Gut microbiota strain richness is species specific and affects engraftment

Abstract

Despite the fundamental role of bacterial strain variation in gut microbiota function^1-6, the number of unique strains of a species that can stably colonize the human intestine is still unknown for almost all species. Here we determine the strain richness (SR) of common gut species using thousands of sequenced bacterial isolates with paired metagenomes. We show that SR varies across species, is transferable by faecal microbiota transplantation, and is uniquely low in the gut compared with soil and lake environments. Active therapeutic administration of supraphysiologic numbers of strains per species increases recipient SR, which then converges back to the population average after dosing is ceased. Stratifying engraftment outcomes by high or low SR shows that SR predicts microbial addition or replacement in faecal transplants. Together, these results indicate that properties of the gut ecosystem govern the number of strains of each species colonizing the gut and thereby influence strain addition and replacement in faecal microbiota transplantation and defined live biotherapeutic products.

Extended Data Fig. 1 |. Determination of SR_j strain threshold and validation of SR_j with deeper sampling of a subset of cultured gut bacterial species.

A, K-mer overlap was calculated for all pairwise combinations of isolates from a species cultured from two unrelated individuals (purple) with no direct microbial transfer between them or an individual’s own microbes from a single timepoint (green). Dotted line shows the threshold of 0.96 k-mer similarity. B, fastANI versus pairwise k-mer overlap for isolates of the same species. Dotted line represents k-mer overlap threshold of 0.96 (k-mer distance of <0.04). C, Comparison of B. fragilis SR_ij for the individuals in this study and in the study by Zhao et al. D, Preliminary calculation of SR_j using genomes isolated from the Broad pipeline (standard pipeline used to create libraries of cultured gut bacteria). For deeper sampling estimates of SR_j, we isolated additional genomes from the same species from E, the original human stool sample and F, mouse stool samples from gnotobiotic mice colonized with the same human stool (N = 2–3 mice per microbiota/diet combination) and given unique diets for strain enrichment. Error bars in D–F represent SEM. Comparison of SR_j calculated with genomes from the broad pipeline or the broad pipeline plus additional genomes from G, human stool samples or H, human and mouse stool samples. I–L, Rarefaction curves for validation species. Dotted line shows the mean isolates/species for each species (overall mean isolates/species across the dataset was 4.7 isolates as demonstrated in Fig. 1b). M,N, Rarefaction curves for a soil species (M) and lake species (N). **p < 0.01, paired Wilcoxon test, each point represents the average SR_j measured from microbiomes of a healthy or disease state. ns: not significant by paired Wilcoxon test. Grey regions on rarefaction curves indicate 95% confidence intervals.

Extended Data Fig. 2 |. The influence of core genome size and disease state on strain richness.

A, Spearman rank correlation for SR_j versus core genome fraction for several genera. B, SR_j was compared for 59 species present in both healthy and CD microbiomes. C, SR_j was compared for 51 species present in both healthy and UC microbiomes. Each point represents the average SR_j of a species as calculated using isolates cultured from healthy microbiomes or isolates cultured from disease microbiomes. Lines in B,C connect the SR_j for each shared species between a healthy subject and a subject with IBD. ns: not significant by paired Wilcoxon test.

Extended Data Fig. 3 |. Metagenomics-quantified SR_j correlates with the cultured SR_j measured across our cohort.

A, Spearman correlation between donor SR_j as measured by metagenomics and SR_j from our original cultured cohort (first panel) and differences in SR_j between the two groups (second panel). B, Spearman correlation between recipient SR_j as measured by metagenomics at week 8 post-FMT and SR_j from our original cohort (first panel) and differences in SR_j between the two groups (second panel). A,B, Each point represents the average SR_j for species. C, Heatmap representing the remaining six donors who donated stool to six different recipients. D, Correlation of SR_j across all recipients and all donors in an independent FMT validation cohort (Leiden).

Extended Data Fig. 4 |. An ecological framework for strain persistence, replacement, or addition based on strain richness.

A, Schematic for FMT experimental design with multi-donor stool batches administered to each recipient. B, Within a species, bacterial strains vary in their engraftment frequency when administered in the context of a multi-donor FMT product. C, Spearman rank correlation between the metagenomics SR_j of the individual FOCUS donors with the overall cultured SR_j measured across our cohort. D, Expected outcomes for donor strain engraftment based on species SR_j.

Fig. 1 |. SR_j of 92 human gut species.

a, SR_j varies by species (Kruskal–Wallis, p <2.2 × 10⁻¹⁶ Data are represented as mean value ± s.e.m. b, SR_j using the isolate genome set in this study (n = 4,773) is highly correlated with SRj estimated from an independent set of bacterial genomes (n = 1,947) isolated from 11 humans. Spearman rank correlation was applied. Grey area, 95% confidence interval. c, The SR_j of human gut species is lower than SR_j of species isolated from lake and soil microbiomes (Kruskal–Wallis test). Blue points, average SR_j for a species found in each of the environments; black points, mean of the environment; error bars, s.e.m.

Fig. 2 |. Factors influencing SR_j.

a, Spearman rank correlation of SR_j versus species frequency in our cohort. No corrections were used. Grey area, 95% confidence interval. b, Pairwise k-mer distances between unique conspecific strains for species with SR_j < 1.1 and SR_j > 1.2. c, Functional categories over- and under-represented in genes that vary between species with SR_j < 1.1 versus SR_j > 1.2.

Fig. 3 |. FMT durably transmits healthy donor SR_j to rCDI patients.

a, Schematic of FMT experimental design with 1:1 donor–recipient pairs. b, Representative heatmap showing the transmission of SR_ij from donor D283 to seven different recipients (R282, R285 and so on). The numbers in the cells of the heatmap indicate the number of strains of the given species detected in the donor or recipient across five time points for donor D283 and up to three time points for the seven recipients. c, Spearman rank correlation between recipient SR_j at week 8 post-FMT and donor SR_j. d, Spearman rank correlation between donor SR_j at 5 years and time 0 (pre-FMT stool sample). e, Spearman rank correlation between recipient SR_j at 5 years post-FMT and 8 weeks post-FMT. c–e, Each datapoint represents the average SR_j for a tracked species in b; grey area, 95% confidence interval, and Spearman rank correlations are two-tailed. Credit: a, © Jeremiah Faith/123rf.com.

Fig. 4 |. Supraphysiologic manipulation of SR_j in FMT recipients converges to the population baseline observed in untransplanted people.

a, Heatmap showing the SR_ij of single donors, donor batches and recipients at post-FMT time points both during and after FMT drug administration. b, SR_j for representative species across the donor batch, recipient post-FMT time points and cultured cohort from Fig. 1a (individual donors). Data are presented as the mean value ± s.e.m. with each point representing the SR_j for a species at each time point or for the cultured cohort. c, Spearman rank correlation (two-tailed) between recipient SR_j at drug week 8 and donor batch SR_j. Each point represents the SR_j of a species as measured in the pooled donor batch versus as measured in the recipient post-FMT week 8. d, SR_j across donors, batches, recipient time points and culture (previously measured in Fig. 1a and Extended Data Table 1). Blue points, tracked SR_j of a species as measured in each donor group (individual donors versus pooled batch) or in each recipient post-FMT time point; black points, mean SR_j across the overall time point or group. Two-sided Wilcoxon tests were used to compare groups. e, Proportional occurrence of addition, persistence and replacement events across species with low SR_j and high SR_j. ***P < 10⁻³, Wilcoxon test; ****P < 10⁻⁴, Wilcoxon test; NS, not significant by Wilcoxon test. Exact P values: individual donors versus recipients 5 years (P = 5.1 × 10⁻⁵), donor batch versus recipients week 4 (P = 6 × 10⁻⁸), recipients week 4 versus week 8 (P = 8.5 × 10⁻⁴), recipients week 8 versus week 16 (P = 1.9 × 10⁻⁵), recipients week 16 versus recipients 5 years (P = 1.2 × 10⁻⁵), recipients 5 years versus cultured SR_j (P = 0.071).