The gut mycobiome of the Human Microbiome Project healthy cohort

Abstract

Background: Most studies describing the human gut microbiome in healthy and diseased states have emphasized the bacterial component, but the fungal microbiome (i.e., the mycobiome) is beginning to gain recognition as a fundamental part of our microbiome. To date, human gut mycobiome studies have primarily been disease centric or in small cohorts of healthy individuals. To contribute to existing knowledge of the human mycobiome, we investigated the gut mycobiome of the Human Microbiome Project (HMP) cohort by sequencing the Internal Transcribed Spacer 2 (ITS2) region as well as the 18S rRNA gene.

Results: Three hundred seventeen HMP stool samples were analyzed by ITS2 sequencing. Fecal fungal diversity was significantly lower in comparison to bacterial diversity. Yeast dominated the samples, comprising eight of the top 15 most abundant genera. Specifically, fungal communities were characterized by a high prevalence of Saccharomyces, Malassezia, and Candida, with S. cerevisiae, M. restricta, and C. albicans operational taxonomic units (OTUs) present in 96.8, 88.3, and 80.8% of samples, respectively. There was a high degree of inter- and intra-volunteer variability in fungal communities. However, S. cerevisiae, M. restricta, and C. albicans OTUs were found in 92.2, 78.3, and 63.6% of volunteers, respectively, in all samples donated over an approximately 1-year period. Metagenomic and 18S rRNA gene sequencing data agreed with ITS2 results; however, ITS2 sequencing provided greater resolution of the relatively low abundance mycobiome constituents.

Conclusions: Compared to bacterial communities, the human gut mycobiome is low in diversity and dominated by yeast including Saccharomyces, Malassezia, and Candida. Both inter- and intra-volunteer variability in the HMP cohort were high, revealing that unlike bacterial communities, an individual's mycobiome is no more similar to itself over time than to another person's. Nonetheless, several fungal species persisted across a majority of samples, evidence that a core gut mycobiome may exist. ITS2 sequencing data provided greater resolution of the mycobiome membership compared to metagenomic and 18S rRNA gene sequencing data, suggesting that it is a more sensitive method for studying the mycobiome of stool samples.

Keywords: Fecal microbiome; Fungal microbiome; Fungi; HMP; ITS2; Metagenomics; Microbiome; Microbiota; There was a high degree o Spacer.

Fig. 1

Fungal and bacteria alpha diversity. a Observed OTUs and Shannon diversity index values of HMP samples with both 16S rRNA gene and ITS2 sequencing data compared. Only visit 1 samples are shown for statistical purposes. Visit 2 and visit 3 comparisons showed similar results. For statistical analysis, only samples with both ITS2 and 16S rRNA gene sequencing data were used. ***P < 0.0001 for both observed OTUs and Shannon diversity index (Mann-Whitney test). b Associations between fungal (ITS2) and bacterial (16S) alpha diversity (observed OTUs and Shannon diversity index values) for a given sample. Shaded gray region represents 95% confidence intervals. Linear regression analysis: P = 0.693 for observed OTUs and P = 0.929 for Shannon diversity. Only samples with ITS2 and 16S rRNA gene sequencing data are plotted and analyzed

Fig. 2

Relative abundance of fungi at the a phylum level and b genus level. a Relative abundance of fungal phyla in each sample. “Fungi sp.” here represents unknown/unidentified fungal phylum. b Relative abundance of fungal genera in each sample. “Fungi sp.” here represents unknown/unidentified fungal genus

Fig. 3

Variability of the mycobiome. a Bacterial (16S) and b Fungal (ITS2) Bray-Curtis dissimilarity shown on principal coordinates analysis (PCoA) plots for a subset of volunteers (20 volunteers, randomly chosen, subsetted for clarity). Samples are colored by volunteer, and each volunteer was assigned the same color in both a and b. Lines connect samples donated by the same volunteer. c Pairwise comparisons of Bray-Curtis dissimilarity values between samples donated by the same volunteer (within volunteers) and between samples donated by different volunteers (between volunteers) for 16S rRNA gene and ITS2 sequencing data. Bray-Curtis dissimilarity values range from 0 to 1, with 0 being the least dissimilar and 1 being the most dissimilar. ***P < 0.0001; ns: not significant

Fig. 4

Correlations occurring between fungal taxa (ITS2) and a fungal taxa (ITS2) or b bacterial taxa (16S). Red squares represent significant (P < 0.05 after FDR adjustment) negative correlations. Blue squares represent significant (P < 0.05 after FDR adjustment) positive correlations. Darker colors represent stronger correlations. Non-significant correlations are not shown