Gut Microbiome //

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).

Publications

  

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. 

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.

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.

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).

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.

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.

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.

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.

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.

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.

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. 

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.

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.

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

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

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.

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.

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.

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.

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.

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.

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

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.

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

 

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.

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. 

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