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Abstract: The enteric bacterial ora play a key role in maintaining health. Inammatory bowel disease is associated with quantitative and
qualitative alterations in the microbiota. Early characterization of the microbiota involved culture-dependent techniques. The advent of metagenomic techniques, however, allows for structural and functional characterization using culture-independent methods. Changes in diversity, together with quantitative alterations in specic bacterial species, have been identied. The functional signicance of these changes, and their pathogenic role, remain to be elucidated. (Inamm Bowel Dis 2012;18:372390) Key Words: enteric bacterial ora, inammatory bowel disease, metagenomic, microbiota
ecent advances in the techniques available to characterize the microbiota and their interaction with the human host have opened up new means of exploring the role of the intestinal microbiota in health and inammatory bowel disease (IBD). The gut microbiota is a complex ecological environment, with each human harboring up to 100 trillion (1014) bacteria,1,2 10-fold the number of human cells in the same individual. It is estimated that the gut microbiota comprises 40,000 or more different species.3 Microbes comprise 50% of our fecal volume.4 There is strong evidence for the involvement of microbes in the development of IBD. Animal models of inammation require bacteria.5,6 Antibiotics have therapeutic efcacy in Crohns disease (CD),7 pouchitis,8,9 and ulcerative colitis (UC).10 Probiotics have therapeutic efcacy in pouchitis11 and UC.12 Diverting the fecal stream from the inamed gut induces healing in CD,13,14 while reinfusion of intestinal content into surgically excluded ileum triggers recurrence.15 There is an ongoing debate as to whether changes in the intestinal microbiota precede or follow the development
Received for publication March 21, 2011; Accepted March 31, 2011. From the *St Vincents Hospital, Melbourne, Australia, University of Melbourne, Australia, Murdoch Childrens Research Institute, Australia, Chinese University of Hong Kong, kPrince of Wales Hospital, Hong Kong, Commonwealth Scientic and Industrial Research Organisation (CSIRO), Australia. The rst two authors contributed equally to this article. Reprints: Professor Michael A. Kamm, St Vincents Hospital, Victoria Parade, Fitzroy 3065, Melbourne, Australia (e-mail: mkamm@unimelb.edu.au). C 2011 Crohns & Colitis Foundation of America, Inc. Copyright V DOI 10.1002/ibd.21751 Published online 20 May 2011 in Wiley Online Library (wileyonlinelibrary. com).
of colitis in IBD. Early work to correlate altered microbiota composition with disease causation has had some success, with changes in the relative densities and spatial distribution of dominant bacterial groups preceding the onset of colitis in interleukin (IL)-10 knockout mice.16 Seventy percent of the gut microbiota have not been cultured by standard, culture-based techniques.17 This has in part given rise to the eld of metagenomics, the study of microbiota using culture-independent techniques.18 At the cornerstone of bacterial classication using these techniques is the bacterial 16S ribosomal (r)RNA gene. The 1.5 kbp bacterial 16S rRNA gene, which consists of nine conserved regions and nine variable regions, provides the basis for bacterial classication. This has revolutionized bacterial taxonomy.19 It has now been almost a decade since the completion of the Human Genome Project. Knowledge of the sequences of the 20,000 genes in the human body, however, is not enough to understand the functional and metabolic capabilities of man. This relates, at least in part, to Mans coevolution with microbiota; Man can be considered a supra-organisma composite of human and microbial genes (the microbiome).20,21 This microbiome, estimated to encode 100fold more genes than the human genome,1 is currently being characterized as part of the Human Microbiome Project.21 Characterization of the microbiota has until now been complicated by large person-to-person variation.22 The normal human bowel community is still being dened.23
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approaches based on 16S rRNA have been employed to detect uncultivable microbiota, using denaturing gradient gel electrophoresis (DGGE),2527 temporal temperature gradient gel electrophoresis (TTGE),28 temperature gradient gel electrophoresis (TGGE),22 uorescent in-situ hybridization (FISH),22,29 and terminal restriction fragment length polymorphism (T-RFLP).3032 These techniques provide information about the predominant microbiota but an incomplete view of microbial composition, only showing the most abundant taxa.33 More recent DNA sequencing technologies and phylogenetic microarray analysis have revolutionized bacterial characterization by increasing sequence sampling depth and throughput, respectively. As with traditional polymerase chain reaction (PCR)-based techniques, the main limitations of these methodologies are sequence-specic PCR amplication differences and biases introduced by DNA extraction.34 Sequencing techniques are used to determine the order of nucleotides in any DNA fragment. Initially, Sanger et al35 described a technique based on chain termination. The more recent technique of next-generation sequencing is based on sequencing by synthesis.36 Compared to Sanger sequencing, next-generation sequencing is higher throughput, less expensive, and reveals less abundant taxa.36 Phylogenetic microarray analysis characterizes the presence and abundance of different species within the bacterial community. It uses species-specic probes to analyze multiple sequences simultaneously. It is high throughput, allows comparison between two groups, and is relatively inexpensive. Its main limitations are that probes are limited to those related to known sequences, and it is not quantitative.33,37 Once a sequence of interest is identied, it can be quantitated using real-time PCR (also called quantitative PCR, qPCR). The method can be used to quantify organisms from the level of species to higher taxonomic groups depending on the specicity of the primer sets being employed. Real-time PCR allows validation of metagenomic data and is fast compared with traditional methods of quantication.38 Through metagenomic approaches the generation of large volumes of bacterial 16S sequence data, of both known and unknown microbes, within our gut is possible. Sequence data are categorized into groups called operational taxonomic units (OTUs), sometimes referred to as ribo-types or phylo-groups. Based on the 16S gene sequence similarity of more than 99% is regarded as a strain; 97% similarity as a species; 95% as genus; more than 90% as family19; and more than 80% as a phyla.39 Previously unidentied bacteria can often be assigned a classication within the taxonomic tree based on their similarity to previously identied bacteria.
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pyrosequencing on one monozygotic twin pair, and broader sequencing on 54 twin pairs and their mothers. Of 134 phylotypes identied in all the twin pairs, only 37 were present in more than 50% of the samples. Only one phylotype, Lachnospiraceae, was present in nearly all individuals. The concept that previously inadequate depth of sampling concealed common species which are in low abundance was reinforced by Qin et al,79 who found that a 23-fold increase in sequencing depth raised by 25% the number of shared species between two individuals. Recently, Sekelja et al84 conrmed the presence of a phylogenetic core by reanalyzing sequence data using a phylogroup- and tree-independent approach. In particular, two highly prevalent core phylogroups were identied which belong to the clostridial family Lachnospiraceae and are believed to be ancient, abundant, and stable in the human gut.84
Functional Core
While the phylogenetic core describes what is commonly present in the gut between individuals, it does not explain the role of these bacteria. An emerging concept is that of a functional core, a core microbiome, at a gene expression and organismal lineage level,78 related to the products and effects of the microbiota. There appears to be extensive functional redundancy within an individuals gut microbiota whereby a number of different organisms, some of which are shared, and some of which differ between individuals, perform similar functions. Qin et al79 recently conrmed the presence of a functional core and extended the size of functional categories by 30%, compared with the previous work of Turnbaugh et al.78 Functional analysis of the microbiota is a rapidly evolving area and refers broadly to community characterization at the level of RNA (metatranscriptomics), protein (metaproteonomics), and metabolites (metabonomics).
A study employing culture of enteric bacteria showed that the fecal-associated microbiota (FAM) of monozygotic twins were much more alike than those of dizygotic twins.88 TTGE on fecal samples from children further demonstrated a greater similarity between monozygotic twins than dizygotic twins or unrelated paired controls.89 In contrast, other studies using DGGE90 and pyrosequencing78,91 have shown no difference between monozygotic and dizygotic twins with respect to FAM. Family members were, however, found to have more similar proles than unrelated individuals.90 Although a study by Willing et al,91 which included CD patients, showed that the effect of disease phenotype was greater than that of host genotype, there was similarity apparent at the genus level, in adult healthy twin pairs who had lived apart for many years. This observation underscores the importance of genes and/or environmental exposure during early childhood in the development of the microbial community.91 Differentiating between the environmental and genetic factors that converge to shape the gut microbiota is a complex task, which was highlighted in a study by Rawls et al92 that used reciprocal mice/zebrash gut microbiota transplantations to germ-free zebrash/mice hosts. The microbiota posttransplantation reected the donor community in terms of the bacterial lineages, but in contrast, microbial community abundances more closely resembling the normal prole of the recipient host. A donor fecal infusion study has been performed in humans; following a healthy donors fecal infusion, a durable benecial change in the patients colonic microbiota (using DGGE) was found, which represented that of the healthy donors microbiota.93
HOST-BACTERIAL MUTUALISM
Mans coevolution with microbiota has resulted in a tight, nely balanced relationship, referred to as host-bacterial mutualism.85 The host inuences or determines the nature and activity of the microbiota and the microbiota inuences or determines aspects of host immunity.17
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dependent activation of TLRs, thereby limiting bacterial penetration of host tissue.96 Paneth cell function and antimicrobial activity is regulated by a number of immune pathways and genes including UPR, autophagy, and NOD2.97 The UPR pathway maintains normal epithelial function and therefore homeostasis between the microbiota and immune system and the epithelial interface.98100 The UPR is activated in response to endoplasmic reticulum (ER) stress and regulates autophagy. Paneth cells, goblets cells, and other highly secretory cells are particularly prone to ER stress and therefore dependent on the UPR pathway to increase the size and protein processing function of the ER.101 Mutations within the UPR, and environmental factors that create disturbances in the UPR, such as microbial products and inammatory cytokines, have been shown to cause or perpetuate intestinal inammation.101 Autophagy, a lysosomal process, is triggered by cellular stress including ER stress.97,101 Two variants of the autophagy protein, ATG16L1 and IRGM, have been discovered in association with CD. Paneth cells have defects in granule content and exocytosis, indicating that autophagy also plays an important role in Paneth cell function.102 Other key innate immunity mediators that allow crosstalk between the host and microbiota include pattern recognition receptors such as NOD2 and TLRs.103 NOD2, highly expressed in Paneth cells, enables detection of a wide variety of bacteria by responding to their cell wall peptidoglycan.104 Mutations in NOD2 alter the antimicrobial activity of Paneth cells in the terminal ileum,105 resulting in impaired bacterial surveillance.106,107 The important role of NOD2 in the regulation of commensal microbiota was recently highlighted in a study which showed that in comparison with wildtype mice, NOD2-decient mice had increased amounts of commensal bacterial in the gut as well as reduced capability to clear newly colonizing bacteria.104 Antimicrobial peptides such as defensins are also key effector molecules in the innate immune systems control of gut microbiota.17 Reduced levels of defensins have been observed in both ileal and colonic CD.97 Reduced defensin production has been associated with NOD2 mutations.108110
(DCs) and gut epithelial cells, thereby regulating the intensity of intestinal B- and T-cell responses.112114 DCs play an important role in mediating the effect of gut bacteria on the host immune system. DCs sample and respond to luminal microbiota via pattern recognition receptors such as TLRs, and therefore modify the nature of subsequent T-cell responses.115 It has recently been shown that IL-6 production by intestinal DCs is increased in CD and correlates with disease activity and C-reactive protein, and intestinal DC function may be inuenced by the composition of the commensal microbiota.116 Commensal microbiota, such as Bacteroides fragilis, express molecules such as polysaccharide A which protect against the proinammatory effects of potentially pathogenic microbiota by stimulating IL-10-producing CD4 T cells.117119 Similarly, the commensal Fecalibacterium prausnitzii has been shown to have antiinammatory effects due to secreted metabolites which block NF-jB activation and IL-8 production.120 Segmented lamentous bacteria (SFB) have also been shown to regulate the balance between T helper 17 (Th17) and regulatory T cells, contributing to the inuence that microbiota composition has on intestinal immunity, tolerance, and susceptibility to IBD.119121 Recently, a method of identication of bacterial genes involved in regulation of NF-jB signaling in intraepipthelial cells has been developed. This may help identify bacteria that directly affect mucosal immunity and inammation.122
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concept of dysbiosis. The candidate microorganism strategy refers to the identication of single specic microorganisms that are thought to be directly pathogenic, such as Escherichia coli129 or Mycobacterium avium subspecies paratuberculosis (MAP).130 These two approaches are not mutually exclusive.
There are conicting data regarding the phylum Bacteroidetes. In the FAM and MAM of CD and UC versus controls, Bacteroidetes and its species has been found to be both increased128,131,133,144,145,151,166,167 and decreased.3,136,142,148,155 Increased numbers of Fusobacteria have been found in the FAM140 and MAM158 of active UC compared with inactive UC and controls. In CD Bidobacteria and Lactobacilli have been found to be decreased in the FAM in both adults28,142,156,168 and children.151 In the MAM of UC patients, Bidobacteria are decreased,164 whereas Lactobacilli have been found to be decreased in the FAM of patients with active UC.169 Recently, TM7 (a subgroup of Gram-positive uncultivable bacteria), previously implicated in oral inammation, has been shown to be increased in diversity in active CD (23 phylotypes) compared with active UC (10 phylotypes) and non-IBD controls (12 phylotypes). The TM7 associated with CD and UC was strongly associated with antibiotic resistance compared with controls.170 Swidsinski et al150 suggested that two features could be useful as a ngerprint to discriminate between CD and UC: a reduction in the concentration of F. prausnitzii in CD to less than 1 109 per mL, and an increase in leukocytes in UC to >30 leukocytes/104lm2. Recent studies have also demonstrated differences in the relative abundance of the Bidobacteriaceae, Coriobacteriaceae, and Ruminococcaceae families among individuals with different CD phenotypes.91,144,153 In a study of FAM using DGGE, Joossens et al171 found a dysbiosis signature associated with CD, characterized by ve bacterial species, namely, a decrease in Dialister invisus, F. prausnitzii, and Bidobacterium adolescentis, and an increase in R. gnavus and an uncharacterized species of Clostridium cluster XIVa. In the same study, the fecal samples of 84 unaffected rst-degree relatives of CD patients were found to have an altered composition of the predominant microbiota compared with controls. No difference was seen when F. prausnitzii was specically targeted by qPCR.171 Prospective studies are needed to identify if overt disease will develop in some of these relatives over time.
MAP
MAP has long been considered a possible causative agent in CD because of the similarity of Johnes disease in cattle, caused by MAP, and CD in humans.172
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Of two recent meta-analyses and systematic reviews of the association of MAP with CD the rst included 28 case-controlled studies of MAP DNA detected by PCR in tissue samples, or antibodies against MAP antigens tested by enzyme-linked immunosorbent assay in serum. MAP was detected more often in patients with CD than in controls and patients with UC.173 The second meta-analysis only included studies of tissue samples using nucleic acidbased techniques, specically PCR or in situ hybridization. In the analysis of the 47 studies, MAP was detected more frequently among CD patients compared with controls.174 This meta-analysis has been criticized on its exclusion of 13 studies in which MAP was not detected in any patients with CD or in controls.175 In a recent large-scale 16S rRNA gene library study which was not included in either of these two meta-analyses, more than 15,000 small subunit ribosomal RNA genes were analyzed and MAP was not detected in CD patients.3 The inuence of IBD medication on MAP should be considered. Methotrexate, 6-mercaptopurine, and 5-aminosalicylic acid have been shown to inhibit MAP growth in vitro.176,177 Kirkwood et al130 investigated pediatric patients not yet treated with any medication and found that MAP was identied more often in mucosal biopsies and peripheral blood mononuclear cells from CD than in nonIBD patients. Difculties in extraction of MAP DNA may explain the failure and detection in many studies. The effect of antimycobacterial therapy has been the subject of a Cochrane review, which cautiously concluded that antimycobacterial therapy may have a role in maintaining remission in CD, but fell short of recommending therapy.178 The result of the largest randomized controlled trial to date does not support the use of antimycobacterial therapy in CD.179
sis, and survive and replicate extensively in large vacuoles within macrophages without triggering host cell death.189191 The type 1 pili of AIEC bind to the specic receptor CEACAM-6 expressed in ileal epithelial cells of patients with CD but not healthy controls.192 CEACAM-6 receptors become overexpressed in response to stimulation of ileal epithelial cells by tumor necrosis factor alpha (TNFa), which is released from macrophages that have taken up CD-associated AIEC.193 AIEC therefore causes an amplication loop of colonization and inammation.190 The early inammatory response to AIEC among patients with CD carrying CARD15 polymorphisms appears to be disturbed.194
Other Organisms
Several other bacteria such as Pseudomonas,195199 Yersinia,200,201 Listeria,155,202204 Burkholderia,205 and Helicobacter206210 have been linked to IBD, but what pathophysiological role, if any, they play remains to be determined. A contribution from viruses211213 and fungi214216 has also not been excluded.
E. coli
Numerous studies have shown increased numbers of mucosa associated E. coli in both CD and UC compared with healthy controls.129,132,133,136,153,163,164,180182 As a commensal organism within the normal gut microbiota, E. coli plays an important role in maintaining intestinal homeostasis and is not implicated in disease unless there is a breach in the intestinal mucosa barrier. Of the colonic-like microbiota that colonize the neoterminal ileum postresectional surgery for CD, E. coli tend to predominate.180,181 The adherent invasive strain of E. coli (AIEC) has developed virulence factors that allow it to adapt and survive in the postoperative environment.183 AIEC has been found in association with early neoterminal ileal lesions in the postoperative CD setting.165 The ability of AIEC to adhere and invade intestinal cells is mediated by a number of virulence factors including type 1 pili, agella, and outer membrane porin C.184188 Moreover, AIEC is able to resist phagocyto-
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In both CD and UC no signicant difference has been identied between the ileum and colon.220,222,223 This observation has been conrmed in CD in a recent study using microarray227 as well as a study which involved pyrosequencing on biopsies of nine twin pairs.91
Other studies of FAM have grouped subjects into active and inactive and then compared their microbiota. In CD, using pyrosequencing, no difference has been seen between active CD versus remission.91 In UC, some studies have shown differences in specic bacterial probes between active disease and remission. In one study, Lactobacilli species was found to be present, or increased, during inactive versus active UC.158,169 In a study using T-RFLP, Andoh et al235 found a decrease in the Clostridium family in the FAM of patients with active UC, and inactive/active CD. In contrast, Bacteroides was signicantly increased in CD patients.
Pouchitis
An etiological role for the microbiota in pouchitis has been proposed by studies indicating that remission of
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pouchitis can be induced by antibiotics,9,236 and maintained by probiotics.237 Initial culture based studies have shown that pouch efuent is characterized by the presence of more anaerobes than ileostomy efuent, and that the pouch and fecal microbiota are similar.238240 However, no trends in specic levels of different bacterial species or the degree of mucosal inammation were observed. A number of studies have suggested that dysbiosis is implicated in the development of pouchitis, but it is unclear whether this is a predisposing factor or the cause of pouchitis. In a study using 16S rRNA analysis on patients with FAP and UC with and without pouchitis, there appeared to be limited differences in bacterial composition between those with and without pouchitis, suggesting dysbiosis as a predisposing rather than causative factor.241 However, a central role for dysbiosis in pouchitis is more likely and has been supported by two studies, one of which compared the MAM of UC pouch patients with and without active pouchitis, and found UC pouchitis was characterized by persistence of Fusobacter and Enteric species and the absence of specic bacteria such as Streptococcus species.242 The other study analyzed the FAM and MAM of UC patients with and without pouchitis compared with Familial Adenomatous Polyposis pouches and found that UC pouchitis patients had substantially fewer Bacteroidetes and more Clostridia compared to the healthy UC pouch and Familial Adenomatous Polyposis groups.243
olite release and production (metabonomics). In that context, Qin et al79 recently described a gut microbial gene catalog of 3.3 million genes which will help advance the utilization of these -omic-based technologies in the future.
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other words, the composition of human gut metagenomes are fast approaching the level of characterization that their proling will extend beyond a select number of taxonomic marker genes, and include a much greater depth of functional complexity, including the approaches described below.
metatranscriptomes, suggesting site-specic alterations in gene expression proles.248 Furthermore, functional gene microarrays have been synthesized using the metagenomic dataset produced by Qin et al79 (2010; Ehrlich, pers. commun.). Accordingly, exciting new results will soon be forthcoming as a result of the successful use of these resources.
Metatranscriptomics: RNA
Transcriptomics and metatranscriptomics seek to characterize the gene expression patterns of select microbes, or the entire microbiome, respectively. Rehman et al247 analyzed the transcriptional activity of MAM by comparing 16S rRNA gene and rRNA proles from biopsies of active IBD patients and healthy subjects. They found that specic bacterial populations were activated in IBD patients, whereas other groups lay dormant. In contrast, among healthy patients, there did not appear to be the same variation in abundance and activity. This conrms that, in disease, it is the microbiotas functional activity or inactivity that is as important as its presence, abundance, or absence. Our collaborative group has recently undertaken studies to examine how the transcriptomes of Australian isolates of Enterococcus fecium and Bacteroides vulgatus may change in media designed to simulate the colonic environment during health and disease. These bacteria were cultured using anaerobic media supplemented with fecal waters prepared from either healthy persons or IBD patients, as well as media supplemented with additional water or a complex nutrient broth. A preliminary assessment of the transcriptomic responses has been obtained for the E. fecium isolate (Klaassens et al, submitted). Approximately 16% of the RNA-sequence reads were expressed under all four growth conditions, suggesting the genes encode general housekeeping functions. However, there was also a similar percentage of RNA-sequence reads that were produced only in response to the bacteriums exposure to the fecal waters; several of these encode proteins that are presumptive extracellular, membrane-bound proteins. These ndings further conrm that the transcriptomes of gut bacteria are dynamic and responsive to components present in the colonic environment. Subtractive enrichment of microbial mRNA from stool or tissue samples, as well as a comprehensive metagenome dataset for interrogation, have been viable constraints on these metatranscriptomics analyses of human gut microbiomes. However, for reasons outlined above, metatranscriptomic studies of the human gut microbiome are now within reach: a recent abstract illustrated a metatranscriptome and metagenome comparison between the luminal and MAM of a single CD patient using pyrosequencing and KEGG (Kyoto Encyclopedia of Genes and Genomes) database analysis.248 Metagenomes of luminal and MAM were more similar to each other than to their respective
Metaproteomics: Proteins
Metaproteomics is the study of the proteome, which refers to the set of all expressed proteins in a cell, tissue, or organism. The proteome is dynamic and subject to changes in health and disease, and reects the interactions between genes and the environment. The uctuation of proteins in response to health and disease make them attractive as both diagnostic biomarkers and targets to guide molecular therapeutics.249 The most commonly used technique in metaproteomics is mass spectrometry, which measures the mass-tocharge ratio of charged particles. These charged particles are created from proteins, peptides, or metabolites, which are subsequently separated according to this mass-to-charge ratio. Klaassens et al250 were among the rst to examine the metaproteome produced from human fecal samples, and also performed DGGE of the gene encoding 16S rRNA to monitor the bacterial communities from two infants. FAM was found to be relatively simple and predominated by bidobacteria. Using mass spectrometry, a peptide sequence was discovered in the stool sample which was similar to a bidobacteria transaldolase.250 It is felt that other functionally active bacteria can be discovered via a similar metaproteomic approaches. Using mass spectrometry-based shotgun proteomics, Verberkmoes et al251 compared the identied proteins in the feces of a healthy monozygotic twin pair to available protein databases, and in so doing, identied the functional activities of the gut microbiota on a large scale. Metaproteomics has not yet been applied to the IBD setting, but it has been employed by Ang et al252 to examine stool samples in pursuit of diagnostic biomarkers for early colorectal cancer diagnosis. Similar to metatranscriptomics, the DNA sequence databases that are being rapidly produced should serve to improve the interrogative potential of proteomic methods, ensuring more of these types of analyses successfully result in the identication of the cognate gene (and microbe[s]) encoding that particular protein.
Metabolomics: Metabolites
Metabolomics (also known as metabonomics) refers to the quantication of metabolites present in cells or organisms that participate in the metabolic reactions required for growth and maintenance of normal function. It also includes metabolites ingested from the external environment and the
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metabolites of the gut microbiota.253 Metabolomics is a rapidly evolving area and involves the characterization of the true endproducts of biological processes within biological tissues and uids such as urine, feces, and blood.254,255 Just as genome-wide association studies (GWAS) have found associations between genotype and disease phenotypes,256,257 the metabolome-wide association study (MWAS) has revealed associations of metabolic phenotypes with disease risk.258,259 Metabolic proling can also be used to determine the impact of diet260265 and medications.266 Metabolomics utilizes mass spectrometry and nuclear magnetic resonance spectroscopy, which exploits the ne differences in the frequency of subatomic particles within organic compounds that depends on their neighboring atoms to differentiate one compound from another.253 There is increasing research on the use of metabolomics in IBD. Using feces and urine from healthy subjects, Li et al83 were able to link the microbiota composition within feces with the metabolic products in the urine. Other studies were able to form similar correlations from feces of patients with and without IBD.267,268 The modulation of the metabolites in the urine by the gut microbiota has also been proven in animal models.269,270 Williams et al271 applied urinary metabolic proling to 86 CD patients, 60 UC patients, and 60 healthy controls. Specic metabolite levels were signicantly different among the three groups, allowing differentiation according to urinary metabolic prole; importantly, colonic CD could be differentiated from UC. A number of other studies on colonic biopsies have also demonstrated the ability of metabolomics to distinguish CD from UC.272,273 A subgroup analysis in the Williams et al study271 did not reveal any signicant variation of the individual diseases by disease location, which is in contrast to Jansson et al,267 who were able to show a difference in the fecal metabolite proles of ileal versus colonic CD. Regarding disease activity, Jansson et al did not nd any inuence of disease activity on the metabolic prole; however, other studies have found that the activity of IBD could be distinguished on the basis of altered metabolite levels.255,272 Metabolic proles of colonic biopsies in IBD has shown that there was little difference between inamed and noninamed tissue, but the metabolic proles differed from that of healthy controls.274 In summary, the rapid development of extensive datasets of the genetic potential resident within various gut microbiomes should soon result in the next wave of highimpact discoveries, which elucidate not only the structural, but also the functional attributes of the gut microbiota that contribute to health and/or disease. Such datasets should support the further establishment of systems biology approaches within select clinical studies, increasing the likelihood of dissecting the roles of the host, diet, and microbiology in gut health and disease.
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other environmental factors. Diet has a major impact on the composition and activity of the gut microbiota.280,281 Changes in gut microbiota, mediated via environmental factors, may contribute to the recent increase in incidence of IBD in developing countries.282
Diet
The relationship between the host, diet, and the gut microbiota is complex. The dynamic impact of diet on the microbiota composition and function has been reected in obesity research. Studies in both mouse models and humans have shown that compared with the lean gut microbiome, the obese gut microbiome is enriched with genes involved in energy extraction from the host diet.78,86,283 The large and abrupt impact of diet on humanized gnotobiotic mice has been demonstrated by Turnbaugh et al,87 who showed that when switching from a low-fat, plant polysaccharide-rich diet to a high-fat/high-sugar diet, the microbiota changed substantially within 1 day, the representation of metabolic pathways in the microbiome changed, and the microbiome gene expression was altered. More recently, glucagon-like peptide 2 (GLP-2), via its effect on gut permeability, has been raised as a potential mechanism by which the microbiota mediate their effect on obesity.284 A key functional contribution of the microbiota is the harvest of otherwise inaccessible nutrients and/or sources of energy from the diet, and the synthesis of vitamins.21 The microbiota metabolize ber and resistant starch to the short chain fatty acids (SCFAs) butyrate, acetate, and propionate, which serve as a major energy source for colonocytes, and implicated in prevention of colitis and colorectal cancer.285287 Butyrate also strengthens and maintains mucosal barrier function via production of mucin, antimicrobial peptides, and tight-junction proteins.288 The antiinammatory property of SCFAs may relate to their binding to a G-protein-coupled receptor (GPR43), leading to downregulation of inammatory responses.125 Growth in number of butyrate producing bacteria such as Eubacterium rectale and Roseburia species is directly stimulated by dietary carbohydrates.289292 Carbohydrate substrates inulin and fructo-oligosaccharide also promote growth of probiotic bacteria Bidobacteria and Lactobacilli.293 IBD is associated with reduced numbers of butyrateproducing bacteria (e.g., clostridia groups IV and XIVa of which F. prausnitzii is a member), and accordingly, reduced concentrations of SCFAs.120,294 A lack of the antiinammatory effects of butyrate could be implicated in the inammatory cascade involved in IBD and butyrate is already considered to be of therapeutic benet in IBD.295297 A class of microbiota, known as sulfate-reducing bacteria (SRB), have been shown to synthesize toxic metabolic products such as hydrogen sulde,298 which is toxic to
colonocytes, blocks butyrate utilization, indices cell hyperproliferation, and inhibits phagocytosis and bacterial killing.299 Overgrowth of SRB has been demonstrated in IBD, particularly among UC and pouch patients.300 A high meat-containing diet has been associated with the toxic products of SRB.298,300304 A diet of low meat, saturated fat, high ber, and resistant starch, through its effects on the microbiota, may be linked to the low incidence of colorectal cancer in Africa.302,305,306 Diet may impact the composition of the gut microbiota in diverse ways. Genes used by marine microbiota to metabolize carbohydrates in seaweed have been found among the gut microbiome in Japanese but not Americans. Acquisition of these novel genes in the Japanese gut microbiome most likely occurred though ingestion of seaweed and marine microbes, with horizontal gene transfer.307 Case-control studies and epidemiological data have yielded inconsistent results for an association between specic foods and the occurrence of CD and/or UC. None of these studies have clearly linked diet to the gut microbiota and the development of IBD. Foods that have been implicated include meat308 and fats/oils,308310 sweets/confectionary,308,311313 and fast food.313 Fatty acids have been suggested to have a protective role,309 as have ber, fruit, and vegetable from IBD,308,309,314317 while other studies have been negative.308,318,319 There is emerging evidence in mouse models and humans that iron may have an impact on the intestinal microbiota and immune response to inammation. Iron has been shown to increase the concentration of iron-dependent E coli, Klebsiella, and Bacteroides species and exacerbate colitis in mouse models.320,321 In a recent study of anemic African children, treatment with iron produced a more pathogenic gut microbiota prole, which was associated with increased gut inammation (measured by fecal calprotectin).322 In contrast, a recent abstract has shown that iron supplementation reduces inammatory lesions in experimental colitis in rats.323
CONCLUSION
The prospects for discovery within the GI microbiota/microbiome is captivating and rapidly evolving. While traditionally characterization of the microbiota has focused on which bacteria are present, it is becoming clear that analysis of bacterial function is as important to establish the complex relationship between the gut microbiota and its host.
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