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The collection of microbes and their genes that exist within and on the human body is collectively known as the microbiome. The human microbiome in different organs has emerged as a principal factor in human health and disease. Advancements in lab technologies have allowed for the characterization of microbiomes throughout the human body including the gut, in a myriad of human diseases and in health. The microbiome within the bowel known as the gut microbiome has especially emerged as being important not only in intestinal diseases but also in other immune-mediated inflammatory diseases (like rheumatoid arthritis or multiple sclerosis) and also in health. Researchers have shown that perturbations in the gut microbiome may impact on obesity and blood sugar control. In the past decade there has been an explosion of research undertaken to understand how alterations in the gut microbiome may lead to intestinal diseases, especially inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS). Our group has been at the forefront of this research. We partnered with a leading group at Stanford University in 2004 and published the first report of the human gut microbiome using sophisticated molecular biology techniques using colon biopsies and stool samples from 3 Manitobans who were healthy and volunteered to participate in our research study. We, identified hundreds of bacterial species in the human gut that had never previously been reported (Eckburg et al, Science 2005). Together with the Denis Krause lab in the Faculty of Agriculture at the University of Manitoba, we reported that a unique type of bacteria known as an adherent invasive E coli was more common in persons with Crohn’s disease including early in diagnosis. This finding was exciting because a handful of other research groups in Europe and North America had found similar bacteria in persons with Crohn’s disease. Then, with Gary van Domselaar’s lab at the National Microbiology Laboratory we reported on differences in the gut microbiome in the bowel lining from persons with Crohn’s disease, persons with ulcerative colitis and in healthy controls. We found that the most important differences were in the non-inflamed tissues from Crohn’s disease compared with the non-inflamed tissues from ulcerative colitis. One message from this study is that analyzing the inflamed tissue from disease states may not give as much information as analyzing the uninflamed tissue, tissue that is at risk of one day becoming inflamed.

 

There are reports in the scientific literature, as well as in the lay press, that changes in the gut microbiome in inflammatory bowel disease are well known and ready to be tapped for treatment. Some changes in the gut microbiome in IBD have been repeatedly reported, such as reduction in microbial diversity. This means that persons with IBD have less varied types of bacteria than healthy persons. However the reports that any one type of bacteria is reduced while other types are increased are variable. Hence, there has yet to be a probiotic developed that includes the specific bacteria thought to be diminished in IBD. It does make sense, though that once it is worked out what the consistently found changes are in the gut microbiome in specific types of IBD (and IBS as well) that drug development may turn to probiotic sidle medications; that is bacteria that can be protective and possibly anti-inflammatory).

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Cadogan K, Shaffer S, Singer A, Reimer A, Knox N, Rumore J, Christianson S, Alexander DC, Forbes JD, Bernstein CN. Physician perspectives of Helicobacter pylori diagnostic and treatment practices in Canada: Results of a Canadian Survey. BMC Gastroenterology 2024; Jun 17;24(1):204.

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Background: Helicobacter pylori infection is prevalent worldwide and can lead to peptic ulcer disease (PUD) and gastric cancer. Effective diagnosis and treatment of H. pylori infection by gastroenterologists and family physicians is crucial. However, there are differing views on optimal diagnosis and treatment. The objective of this study is to understand the impressions of Canadian physicians regarding H. pylori diagnosis and treatment and whether impressions differ between gastroenterologists and family physicians. A second objective is to understand physician perspectives on rising antibiotic resistance and how that guides empiric management.

Methods: A survey facilitated via REDCap was administered to Canadian gastroenterologists and family physicians. A total of 105 participants completed the survey, including 43 gastroenterologists and 62 family physicians. Gastroenterologists were recruited from across the country and family physicians were recruited from Manitoba.

Results: For diagnosis of H. pylori, 67% of gastroenterologists reported endoscopic biopsies for histology assessment as most common and 73% of family physicians reported serology as their main diagnostic test. While nearly all gastroenterologists believed antibiotic resistance to be a problem, nearly one quarter of family physicians did not believe it was a problem.

Conclusions: There is variability in practices among both gastroenterologists and family physicians regarding diagnosis of H. pylori infection. There was consensus that local antibiotic resistance patterns should guide management. If known, the degree and patterns of antibiotic resistance could bring a more uniform consensus to H. pylori management. Greater education of physicians, especially family physicians regarding management of H pylori is needed.

 

 

Raygoza Garay JA, Turpin W, Lee SH, Smith MI, Goethel A, Griffiths AM, Moayyedi P, Espin-Garcia O, Abreu M, Aumais GL, Bernstein CN, Biron IA, Cino M, Deslandres C, Dotan I, El-Matary W, Feagan B, Guttman DS, Huynh H, Dieleman LA, Hyams JS, Jacobson K, Mack D, Marshall JK, Otley A, Panaccione R, Ropeleski M, Silverberg MS, Steinhart AH, Turner D, Yerushalmi B, Paterson AD, Xu W; CCC GEM Project Research Consortium; Croitoru K. Gut Microbiome Composition Is Associated With Future Onset of Crohn's Disease in Healthy First-Degree Relatives. Gastroenterology 2023;165(3):670-681.

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The cause of Crohn's disease is unknown, but the current hypothesis is that microbial or environmental factors induce gut inflammation in genetically susceptible individuals, leading to chronic intestinal inflammation. Case-control studies of patients with Crohn's disease have cataloged alterations in the gut microbiome composition; however, these studies fail to distinguish whether the altered gut microbiome composition is associated with initiation of Crohn's disease or is the result of inflammation or drug treatment. In this prospective cohort study, 3483 healthy first-degree relatives of patients with Crohn's disease were recruited to identify the gut microbiome composition that precedes the onset of CD and to what extent this composition predicts the risk of developing Crohn's disease. We applied a machine learning approach to the analysis of the gut microbiome composition (based on 16S ribosomal RNA sequencing) to define a microbial signature that associates with future development of Crohn's disease. The performance of the model was assessed in an independent validation cohort. Results: In the validation cohort, the microbiome risk score model yielded a hazard ratio of 2.24 (95% confidence interval, 1.03-4.84; P = .04), using the median of the microbiome risk score from the discovery cohort as the threshold. The microbiome risk score demonstrated a temporal validity by capturing individuals that developed CD up to 5 years before disease onset (area under the curve > 0.65). The 5 most important taxa contributing to the MRS included Ruminococcus torques, Blautia, Colidextribacter, an uncultured genus-level group from Oscillospiraceae, and Roseburia. Conclusion: This study is the first to demonstrate that gut microbiome composition is associated with future onset of CD and suggests that gut microbiome is a contributor in the pathogenesis of CD.

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Armstrong HK, Vincent N, Bernstein CN, Bording-Jorgenson M, Veniamin SM, Jovel J, Sobhan S, Torabi M, Wine E, El-Matary W. Gut microbiome composition and metabolic changes are correlated with sleep efficiency and disease phenotype in pediatric inflammatory bowel disease. Journal of Sleep Medicine and Disorders 2023; 8(1): 1131.

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Sleep disturbances are common in patients with IBD, defined by alterations in the quality, timing, and amount of sleep. We hypothesized that disruption of the gut microbiota in pediatric IBD patients may induce sleep disturbances and worsen patient outcomes. Children younger than 17 years with IBD were assessed for sleep disturbances using a sleep diary. Clinical disease activity indices were calculated. Patient stool was collected for calprotectin measurement, and to examine microbiota (metagenomics; Kraken2-R), Short Chain Fatty Acid (SCFA; gas chromatography), and metabolite (metagenomics; Maaslin2) profiles. Regression analyses assessed association of Fecal Calprotectin (FCal), clinical activity indices, sleep efficiency/length, and time in bed.  Results: 15/ 80 (18.6%) children had clinically active disease; 23 (28.8%) had FCAL greater than 100 ug/g. 24/80 (30%) children had abnormal sleep efficiency less than 90% and 22 (27.5%) had subnormal sleep duration. High FCAL was associated with altered sleep efficiency (P = 0.03). Disease phenotype (L3, B2) demonstrated numerous significant correlations with microbiota (e.g., Veillonella, Akkermansia, Allistipes) and metabolites (e.g., H-transport, NADH-dehydrogenase, 6-phospphofruktokinase). Average length of sleep was associated with significant changes in microbiota diversity (e.g., Bacteroides, Enterococcus, Bifidobacterium, Alistipes, Streptococcus, Ruminococcus) and vast metabolic changes, primarily related to energy production. Finally, stool propionate negatively correlated with sleep efficiency and total time in bed (P < 0.05). Conclusions: Our findings suggest that gut microbiota diversity, abundance, and functions (metabolites) are associated with altered sleep patterns in pediatric IBD and more aggressive disease phenotypes. These data support the need to further investigate causal relationships between gut microbiota, sleep efficiency, and disease outcomes in children with IBD.

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Armstrong HK, Bording-Jorgensen M, Santer DM, Zhamg Z, Valcheva R, Rieger AM, Kim JSH, Dijk SI, Mahmood R, Ogungbola O, Jovel J, Moreau F, Gorman H, Dickner R, Jerasi J, Mander IK, Lafleur D, Cheng C, Petrova A, Jeanson TL, Mason A, Sergi CM, Levine A, Chadee K, Armstrong D, Rauscher S, Bermstein CN, Carroll MW, Huynh HQ, Walter J, Madsen KL, Dieleman LA, Wine A. Unfermented B fructans fibers fuel inflammation in select inflammatory bowel disease patients. Gastroenterology 2023; 164:228-240.

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IBD are impacted by dietary factors, including non-digestible carbohydrates (fibers), which are fermented by colonic microbes. Fibers are overall beneficial but not all fibers are alike and some IBD patients report intolerance to fiber consumption. Given reproducible evidence of reduced fiber-fermenting microbes in IBD patients, we hypothesized that fibers remain intact in select patients with reduced fiber-fermenting microbes and can then bind host cell receptors, subsequently promoting gut inflammation. Colonic biopsies cultured ex vivo and cell lines in vitro were incubated with oligofructose (5g/L), or fermentation supernatants (24hr anaerobic fermentation) and immune responses (cytokine secretion [ELISA/MSD] and expression [qPCR]) were assessed. Influence of microbiota in mediating host response was examined and taxonomic classification of microbiota was conducted with Kraken2 and metabolic profiling by HUMAnN2, using R software.  Results: Unfermented dietary β-fructan fibers induced pro-inflammatory cytokines in a subset of IBD intestinal biopsies cultured ex vivo, and immune cells (including peripheral blood mononuclear cells). Results were validated in an adult IBD randomized controlled trial examining β-fructan supplementation. The pro-inflammatory response to intact β-fructan required activation of the NLRP3 and TLR2 pathways. Fermentation of β-fructans by human gut whole-microbiota cultures reduced the pro-inflammatory response, but only when microbes were collected from non-IBD or inactive IBD patients. Fiber-induced immune responses correlated with microbe functions, luminal metabolites, and dietary fiber avoidance. Conclusion: While fibers are typically beneficial in individuals with normal microbial fermentative potential, some dietary fibers have detrimental effects in select patients with active IBD who lack fermentative microbe activities.

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Schoeps VA, Zhou X,  Horton MK, Zhu F,  McCauley KE, Nasr Z,  Virupakshaiah A, Gorman MP,  Benson LA, Weinstock-Guttman B, Waldman A,  Banwell BL, Bar Or A,  Marrie RA, van Domselaar G, O'Mahony J,  Mirza AI,  Bernstein CN,  Yeh EA,  Casper TC,  Lynch SV, Tremlett H, Baranzini S, Waubant E; US Network of Pediatric MS Centers. Short-chain fatty acid producers in the gut are associated with pediatric multiple sclerosis onset. Annals of Clinical and Translational Neurology 2023 Nov 13.

 

The relationship between multiple sclerosis and the gut microbiome has been supported by animal models in which commensal microbes are required for the development of experimental autoimmune encephalomyelitis. However, observational study findings in humans have only occasionally converged when comparing multiple sclerosis cases and controls which may in part reflect confounding by comorbidities and disease duration. The study of microbiome in pediatric-onset multiple sclerosis offers unique opportunities as it is closer to biological disease onset and minimizes confounding by comorbidities and environmental exposures. We undertook a multicenter case-control study in which 35 pediatric-onset multiple sclerosis cases were 1:1 matched to healthy controls on age, sex, self-reported race, ethnicity, and recruiting site. Linear mixed effects models, weighted correlation network analyses, and PICRUSt2 were used to identify microbial co-occurrence networks and for predicting functional abundances based on marker gene sequences. Results: Two microbial co-occurrence networks (one reaching significance after adjustment for multiple comparisons; q < 0.2) were identified, suggesting interdependent bacterial taxa that exhibited association with disease status. Both networks indicated a potentially protective effect of higher relative abundance of bacteria observed in these clusters. Functional predictions from the significant network suggested a contribution of short-chain fatty acid producers through anaerobic fermentation pathways in healthy controls. Consistent family-level findings from an independent Canadian-US study (19 case/control pairs) included Ruminococaccaeae and Lachnospiraceae (p < 0.05). Macronutrient intake was not significantly different between cases and controls, minimizing the potential for dietary confounding. Conclusions: Our results suggest that short-chain fatty acid producers may be important contributors to multiple sclerosis onset.

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Eckenberger J, Butler J, Bernstein CN, Shanahan F, Claesson M. Interactions between medications and the gut microbiome in inflammatory bowel disease. Microorganisms 2022; 10:1-18.

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In view of the increasing evidence that commonly prescribed, non-antibiotic drugs interact with the gut microbiome, we re-examined the microbiota variance in inflammatory bowel disease (IBD) to determine the degree to which medication and supplement intake might account for compositional differences between disease-subtypes and geographic location. We assessed the confounding effects of various treatments on the faecal microbiota composition (16S rRNA gene sequencing) in persons with Crohn’s disease (n=188) or ulcerative colitis (n = 161) from either Cork (Ireland) or Manitoba (Canada) sampled at 3 time points. We confirmed the overall trends of significant differences of microbial composition and diversity across IBD-subtypes and geographic locations from our previous study. Treatments explained more microbiota variance than all other factors combined and more than half of the tested medications and supplements showed significant associations with at least one taxon in the gut microbiota. The medication profiles between patients with Crohn’s disease and ulcerative colitis and from different countries varied in number and type of drugs taken. While treatments accounted for a relatively small proportion of the geographic contribution to microbiome variance between Irish and Canadian patients, additive effects from multiple medications contributed significantly to microbiome differences between Crohn’s disease and ulcerative colitis.

 

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Mirza AI, Zhu F, Knox N, Forbes JD, Bonner C, Van Domselaar G, Bernstein CN, Graham M, Marrie RA, Hart J, Yeh EA, Arnold DL, Bar-Or A, O’Mahony J, Zhao Y, Hsiao W, Banwell B, Waubant E, Tremlett H. The metabolic potential of the paediatric-onset multiple sclerosis gut microbiome.  Multiple Sclerosis and Related Disorders 2022; Jul 63: 103829.

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The aim of this study was to examine the gut microbiome's metabolic potential in paediatric-onset multiple sclerosis patients (symptom onset <18 years).  We included 17 multiple sclerosis participants and 20 controls similar for sex, age, race, and stool consistency from the Canadian Paediatric Demyelinating Disease Network study. Stool-derived gut metagenome gene abundances were used to estimate relative abundances and turnover scores of individual microbial metabolites and the composition and diversity of carbohydrate-active enzymes (CAZymes). Multiple sclerosis participants and controls were compared using the Wilcoxon rank-sum test, as were the disease-modifying drug exposed and naïve multiple sclerosis participants. Results: The median age(s) at multiple sclerosis symptom onset=16.1 years (interquartile range [IQR]=1.7), and at stool sample procurement=16.9/15.8 years (IQR=2.0/1.4), for the multiple sclerosis participants/controls. Most multiple sclerosis and control participants were girls (80-82%). Five (29%) of the multiple sclerosis participants had never been exposed to a disease-modifying drug pre-stool sample and 12 (71%) had (7 to beta-interferon and 5 glatiramer acetate). While the relative abundance of metabolites did not differ between multiple sclerosis participants and controls, turnover scores did. Multiple sclerosis participants had a greater potential to metabolize lipopolysaccharides than controls (score difference=1.6E-04, p = 0.034) but lower potential to metabolize peptidoglycan molecules and starch (score differences<2.2E-02, p<0.040). Further, although CAZymes diversity did not differ (p>0.050), starch-degrading subfamilies were underrepresented in MS participants versus controls (relative abundance differences >-0.34, p<0.040) and in the disease-modifying drug exposed verses disease-modifying drug naïve multiple sclerosis participants (relative abundance differences>-0.20, p<0.049). Conclusion: Paediatric-onset multiple sclerosis participants had an altered gut microbiome-related metabolic potential compared to controls, including higher breakdown of lipopolysaccharide molecules, but lower resistant starch metabolism.

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Leibovitzh H, Lee SH, Xue M, Raygoza Garay JA, Hernandez-Rocha C, Madsen KL, Meddings JB, Guttman DS, Espin-Garcia O, Smith M, Goethel M, Griffiths AM, Moayyedi P, Steinhart AH, Panancionne R, Huynh H, Jacobson K, Aumais G, Mack D, Abreu M, Bernstein CN, Marshall JK, Turner D, Xu W, Turpin W, Croitoru K, on behalf of the Crohn’s and Colitis Canada Genetics Environment Microbial Project Research Consortium. Altered gut microbiome composition and function are associated with gut barrier dysfunction in healthy relatives of patients with Crohn’s disease. Gastroenterology 2022; 163:1364-1376.

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The gut microbiome has been suggested to play a role in gut barrier hemostasis, but data are scarce and limited to animal studies. We therefore aimed to assess whether alterations in gut microbial composition and functional pathways are associated with gut barrier function in a cohort of healthy first-degree relatives of patients with Crohn's disease. Methods: We used the Crohn's and Colitis Canada Genetic Environmental Microbial (CCC-GEM) cohort of healthy first-degree relatives of patients with Crohn's disease. Gut barrier function was assessed using the urinary fractional excretion of lactulose-to-mannitol ratio (LMR). Microbiome composition was assessed by sequencing fecal 16S ribosomal RNA. The cohort was divided into a discovery cohort (n = 2472) and a validation cohort (n = 655). A regression model was used to assess microbial associations with the LMR. A random forest classifier algorithm was performed to assess microbial community contribution to barrier function. Results: Individuals with impaired barrier function (LMR >0.025) had reduced alpha-diversity (Chao1 index, P = 4.0e-4) and altered beta-diversity (Bray-Curtis dissimilarity index, R2 = 0.001, P = 1.0e-3) compared with individuals with an LMR ≤0.025. When taxa were assessed individually, we identified 8 genera and 52 microbial pathways associated with an LMR >0.025 (q < 0.05). Four genera (decreased prevalence of Adlercreutzia, Clostridia UCG 014, and Clostridium sensu stricto 1 and increased abundance of Colidextribacter) and 8 pathways (including decreased biosynthesis of glutamate, tryptophan, and threonine) were replicated in the validation cohort. The random forest approach revealed that the bacterial community is associated with gut barrier function (area under the curve, 0.63; P = 1.4e-6). Conclusions: The gut microbiome community and pathways are associated with changes in gut barrier function. These findings may identify potential microbial targets to modulate gut barrier.

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Liang G, Zhu F, Mirza AI, Arnold D, Bar-Or A, Bernstein CN, Bonner C, Forbes JD, Graham M, Hart J, Knox NC, Marrie RA, Mahony JO, Van Domselaar G, Yeh EA, Zhao Y, Banwell B, Waubant E, Tremlett H, and the Canadian Paediatric Demyelinating Disease Network. Stability of the gut microbiota in persons with pediatric-onset multiple sclerosis and related demyelinating diseases. Multiple Sclerosis Journal 2022; 28:1819-1824.

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We established a biobank of stool from children with pediatric onset multiple sclerosis and monophasic-acquired demyelinating syndromes (monoADS) and unaffected controls. In this study we examined if the gut microbiota composition changes across repeated samples in paediatric-onset multiple sclerosis (MS) or monophasic-acquired demyelinating syndromes (monoADS). A total of 36 individuals (18 MS/18 monoADS) with at least 2 stool samples were included. Stool sample-derived DNA was sequenced. Alpha/beta diversities and genus-level taxa were analysed. Results: Mean ages at first sample procurement (MS/monoADS) = 18.0/13.8 years. Median time (months) between first/second samples = 11.2 and second/third = 10.3. Alpha/beta diversities did not differ between stool samples (p > 0.09), while one genus - Solobacterium did (p = 0.001). Conclusions: The gut microbiota composition in paediatric-onset MS and monoADS exhibited stability, suggesting that single stool sample procurement is a reasonable first approach.

 

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Mirza AI, Zhu F, Knox N, Forbes JD, Van Domselaar G, Bernstein CN, Graham M, Marrie RA, Hart, J Yeh EA, Arnold D, Bar-Or A, O’Mahony J, Zhao Y, Hsiao W, Banwell B, Waubant E, Tremlett H. Metagenomic analysis of the pediatric-onset multiple sclerosis gut microbiome. Neurology 2022; 98(10): e1050-e1063.

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Little is known of the functional potential of the gut microbiome in pediatric-onset multiple sclerosis. More research is being done on the gut microbiome on non-gastrointestinal diseases, like multiple sclerosis. We performed metagenomic analyses using stool samples from individuals with pediatric-onset multiple sclerosis and unaffected controls. Persons 21 years old or younger enrolled in the Canadian Pediatric Demyelinating Disease Network providing a stool sample were eligible. Twenty patients with multiple sclerosis (McDonald criteria) with symptom onset under age 18 years were matched to 20 controls by sex, age (±3 years), stool consistency, and race. Microbial taxonomy and functional potentials were estimated from stool sample-derived metagenomic reads and compared by disease status (multiple sclerosis vs controls) and disease-modifying drug exposure using alpha diversity, relative abundance, and prevalence using Wilcoxon rank sum, ALDEx2, and Fisher exact tests, respectively. Results: Individuals with multiple sclerosis were aged 13.6 years (mean) at symptom onset and 8 were disease-modifying drug-naive. Mean ages at stool sample were 16.1 and 15.4 years for multiple sclerosis and control participants, respectively; 80% were girls. Alpha diversity of enzymes and proteins did not differ by disease or disease-modifying drug status (p > 0.20), but metabolic pathways, gene annotations, and microbial taxonomy did. Individuals with multiple sclerosis (vs controls) exhibited higher methanogenesis prevalence (odds ratio 10, p = 0.044) and Methanobrevibacter abundance (log2 fold change [LFC] 1.7, p = 0.0014), but lower homolactic fermentation abundance (LFC -0.48, p = 0.039). Differences by disease-modifying drug status included lower phosphate butyryl transferase for disease-modifying drug-naive vs exposed patients with multiple sclerosis (LFC -1.0, p = 0.033).  Discussion: The gut microbiome's functional potential and taxonomy differed between individuals with pediatric-onset multiple sclerosis vs controls, including higher prevalence of a methane-producing pathway from Archaea and depletion of the lactate fermentation pathway. Disease-modifying drug exposure was associated with butyrate-producing enzyme enrichment. Together these findings indicate that the gut microbiome of individuals with multiple sclerosis may have a disturbed functional potential.

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Pratt M, Forbes JD, Knox NC, Van Domselaar G, Bernstein CN. Colorectal cancer screening in IBD - can characterization of GI microbiome signatures enhance neoplasia detection? Gastroenterology 2022. Jan 5: S0016-5085.

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Current non-invasive colorectal cancer screening methods are not optimized for persons with IBD, requiring patients to undergo frequent interval screening via colonoscopy. Although colonoscopy-based screening reduces colorectal cancer incidence in IBD patients, rates of interval colorectal cancer remain relatively high, highlighting the need for more targeted approaches. In recent years, the discovery of disease-specific microbiome signatures for both IBD and colorectal cancer has begun to emerge, suggesting that stool-based biomarker detection using metagenomics and other culture-independent technologies may be useful for personalized, early, non-invasive colorectal cancer screening in IBD patients. Here we discuss the utility of the stool microbiome as a non-invasive colorectal cancer screening tool. Comparing the performance of multiple microbiome-based colorectal cancer classifiers, including several multi-cohort meta-analyses, we find that non-invasive detection of colorectal adenomas and carcinomas from microbial biomarkers is an active area of study with promising early results.

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Pratt M, Forbes JD, Knox NC, Bernstein CN, Van Domselaar G. Microbiome-mediated immune signaling in inflammatory bowel disease and colorectal cancer: support from meta-omics data. Frontiers in Cell and Developmental Biology 2021; Nov 16;9:716604.

 

Chronic intestinal inflammation and microbial dysbiosis are hallmarks of colorectal cancer and IBD. However, the mechanistic relationship between gut dysbiosis and disease has not yet been fully characterized. Although the "trigger" of intestinal inflammation remains unknown, a wealth of evidence supports the role of the gut microbiome as a mutualistic pseudo-organ that significantly influences intestinal homeostasis and is capable of regulating host immunity. In recent years, culture-independent methods for assessing microbial communities as a whole (termed meta-omics) have grown beyond taxonomic identification and genome characterization (metagenomics) into new fields of research that collectively expand our knowledge of microbiomes. Metatranscriptomics, metaproteomics, and metabolomics are meta-omics techniques that aim to describe and quantify the functional activity of the gut microbiome. Uncovering microbial metabolic contributions in the context of IBD and colorectal cancer using these approaches provides insight into how the metabolic microenvironment of the GI tract shapes microbial community structure and how the microbiome, in turn, influences the surrounding ecosystem. Immunological studies in germ-free and wild-type mice have described several host-microbiome interactions that may play a role in autoinflammation. Chronic colitis is a precursor to colorectal cancer, and changes in the gut microbiome may be an important link triggering the neoplastic process in chronic colitis. In this review, we describe several microbiome-mediated mechanisms of host immune signaling, such as short-chain fatty acid (SCFA) and bile acid metabolism, inflammasome activation, and cytokine regulation in the context of IBD and colorectal cancer, and discuss the supporting role for these mechanisms by meta-omics data.

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Tremlett H, Zhu F, Arnold D, Bar-Or A, Bernstein CN, Forbes JD, Graham M, Hart J, Knox NC, Marrie RA, Mirza A, O’Mahony J, Van Domselaar G, Yeh EA, Zhao Y, Banwell B, Waubant E, and the US Network of Pediatric MS Centers and the Canadian Paediatric Demyelinating Disease Network. The gut microbiota in pediatric multiple sclerosis and demyelinating syndromes. Annals of Clinical and Translational Neurology 2021; 8(12):2252-2269.

 

This study examined the gut microbiota in individuals with and without pediatric-onset multiple sclerosis (MS). Stool samples were collected across the Canadian Pediatric Demyelinating Disease Network and banked at the University of Manitoba IBD Clinical and Research Centre Biobank. Microbiome analysis was undertaken at the National Microbiology Laboratory, Winnipeg, Canada. Study participants were 21 years old or younger, with MS (disease-modifying drug [DMD] exposed and naïve) or monophasic acquired demyelinating syndrome [monoADS] (symptom onset prior to age 18 years), and unaffected controls. All were at least 30 days without antibiotics or corticosteroids. V4 region 16S RNA gene-derived amplicon sequence variants (Illumina MiSeq) were assessed using negative binomial regression and network analyses; rate ratios were age- and sex-adjusted (aRR). There were 32 MS, and 41 monoADS and 36 control participants Although microbiota diversity (alpha, beta) did not differ between participants (p > 0.1), taxa-level and gut community networks did. MS (vs. monoADS) exhibited > fourfold higher relative abundance of the superphylum Patescibacteria (aRR = 4.2;95%CI:1.6-11.2, p = 0.004, Q = 0.01), and lower abundances of short-chain fatty acid (SCFA)-producing Lachnospiraceae (Anaerosporobacter) and Ruminococcaceae (p, Q < 0.05). DMD-naïve MS cases were depleted for Clostridiales vadin-BB60 (unnamed species) versus either DMD-exposed, controls (p, Q < 0.01), or monoADS (p = 0.001, Q = 0.06) and exhibited altered community connectedness (p < 10-9 Kruskal-Wallis), with SCFA-producing taxa underrepresented. Consistent taxa-level findings from an independent US Network of Pediatric MS Centers case/control (n = 51/42) cohort included >eightfold higher abundance for Candidatus Stoquefichus and Tyzzerella (aRR = 8.8-12.8, p < 0.05) in MS cases and 72%-80% lower abundance of SCFA-producing Ruminococcaceae-NK4A214 (aRR = 0.38-0.2, p ≤ 0.01). 

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We concluded that the gut microbiota community structure, function and connectivity, and not just individual taxa, are of likely importance in MS.

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Clooney AG, Eckenberger J, Laserna-Mendieta EJ, Sexton KA, Bernstein MT, Vagianos K, Sargent M, Moran C, Sheehan D, Sleator RD, Targownik LE, Bernstein CN, Shanahan F, Claesson MJ. Ranking microbiome variance in inflammatory bowel disease: A large longitudinal intercontinental study. Gut 2021; Mar;70(3):499-510. 

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The microbiome contributes to the pathogenesis of IBD but the relative contribution of different lifestyle and environmental factors to the compositional variability of the gut microbiota is unclear. In this study we rank the size effect of disease activity, medications, diet and geographic location of the faecal microbiota composition (16S rRNA gene sequencing) in patients with Crohn's disease (CD; n=303), ulcerative colitis (UC; n = 228) and controls (n=161), followed longitudinally (at three time points with 16 week intervals). This study was conducted in persons from Ireland and persons from Manitoba. Reduced microbiota diversity but increased variability was confirmed in CD and UC compared with controls. Significant compositional differences between diseases, particularly CD, and controls were evident. Longitudinal analyses revealed reduced temporal microbiota stability in IBD, particularly in patients with changes in disease activity. Machine learning separated disease from controls, and active from inactive disease, when consecutive time points were modelled. Geographic location accounted for most of the microbiota variance, second to the presence or absence of CD, followed by history of surgical resection, alcohol consumption and UC diagnosis, medications and diet with most (90.3%) of the compositional variance stochastic or unexplained.

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We concluded that the popular concept of precision medicine and rational design of any therapeutic manipulation of the microbiota will have to contend not only with the heterogeneity of the host response, but also with widely differing lifestyles and with much variance still unaccounted for.

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Sood A, Singh A, Midha V Mahajan R, Kao D, Rubin D, Bernstein CN. Faecal Microbiota Transplantation; for Ulcerative Colitis: An Evolving Therapy. Crohn’s and Colitis 360 2020; in press. 

 

Fecal microbiota transplantation is an established treatment for recurrent and refractory Clostridioides difficile infection. However, its use in inflammatory bowel disease, ulcerative colitis in particular, is at an early stage and significant gaps remain in our understanding of the mechanisms and logistics of practical application. This article aims to assess unsettled issues for fecal microbiota transplantation in UC. Experts with experience in different modes of fecal microbiota transplantation reviewed the published and grey scientific literature to synthesize information regarding methods of performance and outcomes for fecal microbiota transplantation. This includes proposed mechanisms of action, donor and recipient selection, route of administration, need for maintenance therapy, impact of diet, response assessments, outcomes from randomized controlled trials and the current state of fecal microbiota transplantation regulations. The science of the gut microbiome is still rudimentary and clinical use of fecal microbiota transplantation outside clinical trials is not recommended at the moment. Current methods of performing fecal microbiota transplantation are crude and primordial.  Advancements in fecal slurry preparation and mode of delivery are necessary. It remains to be seen whether shifting to selective microbiota transplantation tailored according to a particular disease can substitute the whole stool; or whether donors and recipients should be matched for genotype, diet, or environment. We recommend the term fecal microbiota transplantation should be replaced by intestinal microbiome transfer (IMT).

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The future holds much promise for the potential applications of this approach to management of ulcerative colitis, however more research refining the technique is warranted before it can become an accepted clinical practice.

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Khafipour A, Eissa N, Munyaka PM, Rabbi M, Kapoor K, Kermarrec, L Khafipour E, Bernstein CN, Ghia JE. Denosumab regulates gut microbiota composition and cytokines in dinitrobenzene sulfonic acid (DNBS)-experimental colitis. Frontiers in Microbiology 2020 Jun 25;11:1405. 

 

The pro-inflammatory mediator receptor activator of nuclear factor-kappa B ligand (RANKL) plays a significant role in the development of rheumatoid arthritis; however, its role in IBD is unknown. Genome-wide association meta-analysis for Crohn's disease identified a variant near the TNFSF11 gene that encodes RANKL and Crohn's disease risk allele increased expression of RANKL in specific cell lines. This study aims to elucidate if the RANKL inhibitor denosumab can reduce the severity of experimental colitis and modify the gut microbiota composition using murine dinitrobenzenesulfonic acid (DNBS)-experimental model of colitis mimicking Crohn's disease. In colitic conditions, denosumab treatment significantly decreased the pro-inflammatory cytokines IL-6, IL-1β, and TNF-α within the colonic mucosa. Moreover, colitis was accompanied by disruption of gut microbiota, and preventative treatment with denosumab modulated this disruption. Denosumab treatment also modified the alpha- and beta diversity of colonic mucosa and fecal microbiota. These results provide a rationale for considering denosumab as a future potential therapy in Crohn's disease; however, more detailed experimental and clinical studies are warranted.

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Shaffer SR, Witt J, Targownik LE, Kao D, Lee C, Smielauskas F, Rubin DT, Singh H, Bernstein CN. Cost-effectiveness Analysis of a Fecal Microbiota Transplant Center for Treating Recurrent C.difficile Infection. Journal of Infection 2020; 81:758-65.

 

We assessed the cost-effectiveness of establishing a fecal microbial transplant (FMT) unit in Canada for the treatment of recurrent C.difficile infection. We performed a cost-effectiveness analysis to determine the number of patients with recurrent C.difficile infection needed to treat (NNT) annually to make establishing a FMT unit cost-effective. We compared treating patients for their second recurrence of C.difficile infection with FMT in a jurisdiction with a FMT unit, compared to being treated with antibiotics; then sent to a medical center with FMT available for the third recurrence. We used a willingness to pay threshold of $50,000 per quality-adjusted-life-year gained. The minimum annual NNT was 15 for FMT via colonoscopy, 17 for FMT via capsule, and 44 for FMT via enema compared with vancomycin, and 16, 18, and 47 compared with fidaxomicin, respectively. A medical center's minimum catchment area when establishing a FMT unit would have to be 56,849 if using FMT via colonoscopy, or 64,429 if using capsules.

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We report the minimum number of patients requiring treatment annually with FMT to achieve cost-effectiveness, when including start-up and ongoing costs. FMT is cost-effective in Canada in populations with a sufficient number of eligible patients, ranging from 15 to 47 depending on the FMT modality used. This is crucial for medical jurisdictions making decisions about establishing a FMT unit for the treatment of recurrent C.difficile infection. The cost-effectiveness can be generalized in other countries.

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Shaffer S, Nugent Z, Walkty A, Yu BN, Lix LM Targownik LE, Kao D, Bernstein CN,  Singh H. Time trends and predictors of laboratory confirmed recurrent and severe C.difficile infections in Manitoba: a population-based study. CMAJ Open 2020; Nov 16;8(4):E737-E746. 

 

Many previous studies of Clostridioides difficile infection epidemiology have used hospital discharge data codes, which can have limited accuracy. We used a data set of laboratory-confirmed cases of Clostridioides difficile infection in the province of Manitoba, Canada, to describe the epidemiology of Clostridioides difficile infection over a decade. We conducted a population-based historical cohort study using Manitoba Health's population-wide laboratory-based Clostridioides difficile infection data set linked to administrative health databases. All individuals living in Manitoba and experiencing a Clostridioides difficile infection episode between 2005 and 2015 were included (n = 8471) and followed up from Clostridioides difficile infection diagnosis. We assessed time trends of Clostridioides difficile infection, incidence and predictors of recurrence and severe outcomes, and health care encounters after Clostridioides difficile infection diagnosis. Clostridioides difficile infection episodes were stratified by community versus hospital site of acquiring Clostridioides difficile infection. Between 2005 and 2009, overall Clostridioides difficile infection diagnoses decreased by an average of 12.6% per year (95% confidence interval [CI] -4.4 to -20.0), with no statistically significant change from 2010 to 2015. In stratified analysis, incident and recurrent Clostridioides difficile infection had a similar decrease in the initial study time period and then stabilized. The proportion of community-associated Clostridioides difficile infection cases increased by an average of 4.8% per year (95% CI 2.8 to 6.8) during the study period. Clostridioides difficile infection acquired in a health care facility had a higher recurrence rate and more severe outcomes. Recurrence of CDI increased the likelihood of admission to hospital.

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Between 2005 and 2015, the rates of overall laboratory-confirmed Clostridioides difficile infection, incident Clostridioides difficile infection, recurrent Clostridioides difficile infection and severe outcomes following Clostridioides difficile infection initially decreased before stabilizing, and an increasing proportion of Clostridioides difficile infection cases were community-associated. There is an increasing need to test for Clostridioides difficile infection among outpatients with diarrhea and to increase efforts to prevent recurrent Clostridioides difficile infection.

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Szamosi JC, Forbes JD, Copeland JK, Knox, NC, Shekarriz S, Rossi, L, Graham M, Bonner C, Guttman, DS, Van Domselaar, G, Surette MG, Bernstein CN. Assessment of inter-laboratory variation in the characterization and analysis of the mucosal microbiota in Crohn’s disease and ulcerative colitis. Frontiers in Microbiology 2020; 11: e2072. 

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In studies evaluating the microbiome, numerous factors can contribute to technical variability. These factors include DNA extraction methodology, sequencing protocols, and data analysis strategies. We sought to evaluate the impact these factors have on the results obtained when the sequence data are independently generated and analyzed by different laboratories. To evaluate the effect of technical variability, we used human intestinal biopsy samples resected from individuals diagnosed with IBD, including Crohn's disease ( = 12) and ulcerative colitis ( = 10), and those without IBD ( = 10). Matched samples from each participant were sent to three laboratories and studied using independent protocols for DNA extraction, library preparation, targeted-amplicon sequencing of a 16S rRNA gene hypervariable region, and processing of sequence data. We looked at two measures of interest - Bray-Curtis PERMANOVA 2 values and log2 fold-change estimates of the 25 most-abundant taxa - to assess variation in the results produced by each laboratory, as well the relative contribution to variation from the different extraction, sequencing, and analysis steps used to generate these measures. The 2 values and estimated differential abundance associated with diagnosis were consistent across datasets that used different DNA extraction and sequencing protocols, and within datasets that pooled samples from multiple protocols; however, variability in bioinformatic processing of sequence data led to changes in 2 values and inconsistencies in taxonomic assignment and abundance estimates.

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We concluded that although the contribution of DNA extraction and sequencing methods to variability were observable, we find that results can be robust to the various extraction and sequencing approaches used in our study. Differences in data processing methods have a larger impact on results, making comparison among studies less reliable and the combined analysis of bioinformatically processed samples nearly impossible. Our results highlight the importance of making raw sequence data available to facilitate combined and comparative analyses of published studies using common data processing protocols. Study methodologies should provide detailed data processing methods for validation, interpretability, reproducibility, and comparability.

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Bernstein CN. Is antibiotic use a cause of IBD worldwide? Inflammatory Bowel Diseases 2020; 26: 448-449.

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This editorial reviewed the evidence for antibiotics as a possible culprit in triggering IBD onset.

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Knox N, Forbes JD, Van Domselaar, Bernstein CN.  The gut microbiome in other chronic immune disease: lessons for IBD. American Journal of Gastroenterology 2019; 114:1051-70.

 

There is a growing appreciation for the role of the gut microbiome in human health and disease. Aided by advances in sequencing technologies and analytical methods, recent research has shown the healthy gut microbiome to possess considerable diversity and functional capacity. Dysbiosis of the gut microbiota is believed to be involved in the pathogenesis of not only diseases that primarily affect the gastrointestinal tract, but of other less obvious diseases, including neurologic, rheumatologic, metabolic, hepatic, and other illnesses. Chronic immune-mediated inflammatory diseases represent a group of diseases that share many underlying etiological factors including genetics, aberrant immunological responses, and environmental factors. Gut dysbiosis has been reported to be common to immune-mediated inflammatory diseases as a whole, and much effort is currently being directed towards elucidating microbiome-mediated disease mechanisms and their implications for causality. In this review, we discuss gut microbiome studies in several immune-mediated inflammatory diseases and show how these studies can inform our understanding of the role of the gut microbiome in inflammatory bowel disease.

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Knox N, Forbes JD, Van Domselaar, Bernstein CN. The gut microbiome as a target for IBD treatment: are we there yet? Current Treatment Options in Gastroenterology 2019; 17(1):115-126.

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This review aims to highlight recent research on the gut microbiome in IBD and the application of microbiome-modulating therapies for the treatment of IBD including the use of the microbiome as an indicator for disease severity and treatment response. Despite the high number of gut microbiome studies and emerging evidence supporting the gut microbiome's involvement in disease pathogenesis, no single microorganism has been identified as a pathogenic agent in IBD. Retrospective studies and meta-analyses on antibiotic use in ulcerative colitis and Crohn's disease and long-term outcomes are conflicting. Similarly, the use of probiotics for the treatment of IBD remains inconclusive; however, some encouraging results are emerging as microbial concoctions are optimized to include beneficial bacterial strains. Fecal microbial transplantation is currently emerging as one of the more promising microbiome-modulating IBD therapies. Fecal microbial transplantation studies in ulcerative colitis have shown improved remission rates compared to placebo; however, relatively small study sample sizes and varied treatment methods, limit definitive conclusions. With clear evidence of an IBD gut dysbiosis, novel therapies to treat and prevent disease relapse will undoubtedly require a microbiome-modulating approach. The complexity and variability of IBD disease pathogenesis (disease phenotype, gut microbiome, host genetic susceptibility, and environmental factors) will likely require a personalized and multidimensional treatment approach where microbiome-modulating therapy is coupled with other therapies to target other IBD disease components.

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Forbes JD, Bernstein CN, Tremlett H, Van Domselaar G, Knox NC. A fungal world: could the gut mycobiome be involved in neurological disease. Frontiers in Microbiology 2019; pages 1-13: doi:10.3389/fmicb.2018.03249.

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The human microbiome has received decades of attention from scientific and medical research communities. The human gastrointestinal tract is host to immense populations of microorganisms including bacteria, viruses, archaea, and fungi (the gut microbiota). High-throughput sequencing and computational advancements provide unprecedented ability to investigate the structure and function of microbial communities associated with the human body in health and disease. Most research to date has largely focused on elucidating the bacterial component of the human gut microbiota. Study of the gut "mycobiota," which refers to the diverse array of fungal species, is a relatively new and rapidly progressing field. Though omnipresent, the number and abundance of fungi occupying the human gut is orders of magnitude smaller than that of bacteria. Recent insights however, have suggested that the gut mycobiota may be intricately linked to health and disease. Evaluation of the gut mycobiota has shown that not only are the fungal communities altered in disease, but they also play a role in maintaining intestinal homeostasis and influencing systemic immunity. In addition, it is now widely accepted that host-fungi and bacteria-fungi associations are critical to host health. While research of the gut mycobiota in health and disease is on the rise, little research has been performed in the context of neuroimmune and neurodegenerative conditions. Gut microbiota dysbiosis (specifically bacteria and archaea) have been reported in neurological diseases such as multiple sclerosis, amyotrophic lateral sclerosis, and Alzheimer's, among others. Given the widely accepted bacteria-fungi associations and paucity of mycobiota-specific studies in neurological disease, this review discusses the potential role fungi may play in multiple sclerosis and other neurological diseases. Herein, we provide an overview of recent advances in gut mycobiome research and discuss the plausible role of both intestinal and non-intestinal fungi in the context of neuroimmune and neurodegenerative conditions.

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Forbes JD, Chen CY, Knox NC, Marrie RA, EL Gabalawy H, de Kevit T, Alfa M, Bernstein CN. Van Domselaar G. A comparative study of the gut microbiota in immune-mediated inflammatory diseases - does a common dysbiosis exist? Microbiome 2018 Dec 13: 6 (1): 221: 1-15.

 

Immune-mediated inflammatory disease represents a substantial health concern. It is widely recognized that immune-mediated inflammatory disease patients are at a higher risk for developing secondary inflammation-related conditions. While an ambiguous etiology is common to all immune-mediated inflammatory diseases, in recent years, considerable knowledge has emerged regarding the plausible role of the gut microbiome in immune-mediated inflammatory diseases. This study used 16S rRNA gene amplicon sequencing to compare the gut microbiota of patients with Crohn's disease (N = 20), ulcerative colitis (N = 19), multiple sclerosis (N = 19), and rheumatoid arthritis (N = 21) versus healthy controls (N = 23). Biological replicates were collected from participants within a 2-month interval. This study aimed to identify common (or unique) taxonomic biomarkers of immune-mediated inflammatory disease s using both differential abundance testing and a machine learning approach. Significant microbial community differences between cohorts were observed. Richness and diversity were significantly different between cohorts and were lowest in Crohn’s disease while highest in healthy controls. Abundances of Actinomyces, Eggerthella, Clostridium III, Faecalicoccus, and Streptococcus were significantly higher in all disease cohorts relative to healthy controls, whereas significantly lower abundances were observed for Gemmiger, Lachnospira, and Sporobacter. Several taxa were found to be differentially abundant in immune-mediated inflammatory diseases versus healthy controls including significantly higher abundances of Intestinibacter in Crohn’s disease, Bifidobacterium in UC, and unclassified Erysipelotrichaceae in multiple sclerosis and significantly lower abundances of Coprococcus in Crohn’s disease, Dialister in multiple sclerosis, and Roseburia in rheumatoid arthritis. A machine learning approach to classify disease versus healthy controls was highest for Crohn’s disease followed by multiple sclerosis, rheumatoid arthritis and UC. Gemmiger and Faecalicoccus were identified as important features for classification of subjects to Crohn’s disease and health controls. In general, features identified by differential abundance testing were consistent with machine learning feature importance. This study identified several gut microbial taxa with differential abundance patterns common to immune-mediated inflammatory diseases. We also found differentially abundant taxa between immune-mediated inflammatory diseases. These taxa may serve as biomarkers for the detection and diagnosis of immune-mediated inflammatory diseases and suggest there may be a common component to immune-mediated inflammatory diseases etiology.

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Bernstein CN, Forbes J. Gut microbiome in inflammatory bowel disease and other chronic immune-mediated inflammatory diseases. Intestinal Inflammatory Diseases 2017; 2: 116-123.

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We undertook a review of the current scientific research on the gut microbiome not just in IBD but also in other chronic inflammatory diseases (such as rheumatoid arthritis and multiple sclerosis). Our report in frontiers in Microbiology was one of the highest cited papers in that journal for 2016. In our report in Inflammatory Intestinal Diseases we reviewed what could be learned in regards to IBD by studying the gut microbiome of non gut chronic inflammatory diseases (such as rheumatoid arthritis, multiple sclerosis, psoriasis and ankylosing spondylitis. We also reported on some of our own research comparing the gut microbiome from persons wit IBD, rheumatoid arthritis and multiple sclerosis and healthy controls.

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Forbes J, Van Domselaar G, Bernstein CN. The gut microbiota in immune-mediated inflammatory diseases. Frontiers in Microbiology 2016; 7:1081.

 

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Forbes J, Van Domselaar G, Bernstein CN. Microbiome survey of the inflamed and non-inflamed gut at different compartments within the gastrointestinal tract of inflammatory bowel disease patients. Inflammatory Bowel Diseases 2016; 22:817-25.

 

We compared the bacterial communities of inflamed and noninflamed bowel lining of patients with IBD and by analysis of the microbiota composition at distinct areas of the ileum and colon. There was no variation in any bacteria comparing inflamed to non-inflamed areas of the bowel within Crohn’s disease or within ulcerative colitis. The main differences were in comparing the non-inflamed mucosa between Crohn’s disease and ulcerative colitis: Groups (phyla) of organisms known as Bacteroidetesand Fusobacteria were detected more frequently in inflamed Crohn’s disease areas than in inflamed ulcerative colitis mucosa. Conversely, Proteobacteria and Firmicutes were more frequently observed in the inflamed ulcerative colitis mucosa. At the genus level (a smaller group of bacteria than phyla) the abundance of 13 groups were significantly different within the non-inflamed mucosa between Crohn’s disease and ulcerative colitis.

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Forbes J, Van Domselaar G, Sargent M, Green C, Springthorpe S, Krause D, Bernstein CN. Microbiome profiling of drinking water in relation to incidence of inflammatory bowel disease. Canadian Journal of Microbiology 2016; 18:1-13.

 

For one year we collected water from firehalls and community centres from over 20 communities in Winnipeg and in rural Manitoba. We aimed for areas that were known to have either high or low incidence of IBD. We wanted to determine if there were bacteria in the water that were unique to high or low incidence areas or missing from high or low incidence areas so as to potentially target those bacteria for treatment of IBD. This work lays out a basis for further studies exploring water as a potential environmental source for IBD triggers. 

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See Epidemiology Studies tab for other studies related to gut microbiome and risk for IBD