1. The demographics and Clinical Characteristics of Study subjects
A total of 97 subjects were enrolled in the study including 34 IBS patients, 45 IBD patients in remission, 18 healthy controls. All the subjects met the enrolling criteria from the First Affiliated Hospital of Nanjing Medical University were recruited from August 2018 to September 2019. The mean age was 42.9 years in IBS group, 30.9 years in CDR-IBS+ group, 29.8 years in CDR-IBS- group, 37.1 years in UCR-IBS+ group, 42.4 years in UCR-IBS- group and 37.9 years in control group. The proportion of male subjects is 54.6% (53/97). However, there were more female subjects in IBS group (61.7%, 21/34), which might be closely related to the obvious gender difference in the incidence of this kind of disease. Detailed demographic data and clinical characteristics of all included subjects are listed in Table 1.
Overall sequencing results
The paired end reads were optimized to remove low-quality reads, and clustered into operational taxonomic units (OTUs) for species classification at 97% similarity, and the abundance information of each sample in each OTUs was counted. The abundance preliminarily explains the species richness of the sample. A total of 4869075 high-quality tags were obtained, and the average number of tags for each sample were 50197. According to the 97% similar clustering principle, a total of 1118 OTUs were generated from 97 samples. Compared with the control group, the number of OTUs in IBS, CD patients in remission with IBS-type symptoms (CDR-IBS+), CD patients in remission without IBS-type symptoms (CDR-IBS-), UC patients in remission with IBS-type symptoms (UCR-IBS+) and UC patients in remission without IBS-type symptoms (UCR-IBS-) reduced, but only the OTUs of patients in CDR-IBS- had a statistical difference (163.7±65.98 vs 240.8±66.75, P<0.05), suggesting that this group of patients may have the lowest species abundance. The detailed results were shown in Table 2.
The Characteristics of Microbial diversity in Different Groups
The rarefaction curve was used to reflect the rationality of the amount of sequencing data. Each sample was obtained a rarefaction curve according the number of the bacterial OTUs on sequence counts at different sequencing depths. As shown in Supplementary Figure 1, as the number of sequencing continues to increase, the rarefaction curve of each sample tended to be saturated, indicating that the final sequencing data in the study was reliable. Alpha diversity was used to evaluate the differences in the microbiota of samples in Control, IBS, CDR-IBS+, CDR-IBS-, UCR-IBS+, and UCR-IBS- groups. The observed species index, Chao index and Ace index were calculated to reflect the species richness of the microbial community in the sample, while the shannon index and simpson index reflected species diversity, which is affected by the species richness and species evenness of the sample community. The results of comparison among the Alpha Diversity Index groups indicated that observed species index, chao and Ace of CDR-IBS- were statistically significant decreased compared with Control groups, respectively, while no difference was found among other groups (Figure 1A-C). Meanwhile, the observed species of microbiota in CDR-IBS- was lower than CDR-IBS+ group, Chao index was lower than IBS group. Therefore, the richness of microbiota in fecal samples from CDR-IBS- groups were significantly decreased base on the analysis of alpha diversity. There were no differences in shannon index and simpson index among the six groups (Figure 1D-E). Although there is no statistical difference in a diversity analysis of patients between IBS, CDR-IBS+ and UCR-IBS+ groups, including observed species, chao and Ace index. Furthermore, the overall microbiota structures were analyzed according the number of shared or unique OTUs. The results were displayed in partial least-squares discriminant analysis (PLS-DA) plots and Venn plots (Figure 1F-G) and heat maps in phylum level (Supplementary Figure 2). PLS-DA and heat maps exhibited that the microbiota structure had slight differences among the groups, but majority of bacteria community overlapped.
Overall Taxonomic Composition of IBDR, IBS and Control Groups in Phylum
From the Figure 2, the proportions of different species in phylum were summarized detail for each sample. The phylum level of taxonomic composition in fecal samples of all groups with major microbiota were as following: Firmicutes, Bacteroidetes, Proteobacteria, Actinobacteria, Fusobacteria, and Verrucomicrobia.(Figure 2A-B). There was no statistical difference of microbiota composition in phylum among the six groups. Compared with Control, CDR patients with or without IBS-type symptoms had a trend decrease in abundance of Bacteroidetes, but they did not obtain a statistically differences. A relatively decrease trend in abundance of Bacteroidetes was found in CDR groups compared with IBS (median proportional abundance CDR-IBS+ vs IBS: 30.0% vs 47.6%, CDR-IBS- vs IBS: 34.7% vs 47.6%, Figure 2C), but no statistical difference was obtained (P>0.05). The proportion of Firmicutes displayed a relative trend to increase in CDR-IBS+ and UCR-IBS+ groups. No difference of Fusobacteria in phylum abundance was determined in current populations, although there was an increase trend for IBDR in different type no matter with or without IBS-type symptoms. Overall, there was no statistical difference of microbiota among IBS, CDR-IBS+ and UCR-IBS+ groups. Further analysis showed that there was no statistical difference in the alteration of microbiota community in the phylum level between UCR with or without IBS-type symptoms, as wells as CDR groups.
Comparison of Microbiota Composition among the Different Groups in Genus Level
The overall genera from each sample were displayed in bar-plot of taxonomic analysis. The main microbiota composition in genus level were as follows: Bacteroides, Faecalibacterium, Prevotella, Escherichia, Roseburia, Blautia, Streptococcus, Fusobacterium, Haemophilus, Lachnospira, (Figure 3A). There was no statistical difference in the changes of the genus level between Control and IBS subjects (Figure 3B). The relative abundance of Bacteroides were slightly increased in IBS, UCR-IBS+ and UCR-IBS- patients compared to that in Controls, but the differences was not statistically significant. The mean abundance of Bacteroides was a trend to decrease in CDR-IBS+ while to increase in UCR-IBS+ compared with IBS groups, although the difference was no statistically significant. Compared with CDR-IBS-, the abundance of Faecalibacterium and Roseburia, Streptococcus were a trend to increase while Prevotella, Escherichia, and Fusobacterium decrease in CDR-IBS+ group, of which the number of Faecalibacterium was significantly higher while Fusobacterium lower in CDR-IBS+ compared with CDR-IBS- (mean relative abundance of Faecalibacterium: 20.35% vs 5.18%, P<0.05; Fusobacterium: 1.51% vs 5.2%, P<0.05). In addition, the changes of microbiota community in UCR subjects were not the same as CDR. The results indicated that the abundance of the Fusobacterium, Streptococcus were increased, but Faecalibacterium, Escherichia, Lachnospira and were a slightly decrease in the group of UCR-IBS+ compared to that in UCR-IBS-, despite of all of the difference without statistically significant. Differences were also found between CDR and UCR at the genus level. There was a significantly greater abundance of Fecalibacterium in UCR-IBS- relative to CDR-IBS-(mean proportional abundance 5.4% vs 16.6%, P=0.012) and a greater abundance of Fusobacterium in CDR-IBS- relative to UCR-IBS- (mean proportional abundance 5.6% vs 0.04%, P=0.001). The genera differences between CDR-IBS+ and UCR-IBS+ were not statistically significant. The difference of microbiota community among IBS, CDR-IBS+ and UCR-IBS+ groups were also been analyzed. The results showed that the mean abundance of Faecalibacterium and Streptococcus were a trend to increase while the level of Prevotella and Lachnospira were a trend to decrease in CDR-IBS+ and UCR-IBS+ groups compared with IBS groups. But further analysis of the genera among IBS, CDR-IBS+, UCR-IBS+ did not reveal a statistically significant difference.(Figure 3B). The difference of genera was displayed in detail in Table 3. As shown, other genera including: Butyricimonas, Odoribacter, Enterococcus, Clostridium, Megasphaera, Megasphaera, Ruminofilibacter, Gemmiger, Desulfovibrio, Actinomyces, Akkermansia. The increase and decrease trend of relative abundance in genera were listed in Table 3.