Microbiota dynamics in patients treated with fecal microbiota transplantation for recurrent Clostridium difficile infection

Abstract

Clostridium difficile causes antibiotic-associated diarrhea and pseudomembraneous colitis and is responsible for a large and increasing fraction of hospital-acquired infections. Fecal microbiota transplantation (FMT) is an alternate treatment option for recurrent C. difficile infection (RCDI) refractory to antibiotic therapy. It has recently been discussed favorably in the clinical and scientific communities and is receiving increasing public attention. However, short- and long-term health consequences of FMT remain a concern, as the effects of the transplanted microbiota on the patient remain unknown. To shed light on microbial events associated with RCDI and treatment by FMT, we performed fecal microbiota analysis by 16S rRNA gene amplicon pyrosequencing of 14 pairs of healthy donors and RCDI patients treated successfully by FMT. Post-FMT patient and healthy donor samples collected up to one year after FMT were studied longitudinally, including one post-FMT patient with antibiotic-associated relapse three months after FMT. This analysis allowed us not only to confirm prior reports that RCDI is associated with reduced diversity and compositional changes in the fecal microbiota, but also to characterize previously undocumented post-FMT microbiota dynamics. Members of the Streptococcaceae, Enterococcaceae, or Enterobacteriaceae were significantly increased and putative butyrate producers, such as Lachnospiraceae and Ruminococcaceae were significantly reduced in samples from RCDI patients before FMT as compared to post-FMT patient and healthy donor samples. RCDI patient samples showed more case-specific variations than post-FMT patient and healthy donor samples. However, none of the bacterial groups were invariably associated with RCDI or successful treatment by FMT. Overall microbiota compositions in post-FMT patients, specifically abundances of the above-mentioned Firmicutes, continued to change for at least 16 weeks after FMT, suggesting that full microbiota recovery from RCDI may take much longer than expected based on the disappearance of diarrheal symptoms immediately after FMT.

Figure 1. Overview of analyzed patient and donor samples.

RCDI patient samples are marked in red, post-FMT patient samples in blue and donor samples in green. *Patient #6a experienced antibiotic-induced relapse of C. difficile infection and was treated successfully with a second round of FMT as patient #6b. In the NCBI short read archive, samples referred to as #6b are designated as #7 samples.

Figure 2. Microbiota rarefaction curves showing fecal microbiota diversity in RCDI (red) and post-FMT (blue) patient and donor (green) samples.

Each curve shows the average number of OTUs found in a given number of sampled sequences. OTUs can be treated as equivalent to taxonomic species in the sequence space. RCDI samples are marked from patient #6a (*), who experienced antibiotic-induced relapse and was treated by FMT again as patient #6b (**).

Figure 3. Microbiota diversity (Shannon) and richness (ACE) of RCDI and post-FMT patient and donor samples.

(A) Shannon index; (B) ACE index. Significant differences are shown (*, p<0.01; **, p<0.001) as measured by Wilcoxon rank sum test. RCDI samples from patient #6a (+), who experienced antibiotic-induced relapse and was treated by FMT again as patient #6b (++) are marked.

Figure 4. Unscaled principal coordinate analysis (PCoA) plots showing unweighted (A) and weighted (B) UniFrac analysis of RCDI (red) and post-FMT (blue) patient and healthy donor (green) samples.

RCDI patient samples are circled in red. RCDI samples from patient #6a (*), who experienced antibiotic-induced relapse and was treated by FMT again as patient #6b (**) are marked in dark red. Sample names indicate case numbers, patient or donor source and time point of collection ("0" time point refers to pre-FMT sampling time points; other time points are abbreviated as weeks [w], months [m] and year [y]).

Figure 5. Microbiota changes between RCDI and post-FMT patient and healthy donor sample groups at the taxonomic order level.

Significant differences between sample groups as calculated with the Metastats tool are marked with asterisks (p<0.01).

Figure 6. Microbiota changes between RCDI and post-FMT patient and healthy donor sample groups at the taxonomic family and genus levels.

Significant differences between sample groups as calculated with the Metastats tool are marked with asterisks (p<0.01). Note that standard deviations are smaller for genera that increased in post-FMT relative to RCDI patient samples (e.g., Lachnospiraceae Incertae Sedis) compared to those that decreased (e.g. Streptococcus), which reflects differences in the relative abundances of major microbiota members among RCDI patient samples.

Figure 7. Post-FMT microbiota changes.

Unweighted (A) and weighted (B) UniFrac distances and Jensen-Shannon divergence (C) metrics were calculated between post-FMT and RCDI patient sample pairs (red), post-FMT patient and donor sample pairs (green) and between donor sample pairs collected over time (blue) and plotted on logarithmic scales. R² values and p-values to establish whether the slope of the curve was significantly different from zero are shown with asterisks indicating significance (p<0.05, F-test). The 20-week data point of patient #8 was classified as outlier and not included in the analyses, based on the Bonferroni-adjusted outlier test, and is shown with parentheses. One-year time points (patient and donor #1) were also classified as outliers and omitted from the analysis and plot. A plot showing all data points including those omitted is part of the supplement (Fig. S4).

Figure 8. Post-FMT changes in selected microbiota members by case (genus level).

(A) Lachnospira Incertae Sedis; (B) Ruminococcus; (C) Streptococcus. Genus-specific changes in relative abundance over time were not significant (p>0.05)when samples were grouped by time periods (1 week, 2–4 weeks, 6–8 weeks, 12–20 weeks) and groups compared with a non-parametric statistical test (Wilcoxon rank sum test).