Species and genus level resolution analysis of gut microbiota in Clostridium difficile patients following fecal microbiota transplantation

Abstract

Background: Clostridium difficile is an opportunistic human intestinal pathogen, and C. difficile infection (CDI) is one of the main causes of antibiotic-induced diarrhea and colitis. One successful approach to combat CDI, particularly recurrent form of CDI, is through transplantation of fecal microbiota from a healthy donor to the infected patient. In this study we investigated the distal gut microbial communities of three CDI patients before and after fecal microbiota transplantation, and we compared these communities to the composition of the donor's fecal microbiota. We utilized phylogenetic Microbiota Array, high-throughput Illumina sequencing, and fluorescent in situ hybridization to profile microbiota composition down to the genus and species level resolution.

Results: The original patients' microbiota had low diversity, was dominated by members of Gammaproteobacteria and Bacilli, and had low numbers of Clostridia and Bacteroidia. At the genus level, fecal samples of CDI patients were rich in members of the Lactobacillus, Streptococcus, and Enterobacter genera. In comparison, the donor community was dominated by Clostridia and had significantly higher diversity and evenness. The patients' distal gut communities were completely transformed within 3 days following fecal transplantation, and these communities remained stable in each patient for at least 4 months. Despite compositional differences among recipients' pre-treatment gut microbiota, the transplanted gut communities were highly similar among recipients post-transplantation, were indistinguishable from that of the donor, and were rich in members of Blautia, Coprococcus, and Faecalibacterium. In each case, the gut microbiota restoration led to a complete patient recovery and symptom alleviation.

Conclusion: We conclude that C. difficile infection can be successfully treated by fecal microbiota transplantation and that this leads to stable transformation of the distal gut microbial community from the one abundant in aerotolerant species to that dominated by members of the Clostridia.

Keywords: Clostridium difficile; Fecal microbiota transplantation; Microbiota; Microbiota Array; Microflora.

Figures

Figure 1

Changes in microbiota diversity and composition following fecal transplantation in CDI patients. Microbiota communities were profiled from three CDI patients, healthy donor, and from each patient over a 4-month period following fecal transplantation. Samples were collected periodically as shown in (A). Community diversity and evenness were assessed by calculating the Shannon H’ (diversity, B) and Simpson E (evenness, C) indices based on microarray phylotype abundance data. Community structure in each sample is shown at class level in (D) (distribution is based on microarray data) and (E) (distribution is based on sequencing data). Missing data represent samples that had lower amount of fecal material available; thus not all analyses could be carried out for these samples.

Figure 2

Separation of samples based on ordination multivariate analysis of microarray phylotype abundance data. Principal components analysis (PCA, A) and unweighted (separation is based on phylotype presence, B) and weighted (separation is based on phylotype presence and abundance, C) principal coordinates analysis (PCoA) show separation of recipient samples before transplantation from both donor and recipient samples obtained after transplantation. Percent of dataset variability explained by each principal component/coordinate is shown in brackets in axis titles.

Figure 3

Quantification of bacterial abundances using fluorescentin situhybridization. Select fecal samples from CDI set 1 were profiled using class specific DNA probes as shown. (A) A comparison of class relative abundances measured by Microbiota Array and by FISH. (B) Representative captured image from each sample visualized with Proteobacteria fluorescent probe (green color) and DAPI DNA stain (blue color).

Figure 4

Relative abundances of bacterial genera in all samples as measured by Microbiota Array. All genera were distributed into groups based on the analysis of genus abundances across samples. A group of genera that were not detected consistently in samples or were detected at very low level is not shown (N = 86). A heat-map of genus abundances is shown on the left-hand side of the image. Gradient color scheme and phylum designation are displayed in the legend. An average abundance of all genera in each type of samples is shown for groups 1 and 2 on line graphs on the right-hand side. For groups 3 and 4, the line graphs display individual genus values. The abundances of after-treatment time points were averaged together. Where possible, error bars were calculated to represent standard error of the mean.