An Anaerobic Environment Drives the Harboring of Helicobacter pylori within Candida Yeast Cells.

Helicobacter pylori protects itself from stressful environments by forming biofilms, changing its morphology, or invading eukaryotic cells, including yeast cells. There is little knowledge about the environmental factors that influence the endosymbiotic relationship between bacterium and yeasts. Here, we studied if oxygen availability stimulated the growth of H. pylori within Candida and if this was a bacterial- or yeast strain-dependent relationship. Four H. pylori strains and four Candida strains were co-cultured in Brucella broth plus 5% fetal bovine serum, and incubated under microaerobic, anaerobic, or aerobic conditions. Bacteria-like bodies (BLBs) within yeast cells (Y-BLBs) were detected by microscopy. H. pylori was identified by FISH and by PCR amplification of the 16S rRNA gene of H. pylori from total DNA extracted from Y-BLBs from H. pylori and Candida co-cultures. BLBs viability was confirmed by SYTO-9 fluorescence. Higher Y-BLB percentages were obtained under anaerobic conditions and using H. pylori J99 and C. glabrata combinations. Thus, the H. pylori-Candida endosymbiotic relationship is strain dependent. The FISH and PCR results identified BLBs as intracellular H. pylori. Conclusion: Stressful conditions such as an anaerobic environment significantly increased H. pylori growth within yeast cells, where it remained viable, and the bacterium-yeast endosymbiotic relationship was bacterial strain dependent with a preference for C. glabrata.

Temperatures Outside the Optimal Range for Helicobacter pylori Increase Its Harboring within Candida Yeast Cells.

Helicobacter pylori is capable of entering into yeast, but the factors driving this endosymbiosis remain unknown. This work aimed to determine if temperatures outside the optimal range for H. pylori increase its harboring within Candida. H. pylori strains were co-cultured with Candida strains in Brucella broth supplemented with 5% fetal bovine serum and incubated at 4, 25, 37 or 40 °C. After co-culturing, yeasts containing bacteria-like bodies (Y-BLBs) were observed by optical microscopy, and the bacterium were identified as H. pylori by FISH. The H. pylori 16S rRNA gene was amplified from the total DNA of Y-BLBs. The viability of intra-yeast H. pylori cells was confirmed using a viability assay. All H. pylori strains were capable of entering into all Candida strains assayed. The higher percentages of Y-BLBs are obtained at 40 °C with any of the Candida strains. H pylori also increased its harboring within yeast in co-cultures incubated at 25 °C when compared to those incubated at 37 °C. In conclusion, although H. pylori grew significantly at 40 °C, this temperature increased its harboring within Candida. The endosymbiosis between both microorganisms is strain-dependent and permits bacterial cells to remain viable under the stressing environmental conditions assayed.

Nutrient Deficiency Promotes the Entry of Helicobacter pylori Cells into Candida Yeast Cells.

Helicobacter pylori, a Gram-negative bacterium, has as a natural niche the human gastric epithelium. This pathogen has been reported to enter into Candida yeast cells; however, factors triggering this endosymbiotic relationship remain unknown. The aim of this work was to evaluate in vitro if variations in nutrient concentration in the cultured medium trigger the internalization of H. pylori within Candida cells. We used H. pylori-Candida co-cultures in Brucella broth supplemented with 1%, 5% or 20% fetal bovine serum or in saline solution. Intra-yeast bacteria-like bodies (BLBs) were observed using optical microscopy, while intra-yeast BLBs were identified as H. pylori using FISH and PCR techniques. Intra-yeast H. pylori (BLBs) viability was confirmed using the LIVE/DEAD BacLight Bacterial Viability kit. Intra-yeast H. pylori was present in all combinations of bacteria-yeast strains co-cultured. However, the percentages of yeast cells harboring bacteria (Y-BLBs) varied according to nutrient concentrations and also were strain-dependent. In conclusion, reduced nutrients stresses H. pylori, promoting its entry into Candida cells. The starvation of both H. pylori and Candida strains reduced the percentages of Y-BLBs, suggesting that starving yeast cells may be less capable of harboring stressed H. pylori cells. Moreover, the endosymbiotic relationship between H. pylori and Candida is dependent on the strains co-cultured.

Antibiotics as a Stressing Factor Triggering the Harboring of Helicobacter pylori J99 within Candida albicans ATCC10231.

First-line treatment for Helicobacter pylori includes amoxicillin and clarithromycin or metronidazole plus a proton pump inhibitor. Treatment failure is associated with antibiotic resistance and possibly also with internalization of H. pylori into eukaryotic cells, such as yeasts. Factors triggering the entry of H. pylori into yeast are poorly understood. Therefore, the aim of this study was to evaluate whether clarithromycin or amoxicillin trigger the entry of H. pylori into C. albicans cells. METHODS: H. pylori J99 and C. albicans ATCC 10231 were co-cultured in the presence of subinhibitory concentrations of amoxicillin and clarithromycin as stressors. Bacterial-bearing yeasts were observed by fresh examination. The viability of bacteria within yeasts was evaluated, confirming the entry of bacteria into Candida, amplifying, by PCR, the H. pylori16S rRNA gene in total yeast DNA. RESULTS: Amoxicillin significantly increased the entry of H. pylori into C. albicans compared to the control. CONCLUSION: the internalization of H. pylori into C. albicans in the presence of antibiotics is dependent on the type of antibiotic used, and it suggests that a therapy including amoxicillin may stimulate the entry of the bacterium into Candida, thus negatively affecting the success of the treatment.

The Administration of the Synbiotic Lactobacillus bulgaricus 6c3 Strain, Inulin and Fructooligosaccharide Decreases the Concentrations of Indoxyl Sulfate and Kidney Damage in a Rat Model.

Indoxyl sulfate (IS) is involved in the progression of chronic kidney disease (CKD) and in its cardiovascular complications. One of the approaches proposed to decrease IS is the administration of synbiotics. This work aimed to search for a probiotic strain capable to decrease serum IS levels and mix it with two prebiotics (inulin and fructooligosaccharide (FOS)) to produce a putative synbiotic and test it in a rat CKD model. Two groups of Sprague-Dawley rats were nephrectomized. One group (Lac) received the mixture for 16 weeks in drinking water and the other no (Nef). A control group (C) included sham-nephrectomized rats. Serum creatinine and IS concentrations were measured using high-performance liquid chromatography with diode array detector (HPLC-DAD). Optical microscopy and two-photon excitation microscopy was used to study kidney and heart samples. The Lac group, which received the synbiotic, reduced IS by 0.8% while the Nef group increased it by 38.8%. Histological analysis of kidneys showed that the Lac group increased fibrotic areas by 12% and the Nef group did it by 25%. The synbiotic did not reduce cardiac fibrosis. Therefore, the putative synbiotic showed that function reducing IS and the progression of CKD in a rat model, but no heart protection was observed.

Intracellular Presence of Helicobacter pylori and Its Virulence-Associated Genotypes within the Vaginal Yeast of Term Pregnant Women.

BACKGROUND: Helicobacter pylori transmission routes are not entirely elucidated. Since yeasts are postulated to transmit this pathogen, this study aimed to detect and genotype intracellular H. pylori harbored within vaginal yeast cells. METHODS: A questionnaire was used to determine risk factors of H. pylori infection. Samples were seeded on Sabouraud Dextrose Agar and horse blood-supplemented Columbia agar. Isolated yeasts were identified using and observed by optical microscopy searching for intra-yeast H. pylori. Total yeast DNA, from one random sample, was extracted to search for H. pylori virulence genes by PCR and bacterial identification by sequencing. RESULTS: 43% of samples contained yeasts, mainly Candida albicans (91%). Microscopy detected bacteria such as bodies and anti-H. pylori antibodies binding particles in 50% of the isolated yeasts. Total DNA extracted showed that 50% of the isolated yeasts were positive for H. pylori 16S rDNA and the sequence showed 99.8% similarity with H. pylori. In total, 32% of H. pylori DNA positive samples were cagA+ vacAs1a vacAm1 dupA-. No relationship was observed between possible H. pylori infection risk factors and vaginal yeasts harboring this bacterium. CONCLUSION: H. pylori having virulent genotypes were detected within vaginal yeasts constituting a risk for vertical transmission of this pathogen.

Detection of Helicobacter pylori in oral yeasts from students of a Chilean university.

INTRODUCTION: Nearly 73% of the Chilean population is infected with Helicobacter pylori (H. pylori), a factor predisposing for gastric cancer. Recent studies have demonstrated the presence of this pathogen within yeasts, suggesting that this fact can directly influence the failure of a treatment, transmission, and reinfection. AIM: To detect the presence of H. pylori inside oral yeasts isolated from students of the University of Concepción (Chile). METHODS: 72 samples, obtained from the oral cavity using cotton swabs were incubated in YPD broth for 48h at 37°C and posteriorly seeded in Sabouraud Dextrose agar plus chloramphenicol at the same temperature and for the same time. Yeasts isolated were observed microscopically (wet mounting and Gram-stained) and identified using microbiological techniques. Intracellular H. pylori detection was performed by the amplification of 16S rDNA by PCR. RESULTS: Oral yeasts were detected in 24 samples (33.3%), being C. albicans (79.2%) the most frequent species, followed by C. dubliniensis (12.4%), C. krusei (4.2%), and C. tropicalis (4.2%). When analyzed by PCR, 15 of the 24 oral yeasts 62.5 % were positive for H. pylori 16S rDNA. From the 15 individuals positive for yeast harboring H. pylori, 81% of them reported stomach discomfort, and the presence of the bacteria was diagnosed at some moment in 20% of them. CONCLUSION: The intracellular presence of the H. pylori in oral yeasts suggests an endosymbiotic relationship of these microorganisms, which could favor H. pylori transmission and reinfection in the gastrointestinal tract.

Detection of Helicobacter pylori in oral yeasts from students of a Chilean university

SUMMARY INTRODUCTION: Nearly 73% of the Chilean population is infected with Helicobacter pylori (H. pylori), a factor predisposing for gastric cancer. Recent studies have demonstrated the presence of this pathogen within yeasts, suggesting that this fact can directly influence the failure of a treatment, transmission, and reinfection. AIM: To detect the presence of H. pylori inside oral yeasts isolated from students of the University of Concepción (Chile). METHODS: 72 samples, obtained from the oral cavity using cotton swabs were incubated in YPD broth for 48h at 37°C and posteriorly seeded in Sabouraud Dextrose agar plus chloramphenicol at the same temperature and for the same time. Yeasts isolated were observed microscopically (wet mounting and Gram-stained) and identified using microbiological techniques. Intracellular H. pylori detection was performed by the amplification of 16S rDNA by PCR. RESULTS: Oral yeasts were detected in 24 samples (33.3%), being C. albicans (79.2%) the most frequent species, followed by C. dubliniensis (12.4%), C. krusei (4.2%), and C. tropicalis (4.2%). When analyzed by PCR, 15 of the 24 oral yeasts 62.5 % were positive for H. pylori 16S rDNA. From the 15 individuals positive for yeast harboring H. pylori, 81% of them reported stomach discomfort, and the presence of the bacteria was diagnosed at some moment in 20% of them. CONCLUSION: The intracellular presence of the H. pylori in oral yeasts suggests an endosymbiotic relationship of these microorganisms, which could favor H. pylori transmission and reinfection in the gastrointestinal tract.

RESUMO INTRODUÇÃO: Quase 73% da população chilena estão infectadas pelo Helicobacter pylori (H. pylori), fator predisponente ao câncer gástrico. Estudos recentes demonstraram a presença desse patógeno em leveduras, sugerindo que esse fato pode influenciar diretamente a falha de um tratamento, transmissão e reinfecção. OBJETIVO: Detectar a presença de H. pylori em leveduras orais isoladas de estudantes da Universidade de Concepción (Chile). MÉTODOS: 72 amostras, obtidas da cavidade oral utilizando cotonetes, foram incubadas em caldo YPD por 48h a 37°C e posteriormente sementes em ágar Sabouraud Dextrose mais cloranfenicol na mesma temperatura e ao mesmo tempo. Leveduras isoladas foram observadas microscopicamente (montagem úmida e corada por Gram) e identificadas utilizando técnicas microbiológicas. A detecção intracelular de H. pylori foi realizada pela amplificação do 16S rDNA por PCR. RESULTADOS: Leveduras orais foram detectadas em 24 amostras (33,3%), sendo C. albicans (79,2%), a espécie mais frequente seguida por C. dubliniensis (12,4%), C. krusei (4,2%) e C. tropicalis (4,2 %) Quando analisadas por PCR, 15 das 24 leveduras orais 62,5% foram positivas para o H. pylori 16S rDNA. Dos 15 indivíduos positivos para leveduras que abrigam H. pylori, 81% deles relataram desconforto estomacal e a presença da bactéria foi diagnosticada em algum momento em 20% deles. CONCLUSÃO: A presença intracelular do H. pylori em leveduras orais sugere uma relação endossimbiótica desses microrganismos, o que poderia favorecer a transmissão e a reinfecção do H. pylori no trato gastrointestinal.

In Vitro Incorporation of Helicobacter pylori into Candida albicans Caused by Acidic pH Stress.

Yeasts can adapt to a wide range of pH fluctuations (2 to 10), while Helicobacter pylori, a facultative intracellular bacterium, can adapt to a range from pH 6 to 8. This work analyzed if H. pylori J99 can protect itself from acidic pH by entering into Candida albicans ATCC 90028. Growth curves were determined for H. pylori and C. albicans at pH 3, 4, and 7. Both microorganisms were co-incubated at the same pH values, and the presence of intra-yeast bacteria was evaluated. Intra-yeast bacteria-like bodies were detected using wet mounting, and intra-yeast binding of anti-H. pylori antibodies was detected using immunofluorescence. The presence of the H. pylori rDNA 16S gene in total DNA from yeasts was demonstrated after PCR amplification. H. pylori showed larger death percentages at pH 3 and 4 than at pH 7. On the contrary, the viability of the yeast was not affected by any of the pHs evaluated. H. pylori entered into C. albicans at all the pH values assayed but to a greater extent at unfavorable pH values (pH 3 or 4, p = 0.014 and p = 0.001, respectively). In conclusion, it is possible to suggest that H. pylori can shelter itself within C. albicans under unfavorable pH conditions.