Experimental Study On Intestinal Microbiome Of Patients With Ulcerative Colitis And Ileal Pouch Based On High-throughput Sequencing

Objective: To investigate the structure,abundance and diversity of the intestinal microbiota in patients with ulcerative colitis（UC）,terminal ileostomy and ileal pouch after proctocolectomy,ileal pouch anal anastomosis（IPAA）,and to explore the intestinal flora-mediated microbiological mechanism in patients with UC and pouchitis.Methods: From December 2017 to December 2018,37 UC patients,14 patients with terminal ileostomy and 16 patients with ileal pouch after IPAA were selected from general surgery,gastroenterology ward and outpatient clinic of Tianjin Medical University General Hospital,who underwent colonoscopy and histopathological examination,and were recorded as UC group,stoma group and pouch group respectively.Eighteen healthy people with matching age and age were selected as healthy control group,and the above four groups of stool specimens were collected into sterile tubes and stored in a cryogenic refrigerator for reserve.According to the modified Mayo scoring system for assessing UC activity,UC group was divided into four subgroups: remission period（Aa）,mild activity（Ab）,moderate activity（Ac）and severe activity（Ad）;pouch group was divided into two subgroups: pouchitis group（≥7 points）and non-pouchitis group（<7 points）according to pochitis disease activity index（PDAI）;stoma group was divided into UC stoma（Sa）group and non-UC stoma（Sb）group according to different basic diseases.Subsequently,the DNA of fecal samples was extracted and tested,and the products were amplificated by PCR.The library was constructed and next generation sequencing was carried out.The sequencing data were annotated by Silva 128₁6s database.Finally,alpha diversity was used to analyze species diversity changes,beta diversity was used to analyze the differences of microbial community structure among groups,and LEfSe were used to identify the biomarkers which had significant difference among groups.Results: The results of alpha diversity analysis showed that the intestinal microflora diversity of UC patients in remission stage was similar to that of healthy people.From mild active patients,with the increase of disease activity,the diversity of intestinal microflora decreased gradually.The diversity of moderate and severe active UC patients decreased significantly compared with healthy controls（p<0.05）;the diversity of intestinal microflora in patients with ileal pouchitis was significantly lower than that in non-pouchitis patients（p<0.05）.There was a downward trend in the diversity of intestinal microflora of UC patients with stoma compared with that of non-UC stoma patients（p>0.05）.The diversity of intestinal microflora in patients with moderate UC activity was similar to that in patients with pouchitis,while the diversity of intestinal microflora in patients with mild and severe UC activity was higher and lower than that in patients with pouchitis respectively（p>0.05）.The level of species diversity in pouchitis group was similar to that in UC stoma group,but the species diversity in the non-pouchitis group was higher than that in the UC stoma group（p>0.05）.The results of beta diversity analysis showed that there was no significant difference in intestinal microbial community structure between UC patients among remission period,mild activity group and healthy control group（p>0.05）,but there was significant difference in intestinal microbial community structure between UC patients with moderate and severe activity group and healthy control group（p<0.05）,and there was significant difference in flora structure between patients with pouchitis and those without pouchitis（p<0.05）.There was no significant difference in intestinal microbial community structure between UC stoma patients and non-UC stoma patients（p>0.05）.There was also significant difference in intestinal microbial community structure between patients with active UC and those with ileal pouchitis（p<0.05）;the flora structure of patients with pouchitis was similar to that of UC stoma patients,while there was significant difference in intestinal microbial community structure between patients with non-pouchitis and those with UC stoma（p<0.05）.LEfSe analysis showed that the bacterial flora of UC subgroups was significantly different from that of healthy control group.Compared with healthy controls,the relative abundance of Streptococcus in remission stage of UC was significantly higher（p<0.05）,the relative abundance of Eubacterium_rectale_group was significantly lower（p<0.05）;the relative abundance of Mogibacterium in mild patients was significantly higher（p<0.05）,the relative abundance of Roseburia was significantly lower（p<0.05）;the relative abundance of Mogibacterium and Prevotella₉ in moderate patients was significantly higher（p<0.05）,while the relative abundance of Eubacterium_rectale_group,Blautia and Bacteroides decreased significantly（p<0.05）;the relative abundance of Lachnoclostridium and Peptostreptococcus increased significantly in severe patients（p<0.05）,while the relative abundance of Eubacterium_rectale_group,Roseburia,Blautia,Faecalibacterium and Bacteroides decreased significantly（p<0.05）.The abundance of Escherichia-Shigella in patients with pouchitis was significantly higher than that in patients without pouchitis（p<0.05）,while the abundance of Bacteroides and Faecalibacterium was significantly lower than that in patients without pouchitis（p<0.05）.Compared with non-UC stoma patients,fecal Escherichia coli increased significantly in UC stoma patients（p<0.05）.In addition,with the increase of disease activity,the abundances of Bacteroides,Blautia,Roseburia,Lachnoclostridium and Peptostreptococcus in UC subgroups showed a change trend.Lachnoclostridium,Escherichia-Shigella and Faecalibacterium showed a significant change trend in UC active period and pouchitis patients.There were obvious changes in Roseburia,Blautia,Megamonas,Faecalibacterium,Bacteroides and Escherichia coli in UC stoma and ileal pouch patients.Conclusion: The diversity,structure and abundance of intestinal microbiota in UC patients are significantly different from those in healthy controls.The abundance of Bacteroides,Blautia,Roseburia,Lachnoclostridium and Peptostreptococcus in UC subgroups shows a significant change trend.The diversity,structure and abundance of bacterial flora in patients with pouchitis were significantly different from those in patients without pouchitis.The number of Escherichia-Shigella in patients with pouchitis was higher than that in patients without pouchitis,while the number of Bacteroides and Faecalibacterium decreased.The intestinal microbiota diversity of UC stoma patients was lower than that of non-UC stoma patients,and there was no significant difference in community structure.Compared with non-UC stoma patients,the number of Escherichia coli increased in UC stoma patients.The abundances of Lachnoclostridium,Escherichia-Shigella and Faecalibacterium showed significant changes in patients with active UC and pouchitis.The intestinal microbiota of UC patients and pouchitis patients were disordered.The decrease of beneficial bacteria and the increase of harmful bacteria affected mucosa of intestinal tract and pouch,resulting in the damage of intestinal and pouch microbial barrier function,which mediated the occurrence and development of UC and pouchitis.