Sequencing quality analysis
A total of 62 tongue coating and fecal samples were sequenced. Tongue coating samples yielded 3147661 effective sequences and 2729527 optimized sequences. The average length of optimized sequences was 371bp, while a total of 3185812 effective sequences and 2732592 optimized sequences were obtained for fecal samples, where the optimized sequences had an average length of 371bp. As shown in Figure 1, both the rarefaction curve and the species accumulation curve indicated that the sequencing had reached a plateau, indicating that the sampling and sequencing depth was sufficient for subsequent analysis.
OTU-based diversity analysis
A total of 371 OTUs were obtained from tongue coating samples, 46 of which were unique to the IBS-D group and 28 to the normal control group. Similarly, 525 OTUs were obtained from fecal samples, 194 of which were unique to the IBS-D group and 15 unique to the normal control group. The alpha diversity reflected bacterial abundance and diversity, and the observed index (P = 0.0013), Chao index (P = 0.0245), and ACE index (P = 0.0172) of tongue coating microbiota in IBS-D group were significantly lower than those in the normal control group. As shown in Figure 2 and Table 2, the Shannon and Simpson indexes revealed no significant difference between the two groups (P > 0.05). Table 2 shows that PD whole tree index of fecal microbiota was significantly higher in IBS-D group than in the normal control group (P = 0.00033). However, there were no significant differences in Chao index, ACE index, or Shannon index between the two groups (P > 0.05).
Alpha diversity analysis of IBS-D and normal control groups.
The differences in bacterial community structure are reflected in beta diversity. PCoA plots based on the bray-Curtis method and the unweighted UniFrac method were performed on tongue coating and fecal samples to assess differences between the two groups. As shown in Figure 3, the closer the distance, the more similar the bacterial structure of the two samples. The Anosim test revealed that the beta diversity of tongue coating microbiota was significantly different in IBS-D group compared to the normal control group (Bray-Curtis: R = 0.170, P = 0.013; UniFrac: R = 0.170, P = 0.013). The beta diversity of fecal microbiota differed from the control group (Bray Curtis: R = 0.0008, P = 0.477; UniFrac: R = 0.529, P = 0.001).
Analysis of species composition
The microbiota can be annotated using 16S rRNA sequencing at six different species classification levels (phylum, class, order, family, genus, and species). This paper primarily explained microbiota species composition of phylum and genus classification levels. At the phylum level, the top four tongue coating microbiota of richness in the IBS-D group compared with normal control group were Bacteroidetes (33.8% vs. 30.8%), Firmicutes (30.7% vs. 34.3%), Proteobacteria (21.0% vs. 17.8%), and Fusobacteria (9.9% vs. 9.0%), while the fecal microbiota were Bacteroidetes (65.3% vs 66.1%), Firmicutes (24.1% vs. 28.2%), Proteobacteria (7.5% vs. 4.3%) and Fusobacteria (2.8% vs. 1.3%). Figures 4A and 4B show the composition of tongue coating and fecal microbiota in two groups at the phylum level. At the genus level, the top four tongue coating microbiota of richness in the IBS-D group compared with normal control group were Prevotella (19.0% vs 20.7%), Neisseria (14.4% vs 10.2%), Streptococcus (11.3% vs. 12.7%), and Veillonella (11.3% vs. 12.7%) (9.7% vs. 11.1%). The fecal microbiota of IBS-D group was dominated by Bacteroides (39.5%) and Prevotella (18.8%), while Bacteroides (58.2%) dominated the normal control group. Figures 4C and 4D show the genus-level composition of tongue coating and fecal microbiota.
Analysis of differential microbiota
LEfSe analysis results revealed significant differences in tongue coating and fecal microbiota between control and IBS-D groups, As shown in Figure 5. Pseudomonadales, Pseudomonadaceae, Pseudomonas, Moraxellaceae, Peptostreptococcus, Parvimonas, and Alloprevotella were relatively enriched in the IBS-D group, while Alphaproteobacteria, Sphingobacteria, Micrococcales, and Lentimicrobiaceae in the normal control group. Similarly, the fecal samples of IBS-D group were relatively enriched in Pseudomonadales, Pseudomonadaceae, Pseudomonas, Alphaproteobacteria, Acidaminococcaceae, Phascolarctobacterium, Alloprevotella, and Escherichia, while in Chloroflexi, Thermomicrobia, Rickettsiales, and Phyllobacteriaceae in the normal control group.