Microbiota transplantation restores normal fecal bile acid composition in recurrent Clostridium difficile infection

Abstract

Fecal microbiota transplantation (FMT) has emerged as a highly effective therapy for refractory, recurrent Clostridium difficile infection (CDI), which develops following antibiotic treatments. Intestinal microbiota play a critical role in the metabolism of bile acids in the colon, which in turn have major effects on the lifecycle of C. difficile bacteria. We hypothesized that fecal bile acid composition is altered in patients with recurrent CDI and that FMT results in its normalization. General metabolomics and targeted bile acid analyses were performed on fecal extracts from patients with recurrent CDI treated with FMT and their donors. In addition, 16S rRNA gene sequencing was used to determine the bacterial composition of pre- and post-FMT fecal samples. Taxonomic bacterial composition of fecal samples from FMT recipients showed rapid change and became similar to the donor after the procedure. Pre-FMT fecal samples contained high concentrations of primary bile acids and bile salts, while secondary bile acids were nearly undetectable. In contrast, post-FMT fecal samples contained mostly secondary bile acids, as did non-CDI donor samples. Therefore, our analysis showed that FMT resulted in normalization of fecal bacterial community structure and metabolic composition. Importantly, metabolism of bile salts and primary bile acids to secondary bile acids is disrupted in patients with recurrent CDI, and FMT corrects this abnormality. Since individual bile salts and bile acids have pro-germinant and inhibitory activities, the changes suggest that correction of bile acid metabolism is likely a major mechanism by which FMT results in a cure and prevents recurrence of CDI.

Keywords: Clostridium difficile; bile acids; fecal microbiota transplantation.

Fig. 1.

Changes in fecal bacterial communities following fecal microbiota transplantation (FMT). A: Shannon diversity indexes of fecal samples. *P < 0.05. B: principal coordinate analysis of UniFrac distances between bacterial communities. PC1, principal coordinate 1; PC2, principal coordinate 2. C: relative abundance of operational taxonomic units (OTUs) from bacterial phyla in fecal samples. Colors correspond to phyla (see key at bottom). D: relative abundance of OTUs from bacterial families in fecal samples. Colors correspond to families (see key at right). †Patient who failed initial FMT.

Fig. 2.

Bile acid concentrations before and after FMT. A: overlay of representative chromatograms of bile acid metabolites in pre- and post-FMT fecal extracts. Chromatograms were generated by extraction of mass spectrometry signals within 20 ppm of calculated exact masses (407.2797, 391.2848, and 375.2899 mass-to-charge ratio in negative mode) of primary and secondary bile acids of sterol metabolites [cholic acid (CA, I), chenodeoxycholic acid (CDCA, II), deoxycholic acid (DCA, III), lithocholic acid (LCA, IV), and IsoDCA (V)]. Signal intensity of deoxycholic acid (III) in the post-FMT sample was arbitrarily set as 100%. B–F: concentration of bile acids (I–V) in pre-FMT, post-FMT, and donor samples.

Fig. 3.

Identification and characterization of FMT-responsive sterol metabolites in fecal extracts of Clostridium difficile infection (CDI) patients. A: scores plot of a principal components analysis model on pre-FMT (▲), post-FMT (△), and donor (∗) samples. The t[1] and t[2] values represent scores of each sample in principal components 1 and 2, respectively. B: loadings plot of principal components analysis model. Sterol metabolites contributing to separation of pre- and post-FMT samples (I–IX) were labeled, and their chemical identities are listed in Table 3. C and D: relative abundances of taurine conjugates of primary bile acids [taurocholic acid (TCA, VI) and taurochenodeoxycholic acid (TCDCA, VII)] in pre-FMT, post-FMT, and donor samples. NS, not significant. *P < 0.05.

Fig. 4.

Antibiotics promote recurrence of CDI by decreasing hydrolysis of bile salts and conversion of primary bile acids to secondary bile acids (the model). A: normal bile acid composition in the colon (shown in brown) prevents germination of C. difficile spores. B: antibiotics allow increased levels of bile salts and primary bile acids in the colon (shown in green), which promote germination of C. difficile spores and growth of vegetative forms of bacteria.