Advanced Metagenomic Analysis of Human Colonic Microbiota in Patients With Chronic GI Disorders

Advanced Metagenomic Analysis of Human Colonic Microbiota in Patients With Chronic Gastrointestinal Disorders (IBS, IBD, CRC)

This clinical trial hypothesize that Gut Microbiota (bacteria, viruses, fungi)play a major role in the occurrence and progression of many chronic gastrointestinal diseases like Irritable Bowel Syndrome, Inflammatory Bowel Diseases and Colo-Rectal Cancer.

Hence, aims to study the spectrum of such microbiota in these patients as compared to normal subjects, by utilizing metagenomic techniques rather than cultural methods.

The terms intestinal "microflora" or "microbiota" refer to the microbial ecosystem colonizing the gastrointestinal tract. Recently developed molecular biology instruments suggest that a substantial part of bacterial communities within the human gut still have to be described. Intestinal bacteria play an essential role in the development and homeostasis of the immune system. Most of these important microbiota are unculturable which significantly have limited our understanding of bacterial-host crosstalk. Among the methods designed to gain access to the physiology and genetics of uncultured organisms, metagenomics, the genomic analysis of a population of microorganisms, has emerged as a powerful technology. Metagenomics is the study of genomic content in a complex mixture of microorganisms. Direct isolation of genomic DNA from an environment circumvents culturing the organisms under study. The two primary goals of this approach are to develop a consensus of what populations of microorganisms are present and then to identify what roles each microorganism has within a specific environment. Metagenomics samples are found nearly everywhere, including several microenvironments within the human gut, soil samples, extreme environments such as deep mines and the various layers within the ocean. Therefore, the diversity of microorganisms is thought to be in the range of hundreds of millions to greater than tens of trillions of species. Among the most mysterious microenvironment is the human gastrointestinal tract that harbor greater than thousands of millions of microbial species (at least 1014), including up to 2000 species dominated by anaerobic bacteria.

Many of the gastrointestinal or even other diseases (metabolic as in obesity, or autoimmune as in allergies) involve primarily the human gut microbiota and then according to specific changes in microbiota equilibrium certain effects occur on either bowel motility (as in irritable bowel syndrome: IBS), homeostasis of the GI immune system (as in inflammatory bowel disease: IBD), or mucosal cells proliferation (as in adenoma - colorectal cancer: CRC). These chronic diseases affect all nations worldwide and represent a significant public health burden. They can be seen among children, adolescents, and adults. Currently there is no medical cure for IBS, IBD or CRC once they develop.

The complex interactions between microbial, genetic, immune, and environmental factors seem to play an important role in the pathogenesis of IBS, IBD and CRC. Lately, post infectious - IBS have gained increasing focus, to the extent that whole pathogenesis of IBS might be attributed to a specific triggering factor of microbiota imbalance. The prevailing theory is that IBD is related to an altered mucosal barrier with a deregulated immune response directed against specific modifications in the normal microbiota leading to the alteration of its equilibrium. The etiology of IBD can therefore be conceptualized as an aberrant immunologic response to a modified component or components of the gut microbiota potentially following an environmental insult. Likewise, CRC development process from normal mucosal surface to adenoma and finally to CRC; is probably related to gut microbiota.

The prevalence of these diseases has been documented to go through an obvious increase in Saudi Arabia during the last 3 decades. This would represent a unique model to study the role of GIT microbiota or their metagenomics and their modifications in response to environmental or dietary factors in this community that went into urbanization fairly recently, and then analyze their causative relations to the focus diseases.

Here, we propose to perform a comprehensive analysis of the gastrointestinal tract microbiota and its contribution on gut homeostasis in normal subjects and patients with IBS, IBD and CRC by using state of the art metagenomics technology. This will be done on a Saudi population sample that we believe represent a unique model.

Our specific objectives for this project are:

• Characterize the microbiota composition (microbes and virus) of the mucosa from Saudi patients with IBS, IBD and CRC.
• Characterize the mobile GI metagenomics of Saudi patients with IBS, IBD and CRC.
• Compare the metagenomics of IBS, IBD and CRC patients to each other and to normal subjects from the same population.

Expected outcomes and Significance of research:

Altogether, the results from aims 1 and 2 will provide for the first time a comprehensive and in-depth analysis of the mucosa-associated microbiota of adult patients with IBS, IBD and CRC in Saudi population. The proposed study will define a microbiota "fingerprint" for Saudi norms, IBS, IBD and CRC. The contribution of virome and the mobile metagenome into these diseases development and/or health maintenance will be assessed for the first time and thus has the potential to reveal new paradigms. In addition, the study of the virome and mobile metagenome will help us to understand the selective forces that could contribute to the alteration and evolution of the microbiota community and thus could have important implications for the treatment of the diseases. Certainly, the work proposed here will pave the way toward future hypothesis-driven research which could lead to the design of therapeutic strategies aimed at manipulating the microbial community.

Inclusion Criteria:

IBS: 50 consecutive patients presenting to KFMC GI service after launching the project on July 2010, will be recruited if they meet the following conditions:

1. Meet the diagnostic criteria as per ROME-II classification and as judged by experienced consultants for not less than 5 years.
2. IBS with diarrhea or constipation or mixed predominance pattern will be included.
3. Standard diagnostic tests have to be done to exclude any possible organic lesion to explain the abdominal pain, and all have to be negative.

IBD: from the KFMC GI service database, 50 Ulcerative Colitis (UC) and 50 Crohn's Disease (CD) will be selected according to following:

1. Confirmed diagnosis as CD or UC based on clinical, endoscopic and histological criteria.
2. Specifically did not use antibiotics for last 6 months before enrollment.
3. Detailed information about current treatment regimen has to be provided, and current use of 5-ASA, Steroid or Azathioprine will not hold from enrolling the patients as we cannot have patients off any one or more of these medications. (Obviously, we cannot exclude effects of these medications on microbiota, but it is not the focus of the current study).
4. None of the patients selected would be on anti-TNF medications.

CRC: 50 consecutive cases of confirmed CRC as per histological diagnosis made by 2 experienced pathologists will be included. Feasibility to do full colonoscopy before any surgical resection would be a condition to recruit any patient.

Normal Subjects: 50 normal control groups that are matched for age and sex to the other 3 disease groups will be selected from consecutive CRC screening colonoscopy subjects who get referred to KFMC GI service during the study period, and proved to have no GI disorders. 25 will be selected from Urban and 25 from Rural areas of KSA.

Exclusion Criteria:

1. Treatment with antibiotics for the last 6 months before enrollment.
2. Absence of recent infective colitis, bowel obstruction, or colonic/small intestinal resection surgery.
3. Not on any medication that may affect gut microbiota, like: cholestyramine, ursodeoxycholic acid, gut prokinetic agents.
4. Refusal to comply with the unified bowel preparation instructions for all cases.