The long-term stability of the human gut microbiota

Abstract

A low-error 16S ribosomal RNA amplicon sequencing method, in combination with whole-genome sequencing of >500 cultured isolates, was used to characterize bacterial strain composition in the fecal microbiota of 37 U.S. adults sampled for up to 5 years. Microbiota stability followed a power-law function, which when extrapolated suggests that most strains in an individual are residents for decades. Shared strains were recovered from family members but not from unrelated individuals. Sampling of individuals who consumed a monotonous liquid diet for up to 32 weeks indicated that changes in strain composition were better predicted by changes in weight than by differences in sampling interval. This combination of stability and responsiveness to physiologic change confirms the potential of the gut microbiota as a diagnostic tool and therapeutic target.

Fig. 1. Multiplex bacterial 16S rRNA gene sequencing using LEA-Seq; comparison with previous methods using mock communities composed of sequenced gut bacterial species

(A) Schematic of how the LEA-Seq method is used to redundantly sequence PCR amplicons from a set of linear PCR template extensions of bacterial 16S rDNA. This approach results in amplicon sequences with a higher precision than standard amplicon sequencing at lower abundance thresholds. (B) Performance of 16S rRNA amplicon sequencing methods assayed as the precision obtained for different sequence abundance thresholds. Standard methods for amplicon sequencing using the 454 pyrosequencer and the Illumina MiSeq instrument exhibit increased precision as less abundant reads are filtered out. By redundantly sequencing each amplicon with LEA-Seq, the precision of amplicon sequencing is increased at lower abundance thresholds for both the V1V2 region of the bacterial 16S rRNA gene (compare red and blue lines) and the V4 region (compare magenta and blue lines), thereby enabling detection of lower-abundance bacterial taxa at high precision.

Fig. 2. Measuring the stability of an individual’s fecal microbiota over time with LEA-Seq

(A) The Jaccard Index (fraction of shared strains) was calculated between all possible pairwise combinations of fecal samples collected from each individual, where bacterial strains were considered shared if the nucleotide sequence was 100% identical across 100% of the length of the V1V2 region of their 16S rRNA genes. Jaccard Indexes were binned into intervals of <3 weeks, 3–6 weeks, 6–9 weeks, 9–12 weeks, 12–32 weeks, 32–52 weeks, 52–104 weeks, 104–156 weeks, 156–208 weeks, 208–260 weeks, and >260 weeks apart (mean±SE for each bin is shown). The decay in the Jaccard Index as a function of time between two samples best fits a power law (blue line). (B) Four individuals losing 10% of their body weight in the study involving consumption of a monotonous low calorie liquid diet (magenta) had significantly less stable microbiota than the mean of the 33 remaining individuals (blue). Mean±SE for the Jaccard Index are plotted. (C) At the phylum level, Bacteroidetes (blue) and Actinobacteria (red) were more stable components of the microbiota than the Proteobacteria and Firmicutes (hypergeometric distribution).

Fig. 3. Relationship between weight stability, time, and fecal microbiota stability

(A) The microbiota sampled from a given individual during periods of weight loss or gain has decreased stability (lower Jaccard Index). (B) The Jaccard Index decreased as the time between samples increased (also see Fig. 2). (C) Across samples from 37 individuals, a linear model of microbiota stability as a function of changes in lnBMI and changes in time explained 46% of the variation in the stability of the microbiota (Jaccard Index). Note that changes in lnBMI explained more of the variation in microbiota stability than did the passage of time. Color changes correspond to the Jaccard Index values in the color bar on the right. Blue dots show the change in Jaccard Index, time, and lnBMI between two samples from a given individual.

Fig. 4. Comparison of genome stability in fecal bacterial isolates recovered from individuals over time

The fraction of aligned nucleotides between any two microbial genomes was calculated using the coverage score (see text for definition). (A) Histogram of the fraction of aligned genome content between all sequenced bacterial isolates from unrelated individuals (blue; only coverage scores ≥ 0.01 are shown) shows that the alignable genome content never exceeded 96% (dotted line). However, highly conserved strains with coverage scores exceeding this threshold were readily detected in the microbiota of individuals at a single time point (red) or between samples from an individual taken up to 15 months apart (green). The y-axis “Counts” represent the number of times a sample fell into each coverage score bin. (B,C) Sequencing the genomes of M. smithii strains (panel B) and B. thetaiotaomicron strains (panel C) revealed that no two isolates from unrelated individuals had more than 96% shared (alignable) gene content (blue), while highly conserved strains above this threshold were found between isolates obtained from a single individual’s fecal microbiota at a single time point (red), as well as from isolates taken from different members of the same family (brown).