Intermittent Escherichia coli O157:H7 colonisation at the terminal rectum mucosa of conventionally-reared lambs

In cattle, the lymphoid rich regions of the rectal-anal mucosa at the terminal rectum are the preferred site for Escherichia coli O157:H7 colonisation. All cattle infected by rectal swab administration demonstrate long-term E. coli O157:H7 colonisation, whereas orally challenged cattle do not demonstrate long-term E. coli O157:H7 colonisation in all animals. Oral, but not rectal challenge of sheep with E. coli O157:H7 has been reported, but an exact site for colonisation in sheep is unknown. To determine if E. coli O157:H7 can effectively colonise the ovine terminal rectum, in vitro organ culture (IVOC) was initiated. Albeit sparsely, large, densely packed E. coli O157:H7 micro-colonies were observed on the mucosa of ovine and control bovine terminal rectum explants. After necropsy of orally inoculated lambs, bacterial enumeration of the proximal and distal gastrointestinal tract did suggest a preference for E. coli O157:H7 colonisation at the ovine terminal rectum, albeit for both lymphoid rich and non-lymphoid sites. As reported for cattle, rectal inoculation studies were then conducted to determine if all lambs would demonstrate persistent colonisation at the terminal rectum. After necropsy of E. coli O157:H7 rectally inoculated lambs, most animals were not colonised at gastrointestinal sites proximal to the rectum, however, large densely packed micro-colonies of E. coli O157:H7 were observed on the ovine terminal rectum mucosa. Nevertheless, at the end point of the study (day 14), only one lamb had E. coli O157:H7 micro-colonies associated with the terminal rectum mucosa. A comparison of E. coli O157:H7 shedding yielded a similar pattern of persistence between rectally and orally inoculated lambs. The inability of E. coli O157:H7 to effectively colonise the terminal rectum mucosa of all rectally inoculated sheep in the long term, suggests that E. coli O157:H7 may colonise this site, but less effectively than reported previously for cattle.

Interaction of enterohemorrhagic Escherichia coli O157 : H7 with mouse intestinal mucosa

In this study, we used mouse ileal loops to investigate the interaction of enterohemorrhagic Escherichia coli (EHEC) O157:H7 with the mouse intestinal mucosa. With a dose of 10(9) and 3 h incubation, EHEC O157 was detected in the lumen and to a lesser extent associated with the epithelium. Typical attaching and effacing (A/E) lesions were seen, albeit infrequently. While the effector protein Tir was essential for A/E lesion formation, the bacterial type III secretion system adaptor protein TccP was dispensable. These results suggest that A/E lesions on mouse intestinal mucosa can be formed independently of robust actin polymerization.

The gut microbiota elicits a profound metabolic reorientation in the mouse jejunal mucosa during conventionalisation

Objective: Proper interactions between the intestinal mucosa, gut microbiota and nutrient flow are required to establish homoeostasis of the host. Since the proximal part of the small intestine is the first region where these interactions occur, and since most of the nutrient absorption occurs in the jejunum, it is important to understand the dynamics of metabolic responses of the mucosa in this intestinal region.Design: Germ-free mice aged 8-10 weeks were conventionalised with faecal microbiota, and responses of the jejunal mucosa to bacterial colonisation were followed over a 30-day time course. Combined transcriptome, histology, (1)H NMR metabonomics and microbiota phylogenetic profiling analyses were used.Results: The jejunal mucosa showed a two-phase response to the colonising microbiota. The acute-phase response, which had already started 1 day after conventionalisation, involved repression of the cell cycle and parts of the basal metabolism. The secondary-phase response, which was consolidated during conventionalisation (days 4-30), was characterised by a metabolic shift from an oxidative energy supply to anabolic metabolism, as inferred from the tissue transcriptome and metabonome changes. Detailed transcriptome analysis identified tissue transcriptional signatures for the dynamic control of the metabolic reorientation in the jejunum. The molecular components identified in the response signatures have known roles in human metabolic disorders, including insulin sensitivity and type 2 diabetes mellitus.Conclusion: This study elucidates the dynamic jejunal response to the microbiota and supports a prominent role for the jejunum in metabolic control, including glucose and energy homoeostasis. The molecular signatures of this process may help to find risk markers in the declining insulin sensitivity seen in human type 2 diabetes mellitus, for instance.

Persistence of anticancer activity in berry extracts after simulated gastrointestinal digestion and colonic fermentation.

Fruit and vegetable consumption is associated at the population level with a protective effect against colorectal cancer. Phenolic compounds, especially abundant in berries, are of interest due to their putative anticancer activity. After consumption, however, phenolic compounds are subject to digestive conditions within the gastrointestinal tract that alter their structures and potentially their function. However, the majority of phenolic compounds are not efficiently absorbed in the small intestine and a substantial portion pass into the colon. We characterized berry extracts (raspberries, strawberries, blackcurrants) produced by in vitro-simulated upper intestinal tract digestion and subsequent fecal fermentation. These extracts and selected individual colonic metabolites were then evaluated for their putative anticancer activities using in vitro models of colorectal cancer, representing the key stages of initiation, promotion and invasion. Over a physiologically-relevant dose range (0-50 µg/ml gallic acid equivalents), the digested and fermented extracts demonstrated significant anti-genotoxic, anti-mutagenic and anti-invasive activity on colonocytes. This work indicates that phenolic compounds from berries undergo considerable structural modifications during their passage through the gastrointestinal tract but their breakdown products and metabolites retain biological activity and can modulate cellular processes associated with colon cancer.

In vitro fermentation of potential prebiotic flours from natural sources: impact on the human colonic microbiota and metabolome

Scope: Fibers and prebiotics represent a useful dietary approach for modulating the human gut microbiome. Therefore, aim of the present study was to investigate the impact of four flours (wholegrain rye, wholegrain wheat, chickpeas and lentils 50:50, and barley milled grains), characterized by a naturally high content in dietary fibers, on the intestinal microbiota composition and metabolomic output. Methods and results: A validated three-stage continuous fermentative system simulating the human colon was used to resemble the complexity and diversity of the intestinal microbiota. Fluorescence in situ hybridization was used to evaluate the impact of the flours on the composition of the microbiota, while small-molecule metabolome was assessed by NMR analysis followed by multivariate pattern recognition techniques. HT29 cell-growth curve assay was used to evaluate the modulatory properties of the bacterial metabolites on the growth of intestinal epithelial cells. All the four flours showed positive modulations of the microbiota composition and metabolic activity. Furthermore, none of the flours influenced the growth-modulatory potential of the metabolites toward HT29 cells. Conclusion: Our findings support the utilization of the tested ingredients in the development of a variety of potentially prebiotic food products aimed at improving gastrointestinal health.

Dendritic cells interact with CD4 T cells in intestinal mucosa

Absence of lymph nodes in nonmammalian species, expression of MHCII by APCs in the periphery, and the recent findings that T cells can change their polarization status after presentation in the lymph nodes imply a role for MHCII-mediated presentation outside the organized lymphoid tissue. This study shows that MHCII+ ECs and DCs from the intestinal mucosa of the pig can present antigen to T cells in vitro. In vivo, APCs colocalize with T cells in pig and mouse intestinal mucosa. In the pig, endothelium is involved in these interactions in neonates but not in adults, indicating different roles for stromal and professional APCs in the neonate compared with the adult. The ratio of expression of DQ and DR MHCII locus products was lower on ECs than on other mucosal APCs, indicating that the two types of cells present different peptide sets. Adult nonendothelial APCs expressed a higher ratio of DQ/DR than in neonates. These results suggest that mucosal DCs can present antigen locally to primed T cells and that stromal APCs are recruited to these interactions in some cases. This raises the possibility that local presentation may influence T cell responses at the effector stage after initial presentation in the lymph node.

Investigation of milk proteins binding to the oral mucosa

High protein dairy beverages are considered to be mouth drying. The drying sensation may be due to the product protein content; however the mechanism of this mouth drying is uncertain. This study investigated the potential adhesion of milk proteins to porcine oral mucosal tissues and their resistance to wash out with simulated saliva was monitored using fluorescence microscopy. Cadein was found to be more adhesive to porcine mucosa then lactogloubulin. Some investigation into the reason for this difference in mucoadhesion was conducted by thiol-content analysis, rheology and zeta-potential measurements. The higher viscosity of casein solution and smaller zeta-potential is believed to be responsible for its better retention on mucosal surfaces. These findings suggest that casein and whey protein are both capable of binding and eliciting mouth drying in high protein dairy beverages.

In Vitro Fermentation of NUTRIOSE® FB06, a wheat dextrin soluble fibre, in a continuous culture human colonic model system

Wheat dextrin soluble fibre may have metabolic and health benefits, potentially acting via mechanisms governed by the selective modulation of the human gut microbiota. Our aim was to examine the impact of wheat dextrin on the composition and metabolic activity of the gut microbiota. We used a validated in vitro three-stage continuous culture human colonic model (gut model) system comprised of vessels simulating anatomical regions of the human colon. To mimic human ingestion, 7 g of wheat dextrin (NUTRIOSE® FB06) was administered to three gut models, twice daily at 10.00 and 15.00, for a total of 18 days. Samples were collected and analysed for microbial composition and organic acid concentrations by 16S rRNA-based fluorescence in situ hybridisation and gas chromatography approaches, respectively. Wheat dextrin mediated a significant increase in total bacteria in vessels simulating the transverse and distal colon, and a significant increase in key butyrate-producing bacteria Clostridium cluster XIVa and Roseburia genus in all vessels of the gut model. The production of principal short-chain fatty acids, acetate, propionate and butyrate, which have been purported to have protective, trophic and metabolic host benefits, were increased. Specifically, wheat dextrin fermentation had a significant butyrogenic effect in all vessels of the gut model and significantly increased production of acetate (vessels 2 and 3) and propionate (vessel 3), simulating the transverse and distal regions of the human colon, respectively. In conclusion, wheat dextrin NUTRIOSE® FB06 is selectively fermented in vitro by Clostridium cluster XIVa and Roseburia genus and beneficially alters the metabolic profile of the human gut microbiota.