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BACKGROUND AND AIMS: The pathophysiology of irritable bowel syndrome (IBS) is multifactorial and includes epithelial barrier dysfunction, a key element at the interface between the gut lumen and the deeper intestinal layers. Beneath the epithelial barrier there is the vascular one representing the last barrier to avoid luminal antigen dissemination The aims of this study were to correlate morpho-functional aspects of epithelial and vascular barriers with symptom perception in IBS. METHODS: Seventy-eight healthy subjects (controls) and 223 patients with IBS were enrolled in the study and phenotyped according to validated questionnaires. Sugar test was used to evaluate in vivo permeability. Immunohistochemistry, western blot, and electron microscopy were used to characterize the vascular barrier. Vascular permeability was evaluated by assessing the mucosal expression of plasmalemma vesicle-associated protein-1 and vascular endothelial cadherin. Caco-2 or human umbilical vein endothelial cell monolayers were incubated with soluble mediators released by mucosal biopsies to highlight the mechanisms involved in permeability alteration. Correlation analyses have been performed among experimental and clinical data. RESULTS: The intestinal epithelial barrier was compromised in patients with IBS throughout the gastrointestinal tract. IBS-soluble mediators increased Caco-2 permeability via a downregulation of tight junction gene expression. Blood vessel density and vascular permeability were increased in the IBS colonic mucosa. IBS mucosal mediators increased permeability in human umbilical vein endothelial cell monolayers through the activation of protease-activated receptor-2 and histone deacetylase 11, resulting in vascular endothelial cadherin downregulation. Permeability changes correlated with intestinal and behavioral symptoms and health-related quality of life of patients with IBS. CONCLUSIONS: Epithelial and vascular barriers are compromised in patients with IBS and contribute to clinical manifestations.
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PURPOSE: Garlic consumption has been inversely associated to intestinal adenoma (IA) and colorectal cancer (CRC) risk, although evidence is not consistent. Gut microbiota has been implied in CRC pathogenesis and is also influenced by garlic consumption. We analyzed whether dietary garlic influence CRC risk and bacterial DNA in blood. METHODS: We conducted a case-control study in Italy involving 100 incident CRC cases, 100 IA and 100 healthy controls matched by center, sex and age. We used a validated food frequency questionnaire to assess dietary habits and garlic consumption. Blood bacterial DNA profile was estimated using qPCR and16S rRNA gene profiling. We derived odds ratios (ORs) and the corresponding 95% confidence intervals (CIs) of IA and CRC according to garlic consumption from multiple conditional logistic regression. We used Mann-Whitney and chi-square tests to evaluate taxa differences in abundance and prevalence. RESULTS: The OR of CRC for medium/high versus low/null garlic consumption was 0.27 (95% CI = 0.11-0.66). Differences in garlic consumption were found for selected blood bacterial taxa. Medium/high garlic consumption was associated to an increase of Corynebacteriales order, Nocardiaceae family and Rhodococcus genus, and to a decrease of Family XI and Finegoldia genus. CONCLUSIONS: The study adds data on the protective effect of dietary garlic on CRC risk. Moreover, it supports evidence of a translocation of bacterial material to bloodstream and corroborates the hypothesis of a diet-microbiota axis as a mechanism behind the role of garlic in CRC prevention.
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Neoplasias Colorretais , Alho , Humanos , Alho/genética , DNA Bacteriano/genética , Estudos de Casos e Controles , Neoplasias Colorretais/epidemiologia , Neoplasias Colorretais/prevenção & controle , Neoplasias Colorretais/etiologia , Dieta , Modelos Logísticos , Antioxidantes , Bactérias/genética , Fatores de RiscoRESUMO
PURPOSE: Aging can be characterized by increased systemic low-grade inflammation, altered gut microbiota composition, and increased intestinal permeability (IP). The intake of polyphenol-rich foods is proposed as a promising strategy to positively affect the gut microbiota-immune system-intestinal barrier (IB) axis. In this context, we tested the hypothesis that a PR-dietary intervention would affect the presence of bacterial factors in the bloodstream of older adults. METHODS: We collected blood samples within a randomized, controlled, crossover intervention trial in which older volunteers (n = 51) received a polyphenol-enriched and a control diet. We quantified the presence of bacterial DNA in blood by qPCR targeting the 16S rRNA gene (16S; bacterial DNAemia). Blood DNA was taxonomically profiled via 16S sequencing. RESULTS: Higher blood 16S levels were associated with higher BMI and markers of IP, inflammation, and dyslipidemia. PR-intervention did not significantly change bacterial DNAemia in the older population (P = 0.103). Nonetheless, the beneficial changes caused by the polyphenol-enriched diet were greatest in participants with higher bacterial DNAemia, specifically in markers related to IP, inflammation and dyslipidemia, and in fecal bacterial taxa. Finally, we found that the bacterial DNA detected in blood mostly belonged to γ-Proteobacteria, whose abundance significantly decreased after the polyphenol-rich diet in subjects with higher bacterial DNAemia at baseline. CONCLUSIONS: This study shows that older subjects with higher bacterial DNAemia experienced a beneficial effect from a polyphenol-rich diet. Bacterial DNAemia may be a further relevant marker for the identification of target populations that could benefit more from a protective dietary treatment. REGISTRATION: This trial was retrospectively registered at www.isrctn.org (ISRCTN10214981) on April 28, 2017.
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Doenças Cardiovasculares , Polifenóis , Idoso , Biomarcadores , Doenças Cardiovasculares/prevenção & controle , Dieta , Fezes/microbiologia , Fatores de Risco de Doenças Cardíacas , Humanos , Permeabilidade , Polifenóis/farmacologia , RNA Ribossômico 16S/genética , Fatores de RiscoRESUMO
An immunosuppressive microenvironment in lung concurs to pre-malignant lesions progression to cancer. Here, we explore if perturbing lung microbiota, which contribute to immunosuppression, by antibiotics or probiotic aerosol interferes with lung cancer development in a mouse carcinogen-induced tumor model. Urethane-injected mice were vancomycin/neomycin (V/N)-aerosolized or live or dead L. rhamnosus GG (L.RGG)-aerosolized, and tumor development was evaluated. Transcriptional profiling of lungs and IHC were performed. Tumor nodules number, diameter and area were reduced by live or heat-killed L.RGG, while only a decrease in nodule diameter was observed in V/N-treated lungs. Both L.RGG and V/N reduced Tregs in the lung. In L.RGG-treated groups, the gene encoding the joining chain (J chain) of immunoglobulins was increased, and higher J chain protein and IgA levels were observed. An increased infiltration of B, NK and myeloid-derived cells was predicted by TIMER 2.0. The Kaplan-Meier plotter revealed an association between high levels of J chain mRNA and good prognosis in lung adenocarcinoma patients that correlated with increased B and CD4 T cells and reduced Tregs and M2 macrophages. This study highlights L.RGG aerosol efficacy in impairing lung cancer growth by promoting local immunity and points to this non-invasive strategy to treat individuals at risk of lung cancer.
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Adenoma , Lacticaseibacillus rhamnosus , Neoplasias Pulmonares , Probióticos , Camundongos , Animais , Carcinógenos , Temperatura Alta , Neoplasias Pulmonares/patologia , Probióticos/uso terapêutico , Probióticos/farmacologia , Modelos Animais de Doenças , Microambiente TumoralRESUMO
BACKGROUND: Severe early-onset erythroderma and gut inflammation, with massive tissue infiltration of oligoclonal activated T cells are the hallmark of Omenn syndrome (OS). OBJECTIVE: The impact of altered gut homeostasis in the cutaneous manifestations of OS remains to be clarified. METHODS: We analyzed a cohort of 15 patients with OS and the 129Sv/C57BL/6 knock-in Rag2R229Q/R229Q (Rag2R229Q) mouse model. Homing phenotypes of circulating lymphocytes were analyzed by flow cytometry. Inflammatory cytokines and chemokines were examined in the sera by ELISA and in skin biopsies by immunohistochemistry and in situ RNA hybridization. Experimental colitis was induced in mice by dextran sulfate sodium salt. RESULTS: We show that memory/activated T cells from patients with OS and from the Rag2R229Q mouse model of OS abundantly express the skin homing receptors cutaneous lymphocyte associated antigen and CCR4 (Ccr4), associated with high levels of chemokine C-C motif ligands 17 and 22. Serum levels of LPS are also elevated. A broad Th1/Th2/Th17 inflammatory signature is detected in the periphery and in the skin. Increased Tlr4 expression in the skin of Rag2R229Q mice is associated with enhanced cutaneous inflammation on local and systemic administration of LPS. Likewise, boosting colitis in Rag2R229Q mice results in increased frequency of Ccr4+ splenic T cells and worsening of skin inflammation, as indicated by epidermal thickening, enhanced epithelial cell activation, and dermal infiltration by Th1 effector T cells. CONCLUSIONS: These results support the existence of an interplay between gut and skin that can sustain skin inflammation in OS.
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Dermatite/imunologia , Inflamação/imunologia , Intestinos/imunologia , Imunodeficiência Combinada Severa/imunologia , Pele/patologia , Células Th1/imunologia , Junções Íntimas/patologia , Animais , Estudos de Coortes , Proteínas de Ligação a DNA/genética , Modelos Animais de Doenças , Microbioma Gastrointestinal , Humanos , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Receptores CCR4/metabolismoRESUMO
PURPOSE: Subclinical vitamin D (vitD) deficiency enhances the predisposition to a myriad of acute and chronic pathologies in many people worldwide. Due to the scarcity of vitD-rich foods, the consumption of supplements or fortified foods can be required to maintain healthy serum levels of 25-hydroxyvitamin D [25(OH)D], and the major circulating form of vitD that is commonly measured in serum to determine the vitD status. Since the vitD absorption seems to resemble that of lipids, improved emulsification in the gut could favor vitD permeation through the enterocyte membrane. Contextually, we hypothesized that a microorganism with cholecalciferol (vitD3)-solubilization properties may potentially result in enhanced serum vitD levels. METHODS AND RESULTS: Six probiotic strains were screened for their ability to create a stable suspension of vitD3 in water: Lacticaseibacillus paracasei DG, L. paracasei LPC-S01, L. paracasei Shirota, L. rhamnosus GG, Limosilactobacillus reuteri DSM 17938, and Lactobacillus acidophilus LA5. The DG strain displayed the strongest vitD3 solubilization ability and, consequently, were used in an in vivo trial where a commercial preparation of vitD3 in refined olive oil was administered by gavage to CD-1 mice with or without the concurrent administration of L. paracasei DG. ELISA measurements showed that the DG strain significantly increased the serum levels of 25(OH) D when administered once a day for 1 week in association with the vitD3 supplement. CONCLUSION: This preliminary pre-clinical study suggests that the combined administration of L. paracasei DG with an oil-based cholecalciferol supplement could contribute to the maintenance of the adequate 25(OH) D serum levels in people at risk of vitD deficiency.
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BACKGROUND: During aging, alterations of the intestinal microbial ecosystem can occur contributing to immunosenescence, inflamm-aging and impairment of intestinal barrier function (increased intestinal permeability; IP). In the context of a diet-microbiota-IP axis in older subjects, food bioactives such as polyphenols may play a beneficial modulatory role. METHODS: MaPLE is a project centered on a randomized, controlled cross-over dietary intervention trial [polyphenol-rich diet (PR-diet) versus control diet (C-diet)] targeted to older people (≥ 60 y) living in a well-controlled setting (i.e. nursing home). The 8-week interventions are separated by an 8-week wash-out period. Three small portions per day of selected polyphenol-rich foods are consumed during intervention in substitution of other comparable products within the C-diet. Biological samples are collected before and after each treatment period to evaluate markers related to IP, inflammation, vascular function, oxidative stress, gut and blood microbiomics, metabolomics. A sample size of 50 subjects was defined based on IP as primary outcome. DISCUSSION: Evidence that increasing the consumption of polyphenol-rich food products can positively affect intestinal microbial ecosystem resulting in reduced IP and decreased translocation of inflammogenic bacterial factors into the bloodstream will be provided. The integration of data from gut and blood microbiomics, metabolomics and other IP-related markers will improve the understanding of the beneficial effect of the intervention in the context of polyphenols-microbiota-IP interactions. Finally, findings obtained will provide a proof of concept of the reliability of the dietary intervention, also contributing to future implementations of dietary guidelines directed to IP management in the older and other at risk subjects. TRIAL REGISTRATION: The trial is registered at (ISRCTN10214981); April 28, 2017.
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Polifenóis/administração & dosagem , Idoso , Idoso de 80 Anos ou mais , Dieta , Microbioma Gastrointestinal , Humanos , Microbiota , Pessoa de Meia-Idade , Permeabilidade , Reprodutibilidade dos TestesRESUMO
Surface layers (S-layers) are proteinaceous arrays covering the cell walls of numerous bacteria. Their suggested properties, such as interactions with the host immune system, have been only poorly described. Here, we aimed to elucidate the role of the S-layer from the probiotic bacterial strain Lactobacillus helveticus MIMLh5 in the stimulation of murine bone-marrow-derived dendritic cells (DCs). MIMLh5 induced greater production of interferon beta (IFN-ß), interleukin 10 (IL-10), and IL-12p70, compared to S-layer-depleted MIMLh5 (naked MIMLh5 [n-MIMLh5]), whereas the isolated S-layer was a poor immunostimulator. No differences in the production of tumor necrosis factor alpha (TNF-α) or IL-1ß were found. Inhibition of the mitogen-activated protein kinases JNK1/2, p38, and ERK1/2 modified IL-12p70 production similarly in MIMLh5 and n-MIMLh5, suggesting the induction of the same signaling pathways by the two bacterial preparations. Treatment of DCs with cytochalasin D to inhibit endocytosis before the addition of fluorescently labeled MIMLh5 cells led to a dramatic reduction in the proportion of fluorescence-positive DCs and decreased IL-12 production. Endocytosis and IL-12 production were only marginally affected by cytochalasin D pretreatment when fluorescently labeled n-MIMLh5 was used. Treatment of DCs with fluorescently labeled S-layer-coated polystyrene beads (Sl-beads) resulted in much greater uptake of beads, compared to noncoated beads. Prestimulation of DCs with cytochalasin D reduced the uptake of Sl-beads more than plain beads. These findings indicate that the S-layer plays a role in the endocytosis of MIMLh5 by DCs. In conclusion, this study provides evidence that the S-layer of L. helveticus MIMLh5 is involved in endocytosis of the bacterium, which is important for strong Th1-inducing cytokine production.IMPORTANCE Beneficial microbes may positively affect host physiology at various levels, e.g., by participating in immune system maturation and modulation, boosting defenses and dampening reactions, thus affecting the whole homeostasis. As a consequence, the use of probiotics is increasingly regarded as suitable for more extended applications for health maintenance, not only microbiota balancing. This implies a deep knowledge of the mechanisms and molecules involved in host-microbe interactions, for the final purpose of fine tuning the choice of a probiotic strain for a specific outcome. With this aim, studies targeted to the description of strain-related immunomodulatory effects and the identification of bacterial molecules responsible for specific responses are indispensable. This study provides new insights in the characterization of the food-origin probiotic bacterium L. helveticus MIMLh5 and its S-layer protein as a driver for the cross-talk with DCs.
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Células Dendríticas/fisiologia , Endocitose , Lactobacillus helveticus/química , Probióticos/química , Animais , Medula Óssea , Camundongos Endogâmicos C57BLRESUMO
PURPOSE: Ability to survive the digestive process is a major factor in determining the effectiveness of a probiotic. In this study, the ability of the probiotic L. casei DG® (Lactobacillus paracasei CNCMI1572) to survive gastrointestinal transit in healthy children was investigated for the first time. METHODS: Twenty children aged 3-12 years received L. casei DG® as drinkable solution of 1 × 109 colony forming units (CFU), once daily for 7 consecutive days. Recovery in faecal samples was evaluated at baseline and at different time-points during and after administration. Defecation frequency, faeces consistency, digestive function and product safety were also assessed. RESULTS: Nineteen (95%) of the 20 enrolled children presented viable L. casei DG® cells in their faeces at least once during the study, with a maximum count (mean 4.3 log10 CFU/g ± 2.3) reached between day 4 and 6 from the beginning of consumption. Notably, for 11 (57.9%) of the 19 children with viable cells, L. casei DG® survived in faecal samples up to 3 days after treatment end. Defecation frequency, faeces consistency and digestive function did not change considerably during or after study treatment. Safety of the study product was very good. CONCLUSIONS: This study showed for the first time that L. casei DG® survives the gastrointestinal transit when ingested by children with a paediatric probiotic drinkable solution containing 1 × 109 CFU, and persists in the gut up to 3 days after the end of product intake, demonstrating resistance to gastric juices, hydrolytic enzymes and bile acids.
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Trato Gastrointestinal/metabolismo , Lacticaseibacillus paracasei/metabolismo , Probióticos/administração & dosagem , Probióticos/metabolismo , Criança , Pré-Escolar , Fezes/microbiologia , Feminino , Humanos , MasculinoRESUMO
Irritable bowel syndrome (IBS), a common functional gastrointestinal disorder, is classified according to bowel habits as IBS with constipation (IBS-C), with diarrhea (IBS-D), with alternating constipation and diarrhea (IBS-M), and unsubtyped (IBS-U). The mechanisms leading to the different IBS forms are mostly unknown. This study aims to evaluate whether specific fecal bacterial taxa and/or short-chain fatty acids (SCFAs) can be used to distinguish IBS subtypes and are relevant for explaining the clinical differences between IBS subcategories. We characterized five fecal samples collected at 4-weeks intervals from 40 IBS patients by 16S rRNA gene profiling and SCFA quantification. Finally, we investigated the potential correlations in IBS subtypes between the fecal microbial signatures and host physiological and clinical parameters. We found significant differences in the distribution of Clostridiales OTUs among IBS subtypes and reduced levels of SCFAs in IBS-C compared to IBS-U and IBS-D patients. Correlation analyses showed that the diverse representation of Clostridiales OTUs between IBS subtypes was associated with altered levels of SCFAs; furthermore, the same OTUs and SCFAs were associated with the fecal cytokine levels and stool consistency. Our results suggest that intestinal Clostridiales and SCFAs might serve as potential mechanistic biomarkers of IBS subtypes and represent therapeutic targets.
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Clostridiales/isolamento & purificação , Ácidos Graxos Voláteis/química , Fezes/química , Fezes/microbiologia , Síndrome do Intestino Irritável/microbiologia , Síndrome do Intestino Irritável/patologia , Adulto , Biomarcadores , Clostridiales/genética , Diarreia/microbiologia , Ácidos Graxos Voláteis/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , RNA Bacteriano/isolamento & purificação , RNA Ribossômico 16S/isolamento & purificaçãoRESUMO
We hypothesised that rehabilitation specifically addressing balance in Parkinson's disease patients might improve not only balance but locomotion as well. Two balance-training protocols (standing on a moving platform and traditional balance exercises) were assessed by assigning patients to two groups (Platform, n = 15, and Exercises, n = 17). The platform moved periodically in the anteroposterior, laterolateral, and oblique direction, with and without vision in different trials. Balance exercises were based on the Otago Exercise Program. Both platform and exercise sessions were administered from easy to difficult. Outcome measures were (a) balancing behaviour, assessed by both Index of Stability (IS) on platform and Mini-BESTest, and (b) gait, assessed by both baropodometry and Timed Up and Go (TUG) test. Falls Efficacy Scale-International (FES-I) and Parkinson's Disease Questionnaire (PDQ-8) were administered. Both groups exhibited better balance control, as assessed both by IS and by Mini-BESTest. Gait speed at baropodometry also improved in both groups, while TUG was less sensitive to improvement. Scores of FES-I and PDQ-8 showed a marginal improvement. A four-week treatment featuring no gait training but focused on challenging balance tasks produces considerable gait enhancement in mildly to moderately affected patients. Walking problems in PD depend on postural instability and are successfully relieved by appropriate balance rehabilitation. This trial is registered with ClinicalTrials.gov NCT03314597.
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Terapia por Exercício/métodos , Transtornos Neurológicos da Marcha/reabilitação , Marcha/fisiologia , Doença de Parkinson/reabilitação , Equilíbrio Postural/fisiologia , Idoso , Idoso de 80 Anos ou mais , Feminino , Transtornos Neurológicos da Marcha/diagnóstico , Transtornos Neurológicos da Marcha/fisiopatologia , Humanos , Locomoção/fisiologia , Masculino , Pessoa de Meia-Idade , Doença de Parkinson/diagnóstico , Doença de Parkinson/fisiopatologiaRESUMO
Lactobacillus paracasei DG is a bacterial strain with recognized probiotic properties and is used in commercial probiotic products. However, the mechanisms underlying its probiotic properties are mainly unknown. In this study, we tested the hypothesis that the ability of strain DG to interact with the host is at least partly associated with its ability to synthesize a surface-associated exopolysaccharide (EPS). Comparative genomics revealed the presence of putative EPS gene clusters in the DG genome; accordingly, EPS was isolated from the surface of the bacterium. A sample of the pure EPS from strain DG (DG-EPS), upon nuclear magnetic resonance (NMR) and chemical analyses, was shown to be a novel branched hetero-EPS with a repeat unit composed of l-rhamnose, d-galactose, and N-acetyl-d-galactosamine in a ratio of 4:1:1. Subsequently, we demonstrated that DG-EPS displays immunostimulating properties by enhancing the gene expression of the proinflammatory cytokines tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6), and particularly that of the chemokines IL-8 and CCL20, in the human monocytic cell line THP-1. In contrast, the expression of the cyclooxygenase enzyme COX-2 was not affected. In conclusion, DG-EPS is a bacterial macromolecule with the ability to boost the immune system either as a secreted molecule released from the bacterium or as a capsular envelope on the bacterial cell wall. This study provides additional information about the mechanisms supporting the cross talk between L. paracasei DG and the host. IMPORTANCE: The consumption of food products and supplements called probiotics (i.e., containing live microbial cells) to potentially prevent or treat specific diseases is constantly gaining popularity. The lack of knowledge on the precise mechanisms supporting their potential health-promoting properties, however, greatly limits a more appropriate use of each single probiotic strain. In this context, we studied a well-known probiotic, Lactobacillus paracasei DG, in order to identify the constitutive molecules that can explain the documented health-promoting properties of this bacterium. We found a novel polysaccharide molecule, named DG-EPS, that is secreted by and covers the bacterium. We demonstrated that this molecule, which has a chemical structure never identified before, has immunostimulatory properties and therefore may contribute to the ability of the probiotic L. paracasei DG to interact with the immune system.
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Expressão Gênica , Lacticaseibacillus paracasei/fisiologia , Polissacarídeos Bacterianos/fisiologia , Linhagem Celular , Humanos , Monócitos/microbiologia , Ramnose/químicaRESUMO
The potential influence of insects' feeding behaviour on their associated bacterial communities is currently a matter of debate. Using the major pest of commodities, Plodia interpunctella, as a model and adopting a culture-independent approach, the impact of different diets on the host-associated microbiota was evaluated. An analysis of similarity showed differences among the microbiotas of moths fed with five substrates and provided evidence that diet represents the only tested factor that explains this dissimilarity. Bacteria shared between food and insects provide evidence for a limited conveyance to the host of the bacteria derived from the diet; more likely, the content of carbohydrates and proteins in the diets promotes changes in the insect's microbiota. Moth microbiotas were characterized by two robust entomotypes, respectively, associated with a carbohydrate-rich diet and a protein-rich diet. These results were also confirmed by the predicted metagenome functional potential. A core microbiota, composed of six taxa, was shared between eggs and adults, regardless of the origin of the population. Finally, the identification of possible human and animal pathogens on chili and associated with the moths that feed on it highlights the possibility that these bacteria may be conveyed by moth frass.
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Bactérias/classificação , Bactérias/genética , Dieta , Comportamento Alimentar , Microbiota/genética , Mariposas/microbiologia , Animais , Bactérias/isolamento & purificação , RNA Ribossômico 16S/genéticaRESUMO
UNLABELLED: Modulation of the intestinal microbial ecosystem (IME) is a useful target to establish probiotic efficacy in a healthy population. We conducted a randomized, double-blind, crossover, and placebo-controlled intervention study to determine the impact of Bifidobacterium bifidum strain Bb on the IME of adult healthy volunteers of both sexes. High-throughput 16S rRNA gene sequencing was used to characterize the fecal microbiota before and after 4 weeks of daily probiotic cell consumption. The intake of approximately one billion live B. bifidum cells affected the relative abundance of dominant taxa in the fecal microbiota and modulated fecal butyrate levels. Specifically, Prevotellaceae (P = 0.041) and Prevotella (P = 0.034) were significantly decreased, whereas Ruminococcaceae (P = 0.039) and Rikenellaceae (P = 0.010) were significantly increased. We also observed that the probiotic intervention modulated the fecal concentrations of butyrate in a manner dependent on the initial levels of short-chain fatty acids (SCFAs). In conclusion, our study demonstrates that a single daily administration of Bifidobacterium bifidum strain Bb can significantly modify the IME in healthy (not diseased) adults. These findings demonstrate the need to reassess the notion that probiotics do not influence the complex and stable IME of a healthy individual. IMPORTANCE: Foods and supplements claimed to contain health-promoting probiotic microorganisms are everywhere these days and mainly intended for consumption by healthy people. However, it is still debated what actual effects probiotic products may have on the healthy population. In this study, we report the results of an intervention trial aimed at assessing the modifications induced in the intestinal microbial ecosystem of healthy adults from the consumption of a probiotic product. Our results demonstrate that the introduction of a probiotic product in the dietary habits of healthy people may significantly modify dominant taxa of the intestinal microbiota, resulting in the modulation of short-chain fatty acid concentrations in the gut. The overall changes witnessed in the probiotic intervention indicate a mechanism of microbiota modulation that could have potential effects on human health.
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Bifidobacterium bifidum/fisiologia , Ácido Butírico/metabolismo , Fezes/química , Microbioma Gastrointestinal/fisiologia , Probióticos/administração & dosagem , Adulto , Estudos Cross-Over , Método Duplo-Cego , Fezes/microbiologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , RNA Bacteriano/genética , RNA Ribossômico 16S/genética , Adulto JovemRESUMO
Bifidobacteria represent one of the dominant groups of microorganisms colonizing the human infant intestine. Commensal bacteria that interact with a eukaryotic host are believed to express adhesive molecules on their cell surface that bind to specific host cell receptors or soluble macromolecules. Whole-genome transcription profiling of Bifidobacterium bifidum PRL2010, a strain isolated from infant stool, revealed a small number of commonly expressed extracellular proteins, among which were genes that specify sortase-dependent pili. Expression of the coding sequences of these B. bifidum PRL2010 appendages in nonpiliated Lactococcus lactis enhanced adherence to human enterocytes through extracellular matrix protein and bacterial aggregation. Furthermore, such piliated L. lactis cells evoked a higher TNF-α response during murine colonization compared with their nonpiliated parent, suggesting that bifidobacterial sortase-dependent pili not only contribute to adherence but also display immunomodulatory activity.
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Bifidobacterium/fisiologia , Fímbrias Bacterianas/fisiologia , Aminoaciltransferases/genética , Aminoaciltransferases/metabolismo , Animais , Aderência Bacteriana/genética , Aderência Bacteriana/imunologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/fisiologia , Bifidobacterium/genética , Bifidobacterium/imunologia , Linhagem Celular , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Citocinas/biossíntese , Feminino , Fímbrias Bacterianas/genética , Fímbrias Bacterianas/imunologia , Genes Bacterianos , Humanos , Lactente , Mucosa Intestinal/imunologia , Mucosa Intestinal/microbiologia , Lactococcus lactis/genética , Lactococcus lactis/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Probióticos , Transcriptoma/imunologiaRESUMO
Here, we describe data obtained from transcriptome profiling of human cell lines and intestinal cells of a murine model upon exposure and colonization, respectively, with Bifidobacterium bifidum PRL2010. Significant changes were detected in the transcription of genes that are known to be involved in innate immunity. Furthermore, results from enzyme-linked immunosorbent assays (ELISAs) showed that exposure to B. bifidum PRL2010 causes enhanced production of interleukin 6 (IL-6) and IL-8 cytokines, presumably through NF-κB activation. The obtained global transcription profiles strongly suggest that Bifidobacterium bifidum PRL2010 modulates the innate immune response of the host.
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Bifidobacterium/fisiologia , Imunidade Inata , Intestinos/imunologia , Intestinos/microbiologia , Probióticos/farmacologia , Animais , Células CACO-2/microbiologia , Linhagem Celular , Citocinas/genética , Ensaio de Imunoadsorção Enzimática , Feminino , Perfilação da Expressão Gênica , Células HT29/efeitos dos fármacos , Células HT29/microbiologia , Humanos , Imunidade Inata/genética , Interleucina-8/metabolismo , Intestinos/citologia , Camundongos , Camundongos Endogâmicos BALB C , NF-kappa B/metabolismoRESUMO
Single-chain variable-fragment antibodies (scFvs) have considerable potential in immunological detection and localization of bacterial surface structures. In this study, synthetic phage-displayed antibody libraries were used to select scFvs against immunologically active S-layer protein of Lactobacillus helveticus MIMLh5. After three rounds of panning, five relevant phage clones were obtained, of which four were specific for the S-layer protein of L. helveticus MIMLh5 and one was also capable of binding to the S-layer protein of L. helveticus ATCC 15009. All five anti-S-layer scFvs were expressed in Escherichia coli XL1-Blue, and their specificity profiles were characterized by Western blotting. The anti-S-layer scFv PolyH4, with the highest specificity for the S-layer protein of L. helveticus MIMLh5, was used to detect the S-layer protein in Grana Padano protected-designation-of-origin (PDO) cheese extracts by Western blotting. These results showed promising applications of this monoclonal antibody for the detection of immunomodulatory S-layer protein in dairy (and dairy-based) foods.
Assuntos
Anticorpos Monoclonais/imunologia , Queijo/microbiologia , Análise de Alimentos/métodos , Lactobacillus helveticus/imunologia , Glicoproteínas de Membrana/imunologia , Bacteriófagos/imunologia , Western Blotting , Escherichia coli/genética , Humanos , Glicoproteínas de Membrana/metabolismo , Biblioteca de Peptídeos , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Anticorpos de Cadeia Única/genéticaRESUMO
Bifidobacteria are Gram-positive inhabitants of the human gastrointestinal tract that have evolved close interaction with their host and especially with the host's immune system. The molecular mechanisms underlying such interactions, however, are largely unidentified. In this study, we investigated the immunomodulatory potential of Bifidobacterium bifidum MIMBb75, a bacterium of human intestinal origin commercially used as a probiotic. Particularly, we focused our attention on TgaA, a protein expressed on the outer surface of MIMBb75's cells and homologous to other known bacterial immunoactive proteins. TgaA is a peptidoglycan lytic enzyme containing two active domains: lytic murein transglycosylase (LT) and cysteine- and histidine-dependent amidohydrolase/peptidase (CHAP). We ran immunological experiments stimulating dendritic cells (DCs) with the B. bifidum MIMBb75 and TgaA, with the result that both the bacterium and the protein activated DCs and triggered interleukin-2 (IL-2) production. In addition, we observed that the heterologous expression of TgaA in Bifidobacterium longum transferred to the bacterium the ability to induce IL-2. Subsequently, immunological experiments performed using two purified recombinant proteins corresponding to the single domains LT and CHAP demonstrated that the CHAP domain is the immune-reactive region of TgaA. Finally, we also showed that TgaA-dependent activation of DCs requires the protein CD14, marginally involves TRIF, and is independent of Toll-like receptor 4 (TLR4) and MyD88. In conclusion, our study suggests that the bacterial CHAP domain is a novel microbe-associated molecular pattern actively participating in the cross talk mechanisms between bifidobacteria and the host's immune system.
Assuntos
Amidoidrolases/imunologia , Bifidobacterium/enzimologia , Bifidobacterium/imunologia , Diferenciação Celular , Células Dendríticas/imunologia , Peptidoglicano/metabolismo , Amidoidrolases/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Parede Celular/química , Células Cultivadas , Cisteína/metabolismo , Histidina/metabolismo , Proteínas de Membrana/imunologia , Proteínas de Membrana/metabolismo , Camundongos Endogâmicos C57BL , Peptidoglicano/análiseRESUMO
Bifidobacterium bifidum MIMBb75 is a human intestinal isolate demonstrated to be interactive with the host and efficacious as a probiotic. However, the molecular biology of this microorganism is yet largely unknown. For this reason, we undertook whole-genome sequencing of B. bifidum MIMBb75 to identify potential genetic factors that would explain the metabolic and probiotic attributes of this bacterium. Comparative genomic analysis revealed a 45-kb chromosomal region that comprises 19 putative genes coding for a potential type IV secretion system (T4SS). Thus, we undertook the initial characterization of this genetic region by studying the putative virB1-like gene, named tgaA. Gene tgaA encodes a peptidoglycan lytic enzyme containing two active domains: lytic murein transglycosylase (LT, cd00254.3) and cysteine- and histidine-dependent amidohydrolase/peptidase (CHAP, pfam05257.4). By means of several in vitro assays, we experimentally confirmed that protein TgaA, consistent with its computationally assigned role, has peptidoglycan lytic activity, which is principally associated to the LT domain. Furthermore, immunofluorescence and immunogold labeling showed that the protein TgaA is abundantly expressed on the cell surface of B. bifidum MIMBb75. According to the literature, the T4SSs, which have not been characterized before in bifidobacteria, can have important implications for bacterial cell-to-cell communication as well as cross talk with host cells, justifying the interest for further studies aimed at the investigation of this genetic region.
Assuntos
Sistemas de Secreção Bacterianos/genética , Bifidobacterium/genética , Bifidobacterium/metabolismo , DNA Bacteriano/química , DNA Bacteriano/genética , Genoma Bacteriano , Análise de Sequência de DNA , Genes Bacterianos , Hidrólise , Dados de Sequência Molecular , Peptidoglicano/metabolismoRESUMO
BACKGROUND: The modulation of gut microbiota is considered to be the first target to establish probiotic efficacy in a healthy population. OBJECTIVE: This study was conducted to determine the impact of a probiotic on the intestinal microbial ecology of healthy volunteers. METHODS: High-throughput 16S ribosomal RNA gene sequencing was used to characterize the fecal microbiota in healthy adults (23-55 y old) of both sexes, before and after 4 wk of daily consumption of a capsule containing at least 24 billion viable Lactobacillus paracasei DG cells, according to a randomized, double-blind, crossover placebo-controlled design. RESULTS: Probiotic intake induced an increase in Proteobacteria (P = 0.006) and in the Clostridiales genus Coprococcus (P = 0.009), whereas the Clostridiales genus Blautia (P = 0.036) was decreased; a trend of reduction was also observed for Anaerostipes (P = 0.05) and Clostridium (P = 0.06). We also found that the probiotic effect depended on the initial butyrate concentration. In fact, participants with butyrate >100 mmol/kg of wet feces had a mean butyrate reduction of 49 ± 21% and a concomitant decrease in the sum of 6 Clostridiales genera, namely Faecalibacterium, Blautia, Anaerostipes, Pseudobutyrivibrio, Clostridium, and Butyrivibrio (P = 0.021), after the probiotic intervention. In contrast, in participants with initial butyrate concentrations <25 mmol/kg of wet feces, the probiotic contributed to a 329 ± 255% (mean ± SD) increment in butyrate concomitantly with an â¼55% decrease in Ruminococcus (P = 0.016) and a 150% increase in an abundantly represented unclassified Bacteroidales genus (P = 0.05). CONCLUSIONS: The intake of L. paracasei DG increased the Blautia:Coprococcus ratio, which, according to the literature, can potentially confer a health benefit on the host. The probiotic impact on the microbiota and on short-chain fatty acids, however, seems to strictly depend on the initial characteristics of the intestinal microbial ecosystem. In particular, fecal butyrate concentrations could represent an important biomarker for identifying subjects who may benefit from probiotic treatment. This trial was registered at www.controlled-trials.com/isrctn as ISRCTN56945491.