Growth dynamics of gut microbiota in health and disease inferred from single metagenomic samples

Abstract

Metagenomic sequencing increased our understanding of the role of the microbiome in health and disease, yet it only provides a snapshot of a highly dynamic ecosystem. Here, we show that the pattern of metagenomic sequencing read coverage for different microbial genomes contains a single trough and a single peak, the latter coinciding with the bacterial origin of replication. Furthermore, the ratio of sequencing coverage between the peak and trough provides a quantitative measure of a species' growth rate. We demonstrate this in vitro and in vivo, under different growth conditions, and in complex bacterial communities. For several bacterial species, peak-to-trough coverage ratios, but not relative abundances, correlated with the manifestation of inflammatory bowel disease and type II diabetes.

Trial registration: ClinicalTrials.gov NCT01892956.

Copyright © 2015, American Association for the Advancement of Science.

Figures

Fig. 1. Peak-to-trough coverage ratio (PTR) accurately measures in vitro growth rates of Escherichia coli

(A) Sequenced reads (right) are mapped to complete bacterial genomes and the sequencing coverage across the genome is plotted. Each bacteria cell in a growing population (top) will be at a different stage of DNA replication, generating a coverage pattern that peaks near the known replication origin (green vertical line in graph), and thus produce a prototypical sequencing coverage pattern with a single peak and a single trough. Bacteria from a non-dividing population (bottom) have a single copy of the genome, producing a flat sequencing coverage pattern across the genome. (B) Above: sequencing coverage patterns of a non-replicating (left) and actively replicating (right) E. coli from two human gut metagenomic samples. Below: distribution of PTR of E. coli across 583 different human gut metagenomic samples (, , ; histogram) and 58 in vitro samples from four growth experiments (boxplot). (C) Genome coverage plots of E. coli, measured at different times during an in vitro cell growth experiment, showing that PTRs are highest during exponential growth (timepoints 1–2.5) and lowest in lag (timepoint 0) and stationary (timepoints 3–7) phases. (D) PTRs (red line) correlate (R=0.95, p<10−4) with the growth rate (black line), measured as the derivative of the logged abundance curve (abundance is measured as optical density of the culture; OD; blue line) in the subsequent 30 minutes (23). N=2. Symbols, mean; error bars, SEM.

Fig. 2. Peak-to-trough coverage ratio (PTR) accurately measures in vitro growth rates in multiple conditions

(A–C) Absolute abundance levels (colony forming units per ml; CFU/ml; top y-axis, blue) and PTR (bottom y-axis, red) as a function of time (minutes) of an in vitro culture of Citrobacter rodentium treated with erythromycin (bacteriostatic antibiotic in this setting, N=2), compared to those of (A) An untreated control culture (N=3); (B) A culture treated with nalidixic Acid, a drug to which C. rodentium is resistant (N=3); and (C) A culture treated with kanamycin, a bactericidal drug in this setting (N=3). Background color indicates the treatment period (dark gray, left), recovery period (gray, middle) and early stationary phase (light gray, right). The black vertical line denotes antibiotic washout. PTR changes precede changes in growth. P-values are Mann-Whitney U-test between abundance (top) or PTR (bottom) of the two different cultures show, at times 30–150 minutes (left) or times 210–300 minutes (right). (D) Bacterial abundances (CFU/ml; top panel) and PTR (bottom panel) of L. gasseri and a mixture of six additional bacterial strains that inhabit the human gut. N=4. Symbols, mean; error bars, SEM.

Fig. 3. Peak-to-trough ratios (PTR) reflect the growth dynamics of in vivo microbial communities

(A) Shown are PTRs of virulent (icc169; WT; N=3) and non-virulent (tir mutant; N=3) Citrobacter rodentium 1-5 or 6-9 days post infection (p.i.) of C57BL/6 mice previously depleted of their native microbiota. PTR of stationary and exponential in vitroC. rodentium cultures are shown for reference. P-values are Mann-Whitney U-test. See Fig. S3 for the corresponding measured abundances. (B) Relative abundance (left y-axis, blue) and PTRs (right y-axis, red) of Parabacteroides distasonis from fecal metagenomic samples obtained approximately every 6 hours from one human individual in 4 consecutive days (26). Plotted lines are spline interpolations using the displayed data points. Time is with respect to light cycles (Zeitgeber time, horizontal axis). P-value is for 24 hour oscillations (23). (C, D) Shown are standardized PTRs (top graphs, mean ± SEM) and specific PTRs for species present in the sample (bottom heatmaps), belonging to two human subjects that underwent a radical dietary change. Compared are days in which only white boiled rice was consumed (grey area) and days of normal diet (white area). A global change in bacterial growth dynamics was observed between dietary regimens (** - Mann-Whitney p<0.005, *** - p<0.001).

Fig. 4. Bacterial dynamics correlate with several diseases and metabolic disorders

Peak-to-trough ratios (PTR) of species from Chinese (; N=363; Q) and European (; N=396; M) cohorts are shown (boxplots, left; red-median; boundaries-25–75th percentiles) if its relative abundances or PTRs were significantly associated with clinical parameters. Shown are phylum membership; the number of samples for which PTRs were calculated; and a row with colored entries for each statistically significant (FDR-corrected p<0.05) association between clinical parameters and its PTR (left column block) or relative abundance (right column block). Mann-Whitney U-test and Spearman correlations were used for binary and continuous clinical parameters, respectively. Top-block: species with significant associations between PTR and clinical parameters; bottom-block: species with significant associations only between relative abundance and clinical parameters. A-Actinobacteria, B-Bacteroidetes, E- Euryarchaeota. F-Firmicutes, P-Proteobacteria, V-Verrucomicrobia