We included 37 patients with UC (51% females; average age, 41.6 ± 13 years), 30 patients with UC-pouch (50% females, average age 43.3 ± 11 years), and 18 healthy individuals (50% females; average age, 38.3 ± 13 years). According to their pouchitis disease activity index, 15 patients were included in the pouchitis group.
Intestinal microorganism analysis of ulcerative colitis
The general conditions of the UC and control groups are shown in Table 1. We summarized the changes of intestinal microbiota within different UC activity levels. The abundances of Firmicutes decreased in patients with UC with moderate and severe activity, while that of Proteobacteria increased in patients with severe active UC (Fig. 1a). The compositions of the intestinal microbiota of patients with UC patients were altered at the genus level (Fig. 1b). We used Shannon’s α-diversity index to show that microbial diversities of the remission and mild groups, as well as that of the control group, were similar. In contrast, the diversities of the moderate and severe groups were significantly decreased (4.79 and 4.39 vs 5.04, P = 0.012, P = 0.002, respectively) (Fig. 1c). The results of principal coordinate analysis showed similar results, in that the microbial communities of the moderate and severe groups were significantly different compared with those of the control group (P < 0.001 and P < 0.001, respectively) (Fig. 1d).
Table 1 Demogarphic and clinical characteristics of the UC and HC group
|
Healthy
|
UC(n=37)
|
|
controls
|
Remission
|
Mild
|
Moderate
|
Severe
|
Number of patients
|
18
|
9
|
10
|
10
|
8
|
Sex,n,M/F
|
9/9
|
4/5
|
3/7
|
5/5
|
3/5
|
Age(yr)
|
38.3±13.0
|
35.8±13.3
|
38.0±12.9
|
38.6±14.1
|
47.4±15.1
|
BMI(kg/m²)
|
22.0±3.8
|
22.3±1.8
|
22.1±2.4
|
23.2±3.4
|
21.2±2.1
|
UC duration(yr)
|
NA
|
7.0±1.9
|
10.3±4.9
|
8.3±4.2
|
11.7±5.1
|
Mayo score
|
NA
|
≤2
|
4.3±0.6
|
8.4±1.6
|
11.3±0.6
|
Iron deficiency anemia(%)
|
NA
|
4(44.4)
|
6(60)
|
7(70)
|
7(88)
|
Faecal calprotectin[ug/g±SD]
|
37.3±21.7
|
73.2±31.5
|
180.7±160.8
|
602.1±313.5
|
1740.3±1215.4
|
Smoking(%at recrutiment)
|
6(33.3)
|
1(11)
|
2(20)
|
1(10)
|
1(13)
|
UC: Ulcerative colitis; NA: Not available
When we used LEfSe analysis to further search for different species among the subgroups, we found that Mogibacterium species increased in patients with mildly and moderately active UC, while Lachnoclostridium and Peptostreptococcus were increased in patients with severe, active UC (Fig. 1e,f). Furthermore, Bacteroides, Roseburia, and Blautia gradually decreased with the development of inflammation.
Intestinal microorganism analysis of pouchitis
Certain baseline characteristics of patients with pouch is shown in Table 2. At the level of the Bacteriophyta, compared with the normal pouch, Bacteroidetes decreased while Proteobacteria significantly increased in the pouchitis group (Fig. 2a). The pouchitis group had a lower Shannon α-diversity level (4.09 vs 5.07, respectively, P = 0.049) (Fig. 2b) and a significantly different community structure compared with the normal pouch group (P < 0.001) (Fig. 2c). The relative abundances of Escherichia-Shigella and Bacteroides were respectively over-represented and significantly lower compared with controls (Fig. 2d,e). Furthermore, the results of LEfSe analysis were similar in that Escherichia-Shigella increased while Bacteroides and Faecalibacterium decreased in the pouchitis group (Fig. 2f).
Table 2 Demogarphic and clinical characteristics of the pouch group
|
Pouch(n=30)
|
|
Pouchitis
|
NP
|
Number of patients
|
15
|
15
|
Sex,n,M/F
|
7/8
|
8/7
|
Age(yr)
|
39.0±8.7
|
46.4±13.1
|
Pouch duration(yr)
|
2.7±1.8
|
3.9±2.0
|
BMI(kg/m²)
|
24.5±2.1
|
25.1±1.8
|
PDAI score
|
≥7
|
<7
|
Age at colectomy
|
38.4±12.1
|
42.5±10.9
|
Smoking(%at recrutiment)
|
0(0)
|
0(0)
|
iron deficiency anemia(%)
|
10(67)
|
7(47)
|
Number of patients with chronic pouchitis(%)
|
6(40)
|
0(0)
|
Crohn's-like disease of the pouch(%)
|
4(26)
|
1(7)
|
Primary sclerosing cholangitis
|
1(7)
|
0(0)
|
Faecal calprotectin[ug/g±SD]
|
351±207
|
103±79
|
Pouchitis: Inflammation of ileal pouch;NP:Patients without pouchitis
|
The structure of intestinal microbiota differ between active UC and UC-pouchitis
Colon metaplasia in pouches provides the basis for comparison between UC and pouchitis. We next integrated the intestinal microbiota data of patients with active UC and patients with pouchitis. Though the Shannon α-diversity index of pouchitis was close to that of moderate-activity UC, there was no significant difference between the pouchitis group and the other two subgroups (Fig. 3a). The microbial community of the pouchitis group was significantly different compared with that of the active UC group (P = 0.001) (Fig. 3b).
Functional analysis of intestinal microbiota in ulcerative colitis and pouchitis
The findings described in Sect. 3.1 and 3.3 prompt us to use the PICRUSt2 algorithm to analyze the combined data for the moderate and severe UC subgroups (MSUC).This analysis revealed 77 and 132 functional differences in patients with MSUC and those with pouchitis, respectively (Supplementary Table S1). For example, the Bifidobacterium shunt, inosine − 5'−phosphate biosynthesis, and heterolactic fermentation were enhanced in patients with UC, while the L − alanine and demethylmenaquinol pathways were increased in those with pouchitis. Furthermore, the functional prediction indicates overproduction of Enterobactin in patients with pouchitis(Fig. 4a,b). Further Spearman correlation analysis of different microbiota based on PICRUSt showed that Escherichia-Shigella, as well as other bacteria such as Ruminococcus_gnavus, may be associated with the production of Enterobactin (Fig. 4c).
According to KEGG analysis, (all data was displayed in Supplementary Table S2), the enrichment of pathways associated with the immune system and infectious diseases varied among the bacteria included in the heat map (Fig. 4d). These results show that these pathogens, mainly Escherichia-Shigella, were involved in diverse infection pathways.