P709 Association between genotypes of Bacteroidetes in fecal samples from patients with Crohn’s disease and its assessment using an experimental mouse model
R. Sueiro1, A.P. De Felipe1, L. Urisarri2, R. Ferreiro2, A. Lorenzo2, J.E. Dominguez-Munoz2, J.M. Leiro1, M. Barreiro-de Acosta*2
1University of Santiago de Compostela, Department of Microbiology and Parasitology, Institute of Food Research and Analysis, Santiago, Spain, 2University Hospital Santiago de Compostela, Gastroenterology, Santiago, Spain
The pathogenesis of inflammatory bowel disease (IBD) involves an imbalance of the gut microbiota generating an inappropiate activation of the mucosal immune system in genetically predisposed individuals. The human commensal microbiota contains a large number of Bacteroidetes species that may cause inflammation in animal models. The aim of this study was to detect and evaluate the influence of different Bacteroidetes genotypes on the activity of Crohn disease (CD) patients. In addition, the effect of these bacteria isolated from CD patients on gut inflammation was evaluated in mice.
We performed a case control study on the intestinal bacteria of the phylum Bacteroidetes from faeces of CD and healthy controls (HC) using a polymerase chain reaction (PCR) designed to detect human-specific genetic markers targeting Bacteroidetes-like 16S rRNA genes in fecal DNA samples. The PCR products from the 16S rRNA genes were digested with HinfI, PciI, DpnII and AciI enzymes and restriction fragment length polymorphism (RFLP) were determined. RFLP and sequencing analysis indicated that a total of 6 bacterial genotypes do exist: N1, C1, C2, C3, C4 and C5 (of which N1 genotype is probably a strain of Bacteroides dorei and C1, and maybe C2, strains of B. vulgatus). The relationship between CD activity (CDAI>150) and microbiota was evaluated. Microbiota from CD patients were transplanted into mice gut to evaluate their ability to induce inflammation. Results are shown in percentages.
11 CD patients (8 with active CD -aCD- (CDAI>150), and 3 with inactive CD -iCD-), and 11 HC were included. The predominant Bacteroidetes genotype in feces from HC and iCD was N1 (present in 100% of samples), whereas this genotype was found in only 28% of patients with aCD. 18% aCD patients showed the C1 genotype, 9% the C1 and C3 genotypes together, 18% the C4 genotype, and 27% the C1 and C4 genotypes together. The transplant of bacteria from CD patients to mice led to large bowel inflammation, and the stool of the transplanted mice consisted in 30% C4 genotype and had a high level of Bacteroidetes cluster in comparison with the mice transplanted with bacteria from HC.
The fecal microbiota of CD patients is different from those of HC in that they present a wide variety of Bacteroidetes cluster genotypes. The C4 genotype by itself, or together with the C1 genotype, seems to be intimately related to the activity of the disease. These results were also confirmed in transplanted mice.