The Biodiversity And Composition Of The Dominant Fecal Microbiota In Patients With IBD

BackgroundCrohn s disease (CD) and ulcerative colitis (UC) are chronic immune inflammatory conditions of the alimentary tract referred to collectively as inflammatory bowel diseases (IBD). Several experimental animal models of IBD have led to the theory that the pathogenesis of IBD is the result of an aberrant immune response to normal commensal bacteria in genetically susceptible individuals. The distal gut of humans represents one of the most densely populated microbial ecosystems on Earth. It has been estimated that the microbes in our bodies collectively make up to 100 trillion cells, ten-fold the number of human cells(1). Clinical and experimental observations in animal models indicate that intestinal commensal bacteria are involved in the initiation and amplification of inflammatory bowel disease (IBD). However, no specific pathogen has been identified as being causal, and UC is widely thought to result from a genetically determined but abnormal immune response to bacterial species in the normal gut microflora. As most of the colonic bacteria cannot be identified by culture techniques, the aim of this study was to investigate and characterise the composition of the dominant fecal microbiota in patients with Crohn's disease (CD), ulcerative colitis (UC) and healthy subjects (HS) by using sequence-dependent electrophoresis fingerprinting techniques.MethodsFecal samples were collected from 26 patients with UC(active/inactive11/15) ,10 patients with active CD Patients who were treated in Xijing hospital from March 2009 to August 2009, in accordance with UCAI and CDAI. The patients were individed into active group and remission group. Meanwhile, fecal samples were also gained from 14 matched healthy volunteers. Nucleic-acid based methods of analysis were used to explore the biodiversity and composition of the dominant fecal microbiota in patients with IBD. The DNA extracted from these samples were subjected to two different methods of microbiome analysis. Frist, mucosa-associated bacteria were quantified using real-time quantitative polymerase chain reaction (Q-PCR) targeting the 16S rRNA gene, including Bacteroides-Porphyromonas-Prevotella, Bifidobacterium, Helicobacter, Lactobacillus, Enterococcus, Bacteroides fragilis Subgroup and Clostridium phylogenetic clusters XI and XIVa . Bacterial counts were then transformed to logarithms (Log10CFU) for statistical analysis. Meanwhile, the bacteria diversity of the same DNA extracts was also assessed by polymerase chain reaction-denaturing gradient gel electrophoresis, after that, as much as visible DGGE bands were cut out and sequenced.ResultsThere was no significant difference between numbers of Bacteroides-Porphyromonas-Prevotella between HS group(8.49) and R-UC group(8.33). But it was decreased in the activity groups, which means A-UC and A-CD group.( A-UC:7.03;A-CD:5.52;P =0.014 and P =0.003, respectively; Table 3). Occurrence of Bifidobacterium was low in active ulcerative colitis and Crohn's disease (A-UC:7.59; A-CD:7.51;P =0.016 and P =0.001, respectively), however, the results obtained from R-UC(7.93) and healthy subject samples(7.97) did not differ (P>0.05). The Helicobacter was decreased in the faeces of UC (both in active UC and UC in remission) (R-UC:5.31;A-UC:5.24;A-CD:5.18; P =0.000 and P =0.001, respectively)and CD (5.18;P=0.000) patients compared with healthy subjects (5.76). The number of Lactobacillus CFU per gram tended to decrease, but the difference did not reach significance, the numbers associated within CD and UC groups did not differ from healthy subjects (P>0.05). Similar results were observed concerning Enterococcus in R-UC. The results of obtained from R-UC(4.97;P=0.297) and healthy subject(4.76)did not differ (P>0.05),while they were decreased in A-UC(4.42;P=0.014) and increased in the faeces of CD (5.84;P=0.003) patients. The Bacteroides fragilis Subgroup was decreased in the faeces of active UC and CD (A-UC:6.84; A-CD:6.12; P=0.000 in both groups)patients compared with healthy subjects (9.25). Same result was observed in patients with inactive UC(8.22,P <0.001), but not so apparent. The numbers of Clostridium phylogenetic clusters XI and XIVa were decreased in the faeces of UC patients (P<0.05), especially in active UC(8.22;P=0.000). And also significantly reduced in patients with CD(6.74; P=0.000).DGGE demonstrated a unique bacterial community in the faeces of each individual. The banding pattern was varied greatly between subjects. IBD fecal microbiota harbor specific discrepancies and differ from that of healthy subjects. Most DGGE fingerprints were characterised by the presence of approximately 3-5 dominant bands with background of up to 20 distinct but less intense bands. The community diversity of UC and CD were less than HS. When all the samples of both the IBD group and the healthy group were compared, 504 Dice similarity coefficient values ranged from 3.5 to 88.9%. The mean similarity index between groups were 29.10±13.57%, 35.66±10.38%,25.47±20.50%(A-UC;R-UC;A-CD versus HS, respectively),which were less than the intra-group similarity index.Most sequence from the DGGE bands were similar to environmental sequences from bacteria that have never been cultivated. They extremely belonged to the phylum Proteobacteria, the phylum Bacteroidetes and phylum Firmicutes, especially the phylum Firmicutes was the dominant bacteria in both IBD group and healthy group. These results also suggest that the dominant genera in the intestinal microbiota of IBD group is different compared with that of healthy group. ConclusionThese results allow a better understanding of changes in mucosa-associated bacterial flora in patients with IBD, showing either a predominance of some potentially harmful bacterial groups or a decrease in beneficial bacterial species. By using DGGE, we have demonstrated the complexity and individuality of the human intestinal microflora and shown that this is a confounding factor in determining the possible significance of individual organisms in the pathogenesis of IBD.