PMC6462343_01
Differences in the effects of stools from donors with two feeding patterns on outcomes of diarrhea treatment via FMT in the same recipient. (a) Illustration of this study design, which was a longitudinal observation comparing the effects of stool samples from two donors with different feeding patterns (donor #1 was mixed fed, and donor #2 was exclusively breastfed) on the therapeutic outcomes of diarrhea via FMT in the same recipient (subject #A14). Thinboicing stool is the transitional form between watery stool and musy stool. The stool frequency (b) and weight (c) of the recipient were continuously recorded during the entire study.
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Diarrhea status was routinely monitored via frequency, weight, and shape of bowel movements each day during the entire study period (Figures 1(a)-1(c)). During the B phase (the baseline time point: on day 2 after cessation of medical treatment for diarrhea), patient #A14 had 8 liquid bowel movements a day, and the wet weight of the stool reached 1640 g in 24 h (Figures 1(b) and 1(c)). During the 7-day follow-up after cessation of the first FMT, the shape of the feces was obviously improved (Figure 1(a)), and the frequency of bowel movements gradually decreased from 5 times at 3FMT1st to once at the third sampling time point of the first washout phase (3Washout1st, Figure 1(b)). Similarly, during the 7-day FMT period, we found significant improvements in both the shape and frequency of the patient's stool (Figures 1(a) and 1(b)). DNA from stool samples collected at the baseline (B point) and the indicated time points (Figure 1(a)) with 16S rRNA gene sequencing. During allo-HSCT, patients normally undergo dramatic alterations of their intestinal microbiota.
Dynamic changes in microbial diversity and richness during the entire FMT process. Microbial diversity was measured by the Shannon index at baseline (a) and the indicated time points (b) for both the FMT donors and the FMT recipient. Microbial richness was evaluated via the Chao1 index at baseline (c) and the indicated time points (d) during the entire FMT process. Recipient specimen collection points were rearranged according to the level of microbial diversity, and diarrhea status is labeled on the right. (e) The horizontal stacked bars on the left side represent the phylogenetic composition of the recipient at each sample collection time point. The hierarchy of microbial diversity was determined by the inverse Simpson index. Time bars are shown in the columns; the length of the time bar depends on the sequence of specimen collection.
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In this study, we also found that the overall bacterial diversity (Shannon a-diversity = 0.47) and richness (Chao1 index = 26.75) of patient #A14 were significantly reduced at the beginning of FMT (Figures 2(a) and 2(c)). Although we observed that stool samples from donor #1 were more effective for the treatment of diarrhea than those from donor #2, there were no obvious differences in diversity (Shannon) or richness (Chao1) between the two donors (Figures 2(a) and 2(c)). During the entire FMT process, we observed significantly increased bacterial diversity in the patient after the introduction of stool from either donor (FMT1st, FMT2nd, and FMT3rd; Figures 2(b) and 2(d)). Washout1st, Washout2nd, and Washout3rd time points; Figures 2(b) and 2(d)). We next asked whether the diversity of bacteria affected the occurrence of diarrhea. To this end, we classified the overall samples we sequenced into three different levels based on the inverse Simpson index and correlated diarrhea status with bacterial diversity (Figure 2(e)).
Dynamic changes in composition in the intestinal microbiota. (a) OTU heatmap of all the samples from both donors and the recipient during the entire FMT process. The thermal analysis shows the top 20 OTUs based on their relative abundances. The names on the left are the species annotations for each outlined OTU, and the color value of each square in each row indicates the relative abundance of that OTU. Dynamic changes in 6 OTUs were selected (b-g) from the 114 identified OTUs of patient #A19 based on their sensitivity to FMT.
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Based on OTU abundance, we outlined the top 20 genera for each sample (Figure 3(a)). Notably, similar compositions were observed in the samples from donor #1 in both FMT1st and FMT2nd procedures (Figure 3(a)). Meanwhile, we identified 6 OTUs that were more sensitive to changes during FMT (OTU_01: Enterobacter spp., OTU_29: Lactobacillus spp., OTU_52: Campylobacter spp., OTU_104: Ezakiella spp., OTU_80: Edwardsiella spp., and OTU_86: Luteibacter spp.) among the 114 recognized OTUs (Figures 3(b)-3(g)).
The differences in microbiota species between diarrhea and nondiarrhea stages. (a) Principal coordinates analysis of microbiota species composition of all samples during the whole study. Principal coordinate 1 (PC1, x-axis) explains 38.43% of the variance, and principal coordinate 2 (PC2, y-axis) explains 20.44% of the variation in Bray-Curtis dissimilarity between samples grouped by diarrhea status (pink circle = diarrhea, blue circle = normal). (b) LDA coupled with effect size measurements identifies the most differentially abundant taxa between diarrhea and normal status. Diarrhea-enriched taxa are indicated with a positive LDA score (red). No obvious differentially abundant taxa were observed in samples with normal status (not shown). Only taxa meeting an LDA significance threshold of >2 are shown.
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Washout3rd: 2 and 3) segregated from the other samples (Figure 4(a)). We noted that these samples from the same patient clustered closely and continuously together. A total of 7 genera (Morganella, Dysgonomonas, Prevotella, Fusobacterium, Luteibacter, Lactococcus, and Pyramidobacter; Figure 4(b)) were found to be significantly different between samples with signs of diarrhea and normal samples.