Stability of the human faecal microbiome in a cohort of adult men

Abstract

Characterizing the stability of the gut microbiome is important to exploit it as a therapeutic target and diagnostic biomarker. We metagenomically and metatranscriptomically sequenced the faecal microbiomes of 308 participants in the Health Professionals Follow-Up Study. Participants provided four stool samples-one pair collected 24-72 h apart and a second pair ~6 months later. Within-person taxonomic and functional variation was consistently lower than between-person variation over time. In contrast, metatranscriptomic profiles were comparably variable within and between subjects due to higher within-subject longitudinal variation. Metagenomic instability accounted for ~74% of corresponding metatranscriptomic instability. The rest was probably attributable to sources such as regulation. Among the pathways that were differentially regulated, most were consistently over- or under-transcribed at each time point. Together, these results suggest that a single measurement of the faecal microbiome can provide long-term information regarding organismal composition and functional potential, but repeated or short-term measures may be necessary for dynamic features identified by metatranscriptomics.

Fig. 1. Experimental design

308 participants from the MLVS, nested within the HPFS cohort, were recruited to assess the stability of microbiome communities, metagenomes and metatranscriptomes. Participants provided up to four stool samples using a previously validated self-sampling method. One set of stool samples was collected 24–72 h apart followed by a second set approximately 6 months later. Metagenomic and metatranscriptomic reads were generated to provide taxonomic, functional metagenomic and transcriptional features for stability assessment.

Fig. 2. Inter-individual differences in organismal composition and functional potential appear to be preserved, unlike the more variable metatranscriptomes

a, 1 – Jaccard Index (fraction of shared features) between all possible pairwise combinations of the first faecal sample with the other three samples collected from each individual (n = 308). 95% confidence intervals are shown in grey. b, Bray–Curtis β-diversity scores within and between subjects for short- (24–72 h; n = 308 individuals) and intermediate-term intervals (6 months; n = 160 individuals). Here, species represents taxonomic profile abundances, DNA represents metagenomic functional profiles, and RNA represents metatranscriptomes. Boxplot whiskers include observations within 1.5 interquartile range of the upper and lower quartiles.

Fig. 3. Stability of individual species, genes and transcripts over 6 months is correlated with average baseline relative abundance and prevalence

Each point represents an individual feature—species (n = 139), genes (n = 1,952) or transcripts (n = 1,803). Coloured circles highlight species and gene families discussed in the main text, according to the figure legend.

Fig. 4. Relating metagenomic and metatranscriptomic stability over time

Genomic instability appears to explain a large proportion of transcriptomic instability over intermediate-term (6 month) periods (r²= 0.74); the metatranscriptome should typically be no more stable than the metagenome. In addition, we investigated whether dominant expression of stable genes versus expression of unstable genes varied according to species. For each transcript (n = 1,803), a dominant contributing species was identified based on the maximal average contribution to each transcript. We then ranked the species according to the total number of genes that they dominantly expressed and (for presentation purposes) selected those contributing to 30 or more and overlaid them on the scatterplot in colour (grouped by genus). Each point represents an individual feature.

Fig. 5. Exploring the stability of gene expression

a, For a large proportion of transcriptional pathways over- or under-expressed over time in the stool, this differential expression may be consistent. The Venn diagram shows consistency of differentially transcribed pathways (mean RNA/DNA ratios >2 across at least one time point; n = 218) across 4 stool samples. b, A subset (n = 8) of stably transcribed pathways is highlighted, according to those with the lowest coefficient of variance of mean RNA/DNA expression ratios across the four time points. Many of the stable pathways appear to be involved in cellular housekeeping, such as carbon metabolism (over-expressed; above dashed red line; >1) and amino acid synthesis (under-expressed; below dashed red line; <1). Error bars indicate 95% confidence intervals.