The Long-Term Stability of the Human Gut Microbiota

Background Understanding the dynamics and stability of the human gut microbiota is important if its characterization is to play a role in the diagnosis, treatment, and prevention of disease. To characterize stability in related and unrelated individuals and its responsiveness to physiologic change (weight loss), we developed a method for bacterial 16S rRNA amplicon sequencing at high depth with high precision. We also sequenced the genomes of anaerobic bacteria represented in culture collections prepared from fecal samples collected from individuals over time. Methods Low-error amplicon sequencing (LEA-Seq) is a quantitative method based on redundant sequencing of bacterial 16S rRNA genes. A dilute, barcoded, oligonucleotide primer solution is used to create ~150,000 linear PCR extensions of the template DNA. The labeled, bottlenecked linear PCR pool is amplified with exponential PCR, using primers that specifically amplify only the linear PCR molecules. The exponential PCR pool is sequenced at sufficient depth to obtain ~20× coverage. Multiple reads enable the generation of an error-corrected consensus sequence for each barcoded template molecule. LEA-Seq can be used for a variety of other applications. Relationship among time, physiology, and microbiota stability. (A) Stability of fecal microbiota follows a power-law function (n = 37 females sampled over time; >1 week to <5 years). Dashed lines show 95% confidence bounds over 10- and 50-year extrapolations (inset). (B) Microbiota stability is inversely related to the stability of each individual’s body mass index. Results and Discussion LEA-Seq of fecal samples from 37 healthy U.S. adults sampled 2 to 13 times up to 296 weeks apart revealed that they harbored 195 ± 48 bacterial strains, representing 101 ± 27 species. On average, their individual microbiota was remarkably stable, with 60% of strains remaining over the course of 5 years. Stability followed a power law, which, when extrapolated, suggests that most strains in an individual’s intestine are residents for decades (figure, panel A). Members of Bacteroidetes and Actinobacteria are significantly more stable components than the population average. LEA-Seq of four individuals sampled during an 8- to 32-week period during a calorie-restricted dietary study showed that weight stability is a significantly better predictor of microbiota stability than the time interval between samples (figure, panel B). After generating clonally arrayed collections of anaerobic bacteria from frozen fecal samples collected from six weight-stable individuals sampled 7 to 69 weeks apart, we produced draft genome sequences for 534 isolates representing 188 strains and 75 species. A targeted approach focused on Methanobrevibacter smithii isolates from two sets of twin pairs and their mothers and Bacteroides thetaiotaomicron strains from nine donors including sister-sister and mother-daughter pairs. Strains, defined as isolates sharing >96% of their genome content, were maintained over time within an individual and between family members but not between unrelated individuals. Thus, early gut colonizers, such as those acquired from our parents and siblings, have the potential to exert their physiologic, metabolic, and immunologic effects for most, and perhaps all, of our lives. Inheritance Guts We know little about the stability of the constituent microbiota in the human gut or the extent to which the gut microbiota are a potential target for long-term health interventions. Faith et al. (p. 10.1126/science.1237439) analyzed the fecal microbiota of 37 individuals and found that, on average, 60% of bacterial strains remained stable for up to 5 years and many were estimated to remain stable for decades. Low-error sequencing data suggest that initial microbial colonizers of infant guts could persist over the life span of an individual. 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.