Intestinal Host-Microbe Interactions under Physiological and Pathological Conditions

The intestinal mucosa is unique in that it can be tolerant to the resident, symbiotic microbiota but remaining, at the same time, responsive to and able to fight pathogens. The close interaction between host-symbiotic microbiota at the mucosal level poses important challenges since microbial breaches through the gut barrier can result in the breakdown of gut homeostasis. In this paper, hosts-integrated components that help to preserve intestinal homeostasis including barrier and immune function are discussed. In addition global alterations of the microbiota that can play a role in the initiation of an exaggerated inflammatory response through an abnormal signaling of the innate and adaptive immune response are briefly described

Características probióticas de Bifidobacterium: estudio, selección de cepas y desarrollo

El creciente interés funcional y comercial de los productos probióticos fue la motivación de este trabajo de Tesis que estudiará espectos microbiológicos, propiedades relacionadas y herramientas para la cuantificación de microorganismos pertenecientes al género Bifídobacterium, uno de los géneros actualmente utilizados en la formulación de probióticos. Este trabajo consta de una Introducción que contiene una reseña histórica del concepto de probiótico y los criterios de selección de microorganismos para la formulación de productos. Se describirán aspectos tecnológicos y fisiológicos asociados a esta selección, detallando particularmente los conocimientos y evidencias científicas actuales de dos de ellos: la adherencia a epitelio intestinal y la competencia con microorganismos enteropatógenos. En el Capítulo I se presentará el desarrollo de un ensayo enzimático para la determinación cuantitativa de bacterias del género Bifídobacterium. Se detallarán las condiciones de optimización del ensayo y se demostrará su aplicabilidad en diferentes sistemas, tanto en modelos para estudios de adherencia como en la determinación de bifidobacterias en productos lácteos. En el Capítulo II se desarrollará un estudio sistemático de las propiedades superficiales de las bifidobacterias a fin de obtener información acerca de los mecanismos moleculares involucrados en la adherencia de estos microorganismos, considerada como un criterio de selección de bacterias para la formulación de probióticos. En el Capítulo III se estudiará otro aspecto de selección de bacterias probióticas: la competencia con enteropatógenos. Se analizará el mecanismo de acción de las bifidobacterias sobre bacterias patógenas aisladas de muestras clínicas en un modelo in vitro, compuesto por células epiteliales en cultivo. Finalmente se realizará una Discusión General acerca de las evidencias científicas aportadas en este trabajo de Tesis y las inquietudes para futuras investigaciones sobre probióticos.Tesis digitalizada en SEDICI gracias a la Biblioteca Central de la Facultad de Ciencias Exactas (UNLP).Facultad de Ciencias Exacta

A whole-grain cereal-rich diet increases plasma betaine, and tends to decrease total and LDL-cholesterol compared with a refined-grain diet in healthy subjects

Epidemiological studies have repeatedly found that whole-grain (WG) cereal foods reduce the risk of several lifestyle-related diseases, though consistent clinical outcomes and mechanisms are elusive. To compare the effects of a WG-rich diet with a matched refined-grain (RG) diet on plasma biomarkers and bowel health parameters, seventeen healthy subjects (eleven females and six males) completed an exploratory cross-over study with a 2-week intervention diet based on either WG- or RG-based foods, separated by a washout of at least 5 weeks. Both diets were the same except for the use of WG (150g/d) or RG foods. Subjects undertook a 4h postprandial challenge on day 8 of each intervention diet. After 2 weeks, the WG diet tended to decrease plasma total and LDL-cholesterol (both P=0·09), but did not change plasma HDL-cholesterol, fasting glucose, C-reactive protein or homocysteine compared with the RG diet. Plasma betaine and alkylresorcinol concentrations were elevated after 1 week of the WG diet (P=0·01 and P<0·0001, respectively). Clostridium leptum populations in faeces were increased after the WG diet, along with a trend for decreased faecal water pH (P=0·096) and increased stool frequency (P<0·0001) compared with the RG diet. A short controlled intervention trial with a variety of commercially available WG-based products tended to improve biomarkers of CVD compared with a RG diet. Changes in faecal microbiota related to increased fibre fermentation and increased plasma betaine concentrations point to both fibre and phytochemical components of WG being important in mediating any potential health effect

An Enzymatic–Colorimetric Assay for the Quantification of <i>Bifidobacterium</i>

An enzymatic-colorimetric assay for the quantification of Bifidobacterium was developed. The method, based upon the standard detection of fructose-6-phosphate phosphoketolase activity, was optimized with respect to bacterial cell pretreatment, time of incubation, and substrate concentration. The relationship between bacterial biomass and phosphoketolase activity was linear in a wide spectrum of bacterial densities. Higher sensitivity over the standard method was achieved by using 0.25% Triton X-100 in the reaction mixture to pretreat the bacterial cells. Because autoaggregation is a frequent feature among Bifidobacterium strains, this simple and reproducible method offers good advantage over viable plate count and turbidimetric techniques. The methodology can also be applied to the assessment of adherent Bifidobacterium strains to human epithelial cells.Centro de Investigación y Desarrollo en Criotecnología de Alimento

Enzyme-Based Most Probable Number Method for the Enumeration of <i>Bifidobacterium</i> in Dairy Products

An enzyme-based assay in combination with the most probable number (MPN) technique was developed for the enumeration of bifidobacteria. The assay employs the detection of fructose-6-phosphate phosphoketolase (F6PPK) activity as an indicator of the presence of bifidobacteria. The method was validated against viable counts and optimized with respect to selective media in order to quantitatively assess bifidobacteria in dairy products and other probiotic preparations. Several commercial products and homemade fermented milks were analyzed. Counts of bifidobacteria ranged from 10⁷ to 10⁸ MPN/ml in commercial products and homemade fermented milks Commercial starters provided by Argentinean industries had between 10⁷ and 10¹¹ MPN/ml. The results obtained in this study suggest that the combination of F6PPK activity determination and the MPN methodology allows an accurate determination of Bifidobacterium in pure cultures, dairy products, and other probiotic preparations.Facultad de Ciencias ExactasCentro de Investigación y Desarrollo en Criotecnología de Alimento

Detection of Sialic Acid-Utilising Bacteria in a Caecal Community Batch Culture Using RNA-Based Stable Isotope Probing

Sialic acids are monosaccharides typically found on cell surfaces and attached to soluble proteins, or as essential components of ganglioside structures that play a critical role in brain development and neural transmission. Human milk also contains sialic acid conjugated to oligosaccharides, glycolipids, and glycoproteins. These nutrients can reach the large bowel where they may be metabolised by the microbiota. However, little is known about the members of the microbiota involved in this function. To identify intestinal bacteria that utilise sialic acid within a complex intestinal community, we cultured the caecal microbiota from piglets in the presence of 13C-labelled sialic acid. Using RNA-based stable isotope probing, we identified bacteria that consumed 13C-sialic acid by fractionating total RNA in isopycnic buoyant density gradients followed by 16S rRNA gene analysis. Addition of sialic acid caused significant microbial community changes. A relative rise in Prevotella and Lactobacillus species was accompanied by a corresponding reduction in the genera Escherichia/Shigella, Ruminococcus and Eubacterium. Inspection of isotopically labelled RNA sequences suggests that the labelled sialic acid was consumed by a wide range of bacteria. However, species affiliated with the genus Prevotella were clearly identified as the most prolific users, as solely their RNA showed significantly higher relative shares among the most labelled RNA species. Given the relevance of sialic acid in nutrition, this study contributes to a better understanding of their microbial transformation in the intestinal tract with potential implications for human health

Changes in gut microbiota control metabolic endotoxemia-induced inflammation in high-fat diet-induced obesity and diabetes in mice

Diabetes and obesity are characterized by a low grade inflammation whose molecular origin is unknown. We previously determined first, that metabolic endotoxemia controls the inflammatory tone, body weight gain, and diabetes, second, that high-fat feeding modulates gut microbiota and the plasma concentration of lipopolysaccharide (LPS) i.e. metabolic endotoxemia. Hence, it remained to demonstrate whether changes in gut microbiota control the occurrence of metabolic diseases. Objective: first, to demonstrate that changes in gut microbiota, by the mean of antibiotic treatment, could be responsible for the control of metabolic endotoxemia, the low grade inflammation, obesity, and type 2 diabetes, and second to provide some mechanisms responsible for such effect. Results: We found that changes of gut microbiota induced by an antibiotic treatment reduced metabolic endotoxemia and the ceacal content of LPS in both high-fat fed and ob/ob mice. This effect was correlated with reduced glucose intolerance, body weight gain and fat mass development, lower inflammation, oxidative stress, and macrophages infiltration marker mRNA expression in visceral adipose tissue. Importantly, high-fat feeding strongly increased intestinal permeability and reduced the expression of genes coding for proteins of the tight junctions. Furthermore, the absence of CD14 in ob/ob CD14(-/-) mutant mice mimicked the metabolic and inflammatory effects of antibiotics. Conclusions: This new finding demonstrates that changes in gut microbiota controls metabolic endotoxemia, inflammation and associated disorders by a mechanism which could increase intestinal permeability. It would thus be useful to develop strategies for changing gut microbiota to control, intestinal permeability, metabolic endotoxemia and associated disorders