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The vertebrate gut harbors a coevolved consortium of microbes that plays critical roles in the development and health of this organ. Here we discuss recent insights into the microbial-host molecular dialogs that shape the digestive tracts of the model vertebrates, mice and zebrafish, and consider the parallels between vertebrate-microbial mutualisms and the well-studied squid-Vibrio symbiosis.
Increasingly, epidemiologic and clinical data support the hypothesis that perturbations in the gastrointestinal (GI) microbiota because of antibiotic use and dietary differences in 'industrialized' countries have disrupted the normal microbiota-mediated mechanisms of immunological tolerance in the mucosa, leading to an increase in the incidence of allergic airway disease. The data supporting this 'microflora hypothesis' includes correlations between allergic airway disease and (1) antibiotic use early in life, (2) altered fecal microbiota and (3) dietary changes over the past two decades. Our laboratory has recently demonstrated that mice can develop allergic airway responses to allergens if their endogenous microbiota is altered at the time of first allergen exposure. These experimental and clinical observations are consistent with other studies demonstrating that the endogenous microbiota plays a significant role in shaping the development of the immune system. Data are beginning to accumulate that a 'balanced' microbiota plays a positive role in maintaining mucosal immunologic tolerance long after post-natal development. Other studies have demonstrated that even small volumes delivered to the nasopharynx largely end up in the GI tract, suggesting that airway tolerance and oral tolerance may operate simultaneously. The mechanism of microbiota modulation of host immunity is not known; however, host and microbial oxylipins are one potential set of immunomodulatory molecules that may control mucosal tolerance. The cumulative data are beginning to support the notion that probiotic and prebiotic strategies be considered for patients coming off of antibiotic therapy.
The mucosa-associated microbiota lining the gut epithelium might play a central role in the activation and/or perpetuation of mucosal inflammation in Crohn's disease (CD). We sought for localized dysbiosis by comparing the biodiversity and composition of the microbiotas in ulcerated and nonulcerated mucosal samples from patients with CD. Biopsy samples (n = 75) of ulcerated and adjacent nonulcerated mucosa were collected during colonoscopy from 15 patients, from the ileum, right colon, left colon, and rectum. Temporal temperature gradient gel electrophoresis (TTGE) of bacterial 16S rRNAs was used to evaluate the dominant bacterial species. TTGE profiles were compared using software that calculates similarity percentages. For a given patient, average similarity indexes between ulcerated and nonulcerated mucosal TTGE profiles ranged from 95.2% +/- 4.2% to 97.9% +/- 1.7% (means +/- standard deviations) for the different segments. The mean values did not differ significantly. Average interindividual similarity indexes for a given segment among the different patients ranged from 33.6% +/- 15.5% to 42.0% +/- 25.6%. In CD, the dominant microbiotas do not differ qualitatively between ulcerated and nonulcerated mucosae. Biodiversity remains high in ulcerated mucosa. This argues against a pathogenic role of localized qualitative dysbiosis in CD-associated ulceration.
Numerous studies have reported critical roles for the gut microbiota in obesity. However, the specific microbes that causally contribute to obesity and the underlying mechanisms remain undetermined. Here, we conducted shotgun metagenomic sequencing in a Chinese cohort of 631 obese subjects and 374 normal-weight controls and identified a Megamonas-dominated, enterotype-like cluster enriched in obese subjects. Among this cohort, the presence of Megamonas and polygenic risk exhibited an additive impact on obesity. Megamonas rupellensis possessed genes for myo-inositol degradation, as demonstrated in vitro and in vivo, and the addition of myo-inositol effectively inhibited fatty acid absorption in intestinal organoids. Furthermore, mice colonized with M. rupellensis or E. coli heterologously expressing the myo-inositol-degrading iolG gene exhibited enhanced intestinal lipid absorption, thereby leading to obesity. Altogether, our findings uncover roles for M. rupellensis as a myo-inositol degrader that enhances lipid absorption and obesity, suggesting potential strategies for future obesity management.
The pathogenesis of Crohn's disease (CD), an idiopathic inflammatory bowel disease, is attributed, in part, to intestinal bacteria that may initiate and perpetuate mucosal inflammation in genetically susceptible individuals. Paneth cells (PC) are the major source of antimicrobial peptides in the small intestine, including human alpha-defensins HD5 and HD6. We tested the hypothesis that reduced expression of PC alpha-defensins compromises mucosal host defenses and predisposes patients to CD of the ileum. We report that patients with CD of the ileum have reduced antibacterial activity in their intestinal mucosal extracts. These specimens also showed decreased expression of PC alpha-defensins, whereas the expression of eight other PC products either remained unchanged or increased when compared with controls. The specific decrease of alpha-defensins was independent of the degree of inflammation in the specimens and was not observed in either CD of the colon, ulcerative colitis, or pouchitis. The functional consequence of alpha-defensin expression levels was examined by using a transgenic mouse model, where we found changes in HD5 expression levels, comparable to those observed in CD, had a pronounced impact on the luminal microbiota. Thus, the specific deficiency of PC defensins that characterizes ileal CD may compromise innate immune defenses of the ileal mucosa and initiate and/or perpetuate this disease.
The major gut bacteria of the worker caste of nine species of Australian termites, belonging to four families, were isolated and identified to generic level. All species were either facultative anaerobes or strict aerobes. A correlation appears to exist between the major gut bacterium and the family to which the termite belongs. The major bacterium from the two lowest termites, Mastotermes darwiniensis (family Mastotermitidae) and Cryptotermes primus (family Kalotermitidae), was Streptococcus; from four species belonging to the Rhinotermitidae (Heterotermes ferox, Coptotermes acinaciformis, C. lacteus, Schedorhinotermes intermedius intermedius) it was Enterobacter; and from three species of the Termitidae (Nasutitermes exitiosus, N. graveolus, N. walkeri) it was Staphylococcus. Enterobacter was a minor symbiont of M. darwiniensis, C. primus, and N. graveolus; Streptococcus was a minor symbiont of H. ferox, C. lacteus, S. intermedius intermedius, and N. exitiosus; and Bacillus was a minor symbiont of C. acinaciformis and S. intermedius intermedius. M. darwiniensis possessed another minor symbiont tentatively identified as Flavobacterium. C. acinaciformis from three widely separated locations possessed a similar microbiota, indicating some form of control on the composition of the gut bacteria. Bacteria, capable of growth on N-free medium in the presence of nitrogen gas, were isolated from all termites, except N. exitiosus and N. walkeri, and were identified as Enterobacter. No cellulose-degrading bacteria were isolated.
BACKGROUND & AIMS: Crohn's disease patients can be characterized by antibody responses against Crohn's disease-related bacterial sequence, Escherichia coli outer membrane porin C, Saccharomyces cerevisiae (oligomannan), and neutrophil nuclear antigens. Our aim was to determine whether expression of antibodies against Crohn's disease-related bacterial sequence and Escherichia coli outer membrane porin C is associated with distinct phenotypic manifestations. METHODS: Sera from 303 patients were tested for antibodies to the Crohn's disease-related bacterial sequence (I2), anti-Escherichia coli outer membrane porin C, anti-Saccharomyces cerevisiae, and perinuclear antineutrophil cytoplasmic antibodies and for 3 Crohn's disease-associated variants of the NOD2 gene (R702W, G908R, and 1007fs) and compared with clinical data. RESULTS: Patients expressing I2 were more likely to have fibrostenosing Crohn's disease (64.4% vs. 40.7%; P < 0.001) and to require small bowel surgery (62.2% vs. 37.4%; P < 0.001). Patients with anti-Escherichia coli outer membrane porin C were more likely to have internal perforating disease (50.0% vs. 30.7%; P = 0.001) and to require small bowel surgery (61.4% vs. 44.2%; P = 0.003). Anti-Crohn's disease-related bacterial sequence was independently associated with fibrostenosis (P = 0.027) and small bowel surgery (P = 0.01), whereas anti-Escherichia coli outer membrane porin C was independently associated with internal perforations (P < 0.006). Patients positive for I2, anti-Escherichia coli outer membrane porin C, and anti-Saccharomyces cerevisiae were the most likely to have undergone small bowel surgery (72.0%; odds ratio, 8.6; P < 0.001) compared with patients without reactivity (23.0%). When the presence and magnitude of antibody responses were considered, 90% of patients with small bowel disease who required surgery had high levels of I2, Escherichia coli outer membrane porin C, and oligomannan antibodies, compared with only 18.2% with low-titer responses (P < 0.001). CONCLUSIONS: I2 and anti-Escherichia coli outer membrane porin C are associated with Crohn's disease phenotypes, and patients with the highest level of serum reactivity toward an increasing number of microbiota have the greatest frequency of strictures, internal perforations, and small bowel surgery.
An experiment was designed to evaluate the effects of 3 different additives on the gastrointestinal microbiota of early-weaned pigs. Early-weaned (18 to 22 d; n = 32) pigs (6.0 +/- 0.10 kg of BW) from 8 litters were randomly distributed into 8 pens. Each pen was assigned 1 of 4 dietary treatments: a prestarter or control diet, the control diet with 0.04% avilamycin (AB), with 0.3% sodium butyrate, or with 0.03% plant extract mixture (XT; standardized mixture with 5% (wt/wt) carvacrol extracted from Origanum spp., 3% cinnamaldehyde extracted from Cinnamonum spp., and 2% capsicum oleoresin from Capsicum annum). At the end of the experimental period, 8 pigs per treatment were killed, and samples of their intestinal content were taken. The total bacterial load along the gastrointestinal tract (GIT; stomach, jejunum, cecum, and distal colon) and the lactobacilli and enterobacteria in the jejunum and cecum were measured by quantitative PCR. The total microbial counts along the GIT did not differ among the diets, but there was an increase in the lactobacilli:enterobacteria ratio in the cecum of the piglets on the XT diet (P = 0.003). Restriction fragment length polymorphism of the PCR-amplified V3, V4, and V5 regions of the 16S rDNA gene showed changes in the structure of the microbial community in the jejunum. Dendrograms grouped animals by diets; control with 0.3% sodium butyrate was the treatment that promoted the biggest changes in the microbial ecosystem, followed by AB and then XT. Biodiversity increased when using additives compared with the control diet (P = 0.002). Microbial metabolic activity along the hindgut was studied using the concentration of purine bases and carbohydrase activities. Different patterns for purine bases were observed between diets (diet x intestinal section, P = 0.01). The control diet reached a maximum purine base concentration at the end of the colon, whereas that of the AB diet was reached at the cecum. We could not detect any cellulase or xylanase activities in animals of this age. Appreciable amylase and amylopectinase activities were found, but they did not differ between diets. The results suggest that the effects of avilamycin, butyrate, or the plant extract would not be related to a reduction in the number of total bacteria inhabiting different sections of the GIT but rather to changes in the ecological structure and metabolic activity of the microbial community.
The increased prevalence of atopic diseases, atopic dermatitis, allergic rhinitis, and asthma has been described as an epidemic. New approaches in the fight against allergic diseases are called for, the target being the persistence of the atopic T helper 2-skewed immune responder pattern beyond infancy. Atopic dermatitis, the earliest of these conditions, might act as a portal for the development of IgE-mediated atopic manifestations. Abundant evidence implies that specific strains selected from the healthy gut microbiota exhibit powerful antipathogenic and anti-inflammatory capabilities, and several targets for the probiotic approach have emerged in atopic dermatitis: degradation/structural modification of enteral antigens, normalization of the properties of aberrant indigenous microbiota and of gut barrier functions, regulation of the secretion of inflammatory mediators, and promotion of the development of the immune system. Better understanding of the effects of different probiotic strains and deeper insight into the mechanisms of the heterogeneous manifestations of atopic disease are needed for the validation of specific strains carrying anti-allergic potential.
BACKGROUND: The mucosa-associated microbiota, being very close to the inflammatory process associated with inflammatory bowel disease (IBD), may have a pathogenic role. We used a culture-independent method to analyze the mucosa-associated microbiota in IBD patients at various points of the distal digestive tract. METHODS: Thirty-five patients (20 with Crohn's disease, 11 with ulcerative colitis, and 4 controls) underwent colonoscopy. Biopsies (n = 126) were taken from 4 sites: the ileum, right colon, left colon, and rectum. Fecal samples were also obtained from 7 individuals. Temporal temperature gradient gel electrophoresis (TTGE) of 16S rDNA was used to evaluate dominant species diversity. TTGE profiles were compared using software that measures the degree of similarity. RESULTS: In a given individual, the overall similarity percentage between the 4 segments of the distal digestive tract was 94.7 +/- 4.0%, regardless of clinical status. The average similarity of all profiles for a given segment was 59.3 +/- 18.3% in the overall population. Dendrogram analysis showed that TTGE profiles did not cluster with clinical status. Differences were observed between the dominant fecal microbiota and the mucosa-associated microbiota of all 4 sites, with similarity percentages less than 92%. CONCLUSIONS: These results confirm that the dominant species differ between the mucosa-associated and fecal microbiota. They also show that, in a given individual, the microbiota is relatively stable along the distal digestive tract, showing a slight evolution in dominant species diversity from the ileum to the rectum, in both healthy subjects and patients with IBD.
HLA-B27 transgenic rats develop spontaneous colitis under specific pathogen-free conditions (SPF) but germ-free rats remain disease-free, emphasizing a role for intestinal bacteria in the pathogenesis of chronic intestinal inflammation. Prebiotics are dietary substances that affect the host by stimulating growth and/or activity of potentially health promoting bacteria. The aims of this study were to investigate whether prebiotics can prevent colitis in SPF HLA-B27 rats, and secondly, to explore mechanisms of protection. SPF HLA-B27 transgenic rats received orally the prebiotic combination long-chain inulin and oligofructose (Synergy 1), or not, prior to the development of clinically detectable colitis. After seven weeks, cecal and colonic tissues were collected for gross cecal scores (GCS), histologic inflammatory scores (scale 0-4), and mucosal cytokine measurement. Cecal and colonic contents were collected for analysis of the gut microbiota by PCR-denaturing gradient gel electrophoresis (PCR-DGGE) and fluorescent in-situ hybridization (FISH), and analysis of short-chain fatty acid composition. Prebiotic treatment significantly decreased GCS and inflammatory histologic scores in the cecum and colon. Prebiotic treatment also decreased cecal IL-1beta, but increased cecal TGF-beta concentrations. Inulin/oligofructose altered the cecal and colonic PCR-DGGE profiles, and FISH analysis showed significant increases in cecal Lactobacillus and Bifidobacterium populations after prebiotic treatment compared with water-treated rats. In conclusion, the prebiotic combination Synergy 1 reduced colitis in HLA-B27 transgenic rats, which effect was associated with alterations to the gut microbiota, decreased tissue proinflammatory cytokines and increased immunomodulatory molecules. These results show promise for prebiotics as primary or adjuvant maintenance therapy for chronic inflammatory bowel diseases.
Wistar rats (ten per group) were fed either an oat-free control diet or a dietary fibre-rich test diet containing 500 g oat-based products/kg for 6 weeks. The oat-based products, containing 4-128 g/kg resistant starch, 30-92 g/kg beta-glucan and 122-304 g/kg total dietary fibre, were oat flour extrudate, flour/Novelose (commercial resistant starch) extrudate (80:20 w/w), oat bran, bran/Novelose extrudate (80:20 w/w) and autoclaved oat flour. Serum total cholesterol decreased in the groups fed flour, flour/Novelose and bran/Novelose (P<0.05). In most of the test groups, count numbers of bifidobacteria were higher (P<0.001) and of coliforms were lower (P<0.05). The mass of the caecum walls and contents was greater in groups fed Novelose- and bran-containing diets (P<0.005). In all the test groups, pH values were lower in the intestinal contents (P<0.001), and caecal concentrations of acetate (P<0.001), propionate (P<0.05), butyrate (P<0.005) and total SCFA (P<0.001) were higher. The lowest concentrations of steroids were found in rats fed the autoclaved flour. In the other test groups, more bile acids appeared in the caecal (P<0.001) and colonic contents (P<0.005), as well as in the faeces, at week 6 (P<0.001). The highest bile acid excretion was found after feeding bran-containing diets. In the intestinal contents of all the test groups, more primary bile acids (P<0.001) appeared than in the control group. The excretion of steroids increased within the experimental period. Using extrusion technology, dietary fibre-rich oat-based products, which have beneficial physiological effects in rats, can be produced. Oat flour and bran are excellent sources for the preparation of directly edible oat products. Their nutritional properties can be further improved by the addition of resistant starch.
Increasingly, the microbiological scientific community is relying on molecular biology to define the complexity of the gut flora and to distinguish one organism from the next. This is particularly pertinent in the field of probiotics, and probiotic therapy, where identifying probiotics from the commensal flora is often warranted. Current techniques, including genetic fingerprinting, gene sequencing, oligonucleotide probes and specific primer selection, discriminate closely related bacteria with varying degrees of success. Additional molecular methods being employed to determine the constituents of complex microbiota in this area of research are community analysis, denaturing gradient gel electrophoresis (DGGE)/temperature gradient gel electrophoresis (TGGE), fluorescent in situ hybridisation (FISH) and probe grids. Certain approaches enable specific aetiological agents to be monitored, whereas others allow the effects of dietary intervention on bacterial populations to be studied. Other approaches demonstrate diversity, but may not always enable quantification of the population. At the heart of current molecular methods is sequence information gathered from culturable organisms. However, the diversity and novelty identified when applying these methods to the gut microflora demonstrates how little is known about this ecosystem. Of greater concern is the inherent bias associated with some molecular methods. As we understand more of the complexity and dynamics of this diverse microbiota we will be in a position to develop more robust molecular-based technologies to examine it. In addition to identification of the microbiota and discrimination of probiotic strains from commensal organisms, the future of molecular biology in the field of probiotics and the gut flora will, no doubt, stretch to investigations of functionality and activity of the microflora, and/or specific fractions. The quest will be to demonstrate the roles of probiotic strains in vivo and not simply their presence or absence.
Periodontal disease is an infectious disease manifested by the progressive change of a healthy resident commensal microbiota to a pathogenic one characterized by a specific microbiota. Thus, the prospect for the use of selected bacteria or their antigens as a vaccine to interfere with the microbial changes and resulting progression of periodontal tissue destruction has been proposed. As a first step in examining the use of bacterial antigens as immunogens in periodontitis, this study characterized the humoral immune response in Macaca fascicularis after systemic immunization with intact Porphyromonas gingivalis, Prevotella intermedia and Bacteroides fragilis. Parental immunization of the nonhuman primate with the intact bacteria resulted in the production of specific and significantly elevated levels of antibodies to P. gingivalis and P. intermedia, with the predominant isotype being immunoglobulin G (IgG). In contrast, the principal response to the nonoral, intestinal bacterium, B. fragilis, was of the IgM isotype. Immunization increased IgG, IgM, and IgA antibody by 14-227 fold to P. gingivalis and 8-108 fold to P. intermedia. The level of serum IgA antibody increased (77-227 fold). The kinetics of the antibody response post-immunization and post-ligation differed with respect to each of the bacteria tested. IgG antibody to P. gingivalis increased through week 16 of the experiment and remained elevated above baseline through week 32. The IgG antibody level to P. intermedia peaked at 4 weeks following the third immunization and decreased post-ligation to near baseline levels by week 16. Characterization of the immune response after active immunization in the nonhuman primate has demonstrated a substantial and specific increase in antibody response which was sustained for several weeks. The insights obtained from these studies should help optimize the potential for immunologic interference with progressing periodontitis.
A novel helicobacter with the proposed name Helicobacter aurati (type strain MIT 97-5075c) has been isolated from the inflamed stomachs and ceca of adult Syrian hamsters. The new species is fusiform with multiple bipolar sheathed flagella and periplasmic fibers; it contains urease and gamma-glutamyl transpeptidase. By 16S rRNA sequencing and repetitive element PCR-based DNA fingerprinting, it was found that H. aurati represents a distinct taxon and clusters with Helicobacter muridarum, Helicobacter hepaticus, and Helicobacter sp. MIT 94-022. H. aurati was recovered from hamsters housed in various research and vendor facilities. Further studies are necessary to define its association with disease and other microbiota in hamsters, as well as its impact on research projects involving hamsters. H. aurati (GenBank accession number AF297868) can be used in animal experiments to define the factors that are important for gastric helicobacter pathogenesis.
Studying the cross talk between nonpathogenic organisms and their mammalian hosts represents an experimental challenge because these interactions are typically subtle and the microbial societies that associate with mammalian hosts are very complex and dynamic. A large, functionally stable, climax community of microbes is maintained in the murine and human gastrointestinal tracts. This open ecosystem exhibits not only regional differences in the composition of its microbiota but also regional differences in the differentiation programs of its epithelial cells and in the spatial distribution of its component immune cells. A key experimental strategy for determining whether "nonpathogenic" microorganisms actively create their own regional habitats in this ecosystem is to define cellular function in germ-free animals and then evaluate the effects of adding single or several microbial species. This review focuses on how gnotobiotics-the study of germ-free animals-has been and needs to be used to examine how the gastrointestinal ecosystem is created and maintained. Areas discussed include the generation of simplified ecosystems by using genetically manipulatable microbes and hosts to determine whether components of the microbiota actively regulate epithelial differentiation to create niches for themselves and for other organisms; the ways in which gnotobiology can help reveal collaborative interactions among the microbiota, epithelium, and mucosal immune system; and the ways in which gnotobiology is and will be useful for identifying host and microbial factors that define the continuum between nonpathogenic and pathogenic. A series of tests of microbial contributions to several pathologic states, using germ-free and ex-germ-free mice, are proposed.
Non-digestible oligosaccharides are complex carbohydrates of the non-a-glucan type which, because of the configuration of their osidic bonds, resist hydrolysis by salivary and intestinal digestive enzymes. In the colon they are fermented by anaerobic bacteria. Among the non-digestible oligosaccharides, the chicory fructooligosaccharides occupy a key position and, in most european countries, they are recognised as natural food ingredients. The other major products are the short chain fructooligosaccharides and galactooligosaccharides obtained by enzymatic synthesis using sucrose and lactose as substrates respectively, the soybean oligosaccharides, the xylooligosaccharides produced by partial hydrolysis of xylans and polydextrose or pyrodextrins prepared by a chemical treatment of carbohydrates. The most well known effect of most non-digestible oligosaccharides, and in particular of the fructooligosaccharides, is the selective stimulation of the growth of Bifidobacteria thus modifying significantly the composition of the colonic microbiota. Such a modification, which has clearly been demonstrated in human volunteers, is meant to be benificial in part because it is accompanied by a significant reduction in the number of bacteria reported to have pathogenic potential. Within the framework of research and development of "functional foods", such an effect justifies a "functional claim" for fructooligosaccharides namely "bifidogenesis". They are also typical "prebiotics". Besides their bifidogenic effect, the chicory fructooligosaccharides have additional nutritional properties on digestive physiological parameters like colonic pH and stool bulking which justify their classification as dietary fibers. Moreover, in experimental models, it has also been reported that they improve the bioavailability of essentiel minerals and that they reduce serum triglyceridemia by lowering hepatic lipogenesis. Such effects demonstrate interactions between the chicory fructooligosaccharides and key functions in the body but their significance for humans still need to be proven before being used to justify additional claims.
Salmonella is a Gram negative bacillus that behaves like a facultative intracellular pathogen. Its environment is the human and animal gastrointestinal tracts, it is never found like a normal microbiota. It is associated with gastrointestinal problems, septicaemic disease and abortion, due to its cellular invasion capacity and its intraphagocytic survival. Nowadays, it is known that Salmonella contains five pathogenicity islands. Several genes involved in the cellular invasion of nonphagocytic cells such as epithelial cells, apoptosis of macrophages, activation of routes of MAP kinases and transcription factors are located in centisome 63, constituting the pathogenicity island 1 (SPI-1). The SPI-2 and SPI-3 islands control the intracellular survival and replication. The SPI-4 island encodes a putative type I secretion system and its believed that it participates in the intracellular survival. Finally, the SPI-5 island encodes for factors involved in the fluid secretion and inflammatory reaction in the intestinal mucosa. Due to a coordinated and precise regulation of the Salmonella genes, it allows for adaptation to environmental changes that occur during an inflammatory process.