| Objective Inflammatory bowel disease (IBD), mainly consist of ulcerative colitis (UC) and Crohn’s disease (CD), is chronic intestinal inflammatory disorders with unclear etiology. It is generally accepted that the pathogenesis of IBD involves an aberrant mucosal immune system respond towards intestinal commensal microflora antigens in genetically susceptible host. Substantial studies have reported the dysbiosis in active IBD, however, dysbiosis of microflora in quiescent IBD has not been proven. We therefore specifically sought to study the fecal microbiota of health controls and patients with IBD in different genetical background and diet characteristics from central china and explore the relationship between intestinal microbiota and pathogenesis of inflammatory bowel disease.Methods The study enrolled 53 cases of healthy controls (HC),52 cases of patients with active Crohn’s disease (ACD),57 cases of Crohn’s disease patients in remission (RCD),64 cases of patients with active ulcerative Colitis (AUC) and 18 cases of ulcerative Colitis patients in remission (RUC). We collected feces specimens and based on polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) fingerprint technology and multi-variate statistical method to analyze the composition and abundance of intestinal microbiota, and then verified the reliability of the results of the DGGE by real-time PCR.Results Overall, the gut flora of IBD patients presented a decrease on the level of diversity compared to the healthy individuals. Secondly, the abundance of species-specific bacteria significantly differed from healthy control in patients with IBD. We totally found 72 effective band-classes on DGGE fingerprint profiles using BioNumerics software, and 14 band-classes were discriminant significance in DGGE profiles after normalization with relative intensity, and 11 discriminant band-classes were aligned to different single species respectively. Of which, band-class 2.44 was aligned to Bacteroides vultagus (B. vultagus) belonging to Bacteroides genus. Band-classes 4.06,4.37,4.57,4.76,4.99/5.99 were assigned to Eubacterium eligens, Roseburia hominis A2-183, Eubacterium rectale, Anaerosipes butyraticus, Faecalibacterium prausnitzii respectively, of which belonging to Firmicutes phylum with butyrate-producing property. Both of band-class 6.18 and 6.32 were aligned to Eshcerichia coli-Shigella group. Band-class 14.71 and 16.72 were assigned to Bifidobacterium pseudocatenulatum and Collinsella aerofaciens respectively. Subsequently, we found that median intensity of band-class 2.44 and number of represented species decreased significantly in active CD compared to HC(Pc=1.58 x 10-5, P=0.004, respectively) and median intensity of band-class 2.44 also presented a significant decrease in RCD (Pc=4.65 x 10-6) compared to HC, but not in numbers in patients with RCD; we also found that median intensity of band-class 2.44 and numbers of its represented species decreased in active UC compared to HC (Pc=0.03, P=0.001, respectively), while we found count of this species decreased significantly in patients with RUC (P=0.015) compared to HC, but not relative intensity. Both of the relative intensity (Pc=9.93 x 10-5) of band-class 4.06 and amount (P=8.05 x 10-12) of represented species decreased significantly in ACD compared to healthy control in fecal specimens; although no significant decrease of relative intensity was observed in patients with RCD, number of its represented species decreased significantly in RCD (P=1.36 x 10-7) compared with HC, and a inverse correlation between the amount of E. eligens and disease activity (τ=-0.525, P=1.65 x 10-6); moreover, no significant difference was observed between RCD and ACD in numbers. The relative intensity (Pc=0.3 x 10-3) of band-class 4.06 and amount (P=1.47 x 10-9) of representd species E. eligens also decreased significantly in AUC compared to the HC, and we observed the count of E. eligens decreased significantly in RUC (P=0.006) compared with healthy control, but not relative intensity of E. eligens in patients with RUC. As for the band-class 4.37, relative intensity (Pc=1.88 x 10-7) and count (P=1.36 x 10-5) of represented species E. rectale decreased in ACD compare to HC, we also observed significant decrease of relative intensity (Pc=2.76 x 10-7) and number of represented species (P=0.018) in RCD compared to HC, and no significance of relative intensity and count were found between ACD and RCD; relative intensity of band-class 4.37 decreased significantly in both AUC (Pc= 5.06 x 10-13) and RUC (P=0.017) compared to healthy control, and the amount of represented species E. rectale significantly decreased in AUC (P=5.97 x 10-5) compared to HC, but not in RUC; and we found a inverse correlation between the amount of E. rectale and disease activity (τ=-0.388, P =0.63 x 10-4). We found relative intensity of band-class 4.57 decreased significantly in both ACD (Pc=0.008) and RCD (Pc=0.01) compared to HC, and the amount of represented species R. hominis A2-183 also decreased significantly in ACD (P=1.55 x 10-16) and RCD (P=1.34 x 10-14) respectively; moreover, relative intensity of band-class 4.57 displayed significant decrease in both AUC (Pc=1.24 x 10-5) and RUC (Pc=0.0028) compared to HC, and number of its represented species R. hominis also presented significant decrease in both AUC (P=1.04 x 10-15) and RUC (P=9.70 x 10-8) compared to HC; and a inverse correlation was found between the count of R. hominis and UC disease activity (τ=-0.550, P=1.39 x 10-8). Both of Band-class 4.99 and 5.99 were assigned to Faecalibacterium prausnitzii (F. prausnitzii), while relative intensity of band-class 4.99 presented significant decrease in both ACD (Pc=1.56 x 10-5) and RCD (Pc=0.04) compared to HC, but not in UC patients. We also found relative intensity of band-class 5.99 also decreased in both ACD (Pc=0.047) and RCD (Pc=0.048) comparetd to HC in feces, no significant difference was observed between ACD and RCD; however, no significant decrease in relative intensity of band-class 5.99 was observed in patents with UC. The count of F. prausnitzii significantly decreased in both ACD (P=4.61 x 10-10) and RCD (P=0.002) compared to HC; the numbers of F. prausnitzii significantly decreased in AUC (P=0.003) compare to HC, but no significant decrase was observed in RUC. The amounts of F. prausnitzii displayed inverse correlation with disease activity in both CD (t =-0.502, P=4.50 x 10-6) and UC (t=-0.429, P=9.43 x 10-6). Relative intensity of band-class6.18 presented a significantly increase in both active CD (Pc=0.0024) and active UC (Pc =0.005) compared to HC, while we didn’t find significant increasion in RCD and RUC. We found significant increase of relative intensity of band-class 6.32 in both AUC (Pc=0.9 x 10-3) and RUC (Pc=0.0068) compared to HC, but no significance was observed in patients with CD. Moreover, numbers of represented species of band-classes 6.18/6.32 Escherichia coli-Shigella group displayed dramatically increase in both ACD (P=1.08 x 10’6) and (P=9.50 x 10-14) compared to HC, significant increase also were observed in AUC (P=1.06 x 10-7) and RUC (P=0.03) in number, and no significance were observed in active and quiescent disease。Conclusion Significant structural shifts of fecal microbiota were observed in patients with IBD. These shifts were characterized by reduced diversity in both active and quiescent disease. Dysbiosis of butyrate-producing bacteria occurred in patients with IBD, and amounts of butyrate-producing bacteria presented inverse correlation with disease activity, characterizing by E. eligens in CD and R. hominis, F. prausnitzii and E. rectale in UC. Patients with CD and UC shared same dysbiosis characterized by inverse correlation between the amount of F. prausniitzii and disease activity. Distinct dysbiosis occurred in quiescent IBD characterized by decreased amount of E. eligens in CD and R. hominis in UC, and this dysbiosis could not be restored during disease in remission. |
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