Identification of donor microbe species that colonize and persist long term in the recipient after fecal transplant for recurrent Clostridium difficile

Abstract

Fecal microbiota transplantation has been shown to be an effective treatment for patients with recurrent C. difficile colitis. Although fecal microbiota transplantation helps to re-establish a normal gut function in patients, the extent of the repopulation of the recipient microbial community varies. To further understand this variation, it is important to determine the fate of donor microbes in the patients following fecal microbiota transplantation. We have developed a new method that utilizes the unique single nucleotide variants of gut microbes to accurately identify microbes in paired fecal samples from the same individual taken at different times. Using this method, we identified transplant donor microbes in seven recipients 3-6 months after fecal microbiota transplantation; in two of these fecal microbiota transplantation, we were able to identify donor microbes that persist in recipients up to 2 years post-fecal microbiota transplantation. Our study provides new insights into the dynamics of the reconstitution of the gastrointestinal microbe community structure following fecal microbiota transplantation.

Fig. 1

Window-based SNV similarity for pair wise sample comparisons. a Schemtatic of window-based SNV similarity (WSS). WSS method used to determine the similarity of two samples (a and b) for two different species (Species 1 and 2). The metagenome DNA sequences were aligned to reference genomes to call SNVs. INDELS were not included in the analysis. To calculate the WSS score, the reference genome is divided into sequential, non-overlapping 1 KB windows. A window is defined as similar if the SNV pattern (denoted as orange bars) is exactly the same between two samples for that region or if no SNV (S in the equation) is present in both samples; otherwise, the window is called dissimilar (denoted as D). The WSS score (as a percentage) is calculated for each pairwise comparison for every microbial species. Both sample pairs are required to have minimum coverage ≥ 20% and average depth ≥ 5 to be included for comparison. Low coverage windows with more than 50% of the bases having a read depth < 5 were ignored. The proportion of similar windows across the genome defines the genome-wide SNV similarity between two samples for a given species, and is referred to as the WSS score (see Supplementary Data for further explanation). b Scatter plot of the WSS score for all possible pairwise sample comparisons of selected genomes from the HMP data set. WSS scores for all pairwise sample comparisons of species detected in the HMP dataset were determined. The WSS scores (percent similarity) from unrelated HMP sample pairs are presented as blue points. The sample pairs from the same individual at different times (temporally linked) are displayed as red dots. The number of dots for a given species was proportional to the number of samples where the species is present (at depth > 5X). Although 93 species were analyzed, the results from the 21 species that were present in the FMT samples are shown. For each species, we modeled the WSS of related and non-related samples and constructed a simple binary classifier using logistic regression (Supplementary Data). From the classifier, we identified the WSS cutoff value that differentiates a related sample from a non-related sample (Supplementary Table 7)

Fig. 2

WSS Analysis for FMT donor–recipients. a Scatter plot of the pairwise sample comparisons of species from the FMT. WSS scores were calculated for all possible sample pairs (donor–donor, donor–recipient post-FMT, and recipient post FMT-recipient post FMT). The number of dots for species varies because not all samples contain every species (at depth cutoff > 5X). The blue dots represent FMT-unrelated sample pairs, whereas red and orange points represent FMT-related sample pairs (where both samples were derived from a particular FMT transplant). Orange dots represent related sample pairs with WSS scores below the boundary cutoff, and red dots represent related sample pairs with WSS scores above the boundary cutoff (see Supplementary Data). Grey horizontal bars mark the WSS score boundary cutoff derived from the HMP dataset (see Supplementary Table 7). b The WSS for different FMT donor–recipients post transplant. Donor and recipient post transplant samples from seven different FMT were analyzed (FMT A-F). DA-T1A refers to donor A compared to recipient FMT A at time 1; DA-T2A refers to donor A compared to recipient FMT at time 2. (These 2-year samples were only available for FMT-A and FMT-B). The transplant FMT-FG refers to two FMTs that used the same donor (DF, DG) for two separate recipients (F and G) FMT. The T1A-T2A and T1B-T2B are recipient-recipient comparisons at the two different time points. Red shaded boxes display WSS scores above the WSS boundary cutoff as determined by the classifier, suggesting shared microbial species between the two samples. The orange shaded boxes depict sample pairs where the WSS score was below the boundary cutoff. Empty boxes denote missing data that means sample pairs with sequence coverage or read depth too low to compare