Inflammatory bowel disease (IBD) encompasses a major group of chronic diseases of multi-factoral, largely unknown aetiology. It is therefore not possible to treat their causes, only their symptoms, nor to develop preventive measures. Knowledge of the aetiology is urgently needed. An increasing number of diseases of unknown origin are being demonstrated to have a microbial aetiology, but the problems of unculturable pathogens and pathogenic microbial communities, instead of the classical relationship: one readily culturable microbe: one disease, render the demonstration of causal relationships difficult.
Mucosal surfaces are covered with diverse, dynamic and connected microbial communities, which inimaltey interact with, on one side, the mucosal surface tissues and, on the other side, with the environment. The mucoal barrier thus consists of two layers: the inner host epithelium and the outer microbial “skin”. Mucosal microbial communities play a major role in regulating access, processing and transmitting materials and signals from the environment to the body. These interactions have profound effects on the physiology of both the healthy and diseased mucosa. Though the microbial communities of mucosal surfaces are largely benign, they can harbour pathogens and cause or contribute to a wide spectrum of diesease types, ranging from classical infections, inflammatory reactions, autoimmune conditions, cancer, virus-mediated neurological disease, etc. Interactions of mucosal microbial communities with the underlying mucosal tissue are thus of central importance for health and a variety of diseases, and elucidation of crtitical parameters of such interactions will undoubtedly lead to a new understanding of the causes and mechanisms of important diesease syndromes, and open up new avenues for prevention and therapy. At present, there is little information on microbial communities of the human bowel mucosa.
As part of the KG “Infection and Inflammation” this project addresses the composition and function of microbial communities which are associated with mucosal surfaces using genomic techniques. A key element is the comparison of samples from healthy and diseaesd areas of mucosal surfaces of IBD patients and of non-IBD patients.
Project Status
During the last three years, we investigated the mucosa-asociated bacterial and fungal microflora of patients with active IBD (e.g. Crohn’s disease and ulcerative colitis), inflammatory controls and healthy individuals using 16S rDNA-based global genomic techniques. We found that the mucosa-associated bacterial microflora in IBD patients showed a significant reduction of bacterial diversity compared to the inflammatory controls and healthy individuals. The reduction in bacterial diversity was due to a loss of members of the normal endogenous microflora. Obviously, mucosal inflammation in IBD is associated with a breakdown of the normal mucosa-associated bacterial flora. The observed alteration of the bacterial microflora seems to be a disease-specific phenomenon and may play a key role in etiology of IBD. On the other hand, we could demonstrate that the mucosa-associated bacterial microflora is restored after administreation of probiotic therapy in patients with pouchitis. These findings are the starting point for the exploration of the microbial flora in environmental diseases.
In the initial phase of NGFN2, the existing connections to our project partners have been extended, to guaranty a permament flow of well documented clinical samples for our project. The creation and analysis of the proposed metagenome libraries posed some technical challenges: the classical methods applied so far by other scientists require a minimum amount of DNA which far exceeds the microbial DNA available from one or few biopsie samples. Additionally, these samples are heavily “contaminated” with human DNA. Unlike with 16S rDNA-based methods, the exploration of metagenome sequences would be biased by large amounts of contaminant DNA or unspecific amplification products created by, for example, rolling-cycle amplification. To achieve the suggested down-scaling of metagenome analyses to biopsie-sized samples, and to enrich the metagenome of the mucosa-associated microflora required considerable effort into the development and optimization of adequate methods.
These methods have been evaluated with clinical biopsie samples and are currently applied to the clinical samples from IBD patients we are using to create metagenome libraries. The enriched microbial DNA is being cloned into three different vectors to cover a good range of insert sizes, as well as to provide good insert stability and gene expression in E. coli and other hosts.
Outlook
We will extend our previous work to enable a semiquantitative assessment of specific bacteria within the mucosal microflora by using 16S/18S rDNA and alternative marker genes, like gyrB and parE. The functional exploration of specific strains from the intestinal microbiota in control of gene function of epithelial and immune cells is a major aim of our project, as well as the exploration of the role of changes in the colonic microflora in relation to human host genetics in IBD patients. Another important issue is the exploration of the molecular epidemiology of alterations of the microflora by intra-individual follow-up studies to distingiush secondary from primary events. We also wish to explore the role of host genetic factors versus environmental influence in control of the colonic microflora.
Lit.: 1. Schreiber S et al. Genetics of Crohn disease, an archetypal inflammatory barrier disease Nature Reviews Genetics 6(5) : 376-388 2. Hampe J et al. Association of NOD2 (CARD 15) genotype with clinical course of Crohn's disease: a cohort study. Lancet. 2002 May 11;359(9318):1661-5. 3. Ott S et al. : In vitro alterations of intestinal bacterial microbiota in fecal samples during storage Diagnostic Microbiology and Infectious Disease 50 (4):. 237-245 4. Ott S et al.: Reduction in diversity of the colonic mucosa associated bacterial microflora in patients with active inflammatory bowel disease Gut 53 (5): 685-693.
