Bacterial composition of fecal samples from patients with recurrent CDI becomes healthy and donor-like following FMT

Four patients (CD1 to CD4) with recurrent CDI were treated with FMT using material obtained from a single donor but from different time points, and fecal samples were collected from these patients before and after the procedure as well as from the donor at times of donation. Bacterial communities from these fecal samples were characterized by sequencing the V4 region of the 16S rRNA gene. Following trimming and quality filtering from a total of 12,536,492 sequences, we randomly subsampled to 5,000 sequences/sample in order to normalize read depth across all samples. All further analyses were performed using this rarefied read depth.

To better understand changes in bacterial communities following FMT, we compared the bacterial composition of patient fecal samples to those of microbial communities from various body sites from the 252 healthy individuals characterized in the Human Microbiota Project (HMP) [19] (Figure 1) using unweighted UniFrac [20] followed by principal coordinates analysis (PCoA) [21] (see Additional file 1: Movie supplement). The composition of pre-FMT fecal samples from patients CD1 to CD4 and 10 additional patients with recurrent CDI was distinct from both fecal samples from healthy individuals and microbial communities at other body sites, including mouth, vagina, and skin, demonstrating severe alterations in pre-FMT communities compared to healthy fecal communities as has been previously shown [4,5]. In contrast, microbial communities from the donor fell within the range of healthy fecal samples. Using an animated visualization of FMT-associated changes in patients’ fecal microbial communities, we observed rapid and dramatic shifts after FMT towards the communities found in the feces of healthy individuals and of the original donor (see Additional file 1: Movie supplement).

Figure 1 Fecal bacterial communities of recurrent CDI patients shift towards HMP fecal bacterial communities after FMT. Pre-FMT patient samples (red circle); post-FMT patient samples (green circles); trajectory of patient fecal communities after FMT (blue line). Full size image

Fecal microbial communities remain dynamic following FMT

To more closely examine temporal changes in recipient fecal samples following FMT, we analyzed fecal microbial communities from patients CD1 to CD4 and donor, as well as from 10 additional donor samples, using weighted and unweighted UniFrac [20] followed by PCoA [21]. This analysis demonstrated that fecal bacterial communities continued to undergo compositional fluctuation following FMT (Figure 2A and Additional file 2: Figure S1; individuals OTUs listed in Additional file 3: Table S1).

Figure 2 Microbial communities shift following FMT. (A) Unweighted (left) and weighted (right) UniFrac analyses followed by principal component analysis of bacterial communities of recurrent CDI patient fecal samples before (red) and after FMT and donor samples (blue). (B) Weighted UniFrac analysis followed by principal component analysis of bacterial communities of patients before (red) and after FMT versus HMP fecal communities (purple). PC, principal component. Percentages represent percent variability explained by each principal component. Se key at right for colors associated with samples before FMT (pre-FMT), from HMP and donor, and from patients after FMT (CD1 to CD4). Full size image

To determine whether this dynamic range of post-FMT microbial composition fits within the range seen across healthy individuals, we also compared communities in our samples to those in the HMP via weighted UniFrac and PCoA (Figure 2B). Again, fecal microbial communities prior to FMT were highly distinct from healthy fecal microbial communities, and following the procedure, these communities more closely resembled those of healthy individuals. Similar to the comparison with donor communities above, fecal microbial communities of recurrent CDI patients following FMT shifted within the cluster of communities from healthy individuals.

Rapid and substantial changes to Enterobacteriales in feces following FMT

While overall fecal microbial communities were dramatically altered following FMT, we also examined the effects of the procedure on the abundance and dynamics of individual bacterial taxa within the four original CDI patients. As shown previously [2-8], the relative abundance of bacterial phyla in patient fecal samples shifted substantially following FMT, with relative decreases in Proteobacteria and relative increases in Bacteroidetes and Firmicutes (Figure 3). These Proteobacteria are primarily the order Enterobacteriales, which were also substantially decreased in relative abundance following FMT (Figure 4A).

Figure 3 Changes in fecal microbial communities after FMT. Relative abundance of sequences classified to the level of bacterial phyla before and after FMT in patient fecal samples. Samples after FMT indicated with dashed line. See key at right. Full size image

Figure 4 Changes in the order Enterobacteriales after FMT. (A) Relative abundance of Enterobacteriales in donor and patient samples before and after FMT in samples common across all patients. (B) Control charts of relative abundance of Enterobacteriales in donor (leftmost sample) and patient samples before and after FMT. Patient CD1 (top left), patient CD2 (top right), patient CD3 (bottom left), patient CD4 (bottom right). LCL, lower control limit; UCL, upper control limit; mean relative abundance in all samples (center). LCL and UCL represent three standard deviations in relative abundance below and above the mean, respectively. Dashed lines indicate samples after FMT. Full size image

We focused on these changes by examining the relative abundance of Enterobacteriales alone in each patient before and after FMT. The relative abundance of this taxon ranged from 44% to 82% in all four patient samples prior to FMT and rapidly dropped to undetectable levels within 1 week after the procedure. Moreover, abundance of this taxon remained low at 26 days after FMT, the latest time point shared by all four patients (Figure 4A), although other members of the Proteobacteria remain detectable if decreased in relative abundance (Figure 3). In addition, we generated individual value control charts based on the average abundance of this taxon in recurrent CDI patients. Compared to relative abundance, these control charts displayed the expected variation of the abundance of Enterobacteriales in these fecal samples. In all patients, the abundance of Enterobacteriales was above the expected variation (that is, more than three standard deviations above the mean relative abundance [the standard upper control limit, or UCL] of this order across all samples) prior to FMT, and rapidly fell below the upper control limit within 1 to 2 days after the procedure (Figure 4B). These results suggest that the relative abundance of Enterobacteriales significantly decreased in all patients soon after FMT to levels similar to donor samples and remained within a statistically expected range for the duration of sample collection (up to 151 days post-FMT).

Post-FMT communities are initially similar to donor samples but can later diverge

Next, we compared fecal microbial communities within each patient over time to that of the initial donor sample. We generated heat maps based on Pearson correlations between every sample within a given patient set, including respective donor samples and samples from 10 additional pre-FMT patients (Figure 5A). This analysis revealed that while microbiota in samples from patients after FMT quickly became similar to microbiota in donor samples, the similarity of samples taken at later time points after FMT fluctuated.

Figure 5 Pearson and Spearman correlations between fecal communities before and after FMT. (A) Heat map of Pearson correlation values between each sample within each patient set, corresponding donor, and 10 additional pre-FMT patient samples (far right). (B) Pearson correlation values between donor sample and each patient sample. (C) Spearman correlations between donor sample and each patient sample. (D) Heat maps of Pearson (i) and Spearman (ii) correlation values between earliest donor sample and eleven subsequent samples; days represent collection time of each sample versus earliest donor sample. CD1 to CD4, patients 1 to 4. Dashed lines indicate samples after FMT. Full size image

To further investigate how fecal microbial communities in these patients correlate to donor communities, we examined Pearson and Spearman correlations between donor and patient samples, which were common to each patient (pre-FMT samples and those up to 26 days post-FMT; Figure 5B,C and Additional file 4: Figure S2). While fecal microbial communities from patients before FMT were highly distinct from those in the donor, fecal microbial communities from samples 1 day after the procedure were highly correlated to donor communities via both Pearson and Spearman analyses in all patients. After the initial time point after FMT, the Pearson correlation values of patient to donor samples were highly variable within and across patients, although Spearman correlations remained high for three patients. To examine whether this variation is similar in healthy individuals, we determined Pearson and Spearman correlations within the four donor samples used in FMT, as well as eight additional donor samples from the same individual as a control. Results of this analysis revealed that donor microbiota also changed over time (Figure 5D). These findings suggested that the level of variability seen across patient post-FMT fecal microbial communities was within the range of normal microbiota behavior in a healthy individual.

Normalization and dynamic range of post-FMT patient fecal microbial communities are similar to donor communities

Because of the observed variability in later post-FMT patient fecal communities relative to single donor communities, we compared the communities of these patient samples to an expanded set of 17 samples taken from the same donor. We generated two metrics to evaluate the relationships between these communities: normalization and dynamic range (stability). Normalization refers to the mean between-sample distance for each set of patient samples versus the set of donor samples, while dynamic range is the mean distance between each sample within a single patient set. Effectively, the normality of a post-FMT patient sample set is a measure of how similar it is to the donor (healthy) sample set, while dynamic range is a measure of variability within a given patient sample set. We found that neither the normalization nor the dynamic range of any post-FMT patient sample set was significantly different than the donor set following analysis using unweighted UniFrac (Table 1). This suggested that although fecal microbial communities of patients post-FMT do not remain identical to the donor, they nonetheless fall within expected parameters relative to the healthy donor. Similar results were obtained when these analyses were repeated with other parameters, including weighted UniFrac, Jensen-Shannon and root Jensen-Shannon, and Bray-Curtis (data not shown).