Correlation between intestinal microbiota and urolithin metabolism in a human walnut dietary intervention

Abstract

This study is to investigate the relationship between the intestinal microbiota and urine levels of the ellagic acid-derived polyphenols, the urolithins, in a cohort of subjects following a three-week walnut dietary intervention. We longitudinally collected fecal and urine samples from 39 subjects before and after walnut consumption (2 oz per day for 21 days). 16S RNA gene sequencing was performed on fecal DNA to study the association between microbiota composition and the levels of nine urolithin metabolites, which were measured using UHPLC/Q-TOF-MS/MS. Fecal microbial composition was found to be significantly different between pre- and post-walnut intervention (beta diversity, FDR-p = 0.018; alpha diversity, p = 0.018). Roseburia, Rothia, Parasutterella, Lachnospiraceae UCG-004, Butyricicoccus, Bilophila, Eubacterium eligens, Lachnospiraceae UCG-001, Gordonibacter, Paraprevotella, Lachnospira, Ruminococcus torques, and Sutterella were identified as the 13 most significantly enriched genera after daily intake of walnuts. We observed 26 genera that were significantly associated with 7 urolithin metabolites, with 22 genera positively correlating after walnut supplementation (FDR-p ≤ 0.05). PICRUSt analysis showed that several inferred KEGG orthologs were associated with 4 urolithin metabolites after walnut intake. In this study, we found that walnut supplementation altered urolithin metabolites, which associates with specific changes in bacterial taxa and inferred functional contents.

Keywords: 16S RNA gene sequencing; Clinical study; Human gut microbiota; Urolithin; Walnut.

Fig. 1

The difference in gut microbiota composition before and after walnut consumption. A Principal coordinate analysis (PCoA) showing beta diversity measured by Bray–Curtis dissimilarity between timepoints (p = 0.001, FDR-p = 0.018). B Alpha-diversity measured by the Shannon index for subjects from timepoint A and timepoint B (p = 0.018, FDR-p = 0.159). Samples before walnut dietary intervention represent timepoint A (n = 38), and samples after walnut dietary intervention represent timepoint B (n = 39). P values < 0.05 were considered statistically significant

Fig. 2

Identification of differentially enriched microbial genera between before and after walnut intake. A Volcano plot showing differences in the gut microbiome genera between timepoints (FDR-p < 0.1). See Supplemental Table 1 for full list. B Box plot showing Gordonibacter relative abundance at both timepoints (p = 0.0085, FDR-p = 0.067). Samples before walnut dietary intervention represent timepoint A (n = 38), and samples after walnut dietary intervention represent timepoint B (n = 39). P values < 0.05 were considered statistically significant

Fig. 3

The difference in inferred functional contents before and after walnut consumption. A PCA demonstrating the difference in KEGG orthologs between timepoints (p = 0.005). B Volcano plot of KEGG orthologs at timepoint A and timepoint B (p < 0.01). See Supplemental Table 2 for full list. Samples before walnut dietary intervention represent timepoint A (n = 38), and samples after walnut dietary intervention represent timepoint B (n = 39). P values < 0.05 were considered statistically significant

Fig. 4

Association between genera abundances and measured urolithin levels before and after walnut supplementation. A The correlation heatmap of gut microbiota genera and different urolithin metabolites (FDR-p ≤ 0.05, rho > 0.4 or rho < -0.4). Spearman's correlations between 17 enriched relative abundance of bacterial genera and 8 urolithin metabolites at timepoint A. B Correlation between gut microbiota genera and different urolithin metabolites (FDR-p ≤ 0.05, rho > 0.4 or rho < -0.4). Spearman's correlations between 26 enriched relative abundance of bacterial genera and 7 urolithin metabolites at timepoint B. Blue represents positive correlations, and red represents negative correlations. See Supplemental Table 3 for full list

Fig. 5

Association between inferred functional contents and urolithin metabolites before and after walnut supplementation. A Correlation between KEGG orthologs and urolithin metabolites at timepoint A (FDR-p < 0.01, rho > 0.55 or rho < -0.55) and (B) timepoint B (FDR-p < 0.01, rho > 0.55 or rho < -0.55). Blue represents positive correlations, and red represents negative correlations. See Supplemental Table 4 for full list