[Terminal-restriction fragment length polymorphism analysis of diversity of mucosa-associated microbiota in patients with Crohn's disease].

OBJECTIVE

To explore the differences of colonic mucosal-associate bacterial diversity between the patients with Crohn's disease (CD) and healthy controls.

METHODS

Eight CD patients and 23 healthy controls were recruited from September 2010 to December 2011 at West China Hospital. One biopsy were taken from cecum of every patient with CD and healthy control by endoscopic examination. The diversity of colonic mucosa-associated microbiota was detected by terminal-restriction fragment length polymorphism (T-RFLP) . Hierarchical cluster analysis were performed to compare the similarity of microbial communities between CD patients and healthy controls. Differences of bacterial diversity between two groups were also evaluated. The difference of predominant terminal-restrict fragments (T-RF) were analyzed and the bacterium predicted by predominant T-RFs were identified according to MiCA database.

RESULTS

Hierarchical cluster analysis showed that the mucosal microbial community of CD patients differed from healthy controls. And there were more similarities in the samples of same group than that of different groups. Compared with healthy control group, the richness of mucosal microbiota in CD patients was lower (HaeIII:7 ± 4 vs 10 ± 8, P = 0.048; MspI+HaeIII:20 ± 10 vs 24 ± 12, P = 0.036). Shannon-Wiener index of CD patients was lower than healthy control (1.7 ± 0.7 vs 2.0 ± 0.5, P = 0.220) with no significant difference. Species evenness and Simpson index of CD patients were significantly greater than healthy controls (0.84 ± 0.14 vs 0.77 ± 0.13, P = 0.045; 0.25 ± 0.16 vs 0.22 ± 0.15, P = 0.038) . The T-RF of 37, 40 and 66 bp digested with MspI enzyme predominated in CD patients. Relative quantitative analysis showed 35 bp T-RF digested with MspI was significantly higher in CD patients than that in healthy controls (36.8% (23.0%, 55.4%) vs 14.3% (9.5%, 19.5%), P = 0.001), and 74, 141, 486, 490 bp T-RFs were all significantly lower than healthy controls (3.2% (1.3%, 5.1%) vs 10.2% (5.4%, 17.3%), P = 0.001; 4.5% (1.7%, 7.1%) vs 10.8% (5.9%, 21.1%), P = 0.007; 4.2% (1.6%, 5.3%) vs 7.6% (5.9%, 9.3%), P = 0.022; 3.6% (2.4%, 6.1%) vs 18.3% (9.9%, 43.2%), P = 0.008). The mucosal bacterial community composition in CD patients was predominated by Firmicutes, Proteobacterium and Actinobacterium. Compare with healthy control, Bacteroides were significantly reduced in CD patients while Firmicutes (e.g. Enterobacter sp.) and Actinomycetaceae significantly increased.

CONCLUSIONS

Dysbiosis of mucosal microbiota occurs in CD with decreases of richness and biodiversity. Increased Enterobacter sp., Actinobacterium and decreased Bacteroides may play an important role in the pathogenesis of CD.