Changes in general conditions of rats in each group
To evaluate the effect of FMT in chronic intestinal inflammation conditions similar to in human patients with inflammatory bowel disease, experimental enteritis in rats was induced by administering 2,4,6-trinitrobenzenesulfonic acid (TNBS). After starting TNBS treatment, faeces from normal rats were repeatedly administered to UC model rats by gavage. Figure 1A shows the curve of the average body weight in each group. Before developing the TNBS-induced UC models, there was no significant difference between groups (P > 0.05). After model creation for 72 h, the weight of the rats in each model group decreased to a similar extent, with differences observed between the normal group and other groups (P < 0.05). After intervention, there were significant differences in body weight between the frozen FMT group, mesalazine group, and UC model group (P < 0.05), whereas there was no significant difference between the UC model group and fresh FMT group. There was no difference among the fresh FMT, frozen FMT, and mesalazine groups (P > 0.05).
After model development, each group began showing various degrees of malaise, arched back, yellow coat, diarrhoea, mucus pus, and blood in the stool, accompanied by slow weight gain or even weight loss. After intervention, the activity status, bloody stool, and diarrhoea of the rats improved. The disease activity index (DAI) score is shown in Figure 1B.
We also measured the colon length. As shown in Figure 1D, the colons of each group showed varying degrees of dilatation, oedema, and even bleeding ulcers after model creation. However, dilation and oedema of the colon of rats in the three different intervention groups were not as obvious as those in the model group, and there was no obvious ulcer bleeding. According to the colon length statistics, as shown in Figure 1C, only the normal group significantly differed from the other four groups (P < 0.05). The remaining four groups showed no significant differences (P > 0.05).
Pathological changes and scores of colonic tissues of rats in each group
The colon tissue of rats in the normal group showed a complete colonic epithelium, regular crypt structure, and small amount of inflammatory cell infiltration. The pathological changes in rats in the UC model group were as follows: obvious erosion and ulceration was observed in the mucosal epithelium; the number and structure of epithelial crypts were changed, and their arrangement was disordered; goblet cells were significantly reduced; high inflammatory cell infiltration was observed in the lamina propria, and a large amount of inflammatory cell infiltration and oedema was observed in the submucosa. However, in pathological sections of the fresh and frozen FMT groups, inflammatory infiltration and destruction of the intestinal wall were milder than those in the UC model group.
Levels of inflammatory factors in each group of rats
According to the intervention results of each group, treatment with FMT reduced intestinal inflammation. The expression of TNF-α in the colon of the two FMT groups was significantly lower than that in the model group (P < 0.05) and returned to the level in normal rats. There was no significant difference between the fresh and frozen FMT groups (P > 0.05).
FMT treatment improves intestinal flora in UC rats
The pair end reads obtained by Miseq sequencing were first spliced according to the overlap relationship between the sequences, and the sequence quality was controlled and filtered. The samples are distinguished, after which cluster analysis and species taxonomy analysis were performed. Cluster analysis showed that various diversity index analyses could be performed and the depth of sequencing could be detected. Based on taxonomic information, community structure statistics were performed at each classification level.
Data corresponding to the richness and diversity of the gut microflora are shown in Figure 4. The Chao1 index and Shannon index showed that FMT increased alpha diversity (P < 0.05).
Each group of samples contained 17 known phyla from the kingdom of bacteria with an abundance of more than 0%, namely Acidobacteria, Actinobacteria, Armatimonadetes, Bacteroidetes, Chloroflexi, Cyanobacteria, Deferribacteres, Elusimicrobia, Epsilonbacteraeota, Firmicutes, Fusobacteria, Gemmatimonadetes, Nitrospirae, Patescibacteria, Proteobacteria, Tenericutes, and Verrucomicrobia.
The horizontal analysis of the phylum graph showed that Firmicutes, Bacteroidetes, Actinomycetes, and Proteobacteria were predominant (Figure 5). After TNBS-induced induction of UC, the intestinal flora of rats differed from that of normal rats at the phylum level. The difference was manifested as a decreased abundance of Bacteroidetes, significantly increased relative abundance of Firmicutes, and increased abundance of Actinomycetes and Proteobacteria. After different interventions, at the phylum level, there were significant differences between the frozen FMT group and UC model group. The specific performance was as follows: the relative abundance of Firmicutes decreased, whereas the relative abundance of Actinobacteria and Proteobacteria decreased but that of Bacteroides increased. The fresh FMT group showed a similar trend but did not significantly differ from the UC model group (P > 0.05).
At the genus level, the abundance comparison of a single species in each sample group is shown in Figure 6, and the Wilcoxon fit rank test was used to detect differences between groups. This study showed that after model creation, the abundance of Bacteroides, Christensenellaceae_R_7_group, Fusicatenibacter, and Allobaculum increased, whereas Prevotellaceae_NK3B31_group, Ruminococcaceae_UCG_013, Ruminococcaceae_UCG_014, and Eubacterium_coprostanoligenes_group decreased. After FMT intervention, the abundance of Prevotella_9 increased, and the relative abundance of Coprococcus_2 and Subdoligranulum decreased.
In terms of β diversity, following intervention, the similarity of the composition of the fresh and frozen FMT group sample communities became more consistent with the normal group, as observed by principal coordinate analysis (Figure 7). In the community column chart shown in Figure 9, the composition of the colonic microflora also showed similar results: the colonic microflora composition of the frozen and fresh FMT groups gradually trended toward that in the normal group after intervention, which continued after stopping the intervention for one week.
To identify the specific types of bacteria altered by treatment, we performed linear discriminant analysis effect size (LEfSe) analysis to determine the characteristics of different groups of the gut microbiota. The clade map produced by LEfSe analysis revealed species with significant differences in abundance between groups (Figure 9). The abundance of Coprococcus 2 and Ileibacterium was significantly higher than that in the other groups, which were characteristic bacteria in the UC group. Fusobacteriaceae, c_Alphaproteobacteria, Anaerovibrio, Ruminococcaceae_UCG_008, and Prevotellaceae were characteristic bacteria in the frozen FMT group. Prevotella_9, Acidaminococcaceae, Peptostreptococcaceae, Arcobacter, Phascolarctobacterium, Eubacterium_hallii_group, Candidatus Stoquefichus, Arcobacteraceae, Faecalibaculum, and Sulfurovaceae were characteristic bacterial genera in the fresh FMT group. The abundances of Lachnospiraceae NK4A136, Ruminococcaceae_UCG_005, and Romboutsia were significantly higher than those in the other groups, which are characteristic bacterial genera in the MS group.