Identification and Characterization of Probiotic Lactiplantibacillus plantarum BI-59.1 Isolated from tejuino and Its Capacity to Produce Biofilms.

Tejuino is a popular and traditional beverage consumed in north and western of Mexico, due to its biological properties, it is considered a natural source of probiotics. Nevertheless, few studies have been performed on Tejuino microbiota. In this work, the probiotic potential of the tejuino isolated Lactiplantibacillus plantarum BI-59.1 strain was investigated. Its effectiveness was compared with a commercial Lactobacillus spp and identified by 16S rDNA sequence homology. Lactiplantibacillus plantarum BI-59.1 strain showed probiotic properties, i.e., production of antimicrobial compounds (lactic acid and presence of plantaricin A gene), inhibition of entero-pathogens by planktonic cells and metabolites (Salmonella enterica serovar Typhimurium inhibition to HT29-MTX adhesion), biofilm formation, bacterial adhesion (HT29-MTX, 3.96 CFU/cell), and tolerance to stimulated gastrointestinal conditions (tolerance to pH 3 and bile salts). The strain was gamma hemolytic, susceptible to most antibiotics and negative for gelatinase production; thus, the Lactiplantibacillus. plantarum BI-59.1 strain is suitable for its use as a probiotic for nutraceutical or pharmaceutical formulations.

Can Probiotics, Particularly Limosilactobacillus fermentum UCO-979C and Lacticaseibacillus rhamnosus UCO-25A, Be Preventive Alternatives against SARS-CoV-2?

COVID-19, an infection produced by the SARS-CoV-2 virus in humans, has rapidly spread to become a high-mortality pandemic. SARS-CoV-2 is a single-stranded RNA virus characterized by infecting epithelial cells of the intestine and lungs, binding to the ACE2 receptor present on epithelial cells. COVID-19 treatment is based on antivirals and antibiotics against symptomatology in addition to a successful preventive strategy based on vaccination. At this point, several variants of the virus have emerged, altering the effectiveness of treatments and thereby attracting attention to several alternative therapies, including immunobiotics, to cope with the problem. This review, based on articles, patents, and an in silico analysis, aims to address our present knowledge of the COVID-19 disease, its symptomatology, and the possible beneficial effects for patients if probiotics with the characteristics of immunobiotics are used to confront this disease. Moreover, two probiotic strains, L. fermentum UCO-979C and L. rhamnosus UCO-25A, with different effects demonstrated at our laboratory, are emphasized. The point of view of this review highlights the possible benefits of probiotics, particularly those associated with immunomodulation as well as the production of secondary metabolites, and their potential targets during SARS-CoV-2 infection.

Isolation and characterization of Lactobacillus casei A14.2, a strain with immunomodulating activity on Apis mellifera.

Considering the economic and environmental role played by bees and their present threats it is necessary to develop food supplements favoring their health. The aim of this work was to isolate and characterize an immunomodulating probiotic capable to improve the health of honeybee colonies. For this purpose, bacterial strains were isolated from Apis mellifera bees (N = 180) obtained at three apiaries. A total of 44 strains were isolated and 9 of them were identified as Lactobacillus having the capacity to grow under saccharose osmotic stress, at pH 4.0 and possessing a wide susceptibility to antibiotics. Results allowed to select two strains but finally only one of them, strain A14.2 showed a very significant immunomodulating activity. This strain increased the expression of mRNA codifying the antimicrobial peptides 24 h post-administration. We evaluated its growth kinetics under aerobic and microaerobic conditions and its survival in the presence of high concentrations of saccharose. Results demonstrated that Lactobacillus casei A14.2 strain was highly tolerant to oxygen and that it was able to adapt to saccharose enriched environments (50% and 100% w/v). Finally, L. casei A14.2 strain was administered monthly during summer and early fall to 4 honeybee colonies (2 controls and 2 treatments). The results showed a gradual sustained decrease of infestation (p < 0.05) by the pathogenic Nosema spp. but no reduction in the infestation by the mite Varroa destructor. These results suggest that the administration of this potential probiotic, may increase the resistance of honeybee colonies to infectious diseases caused by Nosema spp.

The Ability of Postimmunobiotics from L. rhamnosus CRL1505 to Protect against Respiratory Syncytial Virus and Pneumococcal Super-Infection Is a Strain-Dependent Characteristic.

Previously, we demonstrated that the non-viable strain Lacticaseibacillus rhamnosus CRL1505 (NV1505) or its purified peptidoglycan (PG1505) differentially modulated the respiratory innate antiviral immune response triggered by Toll-like receptor (TLR)-3 activation in infant mice, improving the resistance to primary respiratory syncytial virus (RSV) infection and secondary pneumococcal pneumonia. In this work, we evaluated the effect of other non-viable L. rhamnosus strains and their peptidoglycans on the respiratory immune response and their impact on primary and secondary respiratory infections. In addition, the duration of the protective effect induced by NV1505 and PG1505 as well as their ability to protect against different Streptococcus pneumoniae serotypes were evaluated. Our results showed that among the five selected L. rhamnosus strains (CRL1505, CRL498, CRL576, UCO25A and IBL027), NV1505 and NVIBL027 improved the protection against viral and pneumococcal infections by modulating the respiratory immune response. Of note, only the PG1505 presented immunomodulatory activities when compared with the other purified peptidoglycans. Studies on alveolar macrophages showed that NV1505 and PG1505 differentially modulated the expression of IL-6, IFN-γ, IFN-ß, TNF-α, OAS1, RNAseL and IL-27 genes in response to RSV infection, and IL-6, IFN-γ, IL-1ß, TNF-α, CCL2, CXCL2, CXCL10 and IL-27 in response to pneumococcal challenge. Furthermore, we demonstrated that NV1505 and PG1505 treatments protected mice against secondary pneumococcal pneumonia produced by different serotypes of S. pneumoniae until 30 days after stimulation with poly(I:C). This work advances the characterization of the protective effect of NV1505 and PG1505 by demonstrating that they increase resistance against the pneumococcal serotypes 3, 6B, 14 and 19F, with an effect that lasts up to 30 days after the primary viral inflammation. The results also confirm that the immunomodulatory properties of NV1505 and PG1505 are unique and are not shared by other members of this species, and suggest the existence of a capacity to stimulate trained immunity in alveolar macrophages.

Selenium Nanoparticle-Enriched and Potential Probiotic, Lactiplantibacillus plantarum S14 Strain, a Diet Supplement Beneficial for Rainbow Trout.

Lactic acid bacteria (LAB), obtained from rainbow trout (Oncorhynchus mykiss) intestine, were cultured in MRS medium and probiotic candidates. Concurrently, producers of elemental selenium nanoparticles (Se0Nps) were selected. Probiotic candidates were subjected to morphological characterization and the following tests: antibacterial activity, antibiotic susceptibility, hemolytic activity, catalase, hydrophobicity, viability at low pH, and tolerance to bile salts. Two LAB strains (S4 and S14) satisfied the characteristics of potential probiotics, but only strain S14 reduced selenite to biosynthesize Se0Nps. S14 strain was identified, by 16S rDNA analysis, as Lactiplantibacillus plantarum. Electron microscopy showed Se0Nps on the surface of S14 cells. Rainbow trout diet was supplemented (108 CFU g-1 feed) with Se0Nps-enriched L. plantarum S14 (LABS14-Se0Nps) or L. plantarum S14 alone (LABS14) for 30 days. At days 0, 15, and 30, samples (blood, liver, and dorsal muscle) were obtained from both groups, plus controls lacking diet supplementation. Fish receiving LABS14-Se0Nps for 30 days improved respiratory burst and plasmatic lysozyme, (innate immune response) and glutathione peroxidase (GPX) (oxidative status) activities and productive parameters when compared to controls. The same parameters also improved when compared to fish receiving LABS14, but significant only for plasmatic and muscle GPX. Therefore, Se0Nps-enriched L. plantarum S14 may be a promising alternative for rainbow trout nutritional supplementation.

Consumption of a Gelatin Supplemented with the Probiotic Strain Limosilactobacillus fermentum UCO-979C Prevents Helicobacter pylori Infection in a Young Adult Population Achieved.

Helicobacter pylori is a bacterium associated with various gastrointestinal diseases of high worldwide prevalence. Since probiotics are an emerging alternative to managing infection by this pathogenic bacterium, the present work evaluated, in a randomized double-blind study controlled by a placebo, if consuming Limosilactobacillus fermentum UCO-979C prevents H. pylori infection in humans. Participants consumed either L. fermentum UCO-979C-supplemented gelatin (67 participants) or placebo-supplemented gelatin (64 participants) once a day, five days per week for 12 weeks. H. pylori infection in the participants was controlled before and after the intervention detecting H. pylori antigens in stools. Regarding H. pylori-infected participants before the study, 100% remained infected at the end of the study in the placebo group, while 96.7% of those receiving the probiotic remained infected after the intervention. Most importantly, of the non-infected participants, 34.2% became infected and 65.8% remained non-infected in the placebo group, while 2.7% became infected and 97.3% remained as non-infected individuals in the intervened group. Therefore, consuming the L. fermentum UCO-979C strain significantly reduced H. pylori infection, demonstrating a 92.6% efficacy in avoiding infection by this pathogen in non-infected individuals; thus, this probiotic is an excellent candidate to prevent H. pylori infections in non-infected individuals.

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.

Selenium Nanoparticles Biosynthesized by Pantoea agglomerans and Their Effects on Cellular and Physiological Parameters in the Rainbow Trout Oncorhynchus mykiss.

The applications of nanoparticles (Nps) as food additives, health enhancers, and antimicrobials in animal production are increasing. The aim of this study was to evaluate the effect of selenium (Se) nanoparticles (Se0Nps) stabilized with L-cysteine (Se0Nps/L-Cys), as a nutritional supplement, on immunological, oxidative status, and productive parameters in O. mykiss. TEM and SEM-EDS showed the accumulation of spherical Se0Nps entirely composed by elemental selenium (Se0) as intracellular and extracellular deposits in Pantoea agglomerans UC-32 strain. The in vitro antioxidant capacity of Se0Nps/L-Cys was significant more efficient ROS scavengers than Se0Nps and Na2SeO3. We also evaluate the effect of Se0Nps/L-Cys on cell viability and oxidative stress in RTgill-W1, RTS-11, or T-PHKM Oncorhynchus mykiss cell lines. Se0Nps/L-Cys showed less toxic and high antioxidant activity than Se0Nps and Na2SeO3. Finally, the dietary Se0Nps/L-Cys had a significant better effect on both plasma lysozyme and respiratory burst activity (innate immune response), on tissular Gpx activity (oxidative status), and on well-being (productive parameter) of O. mykiss when it is compared to Se0Nps and Na2SeO3. Se0Nps/L-Cys is a promising alternative for nutritional supplement for O. mykiss with better performance than Na2SeO3 and Se0Nps, ease to implementation, and reduced environmental impact.

Incorporation of Limosilactobacillus fermentum UCO-979C with Anti-Helicobacter pylori and Immunomodulatory Activities in Various Ice Cream Bases.

Limosilactobacillus fermentum UCO-979C is a probiotic strain possessing anti-Helicobacter pylori and immunomodulatory activity. The aim of this work was to examine if this strain maintains its probiotic properties and its viability when added to dairy-based ice creams (cookies and cream, Greek yogurt, and chocolate with brownie) or to fruit-based ice creams (pineapple and raspberry) stored at -18 °C for 90 days. The probiotic anti-H. pylori activity using the well diffusion test, its immunomodulatory activity was measured using transforming growth factor beta 1 (TGF-ß1) cytokine production by human gastric adenocarcinoma (AGS) cells, and its viability was measured using the microdrop technique. Assays were performed in triplicate. The L. fermentum UCO-979C strain maintained strong anti-H. pylori activity in dairy-based ice creams and mild activity in fruit-based ice cream. The production of pro-inflammatory cytokine TGF-ß1 on AGS cells was higher in the probiotic recovered from Greek yogurt ice cream, maintaining a viability exceeding 107 colony-forming units/mL. The addition of the probiotic to ice creams did not significantly influence the physicochemical properties of the product. These data show the great potential of the L. fermentum UCO-979C strain in producing probiotic dairy-based and fruit-based ice creams.

Gastrointestinal Microbiota and Parasite-Fauna of Wild Dissostichus eleginoides Smitt, 1898 Captured at the South-Central Coast of Chile.

Dissotichus eleginoides has a discontinuous circumpolar geographic distribution restricted to mountains and platforms, mainly in Subantarctic and Antarctic waters of the southern hemisphere, including the Southeast Pacific, Atlantic and Indian oceans and in areas surrounding the peninsular platforms of subantarctic islands. The aim of this work was to determine and characterize the gastrointestinal parasitic and microbial fauna of specimens of D. eleginoides captured in waters of the south-central zone of Chile. The magnitude of parasitism in D. eleginoides captured in waters of the south-central zone of Chile is variable, and the parasite richness is different from that reported in specimens from subantarctic environments. Next-generation sequencing (NGS) of the microbial community associated to intestine showed a high diversity, where Proteobacteria, Firmicutes, and Bacteriodetes were the dominant phyla. However, both parasitic and microbial structures can vary between fish from different geographic regions.

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.

Characterization of Weissella viridescens UCO-SMC3 as a Potential Probiotic for the Skin: Its Beneficial Role in the Pathogenesis of Acne Vulgaris.

Previously, we isolated lactic acid bacteria from the slime of the garden snail Helix aspersa Müller and selected Weissella viridescens UCO-SMC3 because of its ability to inhibit in vitro the growth of the skin-associated pathogen Cutibacterium acnes. The present study aimed to characterize the antimicrobial and immunomodulatory properties of W. viridescens UCO-SMC3 and to demonstrate its beneficial effect in the treatment of acne vulgaris. Our in vitro studies showed that the UCO-SMC3 strain resists adverse gastrointestinal conditions, inhibits the growth of clinical isolates of C. acnes, and reduces the adhesion of the pathogen to keratinocytes. Furthermore, in vivo studies in a mice model of C. acnes infection demonstrated that W. viridescens UCO-SMC3 beneficially modulates the immune response against the skin pathogen. Both the oral and topical administration of the UCO-SCM3 strain was capable of reducing the replication of C. acnes in skin lesions and beneficially modulating the inflammatory response. Of note, orally administered W. viridescens UCO-SMC3 induced more remarkable changes in the immune response to C. acnes than the topical treatment. However, the topical administration of W. viridescens UCO-SMC3 was more efficient than the oral treatment to reduce pathogen bacterial loads in the skin, and effects probably related to its ability to inhibit and antagonize the adhesion of C. acnes. Furthermore, a pilot study in acne volunteers demonstrated the capacity of a facial cream containing the UCO-SMC3 strain to reduce acne lesions. The results presented here encourage further mechanistic and clinical investigations to characterize W. viridescens UCO-SMC3 as a probiotic for acne vulgaris treatment.

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.

Efecto del consumo del probiótico Lactobacillus fermentum UCO-979C en peso, masa grasa, masa magra, circunferencia de la cintura y hábitos alimenticios / Effect of the consumption of the probiotic lactobacillus fermentum uco-979c on weight, fat mass, lean mass, waist circumference and eating habits

Fundamentos: Varios estudios evalúan si el consumo de probióticos puede modificar el peso corporal. La cepa Lactobacillus fermentum UCO-979C es una cepa de origen gástrico y éste constituye el primer estudio en humanos. El objetivo fue evaluar el efecto del consumo de la cepa L. fermentum UCO979C en el peso, el porcentaje de masa grasa, masa magra, circunferencia de la cintura y hábitos alimenticios de un grupo de adultos jóvenes de la Universidad de Concepción, Chile.Métodos: Ciento cuatro estudiantes universitarios se dividieron en dos grupos. El grupo intervenido (n = 56) consumió una gelatina que contenía la cepa probiótica, mientras que elgrupo control (n = 46) consumió una gelatina placebo. Los hábitos alimenticios, la composición corporal se evaluaron al comienzo y al final de la intervención. Resultados: No se observaron diferencias significativas después de 12 semanas entre el grupo control y el grupo intervenido en peso, porcentaje de masa grasa, masa magra y circunferencia de la cintura, pero se observaron diferencias significativas en los hábitos alimenticios. A la semana 12 el grupo intervenido mostró un aumento muy significativo de la masa magra y un mayor consumo de productos lácteos y frutas respecto al inicio del estudio.Conclusiones: El consumo del probiótico no modificó ni el peso ni la composición corporal, pero sí modificó los hábitosalimenticios, aumentando el consumo del desayuno, frutas y productos lácteos. (AU)

Background: Several studies evaluate whether the consumption of probiotics can modify body weight. The Lactobacillus fermentum UCO-979C strain is a strain of gastric origin and this constitutes the first study in humans. Theobjective was to evaluate the effect of the consumption of the L. fermentum UCO-979C strain on weight, percentage of fat mass, lean mass, waist circumference and eatinghabits of a group of young adults from the University of Concepción, Chile.Methods: One hundred and four university students were divided into two groups. The intervened group (n = 56) consumed a gelatin containing the probiotic strain, while thecontrol group (n = 46) consumed a placebo gelatin. Eating habits and body composition were evaluated at the beginning and at the end of the intervention. Results: No significant differences were observed after 12weeks of intervention in weight, percentage of fat mass, lean mass, and waist circumference, but significant differences were observed in eating habits. The intra-group analysis showed a very significant increase in lean mass anda higher consumption of dairy products and fruits in the group that received the probiotic.Conclusions: The consumption of the probiotic did not modify neither the weight nor the body composition, but it did modify the eating habits, increasing the consumption of breakfast, fruits and dairy products. (AU)

Candida albicans, a reservoir of Listeria monocytogenes?

Listeria monocytogenes is a pathogen causing serious or mortal infections in human risk populations. Its infectivity is in part due to its ability to infect diverse eukaryotic cells. Since several bacteria can enter into yeast cells, including Candida albicans, the aims of this work were to evaluate if L. monocytogenes was able to harbor, retaining its viability, within C. albicans cells and to evaluate the effect of temperature and an antibiotic as stressing factors in its rate of entry into yeast cells. Both microorganisms were co-incubated in BHI broth during 48 h and the entry of bacteria into yeast cells was evaluated at different times. Then, yeasts free of extracellular bacteria were obtained seeding samples of the co-culture on YGC agar, which contains chloramphenicol, to obtain extracellular bacteria-free yeasts. These extracellular bacteria free yeasts were used to search for bacterial DNA in total yeast DNA and to evaluate the viability of intra-yeast bacteria. Finally, the effect of temperature and of chloramphenicol as inducers of stress on the rate of bacterial entry into yeast cells were investigated. After co-culturing both microorganisms, wet mount optical microscopy showed the presence of moving bacteria within yeasts and transmission electron microscopy confirmed the presence of intra-yeast bacteria. PCR allowed to amplify L. monocytogenes iap gene in C. albicans total DNA obtained from yeasts free of extracellular bacteria. Moreover, the SYTO 9 green fluorescence observed in bacterial cells within vacuoles of yeasts suggests that intra-yeast bacteria remain viable. Furthermore, the entry of L. monocytogenes into yeasts cells was favored by the presence of stressing factors (chloramphenicol and temperature). Therefore, yeasts may be reservoirs of viable L. monocytogenes and might spread them to the following generations of yeasts.

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.

Evaluation of Porcine Intestinal Epitheliocytes as an In vitro Immunoassay System for the Selection of Probiotic Bifidobacteria to Alleviate Inflammatory Bowel Disease.

The use of in vitro systems that allow efficient selection of probiotic candidates with immunomodulatory properties could significantly minimize the use of experimental animals. In this work, we generated an in vitro immunoassay system based on porcine intestinal epithelial (PIE) cells and dextran sodium sulfate (DSS) administration that could be useful for the selection and characterization of potential probiotic strains to be used in inflammatory bowel disease (IBD) patients. Our strategy was based on two fundamental pillars: on the one hand, the capacity of PIE cells to create a monolayer by attaching to neighboring cells and efficiently mount inflammatory responses and, on the other hand, the use of two probiotic bifidobacteria strains that have been characterized in terms of their immunomodulatory capacities, particularly in mouse IBD models and patients. Our results demonstrated that DSS administration can alter the epithelial barrier created in vitro by PIE cells and induce a potent inflammatory response, characterized by increases in the expression levels of several inflammatory factors including TNF-α, IL-1α, CCL4, CCL8, CCL11, CXCL5, CXCL9, CXCL10, SELL, SELE, EPCAM, VCAM, NCF2, and SAA2. In addition, we demonstrated that Bifidobacterium breve M-16V and B. longum BB536 are able to regulate the C-jun N-terminal kinase (JNK) intracellular signalling pathway, reducing the DSS-induced alterations of the in vitro epithelial barrier and differentially regulating the inflammatory response in a strain-dependent fashion. The good correlation between our in vitro findings in PIE cells and previous studies in animal models and IBD patients shows the potential value of our system to select new probiotic candidates in an efficient way.

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.