Gut fungal dysbiosis correlates with reduced efficacy of fecal microbiota transplantation in Clostridium difficile infection

Abstract

Fecal microbiota transplantation (FMT) is effective in treating recurrent Clostridium difficile infection (CDI). Bacterial colonization in recipients after FMT has been studied, but little is known about the role of the gut fungal community, or mycobiota. Here, we show evidence of gut fungal dysbiosis in CDI, and that donor-derived fungal colonization in recipients is associated with FMT response. CDI is accompanied by over-representation of Candida albicans and decreased fungal diversity, richness, and evenness. Cure after FMT is associated with increased colonization of donor-derived fungal taxa in recipients. Recipients of successful FMT ("responders") display, after FMT, a high relative abundance of Saccharomyces and Aspergillus, whereas "nonresponders" and individuals treated with antibiotics display a dominant presence of Candida. High abundance of C. albicans in donor stool also correlates with reduced FMT efficacy. Furthermore, C. albicans reduces FMT efficacy in a mouse model of CDI, while antifungal treatment reestablishes its efficacy, supporting a potential causal relationship between gut fungal dysbiosis and FMT outcome.

Fig. 1

Fungal alterations in CDI. a Comparison of the total fungal load in the feces of controls and CDI subjects. Statistical significance was determined by Mann–Whitney test, ^***P < 0.001. b Comparison of the fecal mycobiota based on Shannon diversity, evenness, Chao1 richness in controls and CDI subjects. The dots indicate individual values of the studied subjects. Statistical significance was determined by Mann–Whitney test, *P < 0.05, ^**P < 0.01. c Fungal community structure difference between controls and CDI by NMDS (nonmetric multidimensional scaling) plot based upon Bray–Curtis dissimilarities. d Comparison of the fecal mycobiota composition between controls and CDI subjects at the phylum level. e Differentially enriched fungal species between controls and CDI. Statistical significance was determined by LefSe analysis with FDR correction (only those species with q values < 0.05 and LDA effect size > 2 are shown). Heatmap of the presence of these differential fungal species is shown in relative abundance intensity. LDA effect size, log₁₀ transformed q value (FDR-adjusted p value) and species annotation are shown. Green bars and dots indicate species enriched in controls, while red bars and dots indicate species enriched in CDI. f Comparison of the relative abundance of fecal C. albicans in controls and CDI subjects. Statistical significance was determined by Mann–Whitney test, ^**P < 0.01. For box plots, the boxes extend from the first to third quartile (25th to 75th percentile), with the median depicted by a vertical line

Fig. 2

Post-FMT alterations in the enteric mycobiota richness and diversity of CDI recipients in association with FMT response. Fecal fungal richness (a) and diversity (b) alterations in FMT recipients over the course of longitudinal follow-up and in their corresponding donors at baseline. Comparison of the fungal richness and diversity of pre-FMT samples and post-FMT samples collected at the last follow-up are shown in FMT responders and FMT nonresponders, respectively. Statistical significance was determined by paired Wilcoxon signed rank test, ^*p < 0.05. “F” indicates FMT-treated subject. “W” indicates weeks post-treatment

Fig. 3

Post-FMT alterations in the enteric mycobiota composition of CDI recipients in association with FMT response. a Alterations in the fecal fungal composition at the genus level in CDI recipients after FMT at different time-points up to the last follow-up. b Differentially enriched fungal taxa across all post-FMT fecal samples of FMT responders versus nonresponders at the genus and species levels. Statistical significance level was determined by LefSe analysis with FDR correction (only those taxa with q values < 0.05 and LDA effect size > 2 are shown) and adjustment for subject number. Green bars and dots indicate taxa enriched in FMT responders, while red bar and dot indicate taxa enriched in FMT nonresponders

Fig. 4

The presence of C. albicans is linked to FMT outcomes in CDI. a Alterations in the relative abundance of fecal C. albicans before and after FMT at the last follow-up in FMT recipients, assessed by ITS sequencing. The relative abundance of fecal C. albicans was calculated as the proportion of sequences taxonomically binned as C. albicans relative to the sequences classified as fungi. Statistical significance was determined by paired Wilcoxon signed rank test, ^*p < 0.05. b The absolute fecal C. albicans levels before and after FMT at the last follow-up in FMT recipients, assessed by quantitative PCR. For those FMT nonresponders, the last follow-up time-point was a time prior to CDI recurrence after FMT. Comparison of the fecal C. albicans levels between pre-FMT samples and post-FMT samples was performed by paired Wilcoxon signed rank test, ^*p < 0.05. Comparison of the fecal C. albicans levels between the post-FMT samples of FMT responders and FMT nonresponders was performed by Mann–Whitney test, ^$$p < 0.01. ND denotes no detectable C. albicans in the feces

Fig. 5

C. albicans compromises FMT efficacy in eradicating C. difficile infection in mice. a Schematic diagram of C. albicans administration and stool infusion (FMT) in a murine C. difficile infection (CDI) model. Antibiotic treatment was ceased before gavage of C. albicans (CA) and C. difficile. Number denotes day across the timeline of the experiment. b Diarrhea in mice on day 1 after FMT. Stool percentage water was expressed as mean ± s.e.m. Statistical significance represents comparisons by t test, ^*p < 0.05, ^**p < 0.01. c Representative H&E-stained cecum sections on day 1 after stool infusion (asterisk denotes edema). Scale bar, 100 µm. Histological scores were assessed and expressed as mean ± s.e.m. n = 6 mice per group. Statistical significance represents comparisons by t test, ^*P < 0.05, ^**p < 0.01, ^***p < 0.001. d Enumeration of C. difficile in mouse feces on day 0 before FMT and day 1 post-FMT (n = 9 mice per group). Statistical significance represents comparisons of FMT-treated mice with C. difficile infection versus other groups by unpaired Mann–Whitney test. ^*p < 0.05, ^**p < 0.01. e Enumeration of C. albicans in mouse feces both on day 0 before FMT and on day 1 post-FMT (n = 9 mice per group). Statistical significance represents comparison between C. albicans load on day 0 before FMT and day 1 post-FMT, by paired Mann–Whitney test. ^*p < 0.05. Dot graphs show means ± s.e.m, performed at least two times independently