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.

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.

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.

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.

Comparison of the effect of non-antifungal and antifungal agents on Candida isolates from the gastrointestinal tract.

BACKGROUND: Non-antifungal drugs appear promising in treatment of opportunistic infections of Candida spp. that are often resistant to current antifungals. METHODS: 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. RESULTS: 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. CONCLUSION: Trifluoperazine could be considered as an alternative antifungal when encountering Candida spp. resistant to current antifungals.