Detection of peptidoglycan in yeast as a marker for the presence or abundance of intracellular Helicobacter pylori and Staphylococcus.

Peptidoglycan (PG) was targeted as the marker for bacterial occurrence inside yeast. Detection of only few bacteria in old and new generations of yeast raised the question of how yeast controls the abundance of its intracellular bacteria. One gastric C. tropicalis that showed concurrence of H. pylori and Staphylococcus 16S rDNA was stained for assessing the viability of intracellular bacteria. Fluorescein isothiocyanate (FITC)-labeled anti-PG monoclonal antibody (APGMAb) was used for detection of PG inside yeast by direct immunofluorescence. APGMAb-coated magnetic beads were used for separation of bacteria from disrupted yeasts. Bead-bound bacteria were separated, fixed, stained, and examined by scanning electron microscope (SEM). Bead-bound bacteria were cultured and identified by amplification and sequencing of 16S rDNA. Fluorescence microscopy demonstrated occurrence of few live bacteria inside yeast cells. FITC- APGMAb interacted with PG of intracellular bacteria, appearing as few green spots in mother and daughter yeast cells. Interestingly, PG fragments were also detected in the exterior of yeast cells. SEM observations showed separated bead-bound bacilli and cocci. Culture of Staphylococcus was positive. Sequencing results confirmed identity of separated bacteria as H. pylori and Staphylococcus. PG detected inside yeast may have belonged to H. pylori, Staphylococcus or any other intracellular bacteria that coexisted in yeast as its microbiome. Detection of only few intracellular bacteria in old and new generations of yeast as well as PG fragments in their exterior suggested that yeast controls the abundance of its intracellular bacteria at low rate by hydrolysis and exporting of PG.

Coniochaeta fungus benefits from its intracellular bacteria to form biofilm and defend against other fungi.

During cultivation of a gastric fungus, Coniochaeta polymorpha, growth of Nocardia colonies on top of the fungal culture raised the question whether bacteria originated from inside of fungus. In this study, the likelihood of intracellular origin of bacteria as well as interaction of two microorganisms was assessed. Fluorescence and electron microscopy showed occurrence of several bacterial cells in fungal cytoplasm. A thick biofilm was observed on the surface of co-culture compared with thin one on bacterial and none on fungal monocultures. Field emission scanning electron microscopy (FESEM) micrographs of co-culture showed a dense network of fungal and bacterial cells embedded in a slime-like layer. Dual cultures revealed antagonistic activity of both fungus and bacteria against three Candida species. These findings indicate that Nocardia isolate identified in this study originated from the inside of fungus C. polymorpha. Intracellular bacteria could benefit the fungal host by producing a rigid biofilm and an antifungal compound.

Yeast engineered translucent cell wall to provide its endosymbiont cyanobacteria with light.

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.

Mucoid and coccoid Helicobacter pylori with fast growth and antibiotic resistance.

BACKGROUND: In this study, one Helicobacter pylori isolate, from gastric biopsy of a dyspeptic patient that turned into mucoid-coccoid (MC) form upon consecutive subcultures, was identified. The culturability, antibiotic resistance, and lipid contents of MC were compared with those of non-mucoid (NM) spiral H pylori. MATERIALS AND METHODS: Mucoid-coccoid and NM H pylori were subcultured on Brucella blood agar (BBA) and incubated under aerobic and microaerobic atmospheres at 37°C. Cultures were examined for colony characteristics and bacterial morphology after 1-3 days. The isolates were identified by biochemical tests and detection of H pylori-16S rDNA. Antibiogram was performed with currently used antibiotics for H pylori eradication. Cellular lipid contents were extracted and analyzed by gas chromatography. RESULTS: Compared with pin-pointed and glistening colonies of NM H pylori that appeared under microaerobic conditions, MC H pylori grew well in consecutive subcultures under aerobic and microaerobic atmospheres and produced white patches of mucoid colonies. MC exhibited coccoid and NM spiral morphology. Both isolates were catalase, oxidase, and urease positive and contained 16S rDNA. Compared with NM that was susceptible to almost all the antibiotics, MC was resistant to all the antibiotics. Lipid analyses showed high frequency of unsaturated fatty acids and cholesterol in MC. CONCLUSIONS: Coccoid forms with high fatty acid and cholesterol contents that show resistance to antibiotics might resist against other stressful conditions such as gastric acidity and immune response. Moreover, mucoid property may enhance resistance of coccoids to stresses. With mucoid-coccoid lifestyle, H pylori may establish a chronic infection refractory to antimicrobial therapy.

Helicobacter pylori release from yeast as a vesicle-encased or free bacterium.

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.

Effective antimicrobial activity of rifabutin against multidrug-resistant Helicobacter pylori.

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 < 0.05). Of 33 isolates resistant to both MTZ and CLR, 25 (75.7%) were sensitive to RFB and 8 (24.3%) resistant (P < 0.05). DISCUSSION: In vitro antimicrobial effectiveness of RFB on MDR H. pylori including those with resistance to both MTZ and CLR was demonstrated. However, RFB efficacy decreased as the number of antibiotics responsible for MDR increased. Considering that RFB inhibits both extra- and intracellular H. pylori, it can be suggested as an effective antibiotic against of MDR H. pylori.

Natural fruits, flowers, honey, and honeybees harbor Helicobacter pylori-positive yeasts.

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.

Protective effect of essential oil of Pistacia atlantica Desf. on peptic ulcer: role of α-pinene.

OBJECTIVE: 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. METHODS: 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. RESULTS: 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 (P < 0.001) with EC(50) value of 12.32 mg/kg. In microscopic examination, Pistacia atlantica attenuated destruction and necrosis of gastric tissue. CONCLUSION: 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.

Negative Effect of Proton-pump Inhibitors (PPIs) on Helicobacter pylori Growth, Morphology, and Urease Test and Recovery after PPI Removal--An In vitro Study.

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 < .05). OMP (minimum inhibitory concentration, MIC 32 µg/mL) and LPZ (MIC 8 µg/mL) inhibited the growth of 78.6% of H. pylori isolates. OMP and LPZ inhibited urease of 90.3% of isolates between 0 and 40 minutes and 54.4% between 20 and 40 minutes, respectively. PAN did not inhibit H. pylori growth and urease. Three 3-day (9 days) consecutive subcultures of H. pylori on brucella blood agar (BBA) supplemented with OMP resulted in reduced bacterial viability (1+), compared with control (4+), change of spiral morphology to coccoid, and reduction in pink color intensity in urea agar. Bacterial growth (1+), morphology, and urease test did not improve after the first 3-day and second 3-day (6 days) subcultures on BBA. However, relative recovery occurred after the third 3-day (9 days) subculture and complete recovery was observed after the fourth 3-day (12 days) subculture, as confluent growth (4+), 100% spiral cells, and strong urease test. CONCLUSION: Proton-pump Inhibitors exert transient negative effects on H. pylori viability, morphology, and urease test. Accordingly, cessation of PPI consumption at least 12 days before endoscopy could help avoiding false-negative results of H. pylori diagnostic tests.

Relevance of Helicobacter pylori vacA 3'-end Region Polymorphism to Gastric Cancer.

BACKGROUND: Helicobacter pylori vacA genotypes play an important role in the pathogenesis of severe gastrointestinal disease. MATERIALS AND METHODS: We identified a novel polymorphic site in the 3'-end region of H. pylori vacA gene, denoted by c1/-c2 (c1: with deletion of 15 bp), and examined associations of this and the previous four sites as well as cagA status with gastroduodenal diseases, in a total of 217 Iranian H. pylori isolates. Histopathologic evaluations were performed and patients with gastric cancer (GC) were further classified based on the anatomic site of tumor, including cardia and noncardia GC, and the histopathologic type of tumor, including intestinal- and diffuse-type GC. RESULTS: The vacA m1, i1, d1, c1, and cagA genotypes were significantly associated with an increased risk of GC, the odds ratio (95% confidence interval) was 4.29 (2.03-9.08), 6.11 (2.63-14.19), 3.18 (1.49-6.76), 15.13 (5.86-39.01), and 2.59 (1.09-6.12), respectively. The vacA c1 genotype had an increased age- and sex-adjusted risk for GC by the multiple logistic regression analysis; the OR was 38.32 (95% CI, 6.60-222.29). This association was independent of and larger than the associations of the m-, i-, and d-type of vacA or cagA status with GC. No significant correlation was found between s1, whether independently or in combination, and the risk of GC or peptic ulcer disease (PUD). The vacA i1 and cagA genotypes were linked to an increased risk of PUD; the OR (95% CI) was 2.80 (1.45-5.40) and 2.62 (1.23-5.61), respectively. The presence of both the vacA i1 and cagA genotypes further increased the risk of PUD; the OR was 5.20 (95% CI, 1.92-14.03). CONCLUSION: The H. pylori vacA c1 genotype might therefore be one of the strongest risk predictors of GC in male patients aged ≥55 in Iran.

Multiple repeats of Helicobacter pylori CagA EPIYA-C phosphorylation sites predict risk of gastric ulcer in Iran.

Biological activity of Helicobacter pylori oncoprotein CagA is determined by a diversity in the tyrosine phosphorylation motif sites. In the present study, the diversity and the type of the H. pylori CagA EPIYA motifs and their association with gastric ulcer (GU) and duodenal ulcer (DU) in Iranian dyspeptic patients were assessed. PCR amplification, sequencing, and bioinformatic analysis were performed to determine the pattern of CagA EPIYA motifs. Of 168 H. pylori cagA(+) strains, the frequency of ABC was 93.50%, ABCCC 5.40%, ABC + ABCCC 0.6% and ABCC 0.6%. There was no EPIYA-D segment. The ABCCC pattern of EPIYA motif was more frequent in the H. pylori isolates from GU (8/50, 16%) than in those from chronic gastritis (CG) (0/81, 0%) (P = 0). In contrast, The ABC pattern of EPIYA motif was less frequent in the H. pylori isolates from GU (41/50, 82%) than in those from CG (80/81, 98.80%) (Age-sex-adjusted odds ratio (OR) = 0.020, 95% CI = 0.002-0.259; P = 0.003). The distribution of the ABC motif was almost the same in H. pylori isolates from CG (98.80%) and DU diseases (97.30%). There was no significant association between the number of CagA EPIYA-C segment and DU (P > 0.05). We have proposed that CagA from Iranian H. pylori strains were Western type and all strains had active phosphorylation sites. The three EPIYA-C motifs of CagA were more frequently observed in the H. pylori strains from GU; thus it might be an important biomarker for predicting the GU risk in Iran.

Consumption of non-antibacterial drugs may have negative impact on Helicobacter pylori colonization in the stomach.

Background: Nineteen non-antibacterials were examined to show that their consumption for treatment of other diseases may inhibit Helicobacter pylori. Four antibiotics were used for comparison. Materials and methods: Agar dilution method was used to examine the susceptibility of 20 H. pylori isolates to 4 antibiotics; metronidazole (MTZ), clarithromycin (CLR), amoxicillin (AMX), tetracycline (TET) and 19 non-antibacterials; proton pump inhibitors (PPIs), H2-blockers, bismuth subsalicylate (BSS), antifungals, statins, acetaminophen (ACE), aspirin (ASA), B-vitamins (B-Vits; Vit B1, Vit B6 and Vit Bcomplex) and vitamin C (Vit C). Blood agar plates were prepared with different concentrations of drugs and spot-inoculated with bacterial suspensions. Plates were incubated at 37 °C under microaerobic conditions and examined after 3-5 days. The isolate #20 that was mucoid and resistant to 19 drugs, including MTZ and SMV was tested against combined MTZ (8 µg/mL) and SMV (100 µg/mL). Results were analyzed statistically. Results: Minimum inhibitory concentrations (MICs, µg/mL) of drugs and the frequency of susceptible H. pylori were determined as MTZ (8, 80%), CLR (2, 90%), AMX (1, 100%), TET (0.5, 70%), PPIs (8-128, 80%), H2-blockers (2000-8000, 75-80%), BSS (15, 85%), antifungals (64-256, 30-80%), statins (100-250, 35-90%), ACE (40, 75%), ASA (800, 75%), B-Vits (5000-20000, 80-100%) and Vit C (2048, 85%). Susceptibility of H. pylori isolates to 16 out of 19 non-antimicrobials (75-100%) was almost similar to those of antibiotics (70-100%) (P-value >0.05). The highest susceptibility rate (100%) belonged to Vit B1, Vit B6 and AMX. Out of 20 H. pylori isolates, 17 (85%) were susceptible to ≥13 non-antimicrobials and 3 (15%) were susceptible to < 13 (P-value <0.05). Mucoid H. pylori showed susceptibility to combination of MTZ and SMV. Conclusions: Most of non-antibacterials inhibited H. pylori isolates, similar to antibiotics but their MICs exceeded those of antibiotics and their plasma concentrations. At low plasma concentration, non-antimicrobials may act as weak antibacterials, antibiotic adjuvants and immunostimulators.

Synthesis and biological evaluation of novel benzyl piperazine derivatives of 5-(5-nitroaryl)-1,3,4-thiadiazoles as Anti-Helicobacter pylori agents.

BACKGROUND AND THE PURPOSE OF THE STUDY: Helicobacter pylori is recognized as the main cause of gastritis and gastroduodenal ulcers and classified as class 1 carcinogen pathogen. Different 1,3,4-thiadiazole derivatives bearing 5-nitroaryl moiety have been shown considerable anti- H. pylori activity. In attempt to find new and potent derivatives of described scaffold, a new series of 1-(substituted benzyl)-4-(5-(5-nitroaryl-2-yl)-1,3,4-thiadiazol-2-yl)piperazine derivatives were synthesized and evaluated against three metronidazole-resistant isolates of H. pylori using paper disk diffusion bioassay test. METHODS: The title compounds were prepared through the reaction of 1-(5-(5-nitroaryl-2-yl)-1,3,4-thiadiazol-2-yl) piperazine 5a-b and substituted benzyl chloride in DMF. The inhibitory activity of the new derivatives 6a-q against three metronidazole-resistant isolates of H. pylori was evaluated by the disc diffusion method and compared with the commercially available standard drug metronidazole. RESULTS AND DISCUSSION: The results of SAR study indicated that the potency and anti-H. pylori activity profile of synthesized derivatives is mainly attributed to the substituted nitroaryl moiety at the C-5 position of 1,3,4-thiadiazole ring. Most of 1,3,4-thiadiazole derivatives bearing 5-nitrofuran moiety at C-5 position of central thiadiazole ring, demonstrated more promising anti-H. pylori than the 5-nitrothiophen counterpart. CONCLUSION: The most potent nitrofuran derivative containing 3-methoxybenzyl piperazine pendant at the C-2 position of 1,3,4-thiadiazole ring (compound 6i), demonstrated strong anti-H. pylori potential at studied concentrations 100-25 µg/disk (IZD > 20 mm) against all studied metronidazole- resistant isolates of H. pylori.

Physicochemical properties of intact fungal cell wall determine vesicles release and nanoparticles internalization.

Our previous microscopic observations on the wet mount of cultured Candida yeast showed release of large extracellular vesicles (EVs) that contained intracellular bacteria (∼500-5000 nm). We used Candida tropicalis, to examine the internalization of nanoparticles (NPs) with different properties to find out whether the size and flexibility of both EVs and cell wall pores play role in transport of large particles across the cell wall. Candida tropicalis was cultured in N-acetylglucoseamine-yeast extract broth (NYB) and examined for release of EVs every 12 h by the light microscope. The yeast was also cultured in NYB supplemented with of 0.1%, 0.01% of Fluorescein isothiocyanate (FITC)-labelled NPs; gold (0.508 mM/L and 0.051 mM/L) (45, 70 and 100 nm), albumin (0.0015 mM/L and 0.015 mM/L) (100 nm) and Fluospheres (0.2 and 0.02%) (1000 and 2000 nm). Internalization of NPs was recorded with fluorescence microscope after 30 s to 120 min. Release of EVs mostly occurred at 36 h and concentration of 0.1% was the best for internalization of NPs that occurred at 30 s after treatment. Positively charged 45 nm NPs internalized into >90% of yeasts but 100 nm gold NPs destroyed them. However, 70 nm gold and 100 nm negatively-charged albumin were internalized into <10% of yeasts without destroying them. Inert Fluospheres either remained intact on the surface of yeasts or became degraded and internalized into ∼100% of yeasts. Release of large EVs from the yeast but internalization of 45 nm NPs indicated that flexibility of EVs and cell wall pores as well as physicochemical properties of NPs determine transport across the cell wall.

Mucoid Helicobacter pylori isolates with fast growth under microaerobic and aerobic conditions.

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.

Anti-Helicobacter pylori activity of the methanolic extract of Geum iranicum and its main compounds.

Geum iranicum Khatamsaz, belonging to the Rosaceae family, is an endemic plant of Iran. The methanol extract of the roots of this plant showed significant 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 identified 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, beta-sitosterol, beta-sitosteryl acetate, and beta-sitosteryl linoleate, one phenyl propanoid, eugenol, one phenolic glycoside, gein, one flavanol, (+)-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.

Efficacy of Disc Diffusion and Agar Dilution Methods in Evaluating Helicobacter pylori Susceptibility to Antibiotics.

BACKGROUND: In this study, efficacy and consistency of disc diffusion (DD) and agar dilution (AD) methods in determining Helicobacter pylori susceptibility to antibiotics were evaluated using Brucella blood agar (BBA) in both methods and tetrazolium egg yolk agar (TEYA) in AD. METHODS: Twenty H. pylori isolates were tested for susceptibility to nine antibiotics; metronidazole (MTZ), clarithromycin (CLR), amoxicillin (AMX), tetracycline (TET), ofloxacin (OFX), levofloxacin (LVX), ciprofloxacin (CIP), furazolidone (FRZ), and rifampin (RIF). Antibiotics solutions were impregnated into blank paper disks on BBA in the DD method or added to BBA (ADB) or TEYA (ADT) media in the AD method. Suspensions of H. pylori isolates were surface or spot inoculated on solid media. Plates were incubated in CO2 incubator at 37°C for 5-7 days. RESULTS: The highest rate of susceptibility to MTZ (65%) was determined by DD method compared with AD method (ADB: 40%, ADT: 30%). Both methods showed similar CLR (85%) and AMX (100%) susceptibility rates. Susceptibility to remaining antibiotics, determined by DD and ADB/ADT media were in respective order as 95%, 75% / 75% for TET, 100%, 95% / 85% for FRZ, 85%, 85% / 75% for OFX, 90%, 95% / 85% for LVX, 90%, 85% / 85% for CIP, and 100%, 85% / 75% for RIF. CONCLUSION: DD and AD methods showed consistency in determining 161 (89.4%) susceptibility and resistance and inconsistency in determining 19 (10.6%) susceptibility and resistance (P < 0.05). DD is recommended as a cheap and easy method with the efficacy and precision comparable to the AD method in determining H. pylori susceptibility to antibiotics.

5-Nitroimidazole-based 1,3,4-thiadiazoles: heterocyclic analogs of metronidazole as anti-Helicobacter pylori agents.

A series of 5-nitroimidazole-based 1,3,4-thiadiazoles were prepared and tested for antibacterial activity against Helicobacter pylori. The anti-H. pylori activity of target compounds along with the commercially available antimicrobial metronidazole was evaluated by comparing the inhibition-zone diameters determined by the paper disc diffusion bioassay. From our bioassay results against 20 clinical isolates it is evident that piperazinyl, 4-methylpiperazinyl, 3-methylpiperazinyl, and 3,5-dimethylpiperazinyl analogs (6a, 6b, 6e, and 6f, respectively) and pyrrolidine derivative 7 had strong activity at 0.5 µg/disc (average of inhibition zone > 20 mm) while metronidazole had no activity at this dose. Compound 6f containing the 3,5-dimethylpiperazinyl moiety at the 2-position of the 5-(1-methyl-5-nitro-1H-imidazol-2-yl)-1,3,4-thiadiazole skeleton was the most potent compound tested at low concentrations.

Weissella confusa with thermostable ß-hemolytic exopolysaccharide.

Consuming cooked meat contaminated with bacteria that carry thermostable hemolytic exopolysaccharide (ESP), could lead to severe diseases. Culture of a 5- h boiled sample of meat goulash on blood agar showed growth of a gram positive rod-shaped, mucoid and hemolytic bacterium. Biochemical tests and amplification of 1500 bp product of 16S rDNA and sequencing revealed bacterial identity as Weissella confusa. After 1 h boiling of bacterial suspension, they were alive and hemolytic, increased in volume and aggregated. After 8 h boiling of bacterial suspension with coverslip, live bacteria showed hemolysis, clustered and adhered to coverslip. Bacterial bacteriocin and hemolytic activities remained unchanged upon autoclaving. Purified bacterial EPS retained hemolytic activity after autoclaving. Boiling contaminated meat had no negative impact on viability of heat-stable W. confusa and its hemolytic EPS. Thermostable hemolytic EPS protected W. confusa from excessive heat. Hygienic practice in butcheries and kitchens are necessary to eliminate bacterial contaminants.

Individual hosts carry H. pylori isolates with different cagA features - motifs and copy number.

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.