Fecal Microbiome, Metabolites, and Stem Cell Transplant Outcomes: A Single-Center Pilot Study

Abstract

Background: Accumulating evidence suggests that the intestinal microbiome may dramatically affect the outcomes of hematopoietic stem cell transplant (HSCT) recipients. Providing 16S ribosomal RNA based microbiome characterization in a clinically actionable time frame is currently problematic. Thus, determination of microbial metabolites as surrogates for microbiome composition could offer practical biomarkers.

Methods: Longitudinal fecal specimens (n = 451) were collected from 44 patients before HSCT through 100 days after transplantation, as well as 1-time samples from healthy volunteers (n = 18) as controls. Microbiota composition was determined using 16S ribosomal RNA V4 sequencing. Fecal indole and butyrate levels were determined using liquid chromatography tandem mass spectrometry.

Results: Among HSCT recipients, both fecal indole and butyrate levels correlated with the Shannon diversity index at baseline (P = .02 and P = .002, respectively) and directly after transplantation (P = .006 and P < .001, respectively). Samples with high butyrate levels were enriched for Clostridiales, whereas samples containing high indole were also enriched for Bacteroidales. A lower Shannon diversity index at the time of engraftment was associated with increased incidence of acute intestinal graft-vs-host disease (iGVHD) (P = .02) and transplant-related deaths (P = .03). Although fecal metabolites were not associated with acute iGVHD or overall survival, patients contracting bloodstream infections within 30 days after transplantation had significantly lower levels of fecal butyrate (P = .03).

Conclusions: Longitudinal analysis of fecal microbiome and metabolites after HSCT identified butyrate and indole as potential surrogate markers for microbial diversity and specific taxa. Further studies are needed to ascertain whether fecal metabolites can be used as biomarkers of acute iGVHD or bacteremia after HSCT.

Keywords: butyrate; graft-vs-host disease (GVHD); hematopoietic stem cell transplant (HSCT); indole; microbiome.

Figure 1.

Differences in microbial diversity between healthy volunteers and hematopoietic stem cell transplant (HSCT) recipients at baseline. A, Box plots of the number of observed operational taxonomic units (OTUs) and the Shannon diversity index for each group are shown, where blue represents 1-time samples from healthy volunteers and orange, samples from HSCT recipients at baseline. †P < .001 (nonparametric Mann-Whitney U test). B, Principal coordinate analyses using Bray-Curtis distances. Biplot displays the dominant taxa (listed on the diagram where average genera abundance across samples is denoted by size of the gray sphere in inlaid legend), which explain the variation seen in the principal component analysis plot. Again, blue represents 1-time samples from healthy volunteers, and orange, samples from HSCT recipients at baseline. Permutational multivariate analysis of variance test was used to determine P and R² values for the differences between the 2 groups. C, Box plots of the differences in relative abundance of the top 10 most abundant genera, with a minimum average abundance of >0.01. P values were determined using the nonparametric Mann-Whitney U test and were adjusted to account for multiple testing using the Benjamini-Hochberg correction. *P < .01; †P < .001.

Figure 2.

Fecal metabolite differences and correlations in healthy volunteers and hematopoietic stem cell transplant (HSCT) recipients at baseline. A, B, Fecal indole (A) and butyrate (B) levels in healthy volunteers and HSCT recipients at baseline. P values refer to pairwise comparisons using Mann-Whitney U test. For the purpose of visualization, metabolite abundance is expressed in log scale, and samples with an abundance of zero were set to 0.001. C–F, Longitudinal HSCT recipient cohort. Fecal indole (C, E) and butyrate (D, F) levels at baseline and first sample after transplantation, respectively, are correlated with the Shannon diversity index. Pearson r and P values for the linear regression coefficient are shown.

Figure 3.

Linear discriminant analysis (LDA) scoring taxa associations with levels of fecal butyrate and indole. Histograms represent LDA scores for differentially abundant features among groups. The threshold on the logarithmic LDA score for discriminative features was set to 4.0. A, Taxa differences associated with high levels of butyrate (>0.02 μmol/g) at baseline [1]. B, Taxa enriched in samples with high indole levels (>0.1 μmol/g) at baseline.

Figure 4.

Fecal butyrate levels 2 weeks after transplantation are significantly decreased among patients in whom bloodstream infection (BSI) develops within 30 days after transplantation. All data points represent fecal butyrate levels at week 2, visit 1, and are segmented according to BSI status. (P value determined with Mann-Whitney U test.)