A typical human intestinal microbiota contains 100–1,000 bacterial species with tremendous compositional diversity between individuals, such that each individual’s microbiota is as unique as a fingerprint1,4. Despite the taxonomic diversity, metagenomic sequencing has highlighted that a health-associated intestinal microbiome codes for highly conserved gene families and pathways associated with basic bacterial physiology and growth2. However, many basic microbiota functions related to homeostasis, immune system development, digestion, pathogen resistance and microbiota inheritance have yet to be discovered5. This formidable challenge to validate and decipher the functional attributes of the microbiota has been hindered because the majority of intestinal bacteria are widely considered to be ‘unculturable’ and have never been isolated in the laboratory3,6.

We sought to establish a genomic-based workflow that could be used as a platform for targeted culturing of specific bacterial phenotypes (Extended Data Fig. 1). Accordingly, we collected fresh faecal samples from six healthy humans and defined the resident bacterial communities with a combined metagenomic sequencing and bacterial culturing approach. Applying shotgun metagenomic sequencing, we profiled and compared the bacterial species present in the original faecal samples to those that grew as distinct colonies on agar plates containing the complex, broad-range bacteriological medium, YCFA7. Importantly, we observed a strong correlation between the two samples at the species level (Spearman’s ρ = 0.75, P < 0.01) (Fig. 1a). When sequenced, the original faecal sample and the cultured bacterial community shared an average of 93% of raw reads across the six donors. This overlap was 72% after de novo assembly (Extended Data Fig. 2). Comparison to a comprehensive gene catalogue that was derived by culture-independent means from the intestinal microbiota of 318 individuals4 found that 39.4% of the genes in the larger database were represented in our cohort and 73.5% of the 741 computationally derived metagenomic species identified through this analysis were also detectable in the cultured samples.

Figure 1: Targeted phenotypic culturing facilitates bacterial discovery from healthy human faecal microbiota. a, Relative abundance of bacteria in faecal samples (x axis) compared with relative abundance of bacteria growing on YCFA agar plates (y axis) as determined by metagenomic sequencing. Bacteria grown on YCFA agar are representative of the complete faecal samples as indicated by Spearman ρ = 0.75 (n = 6). b, Principal component analysis plot of 16S rRNA gene sequences detected from six donor faecal samples (n = 6), representing bacteria in complete faecal samples (green), faecal bacterial colonies recovered from YCFA agar plates without ethanol pre-treatment (black) or with ethanol pre-treatment to select for ethanol-resistant spore-forming bacteria (red). Culturing without ethanol selection is representative of the complete faecal sample, ethanol treatment shifts the profile, enriching for ethanol-resistant spore-forming bacteria and allowing their subsequent isolation. c, Phylogenetic tree of bacteria cultured from the six donors constructed from full-length 16S rRNA gene sequences. Novel candidate species (red), genera (blue) and families (green) are shown by dot colours. Major phyla and family names are indicated. Proteobacteria were not cultured, but are included for context. PowerPoint slide Full size image

Together, these results demonstrate that a considerable proportion of the bacteria within the faecal microbiota can be cultured with a single growth medium. However, more than 8 × 106 distinct colonies would need to be picked from YCFA agar plates to match the species detection sensitivity of metagenomic sequencing. Thus, we established a broad-range culturing method that, when combined with high-throughput archiving or specific phenotypic selection, can be used to isolate and identify novel bacteria from the gastrointestinal tract.

The human intestinal microbiota is dominated by strict anaerobic bacteria that are extremely sensitive to ambient oxygen, so it is not known how these bacteria survive environmental exposure to be transmitted between individuals. Certain members of the Firmicutes phylum, including the diarrhoeal pathogen Clostridium difficile, produce metabolically dormant and highly resistant spores during colonization that facilitate both persistence within the host and environmental transmission8,9,10. Relatively few intestinal spore-forming bacteria have been cultured to date, and while metagenomic studies suggest that other unexpected members of the intestinal microbiota possess potential sporulation genes, these bacteria remain poorly characterized11,12,13,14.

We hypothesized that sporulation is an unappreciated basic phenotype of the human intestinal microbiota that may have a profound impact on microbiota persistence and spread between humans. Spores from C. difficile are resistant to ethanol and this phenotype can be used to select for spores from a mixed population of spores and ethanol-sensitive vegetative cells15. Faecal samples with or without ethanol treatment were processed using our combined culture and metagenomics workflow (Extended Data Fig. 1). Principle component analysis demonstrated that ethanol treatment profoundly altered the culturable bacterial composition and, when compared to the original profile, efficiently enriched for ethanol-resistant bacteria, facilitating their isolation (Fig. 1b). We picked ~2,000 individual bacterial colonies from both ethanol-treated and non-ethanol-treated conditions, re-streaked them to purity, and performed full-length 16S ribosomal RNA gene sequencing to enable taxonomic characterization. Unique taxa were then archived as frozen stocks for future phenotypic analysis.

In total, we archived bacteria representing 96% of the bacterial abundance at the genus level and 90% of the bacterial abundance at the species level based on average relative abundance across the six donors (Extended Data Fig. 3a, b). Even genera that were present at low average relative abundance (<0.1%) were isolated (Extended Data Fig. 3c). Overall, we archived 137 distinct bacterial species including 45 candidate novel species (Fig. 1c, Extended Data Fig. 3d and Supplementary Table 1), and isolates representing 20 candidate novel genera and 2 candidate novel families. Our collection contains 90 species from the Human Microbiome Project’s ‘most wanted’ list of previously uncultured and unsequenced microbes16 (Supplementary Table 1). Thus, our broad-range YCFA-based culturing approach led to massive bacterial discovery, and challenges the notion that the majority of the intestinal microbiota is unculturable.

We isolated and purified bacteria representing 66 distinct ethanol-resistant species that are distributed across 5 known families and 2 newly identified candidate families (Extended Data Fig. 3d and Extended Data Fig. 4). The identification of these new and unexpected spore-formers highlights the broad taxonomic distribution of this phenotype among the enteric species of the Firmicutes. To define the conserved genetic pathways underlying sporulation and germination within the intestinal microbiota, we sequenced, assembled and annotated the whole genomes of 234 archived ethanol-resistant and ethanol-sensitive bacteria. Previously, the gene markers used to identify spore-forming bacterial species have been based on underlying genetic assumptions13,17,18; here we applied an unbiased computational approach to define 66 conserved genes linked to an ethanol-resistance phenotype (Extended Data Fig. 5 and Supplementary Table 1). This gene set allows for the prediction of the sporulation capabilities of bacterial species isolated from diverse environments with a high degree of accuracy (Extended Data Fig. 6a and Supplementary Table 1) and consists of genes from a wide range of functional classes (Extended Data Fig. 6b and Supplementary Table 1).

To test whether commensal spore formation facilitates long-term environmental survival, we exposed a phylogenetically diverse selection of commensal spore-forming and non-spore-forming bacteria and C. difficile to ambient oxygen for increasing periods of time. Under these conditions, non-spore-forming bacteria remained viable for 2–6 days (48–144 h) (Fig. 2a). In contrast, commensal spore-forming bacteria, C. difficile and the facultative anaerobe Escherichia coli were able to survive stably to the end of the experiment on day 21 (504 h). In addition, spore-forming commensals and C. difficile, but not non-spore-forming commensals, survived prolonged exposure to the common disinfectant ethanol (Extended Data Fig. 7). These results demonstrate that commensal spore-formers and C. difficile share a core set of sporulation genes that confer a highly resistant phenotype that is associated with environmental spread between humans.

Figure 2: Phenotypic characterization of phylogenetically diverse intestinal spore-forming bacteria. a, Spore-formers are more aero-tolerant than non-spore-formers, which is expected to facilitate host-to-host transmission. Once exposed to oxygen, only 1% of the original inoculum of non-spore-forming bacteria (dashed lines) were viable after 96 h (4 days) and none were viable after 144 h (6 days). Spore-forming bacteria (solid lines) persist owing to spore formation. The experiment was stopped after 504 h (21 days). Taxonomic families of each species tested are shown in brackets (n = 3 biological replicates for each strain). b, Intestinal spore-formers respond to bile-acid germinants. The number of colony-forming units (c.f.u.) (representing germinated spores) present on plates in the presence of a particular germinant is expressed as a fold change with respect to the number of c.f.u. recovered on plates in the absence of a germinant. Spore-formers and non-spore-formers were subjected to ethanol shock before being plated (n = 6 biological replicates for each strain). Only spore-formers survived. A fold change of one (dashed line) would indicate that a germinant had no effect on the number of c.f.u. recovered. Schematic summarizes the cholate-derived bile acid metabolism in the mammalian intestine. Mean and range, Welch’s unpaired two-tailed t-test (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001). PowerPoint slide Full size image

C. difficile spores have evolved mechanisms to resume metabolism and vegetative growth after intestinal colonization by germinating in response to digestive bile acids released into the small intestine from the gall bladder9. We exposed enteric spore-formers and non-spore-formers to common bile acids (taurocholate, glycocholate and cholate) to assess their response to germinants after ethanol-shock treatment (Fig. 2b). Taurocholate was a potent germinant for all spore-formers, increasing the culturability of spores from commensal bacteria by between 8- and 70,000-fold (P < 0.05 for all spore-formers tested), whereas the other cholate derivatives had varying efficacy in germinating commensal spore-formers (Fig. 2b). Taurocholate and the other bile acids had no impact on the culturability of non-spore-formers, demonstrating that the effect is specific to spore-formers (Extended Data Fig. 8). We propose that this bile-acid-triggered ‘colonizing germination’ mechanism serves as a conserved in vivo cue to promote colonization by intestinal spore-forming bacteria. Thus, a duality of purpose exists in the modus operandi of intestinal spore-forming bacteria; spore formation ensures their survival and transmission while germination in response to in vivo cues ensures their persistence in the human population.

We next sought to estimate the proportions of spore-forming bacteria within the intestinal microbiota. Interrogation of the metagenomic data sets with the spore gene signature predicted that, on average, 60% of the genera contained spore-forming bacteria (Fig. 3a). These genera represent 30% of the total intestinal microbiota (Fig. 3b). We independently validated these observations with 16S rRNA gene amplicon sequencing (Extended Data Fig. 9). Importantly, these proportions of spore-forming bacteria were also observed in 1,351 publicly available faecal metagenomic data sets generated from healthy individuals19 (Fig. 3a, b). We also found the same proportion of spore-formers (61.3%) within the ‘metagenomic species’ derived from 318 healthy individuals4.

Figure 3: Extensive and dynamic sporulation capacity within the human intestinal microbiota. a, b, Using the genomic signature to interrogate public (n = 1,351) and complete faecal sample metagenomic data sets from this study (n = 6) reveals the proportion of spore-formers as a count of the total number of genera (a) and as total microbial abundance (b). c, d, Metagenomic sequencing of donor faecal samples (n = 6) 1 year later demonstrates that spore-forming bacteria are more diverse than non-spore-forming bacteria (c) and that a significantly increased proportion of species show twofold or greater change over the same time period (d). Mean ± standard deviation (s.d.), two-tailed paired t-test (*P < 0.05, ***P < 0.001). PowerPoint slide Full size image

While the intestinal microbiota is considered to be relatively stable over time20, evidence suggests that close contact of family members promotes sharing of Ruminococcaceae and Lachnospiraceae bacteria21, families that we describe as spore-formers (Extended Data Fig. 4). We noted that in our cohort, the spore-forming bacteria of the microbiota were significantly more diverse than the non-spore-forming bacteria (Fig. 3c). To test the dynamics of the spore-forming and non-spore-forming bacteria over time, we analysed the metagenomic profiles of faecal samples collected from the same healthy subjects one year after the original sampling. Interestingly, we noted a significantly increased variability in the proportion of spore-forming bacteria compared with non-spore-forming bacteria over this period. This suggests a higher species turnover or a greater shift in relative abundance in the spore-forming bacterial species (Fig. 3d). Taken together, our phenotypic and genome analyses demonstrate that the spore-forming and non-spore-forming bacteria represent major, distinct phenotypic components of our microbiota, each with unique colonization dynamics.

We show that spore formation is a widespread, although previously unappreciated function of the human intestinal microbiota, with important implications for microbiota transmission and inheritance. On the basis of the shared phylogeny and common evolutionary and phenotypic characteristics of sporulation and germination, we propose that the abundant commensal intestinal spore-formers identified here rely on the same transmission and colonization strategy as C. difficile22. In brief, environmental C. difficile spores are highly transmissible for long periods after they are shed, commonly transmit within a local environment but also have the potential to spread rapidly over long distances23. The transmission dynamics and geographical range of commensal spore-formers has yet to be determined, but we anticipate that this type of information will provide great insight into the heritability and the selective factors that shape the composition of the human intestinal microbiota.

Our workflow enables large-scale culturing, archiving, genome sequencing and phenotyping of novel bacteria from the human gut microbiota that were formerly considered to be unculturable. We have generated a sizable whole-genome-sequence data set that corresponds to 39% of the total number of intestinal bacterial genomes generated by the Human Microbiome Project. Our streamlined, single-medium approach, builds on the considerable efforts of others24,25 and unlocks the human intestinal microbiota for phenotypic characterization.