Assessment of bacterial biodiversity and activity in intestinal microbiota of ulcerative colitis and Crohn's Disease patients through the analysis of the 16SrDNA V6 region

Abstract

Ulcerative colitis (UC) and Crohn's disease (CD) are clinical variants of idiopathic inflammatory bowel diseases (IBD), which manifest as focal mucosa lesions in any part of gastrointestinal tract in CD or diffusely in the colon in UC. These lesions result from anomalous immune responses to bacterial antigens and are associated with genetic and environmental factors. Diet, which is included among the later, has strong influence on species composition of the intestinal microbiota. Several studies have shown the occurrence of dysbiosis in the gut of IBD patients. Yet, in some cases their conclusions are limited by the fact that most of intestinal bacterial species are unknown. Employment of Molecular Biology techniques has contributed for bacterial identification. One of these techniques is based on the comparative analysis of feces or intestinal tissues 16SrDNA base sequences with corresponding bacterial sequences available on DNA databases. In this project, by analyzing the V6 region of 16SrDNA, we intend to assess the biodiversity and activity of mucosa associated bacteria from IBD patients (20 diagnosed with CD and 20 with UC) and 20 control subjects. For this purpose, amplicons of the 16SrDNA V6 region and cDNA of the 16SrRNA V6 region of intestinal biopsies will be resolved by temperature gradient gel electrophoresis (TGGE) and the resulting band profiles, which correspond to the bacterial communities present will be analyzed in relation to variables as group of study (CD, UC, or controls), intestinal segment, disease activity, diet, etc. Subsequently, these DNA amplicons will be submitted to pyrosequencing, in order to identify the bacterial groups detected. (AU)