Baseline clinical characteristics of study participants
The characteristics of 14 patients with UC, 6 patients with CD and 48 healthy controls were demonstrated in Table 1. The mean age was 49.8 ± 14.1 years old in IBD group and 49.9 ± 11.6 in controls. Among the 14 patients with UC, 9 patients were in remission (Mayo score 0-2), 4 patients had active disease with mild inflammation (Mayo score 3-5), and 1 patient had active disease with moderate inflammation (Mayo score 6-10). For the 6 patients with CD, 5 patients were in remission (CDAI < 150) and 1 patient had moderate inflammation (CDAI=300) when entered the study. In the 20 study subjects, 10 patients had taken azathioprine, five had received biological agents (including one CD patient for concurrent rheumatic arthritis) at the data and samples collection. Only one patient with UC did not receive any medication for disease control at the time of stool collection.
Table 1. Baseline clinical characteristics of study subjects
|
IBD (n=20)
|
Control (n=48)
|
Women/ men, n (%)
|
3/17
|
19/29
|
Age, years
|
49.8 ± 14.1
|
49.9 ± 11.6
|
BMI, kg/m2
|
21.5 ± 9.4
|
23.2 ± 2.8
|
CRP, mg/dL
|
0.54 ± 0.51
|
0.05± 0.07
|
WBC, /ul
|
6266.7 ± 1822.4
|
5313.2 ± 1267.9
|
Hb, g/dl
|
13.6 ± 2.2
|
13.7 ± 1.30
|
Mayo score for UC n (%)
0-2
3-5
6-10
>10
|
total:14 (100)
9 (64.3)
4 (28.6)
1 ( 7.1)
0 ( 0)
|
|
CDAI for CD n=6
|
78.5 ± 111.9 (11-300)
|
Mean± SD (range)
|
Medication
Mesalazine, n (%)
Steroid, n (%)
Immunotherapy, n (%)
Biologic agents, n (%)
Enema, n (%)
|
18 (90)
2 (10)
10 (50)
5 (25)
9 (45)
|
|
Data are expressed as the mean ± standard deviation (SD); IBD: inflammatory bowel disease; CD, Crohn’s disease; UC, ulcerative colitis; BMI, body–mass index; CRP, C-Reactive protein; WBC, white blood cell; Hb, hemoglobin; CDAI, Crohn's disease activity index.
The microbiota composition in patients with IBD compared with healthy controls
After 16S rRNA gene sequencing and quality filtering, 5.4 million reads from a total of 6.5 million pair-end reads were obtained. A total of 835 OTUs were identified from 68 stool samples.
The α-diversity was estimated using different indices. The richness of microbiota was estimated by observed OTUs and Chao1 indices and the evenness was evaluated by Shannon and Simpson index. Compared to the control group, the IBD group had significantly lower richness (observed OTUs, p = 0.02; Chao1, p = 0.03). The IBD group was also less evenness than the control (Shannon, p< 0.01; Simpson, p< 0.01) (Fig.1).
The overall structure of the fecal microbiome among the two groups were evaluated. PCoA showed a separation between the control group and the IBD group, which revealed significant differences in bacterial genera abundance (Fig. 2). Significant intercommunity differences among the two groups were demonstrated by ANOSIM (R = 0.3344, p= 0.001) and PERMANOVA analyses (p< 0.001).
Phyla Bacteroidetes, Firmicutes and Proteobacteria consisted more than 90% of stool microbiota in both control and IBD groups. Although the IBD group had higher percentage of Firmicutes (41.5% vs. 37.9%) and lower proportion of Bacteroidetes (40.9% vs. 51.3%) compared to the control group, the Firmicutes to Bacteroidetes ratio was not statistically different between the two groups.
There were nine dominant classes were found in both groups, including Bacteroidia, Clostridia, Negativicutes, Gammaproteobacteria, Bacilli, Fusobacteriia, Verrucomicrobiae, Actinobacteria, Coriobacteriia, and Deltaproteobacteria. Bacterial class Bacilli belonging to Firmicutes (5.5% vs. 0.2%, p = 0.03) and Actinobacteria belonging to Actinobacteria (2.4% vs. 0.5%, p< 0.01) were significantly higher in the IBD group compared to the control group. In the contrast, bacterial class Bacteroidia belonging to Bacteroidetes (40.9% vs. 51.3%, p = 0.04) and Deltaproteobacteria belonging to Proteobacteria (0.2% vs. 0.7%, p< 0.01) were significantly lower in the IBD group compared to the control group.
In genus level, Faecalibacterium (2.6% vs. 7.6%, p< 0.01), Subdoligranulum (0.1% vs. 1.9%, p< 0.01), Parabacteroides (1.6% vs. 3.2%, p = 0.03), Ruminococcaceae UCG-002 (0.4% vs. 1.3%, p < 0.01) and Paraprevotella (0.1% vs. 0.9%, p = 0.03) were significant lower in the IBD group compared to the controls. Bifidobacterium (2.3% vs. 0.5%, p< 0.01), Ruminococcus gnavus group (1.1% vs. 0.2%, p< 0.01), Streptococcus (1.0% vs. 0.2%, p = 0.02) and Blautia (0.9% vs. 0.3%, p = 0.01) were significant higher in the IBD group compared to the control group. Analysis of composition of microbiomes (ANCOM) in genus level revealed that Lactobacillus, Sellimonas and Bifidobacterium were dominant in the IBD group and Subdoligranulum was dominant the control group. Bifidobacterium longum subsp. Longum was significantly increased in the IBD group compared to controls.
The discriminant analysis by using LEfSe method was applied to recognize the key taxa accountable for the difference between the two groups. The recognized taxa were emphasized on the cladogram along with their LDA scores. Lactobacillus and Veillonella were identified as dominant genera in the stool microbiome of the IBD group, whereas genera Faecalibacterium was dominant in the control group (Fig.3).
The microbiota composition analysis with ASV
A total of 2,034 ASV were identified from 68 stool samples with total frequency of 976,619. The mean frequency per feature was 480.1. Similar to the analysis based on OTU clusters, the IBD group had lower richness and lower evenness compared to control group (observed OTUs, p< 0.01; Shannon, p< 0.01). The β-diversity between the two groups also had significant difference in Unweighted UniFrac distance (p = 0.03) and Bray-Curtis distance (p< 0.01). Furthermore, PCoA presented a significant separation between the control group and the IBD group, which revealed significant differences in bacterial genera abundance (Fig.4 A). ANCOM revealed that Lactobacillus and Subdoligranulum were the most dominant genera in the IBD group and the control group, respectively.
Additionally, the α-diversity of the CD, UC and control groups were analyzed. The CD group remained significantly lower α-diversity compared to the control group (observed OTUs, p< 0.01; Shannon, p< 0.01). However, the α-diversity revealed no significant difference between the UC and control group (observed OTUs, p = 0.08; Shannon, p = 0.06), and between the CD and UC group (observed OTUs, p = 0.06; Shannon, p = 0.32).
The β-diversity between the CD group and the control group also had significant difference in Unweighted UniFrac distance (p< 0.01) and Bray-Curtis distance (p< 0.01). Similar results were found between the UC group and the control group (Unweighted UniFrac distance, p< 0.01; Bray-Curtis distance, p< 0.01). Yet, the β-diversity showed no significant difference between the CD group and the UC group. PCoA revealed significant separation between the control group and the CD group, and also between the control group and the UC group (Fig. 4B).
By using the LEfSe method, Lactobacillus, Bifidobacterium and Enterococcus were identified as dominant genera in the UC group. Fusobacterium and Escherichia_Shigella were dominant in the CD group. Faecalibacterium and Subdoligranulum were dominant in the control group.