNA vaccination with plasmid encoding bacterial, viral, and parasitic immunogens has been shown to be an attractive method to induce efficient immune responses. Bacteria of the genus Brucella are facultative intracellular pathogens for which new and efficient vaccines are needed. METHODS: To evaluate the use of a DNA immunization strategy for protection against brucellosis, a plasmid containing the DNA encoding the Brucella melitensis (B. melitensis) 31 kDa outer membrane protein, as a potent immunogenic target, was constructed. RESULTS: The constructed plasmid, pcDNA3.1+omp31, was injected intramuscularly into mice and the expression of omp31 RNA was assessed by RT-PCR. The integrity of the pcDNA3.1+omp31 construct was confirmed with restriction analysis and sequencing. Omp31 mRNA expression was verified by RT-PCR. CONCLUSION: Our results indicate that the pcDNA3.1+omp31 eukaryotic expression vector expresses omp31 mRNA and could be useful as a vaccine candidate.

Helicobacter hepaticus, a new pathogenic species of the Helicobacter genus: Similarities and differences with H. pylori.

Helicobacter hepaticus was discovered in 1992 as a cause of liver cancer in the A/JCr mouse model. In susceptible mice, infection by H. hepaticus causes chronic gastrointestinal inflammation leading to neoplasia. It can also cause morphological changes in breast-glands leading to neoplasm and adenocarcinoma in mouse models. Studies performed on humans have revealed that H. hepaticus may also be a human pathogen since infection by H. hepaticus can be associated with cholecystitis, cholelithiasis and gallbladder cancer. H. hepaticus is a close relative of H. pylori, but it lacks the major virulence factors of H. pylori including vacoulating cytotoxin A (VacA) and cytotoxin associated gene (cagA). Moreover, SabA, AlpA, and BabA, three important adhesin proteins of H. pylori, are absent in its genome. In contrast, the genome of H. hepaticus contains genes encoding some orthologus virulence factors of Campylobacter jejuni such as cytolethal distending toxin (CDT), and PebI adhesin factor. Other genes including 16S rRNA, 18 KDa immunogenic protein, and urease structural subunits are related to H. pylori. Its genome contains a small island consisting of 71 Kbp named HHGI1, which probably encodes a secretion system type IV (T4SS), and some other virulence factors. As far as the immunogenic antigens are concerned, the lipopolysaccharide (LPS) and flagellin of H. hepaticus are weak stimulants of the immune system, while pro-inflammatory responses are mainly induced by its lipoproteins and most likely by the peptidoglycan. Concerning the multidrug efflux pumps, a homologue of H. pylori TolC, HefA, has been observed in H. hepaticus which contributes to resistance to amoxicillin and bile acids.

Proton motive force-dependent efflux of tetracycline in clinical isolates of Helicobacter pylori.

The aim of this study was to evaluate the role of proton motive force (PMF)-dependent efflux in resistance of Helicobacter pylori to tetracycline (Tet). Tet MIC was determined by agar dilution in the presence and absence of carbonyl cyanide m-chlorophenylhydrazone (CCCP), an inhibitor of PMF. Antibiotic accumulation was conducted in the presence or absence of CCCP and the fluorescence of the accumulated antibiotic was measured by spectrofluorometry. In the presence of CCCP, antibiotic accumulation was increased by 2-17-fold in 17/20 Tet(r) isolates and by 3-10-fold in four of five high-level-resistant mutants. Correlation was observed between this increase and diminution of MIC with CCCP. PMF-dependent efflux mechanisms therefore appear to play an important role in the resistance of clinical isolates of H. pylori to Tet.

Application of stool-PCR test for diagnosis of Helicobacter pylori infection in children.

AIM: To evaluate the usefulness of stool-PCR test for diagnosis of Helicobacter pylori (H pylori) infection in pediatric populations. METHODS: Based on endoscopic features (including nodular gastritis, erosive duodenitis and ulcer) and/or a positive rapid urease test (RUT) obtained during endoscopy, 28 children from a group of children admitted to the Children's Medical Center of Tehran for persistent upper gastrointestinal problems were selected to compare biopsy-based tests with stool-PCR. Their gastric activity and bacterial density were graded by the updated Sydney system, and their first stool after endoscopy was stored at -70 degrees C. Biopsies were cultured on modified campy-blood agar plates and identified by gram-staining, biochemical tests, and PCR. Two methods of phenol-chloroform and boiling were used for DNA extraction from H pylori isolates. Isolation of DNA from stool was performed using a stool DNA extraction kit (Bioneer Inc, Korea). PCR was performed using primers for detection of vacA, cagA, and 16srRNA genes in both isolates and stool. RESULTS: Sixteen out of 28 child patients (57%) were classified as H pylori positive by biopsy-based tests, of which 11 (39%) were also positive by stool-PCR. Sensitivity and specificity of stool-PCR was 62.5% and 92.3% respectively. H pylori was observed in histological sections for 10 out of 11 stool-positive patients. Association was observed between higher score of H pylori in histology and positivity of stool-PCR. Also association was observed between the more severe form of gastritis and a positive stool-PCR. CONCLUSION: Association between higher score of H pylori in histology and a positive stool-PCR make it a very useful test for detection of H pylori active infection in children. We also suggest that a simple stool-PCR method can be a useful test for detection of H pylori virulence genes in stool.