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.

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.

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.

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.

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.

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.

Sugar-Rich Foods Carry Osmotolerant Yeasts with Intracellular Helicobacter Pylori and Staphylococcus spp.

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.

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.

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.

Proton Pump Inhibitor-Treated H. pylori Adjust Cell Envelope Fatty Acid and Cholesterol Content to Survive.

BACKGROUND: Proton pump inhibitors (PPIs) with lipophilic nature may interact with lipid components of H. pylori cell membrane, disrupting cell structure and viability. In this study, the effect of PPIs on fatty acid and cholesterol components of H. pylori cell membrane was assessed. METHODS: One H. pylori isolate was treated with 1X and 2X MICs (µg/mL) of lansoprazole (LPZ: 8 and 16) and pantoprazole (PAN: 128 and 256) in brain heart infusion broth plus serum. Treated H. pylori was cultured on brucella blood agar (BBA) and tetrazolium egg yolk agar (TEYA). Bacterial cells stained with Live/Dead kit were examined by fluorescent microscopy. Fatty acid and cholesterol contents of treated H. pylori were measured by gas chromatography. RESULTS: PPI-treated H. pylori did not grow on BBA but grew on TEYA. Fluorescent microscopy showed H. pylori stained red. Analyses showed high frequency of saturated fatty acids, C14:0, C16:0 and C18:0. Among unsaturated fatty acids, C18:1 and C18:2c were increased, while five were eliminated and five were synthesized de novo. Cholesteryl-6-O-tetradecanoyl-α-D- glucopyranoside was detected as the only glycosylated cholesterol in treated H. pylori. Growth of PPI-treated H. pylori on cholesterol-rich TEYA showed that occurrence of cholesterol can reverse the growth inhibition by PPIs. Red- bacilli form of H. pylori showed dye entry through damaged cell membrane without lysis. CONCLUSION: Incorporation of lipophilic PPI into H. pylori cell membrane disrupted lipids and inhibited growth. However, H. pylori adjusted the defected membrane by replacing the lipid components and resisted lysis.

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.

Excision of endosymbiotic bacteria from yeast under aging and starvation stresses.

Although infrequent in our laboratory, growth of bacterial colonies has been observed on top of the purified cultures of yeasts. In this study, the likelihood of bacterial excision from yeast under aging and starvation stresses was assessed using 10 gastric and 10 food-borne yeasts. Yeasts were identified as members of Candida or Saccharomyces genus by amplification and sequencing of D1/D2 region of 26S rDNA. For aging stress, yeasts were cultured on brain heart infusion agar supplemented with sheep blood and incubated at 30 °C for 3-4 weeks. For starvation stress, yeasts were inoculated into distilled water and incubated similarly. After seven days, starved yeasts were cultured on yeast extract glucose agar, incubated similarly and examined daily for appearance of bacterial colonies on top of the yeast's growth. Outgrowth of excised bacteria was observed on top of the cultures of 4 yeasts (Y1, Y3, Y13 and Y18) after 3-7 days. The excised bacteria (B1, B3, B13 and B18) were isolated and identified at the genus level according to their biochemical characteristics as well as amplification and sequencing of 16S rDNA. B1 (Arthrobacter) were excised from Y1 (Candida albicans) upon aging and B3 (Staphylococcus), B13 (Cellulomonas) and B18 (Staphylococcus) were excised from their respective yeasts; Y3 (Candida tropicalis), Y13 (Saccharomyces cerevisiae) and Y18 (Candida glabrata) upon starvation. DNA from yeasts was used for detection of 16S rDNA of their intracellular bacteria and sequencing. Amplified products from yeasts showed sequence similarity to those of excised bacteria. Under normal conditions, yeast exerts tight control on multiplication of its intracellular bacteria. However, upon aging and starvation the control is no longer effective and bacterial outgrowth occurs. Unlimited multiplication of excised bacteria might provide yeast with plenty of food in close vicinity. This could be an evolutionary dialogue between yeast and bacteria that ensures the survival of both partners.

Localization of Staphylococcus inside the vacuole of Candida albicans by immunodetection and FISH.

In our previous study, two bacteria B1 and B2 were excised from two amphotericin B-treated Candida albicans Y1 and Y2, respectively. Bacteria were identified as B1: Staphylococcus hominis and B2: Staphylococcus haemolyticus according to their biochemical characteristics and detection and sequencing of Staphylococcus-specific genes. In this study the intracellular origin of staphylococci inside the vacuole of yeast was examined. Polyclonal antibodies against S. hominis and S. haemolyticus were raised in rabbit and used for detection of staphylococcal proteins in protein pool of yeasts by western blotting (WB). Fluorescein-isothiocyanate (FITC)-conjugated antibodies were used for bacterial localization inside yeast's vacuole by direct immunofluorescence (DIF). Fluorescent in situ hybridization (FISH) with Staphylococcaceae -specific probe was performed for validation of immunodetection results. WB results showed occurrence of several proteins in protein pool of yeasts that were similar to staphylococcal proteins such as those with molecular weight of 57.5 and 66 kDa. Fluorescent microscopy showed interactions of FITC-antibodies with intracellular staphylococci which appeared as green spots. Hybridization of staphylococcal- specific probe with bacteria inside yeasts' vacuole confirmed immunodetection results. Detection of staphylococcal proteins and genes inside Candida albicans yeast indicates existence of intracellular bacteria inside the vacuole of yeast. These results suggest C. albicans as the potential reservoir of medically important bacteria.

Sequestration inside the yeast vacuole may enhance Helicobacter pylori survival against stressful condition.

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.

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.

Helicobacter pylori Multidrug Resistance Due to Misuse of Antibiotics in Iran.

BACKGROUND: 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. METHODS: 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. RESULTS: 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 female <40 and >40 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%). CONCLUSION: 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.

Candida albicans Release Intracellular Bacteria When Treated With Amphotericin B.

BACKGROUND: Coexistence of bacteria and yeast in a myriad of microbial communities is indicative of their intimate relationship, which could be the intracellular existence of bacteria inside yeast. In this study, the intracellular existence of bacteria inside yeast and bacterial release from amphotericin B-treated yeasts was examined using polymerase chain reaction (PCR) and microscopy. METHODS: Released bacteria, B1 from Y1 (a gastric yeast) and B2 from Y2 (an oral yeast) were identified as Staphylococcus hominis and Staphylococcus haemolyticus by biochemical tests as well as amplification of Staph-specific tuf and 16S rRNA genes. PCR products were sequenced and matched with Staphylococcus published sequences in GenBank. PCR was also used for amplification of Staph-tuf and Helicobacter pylori-16S rRNA genes from DNAs of 50 yeasts (20 oral, 20 gastric and 10 fecal). Microscopy was used for observing bacterium-like bodies (BLBs) inside the yeasts vacuole. RESULTS: Thirty-two yeasts (64%) carried Staph-tuf gene, 20 yeasts (40%) carried H. pylori 16S rRNA gene, 14 yeasts (28%) carried both genes, 12 yeasts (24%) carried neither, 6 yeasts (12%) carried only H. pylori 16S rRNA gene, and 18 yeasts (36%) carried only tuf gene. Amplified products of tuf (370 bp) and 16S rRNA (756 bp) genes from B1 and Y1, and B2 and Y2 showed high similarity to S. hominis and S. hemolyticus, respectively. Microscopic observations showed BLBs inside the yeasts vacuoles, which could be related to the released bacteria. These BLBs were alive and could be observed in successive generations of yeasts. CONCLUSION: Amplification of Staphylococcus- and H. pylori- specific genes from yeasts showed that the intracellular BLBs could belong to Staphylococcus species and H. pylori. Release of culturable staphylococci from 2/50 (4%) yeasts showed that not all yeasts release bacteria, and bacterial release takes place under unknown conditions. However, it could be triggered by amphotericin B or hydrolytic enzymes. Coexistence of staphylococci and H. pylori genes could represent a mixed endosymbiotic bacterial population in Fungi such as yeast. By selecting certain bacterial associates, the diversity of microbial communities could be determined. These selected bacteria could have an intracellular origin, being released under certain conditions.

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.