There are now many experimental models of inflammatory bowel disease (IBD), most of which are due to induced mutations in mice that result in an impaired homeostasis with the intestinal microbiota. These models can be clustered into several broad categories that, in turn, define the crucial cellular and molecular mechanisms of host microbial interactions in the intestine. The first of these components is innate immunity defined broadly to include both myeloid and epithelial cell mechanisms. A second component is the effector response of the adaptive immune system, which, in most instances, comprises the CD4+ T cell and its relevant cytokines. The third component is regulation, which can involve multiple cell types, but again particularly involves CD4+ T cells. Severe impairment of a single component can result in disease, but many models demonstrate milder defects in more than one component. The same is true for both spontaneous models of IBD, C3H/HeJBir and SAMPI/Yit mice. The thesis is advanced that 'multiple hits' or defects in these interacting components is required for IBD to occur in both mouse and human.

The gastrointestinal (GI) tract is a complex ecosystem generated by the alliance of GI epithelium, immune cells and resident microbiota. The three components of the GI ecosystem have co‐evolved such that each relies on the presence of the other two components to achieve its normal function and activity. Experimental systems such as cell culture, germ‐free animal models and intestinal isografts have demonstrated that each member of the GI ecosystem can follow a predetermined developmental pathway, even if isolated from the other components of the ecosystem. However, the presence of all three components is required for full physiological function. Genetic or functional alterations of any one component of this ecosystem can result in a broken alliance and subsequent GI pathology. A more detailed understanding of the interactions among microbiota, GI epithelium and the immune system should provide insight into multiple human disease states.

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The diversity and stability of the fecal bacterial microbiota in weaning pigs was studied after introduction of an exogenous Lactobacillus reuteri strain, MM53, using a combination of cultivation and techniques based on genes encoding 16S rRNA (16S rDNA). Piglets (n ‫؍‬ 9) were assigned to three treatment groups (control, daily dosed, and 4th-day dosed), and fresh fecal samples were collected daily. Dosed animals received 2.5 ؋ 10 10 CFU of antibiotic-resistant L. reuteri MM53 daily or every 4th day. Mean Lactobacillus counts for the three groups ranged from 1 ؋ 10 9 to 4 ؋ 10 9 CFU/g of feces. Enumeration of strain L. reuteri MM53 on MRS agar (Difco) plates containing streptomycin and rifampin showed that the introduced strain fluctuated between 8 ؋ 10 3 and 5 ؋ 10 6 CFU/g of feces in the two dosed groups. Denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rDNA fragments, with primers specific for variable regions 1 and 3 (V1 and V3), was used to profile complexity of fecal bacterial populations. Analysis of DGGE banding profiles indicated that each individual maintained a unique fecal bacterial population that was stable over time, suggesting a strong host influence. In addition, individual DGGE patterns could be separated into distinct time-dependent clusters. Primers designed specifically to restrict DGGE analysis to a select group of lactobacilli allowed examination of interspecies relationships and abundance. Based on relative band migration distance and sequence determination, L. reuteri was distinguishable within the V1 region 16S rDNA gene patterns. Daily fluctuations in specific bands within these profiles were observed, which revealed an antagonistic relationship between L. reuteri MM53 (band V1-3) and another indigenous Lactobacillus assemblage (band V1-6).

Gaskins (2020) The 'invivo lifestyle' of bile acid 7αdehydroxylating bacteria: comparative genomics, metatranscriptomic, and bile acid metabolomics analysis of a defined microbial community in gnotobiotic mice,

Intestinal sulfate-reducing bacteria (SRB) growth and resultant hydrogen sulfide production may damage the gastrointestinal epithelium and thereby contribute to chronic intestinal disorders. However, the ecology and phylogenetic diversity of intestinal dissimilatory SRB populations are poorly understood, and endogenous or exogenous sources of available sulfate are not well defined. The succession of intestinal SRB was therefore compared in inbred C57BL/6J mice using a PCR-based metabolic molecular ecology (MME) approach that targets a conserved region of subunit A of the adenosine-5-phosphosulfate (APS) reductase gene. The APS reductase-based MME strategy revealed intestinal SRB in the stomach and small intestine of 1-, 4-, and 7-day-old mice and throughout the gastrointestinal tract of 14-, 21-, 30-, 60-, and 90-day-old mice. Phylogenetic analysis of APS reductase amplicons obtained from the stomach, middle small intestine, and cecum of neonatal mice revealed that Desulfotomaculum spp. may be a predominant SRB group in the neonatal mouse intestine. The toxic gas hydrogen sulfide (H 2 S) is generated from sulfate during anaerobic respiration by sulfate-reducing Archaea and Bacteria (21, 58). A possible link between H 2 S and chronic intestinal disorders has been evoked by data indicating increased numbers of intestinal sulfate-reducing bacteria (SRB) and rates of sulfidogenesis in inflammatory bowel disease (IBD) patients compared to healthy humans (12, 37). Hydrogen sulfide selectively impairs the oxidation of n-butyrate by colonic epithelial cells (42). Because membrane lipid biosynthesis, ion absorption, mucin synthesis, and detoxification processes in colonocytes depend on the oxidation of n-butyrate, diminished n-butyrate metabolism is likely to compromise the epithelial cell barrier (42). Sulfide-induced damage of the epithelial barrier function would promote translocation of bacterial and food antigens, resulting in local inflammatory responses to normally benign antigens, an outcome consistent with histopathological features of IBD (16, 61). Chronic exposure to H 2 S might also perturb normal cycles of epithelial renewal in the intestine, thereby predisposing to proliferative disorders such as colon cancer.Intestinal sulfate can be derived either from exogenous sources, namely sulfate in drinking water and dietary foodstuffs, or from endogenous sources such as sulfated mucins (sulfomucins), sulfate-conjugated bile, and chondroitin sulfate. Use of chemically bound, endogenous sulfate by SRB is facilitated through interactions with sulfatase-harboring bacteria (e.g., Bacteroides spp. [56]). Most goblet cells, a differentiated epithelial cell subtype that produces mucins, generate sulfomucins (22). The degree of sulfation, however, increases from proximal to distal segments of the intestine and is highest in those segments harboring dense bacterial populations, such as the cecum and colon (9,20).The ecology and taxonomy of intestinal SRB and their metabolic activities remain uncharacterized. Most studies of...

A cultivation-independent approach, polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE), was used to characterize changes in fecal bacterial populations resulting from consumption of a low residue diet or oral administration of a broad-spectrum antibiotic. C57BL/6NHsd mice were weaned to either a standard nonpurified diet (LC-diet) or a low residue diet (LR-diet) and at 17 wk of age were randomly assigned to receive drinking water with or without 25 ppm cefoxitin for 14 d. On d 1, 2, 7 and 14, microbial DNA was extracted from feces, and the V3 region of the 16S rDNA gene was amplified by PCR and analyzed by DGGE. The diversity of fecal microbial populations, assessed using Shannon's index (H'), which incorporates species richness (number of species, or in this case, PCR-DGGE bands) and evenness (the relative distribution of species), was not affected by cefoxitin. However, use of Sorenson's pairwise similarity coefficient (C(s)), an index that measures the species in common between different habitats, indicated that the species composition of fecal bacterial communities was altered by cefoxitin in mice fed either diet. Dietary effects on fecal microbial communities were more pronounced, with greater H' values (P < 0.05) in mice fed the LR-diet (1.9 +/- 0.1) compared with the LC-diet (1.6 +/- 0.1). The C(s) values were also greater (P < 0.05) in fecal bacterial populations from mice fed the LR-diet (C(s) = 69.8 +/- 2.0%) compared with mice fed the LC-diet (C(s) = 50.1 +/- 3.8%), indicating greater homogeneity of fecal bacterial communities in mice fed the LR-diet. These results demonstrate the utility of cultivation-independent PCR-DGGE analysis combined with measurements of ecological diversity for monitoring diet- and antibiotic-induced alterations of the complex intestinal microbial ecosystem.

The ability of the proinflammatory cytokine tumor necrosis factor-α (TNF-α) to influence epithelial interleukin (IL)-8 responses to the intestinal bacterium Lactobacillus plantarum 299v was analyzed in the human HT-29 colonic epithelial cell line. In the absence of TNF-α, IL-8 mRNA expression was not detectable by Northern blot analysis in HT-29 cells alone or in HT-29 cells co-cultured with L. plantarum 299v. However, TNF-α induced IL-8 mRNA expression, and co-culture of TNF-α-treated HT-29 cells with L. plantarum 299v significantly increased IL-8 mRNA expression above levels induced by TNF-α alone in an adhesion-dependent manner. The increase in IL-8 mRNA expression was not observed in TNF-α-treated HT-29/L. plantarum 299v co-cultures using heat-killed lactobacllli or when L. plantarum adhesion was prevented using mannoside or a trans-well membrane. Paradoxically, IL-8 secretion was decreased in TNF-α-treated HT-29 cells with L. plantarum 299v relative to cells treated with TNF-α alone. TNF-α-mediated responsiveness to L. plantarum 299v was further investigated by analyzing expression of a coreceptor for bacterial cell wall products CD14. HT-29 cells expressed CD14 mRNA and cell-surface CD14; however, TNF-α did not alter CD14 mRNA or cell-surface expression, and blockade of CD14 with monoclonal antibody MY4 did not alter the IL-8 response to L. plantarum 299v in TNF-α-treated HT-29 cells. These results indicate that although TNF-α sensitizes HT-29 epithelial cells to intestinal lactobacilli, the bacteria exert a protective effect by downregulating IL-8 secretion.