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BACKGROUND Sugar-rich foods are of the main components of daily human meals. These foods with high sugar and low water content kill bacteria. However, osmotolerant yeasts survive and multiply. The aim of this study was to examine the occurrence of intracellular Helicobacter pylori(H. pylori) and Staphylococcus spp. in yeast isolates from sugar-rich foods. METHODS Thirty-two yeast isolates from fresh fruits, dried fruits, commercial foods, and miscellaneous foods were identified by the sequencing of amplified products of 26S rDNA. Fluorescence microscopy and LIVE/DEAD bacterial viability kit were used to examine the occurrence of live bacteria inside the yeast’s vacuole. Immunofluorescence assay was used to confirm the identity of intracellular bacteria as H. pylori and Staphylococcus. Polymerase chain reaction (PCR) was used for the detection of 16S rDNA of H. pylori and Staphylococcus in the total DNA of yeasts. RESULTS Yeasts were identified as members of seven genera; Candida, Saccharomyces, Zygosaccharomyces, Pichia, Meyerozyma, Metschnikowia, and Wickerhamomyces. Intravacuolar bacteria were stained green with a bacterial viability kit, revealing that they were alive. Immunofluorescence assay confirmed the identity of intracellular H. pylori and Staphylococcus spp. PCR results revealed that among the 32 isolated yeasts, 53% were H. pylori-positive, 6% were Staphylococcus-positive, 18.7% were positive for both, and 21.8% were negative for both. CONCLUSION Detection of H. pylori- and Staphylococcus-16S rDNA in yeast isolates from dried fruits, and commercial foods showed the occurrence of more than one kind of endosymbiotic bacterium in yeasts’ vacuoles. While the establishment of H. pylori and Staphylococcus in yeast is a sophisticated survival strategy, yeast serves as a potent bacterial reservoir.

In this work, we have synthesized twenty five new 2-(5-(5-nitrofuran-2-yl)-1,3,4-thiadiazol-2-ylimino)thiazolidin-4-one derivatives bearing an aryl or heteroaryl methylene group on position 5 of thiazolidinone and evaluated their antimicrobial activity against Gram-positive and -negative bacteria as well as three metronidazole resistant Helicobacter pylori strains. Most of the compounds were very potent towards tested Gram-positive bacteria and showed an antibacterial efficacy substantially greater than ampicillin as the reference drug. However, no effectiveness was observed for the Gram-negative microorganisms. The compounds 9, 20 and 29 exhibited strong antimicrobial activity against Helicobacter pylori strains (inhibition zone > 30 mm) in 100 μg/disc and (inhibition zone > 20 mm) in 50 μg/disc. Taking these findings together, it seems that these potent antibacterial derivatives could be considered as promising agents for developing new anti-infectious drugs against microorganisms resistant to currently available antibiotics. Graphical Abstract

Observing release of large extracellular vesicles (EVs) from yeast cell raised the question about size and flexibility of cell wall pores. To estimate the approximate size of pores, internalization of nanoparticles (NPs) with different sizes, into the intact yeast cell was examined. Candida tropicalis was cultured in N-acetylglucoseamine-yeast extract broth (NYB) and examined every 12 hr by light microscopy for observing release of EVs. Yeast culture in NYB was treated with Fluorescein isothiocyanate (FITC) -labelled NPs; gold (45, 70 and 100 nm), albumin (100 nm) and Fluospheres (1000 and 2000 nm). Fluorescence microscope was used for recording internalization of NPs with different concentrations (0.1-10%) after few seconds to 120 min of treatment. Release of EVs with different size and morphology from yeast mostly occurred at 36 hr. Best NPs’ concentration was 0.1% and internalization occurred within few seconds after treatment. Positively charged 45 nm gold NPs internalized into yeast cell and accumulated in the vacuole and 100 nm gold NPs destroyed the yeast cell. Gold NPs with 70 nm size and 100 nm negatively charged albumin NPs were internalized into the vacuole of a few yeast cells. Inert Fluospheres were first degraded outside the yeast’s cell and then absorbed into yeast’s cell and finally reached the vacuole. Release of large EVs from yeast indicated flexibility of cell wall pores and EVs for remodelling. Furthermore, physicochemical properties of internalizing NPs and those of cell wall determined uptake of NPs by yeast cell.

BACKGROUND Sugar-rich foods are of the main components of daily human meals. These foods with high sugar and low water content kill bacteria. However, osmotolerant yeasts survive and multiply. The aim of this study was to examine the occurrence of intracellular Helicobacter pylori (H. pylori) and Staphylococcus spp. in yeast isolates from sugar-rich foods. METHODS Thirty-two yeast isolates from fresh fruits, dried fruits, commercial foods, and miscellaneous foods were identified by the sequencing of amplified products of 26S rDNA. Fluorescence microscopy and LIVE/DEAD bacterial viability kit were used to examine the occurrence of live bacteria inside the yeast’s vacuole. Immunofluorescence assay was used to confirm the identity of intracellular bacteria as H. pylori and Staphylococcus . Polymerase chain reaction (PCR) was used for the detection of 16S rDNA of H. pylori and Staphylococcus in the total DNA of yeasts. RESULTS Yeasts were identified as members of seven genera; Candida, Saccharomyces, Zygosaccharomyces, Pichia, Meyerozyma, Metschnikowia, and Wickerhamomyces. Intravacuolar bacteria were stained green with a bacterial viability kit, revealing that they were alive. Immunofluorescence assay confirmed the identity of intracellular H. pylori and Staphylococcus spp. PCR results revealed that among the 32 isolated yeasts, 53% were H. pylori -positive, 6% were Staphylococcus -positive, 18.7% were positive for both, and 21.8% were negative for both. CONCLUSION Detection of H. pylori - and Staphylococcus -16S rDNA in yeast isolates from dried fruits, and commercial foods showed the occurrence of more than one kind of endosymbiotic bacterium in yeasts’ vacuoles. While the establishment of H. pylori and Staphylococcus in yeast is a sophisticated survival strategy, yeast serves as a potent bacterial reservoir.

Background Yeast has been suggested as a potent reservoir of H. pylori that facilitates bacterial spread within human populations. What mechanism ensures effective H. pylori release from yeast? Here, H. pylori release from yeast as a vesicle-encased or free bacterium was studied. Materials and methods Liquid culture of Candida yeast was examined by light, fluorescence and transmission electron microscopy methods to observe the released vesicles. Vesicles were isolated and examined by TEM. Immunogold labeling was used for detection of H. pylori-specific proteins in vesicles' membrane. Free bacterial cells, released from yeast, were separated by immunomagnetic separation and observed by field emission scanning electron microscopy (FESEM). DNA of bead-bound bacteria was used for amplification of H. pylori-16S rDNA. Viability of bead-bound bacteria was examined by live/dead stain and cultivation on Brucella blood agar. Results Microscopic observations showed that vesicles contained bacterium-like structures. Thin sections showed release of vesicle-encased or free bacterium from yeast. Immunogold labeling revealed occurrence of H. pylori proteins in vesicles' membrane. FESEM showed attachment of H. pylori cells to magnetic beads. Sequencing of 521 bp PCR product confirmed the identity of bead-bound H. pylori. Live/dead staining showed viability of bead-bound H. pylori but the result of culture was negative. Conclusions Escape of intracellular H. pylori from yeast as a membrane-bound or free bacterium indicates that H. pylori uses safe exit mechanisms that do not damage the host which is the principle of symbiotic associations. In human stomach, certain conditions may stimulate yeast cells to release H. pylori as a vesicle-encased or free bacterium.

The following are the responses to the “letter to the editor” (“Helicobacter is preserved in yeast vacuoles! Does Koch’s postulates confirm it?”) authored by Nader Alipour and Nasrin Gaeini that rejected the methods, results, discussions and conclusions summarized in the review article authored by Siavoshi F and Saniee P. In the article, 7 papers, published between 1998 and 2013, were reviewed. The 7 papers had been reviewed and judged very carefully by the assigned expertise of the journals involved, including the reviewers of the World Journal of Gastroenterology (WJG), before publication. In the review article, 121 references were used to verify the methods, results and discussions of these 7 papers. The review article was edited by the trustworthy British editor of the (WJG), and the final version was rechecked and finally accepted by the reviewers of (WJG). None of the reviewers made comments like those in this “letter to the editor”, especially the humorous comments, which seem unprofessional and nonscientific. Above all, the authors’ comments show a lack of understanding of basic and advanced microbiology, e.g. bacterial endosymbiosis in eukaryotic cells. Accordingly, their comments all through the letter contain misconceptions. The comments are mostly based on personal conclusions, without any scientific support. It would have been beneficial if the letter had been reviewed by the reviewers of the article by Siavoshi and Saniee.

Background H. pylori strains with different genetic contents may infect different or an individual human host. Genetic diversity of cagA is thought to contribute to differences in H. pylori strains pathogenicity. In this study, diversity of cagA genotype, EPIYA motif and copy number was assessed in H. pylori single colonies isolated from individual patients. Materials and methods Gastric biopsies from 14H. pylori-positive dyspeptic patients were cultured on selective brucella blood agar and incubated at 37 °C under microaerobic conditions. Four single colonies were obtained from each biopsy subculture on brucella blood agar under similar incubation condition. Presence of cagA and types of EPIYA motifs was determined by polymerase chain reaction (PCR) and cagA copy number by quantitative real-time (RT) PCR. Results Single colonies of 5 patients showed no variation in cagA genotype, EPIYA motif and copy number. Out of the remaining 9 patients, 1 patient showed presence or absence of cagA gene, 2 patients had mixed EPIYA motifs, 2 patients had different cagA copy number, 1 patient showed absence or presence of cagA and mixed motifs, 2 patients had cagA genes with different nucleotide sequences, 1 patient showed presence or absence of cagA and difference in cagA nucleotide sequence. Four isolates that contained multiple copies of cagA, carried EPIYA-ABC motif. Conclusion Genetic diversity of cagA among single colonies isolated from individual patients represents evidence that gastric mucosa of every individual is colonized with a specific and heterogeneous population of H. pylori. Future studies on patients in different disease groups may elucidate the role of mixed populations of H. pylori in development of gastric diseases.

With the increasing prevalence of obesity, non-alcoholic fatty liver disease (NAFLD), has become a frequent cause of chronic liver disease, often leading to cirrhosis. In recent decades, gut microbiota have been evaluated as an effective factor in NAFLD pathogenesis, causing steatohepatitis by involving the host immune system. The aim of this study is to evaluate gut microbiota dysbiosis in NAFLD/NASH patients in comparison to healthy controls.We conducted a systematic search of published studies that have examined the composition of gut microbiota in relation to NAFLD. PubMed, Scopus and ISI Web of Science were searched. After the exclusion of irrelevant studies, 15 eligible studies were included and summarized.Overall, some studies reported the composition of microbiota at the phyla level, while others reported them at smaller subgroups; the results of studies were contradictory in some cases.Overall, study findings indicate a relationship between microbial composition and NAFLD. Study methods and sequencing techniques influenced these results.

In this study, relationship between translucent property of yeast cell wall and occurrence of cyanobacteria inside the yeast vacuole was examined. Microscopic observations on fruit yeast Candida tropicalis showed occurrence of bacterium-like bodies inside the yeast vacuole. Appearance of vacuoles as distinct cavities indicated the perfect harvesting of light by the yeast's cell wall. Transmission electron microscopy observation showed electron-dense outer and electron-lucent inner layers in yeast cell wall. Cyanobacteria-specific 16S rRNA gene was amplified from total DNA of yeast. Cultivation of yeast in distilled water led to excision of intracellular bacteria which grew on cyanobacteria-specific medium. Examination of wet mount and Gram-stained preparations of excised bacteria showed typical bead-like trichomes. Amplification of cyanobacteria-specific genes, 16S rRNA, cnfR and dxcf, confirmed bacterial identity as Leptolyngbya boryana. These results showed that translucent cell wall of yeast has been engineered through evolution for receiving light for vital activities of cyanobacteria.

To evaluate the efficacy of Pistacia atlantica Desf. oleoresin essential oil on peptic ulcer (PU) and its antibacterial effect on metronidazole-resistant Helicobacter pylori, as well as chemical composition of the essential oil.The essential oil was standardized using gas chromatography mass spectrometry (GC/MS) analysis. Acute toxicity of the essential oil was assessed in animal model. In vitro anti-Helicobacter pylori activity was performed through disc diffusion and minimum inhibitory concentration method. For gastroprotective assay, rats received Pistacia atlantica Desf. essential oil (25, 50 and 100 mg/kg orally) 1 h before induction of ulcer by ethanol. Macroscopic (ulcer index and protection rate) and microscopic examination were performed.The GC/MS analysis of the essential oil led to the identification of twenty constituents and α-pinene is predominant constituent. The essential oil was safe up to 2000 mg/kg. All Helicobacter pylori strains were susceptible to the essential oil and the MIC ranged from 275 to 1100 μg/mL. The ulcer index for treated groups was significantly reduced compared to control (P0.001) with EC(50) value of 12.32 mg/kg. In microscopic examination, Pistacia atlantica attenuated destruction and necrosis of gastric tissue.Current study exhibited protective effect of standardized Pistacia atlantica essential oil against ethanol-induced gastric ulcer and its antibacterial activity on Helicobacter pylori. α-pinene might be the responsible agent.

Vacuole of eukaryotic cells, beyond intracellular digestion plays additional roles such as storage of nutrients that provide favorable conditions for bacterial survival. In this study, occurrence of H. pylori inside the vacuole of Candida yeast was studied and the role of vacuolating cytotoxin A (VacA) in constructing the vacuole was discussed. One gastric Candida yeast was used for Live/Dead stain and fluorescence in situ hybridization (FISH) with universal bacterial probe. Yeast total DNA was used for amplification of full-length bacterial 16S rDNA as well as H. pylori-specific 16S rDNA and vacA alleles. Vacuoles were isolated from yeast cells and stained with fluorescent yeast vacuole membrane marker MDY-64. DNA extracted from vacuoles was used for amplification of H. pylori-specific 16S rDNA. Fluorescent microscopy showed occurrence of viable bacteria inside the vacuole of intact Candida yeast cells. FISH showed intracellular bacteria as fluorescent spots inside the vacuole of mother and daughter yeast cells, suggesting bacterial transmission to next generations of yeast. Sequencing of amplified products of bacterial 16S rDNA and amplification of H. pylori 16S rDNA and vacA confirmed the identity of intracellular bacteria as H. pylori. Isolated vacuoles were stained with membrane-specific marker and H. pylori 16S rDNA was amplified from their DNA content. Results of this study suggest yeast vacuole as a specialized niche for H. pylori. It appears that sequestration inside the vacuole may enhance bacterial survival.

BACKGROUND Helicobacter pylori resistance to more than one antibiotic is the main reason for failure in bacterial eradication in a considerable number of patients. Rifabutin (RFB) with a broad-spectrum of antimicrobial therapy has been suggested for treatment of refractory multidrug-resistant infections. METHODS Helicobacter pylori isolates from 104 patients were examined for resistance to 5 currently used antibiotics and RFB, using agar dilution method. Twofold serial dilutions of antibiotics were used and MICs (μg/mL) determined as metronidazole (MTZ 8), clarithromycin (CLR 2), amoxicillin (AMX 1), tetracycline (TET 0.5), furazolidone (FRZ 0.5), and RFB (0.06). RESULTS Of 104 H. pylori isolates, only 7 (6.7%) were sensitive to all the 6 antibiotics. However, 30 (28.8%) were resistant to one antibiotic, 28 (26.9%) to two, 19 (18.2%) to three, 14 (13.4%) to four, and 6 (5.7%) to five currently used antibiotics. Overall, 67(64.4%) of isolates were resistant to 2-5 currently used antibiotics and considered as multidrug-resistant (MDR), with 59 (88.1%) showing sensitivity to RFB and 8 (11.9%) resistance (P

Helicobacter pylori might become highly resistant to antibiotics taken through the life time of patients. This study examined the change in antibiotic resistance of H. pylori by time.Out of 985 dyspeptic patients who were referred to the endoscopy unit of Shariati hospital during 2010-2017, 218 patients with gastric biopsies positive for rapid urease test (RUT) and H. pylori culture were recruited in the study. H. pylori isolates were examined for resistance to 8 currently used antibiotics by the disc diffusion method. Results were compared with those from our three previous studies. The frequency of multidrug resistance (MDR) was also assessed.The highest resistance rate was to metronidazole (MTZ) (79.4%) followed by ofloxacin (OFX) (58.7%), ciprofloxacin (CIP) (46.8%), levofloxacin (LVX) (45%), tetracycline (TET) (38.5%), clarithromycin (CLR) (34.4%), amoxicillin (AMX) (27.1%) and furazolidone (FRZ) (23.9%). No significant difference was found between resistance of H. pylori isolates from male and female40 and40 years old and patients with gastritis and peptic ulcer. The highest rates of MDR were to MTZ+OFX (4.6%), MTZ+OFX+TET (2.8%), MTZ+OFX+CIP+LVX (6.4%) and MTZ+OFX+TET+ CIP+LVX (5%).Resistance to MTZ increased from 33%-55.6% in previous studies to 79.4% by time, to CLR increased from 1.4-7.3% to 34.4%, to TET increased from 0-38.1% to 38.5%, to AMX increased from 1.4%-7.3% to 27.1% and to FRZ increased from 0%-4.5% to 23.9%. Resistance to FQs was 45%-58.7%. Increase in H. pylori antibiotic resistance indicates antibiotic misuse. In Iran, with a considerable number of H. pylori- infected patients, antibiotic therapy should be saved for high risk patients and according to local antibiotic resistance patterns.

Background: Bacterial resistance to the available antibiotics is a life threatening issue and researchers are trying to find new drugs to overcome this problem. Amongst the different structural classes, thiazolidinone-4-one, as a new effective pharmacophore against various bacteria, has been introduced. Objective: A new series of 2-(5-(5-nitrothiophene-2-yl)-1,3,4-thiadiazole-2-ylimino)-5-arylidenethiazolidin- 4-one derivatives were designed and synthesized as new antibacterial agents. Method: Target compounds were synthesized during 5 steps and their in vitro antibacterial and anti-H. pylori activities were evaluated. The interaction of the most active derivatives with the probable targets was assessed by Auto Dock 4.2 Program. Results: The results showed that the most potent compounds, 18, 22 and 23, displayed antibacterial activity versus S.aureus, S.epidermidis, B.cereus and B.subtilis (MIC, 1.56-12.5 µg/mL) and none of the derivatives were active on tested Gram-negative bacteria. Compound 12 in all considered doses and compounds 10, and 27 had strong anti-H. pylori activity (inhibition zone >20 mm) in 25 μg disc. Docking studies determined suitable interactions and affinity of potent compounds with bacterial MUR B and H. pylori urease enzymes. Conclusion: According to the results most of the derivatives are effective anti-bacterial agents and docking evaluation confirmed their possible mechanisms of actions as MURB and Urease inhibitors.

Background Proton-pump inhibitor (PPI) consumption does lead to false-negative results of Helicobacter pylori diagnostic tests such as biopsy culture and rapid urease test (RUT). Materials and Methods Helicobacter pylori isolates from 112 dyspeptic patients with (56.5%) or without (43.5%) PPI consumption were recruited for examining the negative effects of omeprazole (OMP), lansoprazole (LPZ), and pantoprazole (PAN) on H. pylori viability, morphology, and urease, in vitro. The effect of a sublethal concentration of OMP on bacterial features and their recovery after removal of OMP was also assessed. Results Of 112 culture-positive gastric biopsies, 87.5% were RUT positive and 12.5% RUT negative. There was a significant correlation between negative RUT results and PPI consumption (p

BACKGROUND For controlling Helicobacter pylori infection in humans, its environmental reservoir should be determined. In this study, yeast isolates from an isolated village in Iran were studied for the intracellular occurrence of H. pylori. MATERIALS AND METHODS In this study, yeasts were isolated from 29 samples, including oral swabs from villagers (n = 7), flowers and fruits (n = 6), honey and honeybees (n = 12) and miscellaneous samples (4). Yeasts were classified into 12 RFLP groups and identified by amplification of 26S rDNA and sequencing. DNA extracted from the yeast cells was examined for the presence of H. pylori using PCR. RESULTS Of the 29 yeasts, 27 were members of different genera of Ascomycete. H. pylori was detected in 5 of 9 Candida (55.5%), 4 of 5 Komagataella (80%), 3 of 4 Pichia (100%), 2 of 2 Cytobasidia (100%), 2 of 2 Hansenia (100%), 1 of 1 Meyerozyma (100%) and 2 of 3 not sequenced (66.6%) yeasts. Distribution of 19 of 29 (65.5%) H. pylori-positive yeasts within 4 groups was as follows: 1 of 7(14.3%) in oral swabs, 5 of 6 (83.3%) in flowers and fruits, 10 of 12 (83.3%) in honey and the bee group and 3 of 4 (75%) in miscellaneous. CONCLUSIONS Different genera of osmotolerant yeasts from flowers, fruits, honey, and honeybees contained H. pylori in their vacuole. High frequency of H. pylori-positive yeasts in these samples might be related to their high sugar content. Insects such as honeybees that facilitate transfer and easy access of these yeasts to nectars serve as the main reservoirs of these yeasts, playing an important role in their protection and dispersal. Accordingly, H. pylori inside these yeasts can be carried by honeybees to different sugar- and nutrient-rich environments. Sugar-rich environments and honeybees play an important role in distribution of H. pylori-positive yeasts in nature.

In the pursuit of novel antibacterial agents with the 2-thiazolylimino-4-thiazolidinone as a core structure, a series of 5-nitro-heteroarylidene and 5-(2-oxoindolin-3-ylidene) analogs of 2-thiazolylimino-5-arylidene-4-thiazolidinone were synthesized and their antibacterial activities were evaluated against some strains of Gram-positive and Gram-negative bacteria, as well as Helicobacter pylori strains. Biological data indicated that 5-nitrofuran analog 5a and 5-nitroimidazole analog 7a containing no substitutions on the thiazole ring were the most potent compounds.

Background: Helicobacter pylori is microaerobic and turns into coccoid under aerobic conditions. In this study, two mucoid strains, A and D, were isolated from gastric biopsies which grew well on blood agar after 24-hour incubation under aerobic as well as microaerobic conditions. The aim of this study was to identify these strains and compare their growth under aerobic and microaerobic conditions with that of control H. pylori. Materials and Methods: The two isolates A and D were identified as H. pylori according to microscopic morphology, urease, catalase and oxidase tests. Their growth under humidified aerobic and microaerobic conditions was compared with that of control H. pylori which grew only under microaerobic conditions. They were further identified by amplification of 16S rRNA, vacA alleles, cagA and ureAB genes by PCR. Their susceptibility to current antimicrobials was also examined. Results: The strains A and D produced mucoid colonies under aerobic and microaerobic conditions after 24-hour, exhibiting the typical spiral morphology of H. pylori. The results of urease, catalase and oxidase tests were positive. Sequencing of amplified products showed 99‐100% homology with those of the reference H. pylori strains in GenBank. Both strains exhibited resistance to the high concentrations of antimicrobials. Conclusions: This study reports the isolation of two mucoid strains of H. pylori with confluent growth under aerobic and microaerobic conditions. It appears that production of exopolysaccharide (EXP) could serve as a physical barrier to reduce oxygen diffusion into the bacterial cell and uptake of antibiotics. EXP protected the mucoid H. pylori isolates against stressful conditions, the result of which could be persistence of bacterial infection in the stomach.

Despite the rich literature on GERD, its cause and reason for increased prevalence remain obscure. Currently accepted mechanisms leave many questions unanswered. Nitrite chemistry at the GEJ is well described for carcinogenesis. Recent epidemiological and animal data have linked nitrates to GERD. “Nitrate reductase” of oral bacteria converts nitrates to nitrites. We hypothesized that nitrate reductase activity is higher in patients with erosive GERD, delivering more nitrite at the gastroesophageal-junction for a given nitrate intake. To compare oral nitrate reductase activity of erosive GERD patients with controls. Patients with erosive GERD and controls without GERD were enrolled. After overnight fasting, nitrite of oral cavity contents was measured at 1-min intervals for 3 min while incubating a 10-mg nitrate-N/L solution in the mouth. Nitrate reductase activity was calculated and compared between groups. Eleven cases (ten males, mean age: 42.6 ± 11.7 year) and ten controls (eight males, mean age: 37.6 ± 9.2 year) were enrolled. Mean nitrate reductase activity was 3.23 ± 0.99 vs. 2.30 ± 0.83 “μg nitrite-N formed/person/minute” in cases and controls, respectively (p = 0.03). Oral nitrate reductase activity in erosive GERD patients is higher than controls. Therefore, any dietary nitrate load generates more nitrite in these patients. This excess nitrite at the gastroesophageal junction, may potentially contribute to the development of GERD. This is the first report linking oral nitrite production to erosive GERD in man. We suggest that a “nitrate hypothesis” may answer yet unanswered questions about GERD pathogenesis. If confirmed, it may change our understanding of mechanisms of GERD and provide novel therapeutic targets.

Helicobacter pylori infection causes lifelong chronic gastritis, which can lead to peptic ulcer, mucosa-associated lymphoid tissue (MALT) lymphoma and gastric cancer. The growing problem of antibiotic resistance by the organism demands the search for novel candidates from plant-based sources. In the present study, we evaluated the in vitro anti-H. pylori activity of some selected medicinal plants on clinical isolates of H. pylori. Gastric biopsy samples were obtained from patients presenting with gastroduodenal complications. Helicobacter pylori was isolated from the specimens following standard microbiology procedures. The disc-diffusion method was used to determine the susceptibility of three H. pylori isolates to methanol extracts of 23 Iranian plants. All tests were performed in triplicate. Among them, the extracts of Punica granatum and Juglans regia had remarkable anti-H. pylori activity with mean of inhibition zone diameter of 39 and 16 mm at 100 µg disc⁻¹, respectively. In view of the results obtained with P. granatum (pomegranate), the peel extracts of nine cultivars of pomegranate (Shirin-e-Pust Sefid, Agha Mohammad Ali-e-Shirin, Sefid-e-Shomal, Sefid-e-Torsh, Shirin-e-Malase, Tabestani-e-Torsh, Shirin-e-Saveh Malase, Alak-e-Shirin, Pust Siyah) were further assayed against the clinical isolates of H. pylori. The results revealed that all Iranian pomegranate cultivars, except for Alak-e-Shirin, showed significant in vitro anti-H. pylori activity against the clinical isolates of H. pylori (mean of inhibition zone diameter ranging from 16 to 40 mm at 50 µg disc⁻¹).

Reports indicate that H.pylori is able to invade the eukaryotic cells and establish inside their vacuoles. In this study, FITC-conjugated IgY-Hp was used to localize H.pylori inside the vacuole of Candida yeast. Presence of intracellular H.pylori inside the new generations of yeast cells was also examined by light microscopy and Live/Dead BacLight staining method.A single colony of fresh yeast culture was cultivated in a 100-µl medium containing yeast extract and N-acetylglucoseamine supplemented with fetal bovine serum. After 12-hr incubation at 37℃, FITC-conjugated IgY-Hp was added. After 3 hours, 10 µL of yeast suspension was smeared on a glass slide, air-dried and examined by fluorescent microscopy. Wet mounts of yeast culture and Live/Dead BacLight stained preparations were examined by light and fluorescent microscopy, respectively. Photographs were taken from the fast-moving H.pylori inside the yeast vacuoles.Fluorescent microscopy showed that FITC-conjugated IgY-Hp could enter yeast cells and specifically react with H.pylori, localizing the bacterium inside the yeast vacuole. Photographs taken from wet mounts observed by light and fluorescent microscopy showed fast-moving H.pylori cells in the vacuole of mother as well as daughter yeast cells. The intravacuolar H.pylori cells stained green, showing their viability.Intracellular life of prokaryotes inside eukaryotes has been described as an evolutionary phenomenon with a great impact on bacterial persistence despite environmental stresses. Results of this study demonstrated the specific interaction of FITC-conjugated IgY-Hp with H.pylori cells and the bacterial localization inside the Candida yeast vacuole. The intracellular bacteria were viable and existed in the vacuole of next generations of yeast cells. It appears that H.pylori is well-equipped to dwell within the vacuole of eukaryotic cells where it is protected from stressful conditions, including antibacterial therapy. Presence of H.pylori inside the vacuole of new generations of yeasts demonstrates the intimate relationship between the two microorganisms, resulting in bacterial inheritance as part of the vacuolar content of yeast cells.

Nitro-containing heteroaromatic derivatives structurally related to nitroimidazole (Metronidazole) are being extensively evaluated against Helicobacter pylori isolates. On the other hand, 1,3,4-thiadiazole derivatives have also demonstrated promising antibacterial potential. In present study, we evaluated anti-H. pylori activity of novel hybrid molecules bearing nitroaryl and 1,3,4-thiadiazole moieties. Anti-H. pylori activity of novel 5-(5-nitroaryl)-1,3,4-thiadiazole derivatives bearing different bulky alkylthio side chains at C-2 position of thiadiazole ring, were assessed against three different metronidazole resistant H. pylori isolates by paper disk diffusion method. Most of the compounds demonstrated moderate to strong inhibitory response especially at 25 μg/disk. The structure-activity relationship study of the compounds demonstrated that introduction of different alkylthio moieties at C-2 position of thiadiazole ring; alter the inhibitory activity which is mainly dependent on the type of C-5 attached nitrohetercyclic ring. The promising compound of this scaffold, bearing 1-methyl-5-nitroimidazole moiety at C-5 and α-methylbenzylthio side chain at C-2 position of thiadiazole ring, showed strong inhibitory response against metronidazole resistant H. pylori isolates at 12.5 μg/disk (the inhibition zone diameter at all evaluated concentrations (12.5- 100 μg/disk) is >50 mm). Novel 5-(5-nitroaryl)-1,3,4-thiadiazole scaffold bearing different C-2 attached thio-pendant moieties with promising anti-H. pylori potential were identified. Among different nitroheterocycles, 5-nitrofuran and 5-nitroimidazole moieties were preferable for the substitution at C-5 position of 1,3,4-thiadiazole ring. Introduction of different alkylthio side chains at C-2 position of central ring alter the inhibitory activity which is mainly dependent on the type of C-5 attached nitrohetercyclic ring.

Human gastric epithelium and immunocytes have been recognized as the sole specialized eukaryotic cells that host Helicobacter pylori (H. pylori). The aim of this study was to provide further evidence for our previous proposal regarding the occurrence of H.pylori inside the yeast vacuole, verifying the viability of the intravacuolar H.pylori by western blotting.Light microscopy and polymerase chain reaction (PCR) were used for primary detection of nonculturable H.pylori in 11 Candida yeasts (six oral and five gastric). Boiling was used for extraction of proteins from yeasts and the control H.pylori. Western blot analysis was recruited to assess the occurrence of H.pylori-specific proteins in protein pool of yeasts, using IgY-Hp raised in hens and IgG1-Hp raised in mice.The fast-moving bacterium-like bodies (BLBs) were identified as H.pylori by amplification of H.pylori 16S rRNA, ureAB, vacA s1, and ahpC genes from the whole DNA of yeasts. Analysis of the sequenced products of 16S rRNA gene amplified from the yeast and H.pylori isolates of patient #2 showed 100 % homology with the corresponding sequences of the reference H. pylori strains in GenBank. According to published data, it was plausible to assign the H.pylori-specific proteins, detected by western blot analysis, as thiol peroxidase (21 kDa), peroxiredoxin (AhpC) (26 kDa), urease-A subunit (UreA) (32 kDa), vacuolating cytotoxin A (VacA) small subunit (36 kDa), and VacA large subunit (56 kDa).Results of this study show that inside yeast, H.pylori expresses proteins and is viable. These proteins appear to serve as powerful tools to help H.pylori to establish in the vacuole of yeast where it can reach nutrients and multiply. The intimate relationship between H.pylori and Candida yeast which began long time ago, could have led to the establishment of H.pylori inside the yeast vacuole before invading human cells.

Oral cavity has been proposed as an important reservoir of H.pylori, being implicated in bacterial transmission through oral-oral route. However, some investigators believe that the newborn acquires H.pylori from mother through vaginal delivery. In this study, oral and vaginal yeasts were examined for the intracellular occurrence of H.pylori and their possible role in bacterial transmission. Sixty nine oral and vaginal yeasts from expecting mothers (39 oral and 30 vaginal) and seven oral yeasts from neonates(6/46 vaginal delivery, 1/43 cesarean) were identified and studied by light and fluorescent microscopy for observing the intracellular bacterium-like bodies(BLBs). Whole DNAs of yeasts were recruited for detection of H.pylori-specific genes. Urea breath test (UBT) was performed for detection of H.pylori infection in mothers. Stool antigen test (SAT) was used for detection of H.pylori antigens in infants' stool at birth and six months of age. Oral yeasts were isolated more frequently from normally-delivered neonates. The frequency of H.pylori genes in mothers' vaginal yeasts was significantly higher than in mothers' oral yeasts. A significant correlation was found between the occurrence of H.pylori genes in vaginal yeasts and that in neonates' oral yeasts, occurrence of H.pylori genes in mothers' vaginal yeasts or neonates' oral yeasts, and UBT+ results in mothers.C.albicans which colonizes the oral cavity of neonates through vaginal delivery or contact with environment or healthcare workers could be an important reservoir of H.pylori. Vaginal yeasts are more potent in accommodating H.pylori than oral yeasts. Accordingly, vaginal yeast is proposed as the primary reservoir of H.pylori which facilitates H.pylori transmission to neonates.

Helicobacter pylori is microaerobic and turns into coccoid under aerobic conditions. In this study, two mucoid strains, A and D, were isolated from gastric biopsies which grew well on blood agar after 24-hour incubation under aerobic as well as microaerobic conditions. The aim of this study was to identify these strains and compare their growth under aerobic and microaerobic conditions with that of control H. pylori.The two isolates A and D were identified as H. pylori according to microscopic morphology, urease, catalase and oxidase tests. Their growth under humidified aerobic and microaerobic conditions was compared with that of control H. pylori which grew only under microaerobic conditions. They were further identified by amplification of 16S rRNA, vacA alleles, cagA and ureAB genes by PCR. Their susceptibility to current antimicrobials was also examined.The strains A and D produced mucoid colonies under aerobic and microaerobic conditions after 24-hour, exhibiting the typical spiral morphology of H. pylori. The results of urease, catalase and oxidase tests were positive. Sequencing of amplified products showed 99-100% homology with those of the reference H. pylori strains in GenBank. Both strains exhibited resistance to the high concentrations of antimicrobials.This study reports the isolation of two mucoid strains of H. pylori with confluent growth under aerobic and microaerobic conditions. It appears that production of exopolysaccharide (EXP) could serve as a physical barrier to reduce oxygen diffusion into the bacterial cell and uptake of antibiotics. EXP protected the mucoid H. pylori isolates against stressful conditions, the result of which could be persistence of bacterial infection in the stomach.

Non-antifungal drugs appear promising in treatment of opportunistic infections of Candida spp. that are often resistant to current antifungals.The broth macrodilution method (NCCLS M27-P document) was used to compare the antifungal activity of trifluoperazine, propranolol, and lansoprazole with that of ketoconazole and amphotericin B, using 50 yeast isolates from the GI tract. The minimum fungicidal concentrations (MFCs), resistance rates and the time required for fungicidal activity of the drugs (2 - 48 hours) were determined.The most effective antifungal activity was exhibited by trifluoperazine. Its MFC was 32 µg/mL for Candida albicans (3.3% resistance) and Candida spp. (0% resistance) yeasts, and 64 µg/mL for Candida tropicalis with 10% resistance. The MFC for C. albicans and Candida spp. was comparable to that of ketoconazole. However, the time required for the inhibitory effect (6 hr) was shorter than that of ketoconazole (48 hr) or amphotericin B (24 hr). The time required for the inhibitory activity on C. tropicalis was 24 hr, which was shorter than that of ketoconazole and amphotericin B (48 hr). A considerable number (40%) of Candida spp. showed resistance to ketoconazole, and 20% of C. tropicalis showed resistance to amphotericin B. Trifluoperazine, an antipsychotic drug, exhibited effective antifungal activity with the MFC, comparable to ketoconazole (32 µg/mL). Among the three yeast groups, C. tropicalis showed resistance to trifluoperazine and amphotericin B, and Candida spp. was considerably resistant to ketoconazole.Trifluoperazine could be considered as an alternative antifungal when encountering Candida spp. resistant to current antifungals.

Antimicrobials have been useful in the treatment of H. pylori-related dyspeptic diseases. However, emergence of resistant strains often decreases the eradication rates of H. pylori infections. Large-scale use of antimicrobials will lead to the diminishment of susceptible strains while allowing resistant survivors to outgrow and spread resistance genes. The aim of this study was to assess the change in antimicrobial resistance rate of H. pylori isolates from 2005 to 2008 and indicate the consequences of indiscriminate and widespread use of antimicrobials against H. pylori- and non-H. pylori-related infections.A total of 110 H. pylori strains were isolated from dyspeptic patients during 2005 to 2008 and tested for their susceptibility to antimicrobials using the disk diffusion method. MICs were determined for metronidazole (8 microg/mL), tetracycline (0.5 microg/mL), clarithromycin (2 microg/mL), amoxicillin (1 microg/mL) and furazolidone (0.5 microg/mL). Since the rates of resistance to metronidazole and tetracycline were remarkably high, another 50 isolates were tested for their susceptibility to metronidazole at the same MIC (8 microg/mL) and tetracycline at MICs of 0.5,1 and 2 microg/mL. Resistance rates were compared to those obtained in our two previous studies between 1997-2000 and 2001-2004.The resistance rates of 110 H. pylori isolates to clarithromycin, amoxicillin and furazolidone were 7.3%, 7.3%, and 4.5%, respectively. Among 160 H. pylori isolates, 55.6% exhibited resistance to metronidazole and 38.1% to tetracycline.Compared to our two previous studies, the resistance rates of H. pylori isolates to current antimicrobials has changed over time. The change in resistance rates of clarithromycin, amoxicillin and furazolidone was not statistically significant. However, resistance to metronidazole and tetracycline showed a considerable increase from 33-36.3% to 55.6% and 0-0.7% to 38.1%, respectively. Emergence of resistance due to the intensive use of antibiotics has become a global public health problem. It appears that plasmid-carried genes are involved in the spread of resistance traits among bacteria. Results obtained in this study indicate that the increase in resistance rates of H. pylori isolates to metronidazole and tetracycline could be the indication of indiscriminate and frequent use of antibiotics in Iran.

Geum iranicum Khatamsaz, belonging to the Rosaceae family, is an endemic plant of Iran. The methanol extract of the roots of this plant showed signifi cant activity against one of the clinical isolates of Helicobacter pylori which was resistant to metronidazole. The aim of this study was the isolation and evaluation of the major compounds of G. iranicum effective against H. pylori. The compounds were isolated using various chromatographic methods and identifi ed by spectroscopic data (1H and 13C NMR, HMQC, HMBC, EI-MS). An antimicrobial susceptibility test was performed employing the disk diffusion method against clinical isolates of H. pylori and a micro dilution method against several Gram-positive and Gram-negative bacteria; additionally the inhibition zone diameters (IZD) and minimum inhibitory concentrations (MIC) values were recorded. Nine compounds were isolated: two triterpenoids, uvaol and niga-ichigoside F1, three sterols, β-sitosterol, β-sitosteryl acetate, and β-sitosteryl linoleate, one phenyl propanoid, eugenol, one phenolic glycoside, gein, one fl avanol, (+)-catechin, and sucrose. The aqueous fraction, obtained by partitioning the MeOH extract with water and chloroform, was the most effective fraction of the extract against all clinical isolates of H. pylori. Further investigation of the isolated compounds showed that eugenol was effective against H. pylori but gein, diglycosidic eugenol, did not exhibit any activity against H. pylori. The subfraction D4 was the effective fraction which contained tannins. It appeared that tannins were probably the active compounds responsible for the anti-H. pylori activity of G. iranicum. The aqueous fraction showed a moderate inhibitory activity against both Gram-positive and Gram-negative bacteria. The MIC values indicated that Gram-positive bacteria including Staphylococcus aureus, Staphylococcus epidermidis, and Bacillus subtilis are more susceptible than Gram-neagative bacteria including Escherichia coli and Pseudomonas aeruginosa.

Tragopogon graminifolius DC., family Compositae, is widely consumed as a green vegetable in the west of Iran and for the treatment of gastrointestinal and hepatic ailments. In this study, the chemical composition of the essential oil from T. graminifolius aerial parts was evaluated by gas chromatography and gas chromatography mass spectrometry. Moreover, antioxidant and antimicrobial activity of the essential oil and various extracts of T. graminifolius were determined. Fifty-eight compounds representing 87.2% of the essential oil were identified. The main components were n-hexadecanoic acid (22.0%), β-caryophyllene (7.5%), heneicosane (6.6%), and nonanal (5.2%). The essential oil demonstrated the highest DPPH radical scavenging activity (56.6 ± 8.8ug/mL) and the 80% ethanolic extract the highest ferric reducing antioxidant activity (908.2±79.5 mmol Fe2+ ion/g extract). The total phenolic content of the 80% ethanolic extract from the aerial parts was the highest (560.7±18.8 mg/g gallic acid equivalent). In the antimicrobial test, Shigella dysenteriae was the most vulnerable microorganism, followed by Proteus vulgaris, and the essential oil exhibited the highest antibacterial activity among the samples. The results indicated that the essential oil and extracts of T. graminifolius could be consumed as a natural herbal preservative or complementary supplement in the food and pharmaceutical industries.