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Cited by 1,299Gut microbiota and healthMicrobial Community Ecology and PhysiologyGenomics and Phylogenetic Studies

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Culturing of ‘unculturable’ human microbiota reveals novel taxa and extensive sporulation

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Browne, Hilary P., Forster, Samuel C., Anonye, Blessing O., Kumar, Nitin, Neville, B. Anne, Stares, Mark D., Goulding, David, Lawley, Trevor D.·2016·Nature
DOI 10.1038/nature17645PMC PMC4890681
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Abstract

Our intestinal microbiota harbours a diverse bacterial community required for our health, sustenance and wellbeing. Intestinal colonization begins at birth and climaxes with the acquisition of two dominant groups of strict anaerobic bacteria belonging to the Firmicutes and Bacteroidetes phyla. Culture-independent, genomic approaches have transformed our understanding of the role of the human microbiome in health and many diseases. However, owing to the prevailing perception that our indigenous bacteria are largely recalcitrant to culture, many of their functions and phenotypes remain unknown. Here we describe a novel workflow based on targeted phenotypic culturing linked to large-scale whole-genome sequencing, phylogenetic analysis and computational modelling that demonstrates that a substantial proportion of the intestinal bacteria are culturable. Applying this approach to healthy individuals, we isolated 137 bacterial species from characterized and candidate novel families, genera and species that were archived as pure cultures. Whole-genome and metagenomic sequencing, combined with computational and phenotypic analysis, suggests that at least 50-60% of the bacterial genera from the intestinal microbiota of a healthy individual produce resilient spores, specialized for host-to-host transmission. Our approach unlocks the human intestinal microbiota for phenotypic analysis and reveals how a marked proportion of oxygen-sensitive intestinal bacteria can be transmitted between individuals, affecting microbiota heritability.

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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
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 slideCC BY · PubMed Central
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
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 slideCC BY · PubMed Central
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
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 slideCC BY · PubMed Central
Extended Data Figure 1 A workflow for culturing, archiving and characterization of the intestinal microbiota. a–d, Schematic diagram of the workflow, encompassing bacterial culturing and genomics to isolate and characterize bacterial species from the human intestinal microbiota. The process incorporates several steps, which are culture, re-streak, archive and phenotype. a, Fresh faecal samples are left untreated or are treated to select for bacteria with a desired phenotype (such as sporulation). The stool is homogenized and then serially diluted and then aliquots of the homogenate are inoculated on YCFA agar to culture bacteria. b, Isolates are identified by selecting single colonies that are streaked to purity and full-length 16S rRNA genes are amplified and sequenced. c, Each unique, novel and desired isolate is archived frozen in a culture collection and a whole-genome sequence is generated for each. d, Phenotypic characterization and functional validation of metagenomics studies can be performed in vitro and in vivo.
Extended Data Figure 1 A workflow for culturing, archiving and characterization of the intestinal microbiota. a–d, Schematic diagram of the workflow, encompassing bacterial culturing and genomics to isolate and characterize bacterial species from the human intestinal microbiota. The process incorporates several steps, which are culture, re-streak, archive and phenotype. a, Fresh faecal samples are left untreated or are treated to select for bacteria with a desired phenotype (such as sporulation). The stool is homogenized and then serially diluted and then aliquots of the homogenate are inoculated on YCFA agar to culture bacteria. b, Isolates are identified by selecting single colonies that are streaked to purity and full-length 16S rRNA genes are amplified and sequenced. c, Each unique, novel and desired isolate is archived frozen in a culture collection and a whole-genome sequence is generated for each. d, Phenotypic characterization and functional validation of metagenomics studies can be performed in vitro and in vivo.CC BY · PubMed Central
Extended Data Figure 2 Comparison of sequence read content of faecal samples and cultured samples for six donors. The majority of sequence reads from the original donor faecal samples (n = 6) are present in culture samples both as raw reads (93% shared on average across the six donors) and after de novo assembly (72% shared on average across the six donors).
Extended Data Figure 2 Comparison of sequence read content of faecal samples and cultured samples for six donors. The majority of sequence reads from the original donor faecal samples (n = 6) are present in culture samples both as raw reads (93% shared on average across the six donors) and after de novo assembly (72% shared on average across the six donors).CC BY · PubMed Central
Extended Data Figure 3 Archiving of bacterial diversity and novelty through anaerobic culturing. a, b, Representative species from 21 of the 25 most abundant bacterial genera (a) and 23 of the 24 most abundant species (b) were isolated and archived (abundance was determined by metagenomic sequencing and based on average relative abundance across the six donors (n = 6)). This represents 96% of the average relative abundance at the genus level and 90% of the average relative abundance at the species level across the six donors. A red dot in a indicates the number of species archived from each genus. Lachnospiraceae incertae sedis, unclassified Lachnospiraceae, Clostridium IV and Clostridium XI are not strict genera and represent currently unclassified species. Odoribacter splanchnicus in b was the only species not archived. c, Lowly represented intestinal microbiota members were also cultured. At least one representative species from each of the genera presented were cultured. Median and range is presented for the above with taxa ranked by median value. d, The number of bacterial species cultured in this study. At least 40% from each category were previously unknown.
Extended Data Figure 3 Archiving of bacterial diversity and novelty through anaerobic culturing. a, b, Representative species from 21 of the 25 most abundant bacterial genera (a) and 23 of the 24 most abundant species (b) were isolated and archived (abundance was determined by metagenomic sequencing and based on average relative abundance across the six donors (n = 6)). This represents 96% of the average relative abundance at the genus level and 90% of the average relative abundance at the species level across the six donors. A red dot in a indicates the number of species archived from each genus. Lachnospiraceae incertae sedis, unclassified Lachnospiraceae, Clostridium IV and Clostridium XI are not strict genera and represent currently unclassified species. Odoribacter splanchnicus in b was the only species not archived. c, Lowly represented intestinal microbiota members were also cultured. At least one representative species from each of the genera presented were cultured. Median and range is presented for the above with taxa ranked by median value. d, The number of bacterial species cultured in this study. At least 40% from each category were previously unknown.CC BY · PubMed Central
Extended Data Figure 4 Phylogeny of intestinal spore-forming bacteria. Full length 16S rRNA gene phylogeny illustrating the taxonomic relationship of ethanol-resistant bacteria within the Firmicutes cultured from the donor faecal samples. Branch colours indicate distinct families. Shaded text indicates species cultured from an ethanol-treated faecal sample and unshaded text indicates species cultured from a non-ethanol-treated faecal sample. Percentage values represent closest identity to a characterized species. Transmission electron micrographs (TEMs) of spore ultrastructures for a phylogenetically diverse selection of cultured bacteria are shown with an arrow in images and include a candidate novel family with 86% identity to the 16S rRNA gene sequence from Clostridium thermocellum. Typical spore structures are defined and illustrated in the same image. TEMs are ordered according to boxes next to the species name. Scale bars are shown at the bottom of each image. C. difficile is included for context. Bacteria displaying an ethanol-resistant phenotype represent species previously classified as non-spore-formers (Turicibacter sanguinis38 and closely related candidate novel species), species closely related to non-spore-formers (Roseburia intestinalis39 and Oscillibacter valericigenes40 and closely related candidate novel species) or species suspected of forming spores but which, to our knowledge, have never been demonstrated to do so until now (Eubacterium eligens, Eubacterium rectale, Coprococcus comes41 and related candidate novel species).
Extended Data Figure 4 Phylogeny of intestinal spore-forming bacteria. Full length 16S rRNA gene phylogeny illustrating the taxonomic relationship of ethanol-resistant bacteria within the Firmicutes cultured from the donor faecal samples. Branch colours indicate distinct families. Shaded text indicates species cultured from an ethanol-treated faecal sample and unshaded text indicates species cultured from a non-ethanol-treated faecal sample. Percentage values represent closest identity to a characterized species. Transmission electron micrographs (TEMs) of spore ultrastructures for a phylogenetically diverse selection of cultured bacteria are shown with an arrow in images and include a candidate novel family with 86% identity to the 16S rRNA gene sequence from Clostridium thermocellum. Typical spore structures are defined and illustrated in the same image. TEMs are ordered according to boxes next to the species name. Scale bars are shown at the bottom of each image. C. difficile is included for context. Bacteria displaying an ethanol-resistant phenotype represent species previously classified as non-spore-formers (Turicibacter sanguinis38 and closely related candidate novel species), species closely related to non-spore-formers (Roseburia intestinalis39 and Oscillibacter valericigenes40 and closely related candidate novel species) or species suspected of forming spores but which, to our knowledge, have never been demonstrated to do so until now (Eubacterium eligens, Eubacterium rectale, Coprococcus comes41 and related candidate novel species).CC BY · PubMed Central
Extended Data Figure 5 Genomic signature of sporulation within the human intestinal microbiome. A genomic signature for identifying spore-forming bacterial species contains sporulation- and germination-associated genes and genes not previously associated with sporulation. Characterized sporulation genes are on the outer circle, genes not associated with a specific sporulation cycle or uncharacterized genes are in the inside rectangle. C. difficile strain 630 gene names are used when possible, otherwise locus tag identifiers are shown. Bacillus subtilis gene names are used when no C. difficile homologue is available. The signature is enriched with known sporulation-associated genes from stages I–V of the spore formation and germination cycles (significant at q < 3.0 × 10−37, Fisher’s exact test). Genes associated with regulation are present with at least 10 genes coding for regulatory or DNA-binding roles (q < 1.4 × 10−5, Fisher’s exact test). Genes not previously associated with sporulation are also present and these have putative roles as heat shock, membrane-associated proteins and DNA-polymerase-associated proteins.
Extended Data Figure 5 Genomic signature of sporulation within the human intestinal microbiome. A genomic signature for identifying spore-forming bacterial species contains sporulation- and germination-associated genes and genes not previously associated with sporulation. Characterized sporulation genes are on the outer circle, genes not associated with a specific sporulation cycle or uncharacterized genes are in the inside rectangle. C. difficile strain 630 gene names are used when possible, otherwise locus tag identifiers are shown. Bacillus subtilis gene names are used when no C. difficile homologue is available. The signature is enriched with known sporulation-associated genes from stages I–V of the spore formation and germination cycles (significant at q < 3.0 × 10−37, Fisher’s exact test). Genes associated with regulation are present with at least 10 genes coding for regulatory or DNA-binding roles (q < 1.4 × 10−5, Fisher’s exact test). Genes not previously associated with sporulation are also present and these have putative roles as heat shock, membrane-associated proteins and DNA-polymerase-associated proteins.CC BY · PubMed Central
Extended Data Figure 6 Validation and characterization of the sporulation signature. a, The signature accurately distinguishes spore-forming and non-spore-forming bacteria cultured from this study and from across different environments (known spore-formers n = 57, known non-spore-formers n = 50, cultured after ethanol treatment n = 69, cultured after no ethanol treatment n = 149). Refer to Supplementary Table 1 for signature scores of the bacteria tested. Mean ± s.d. b, Assignment of functional classes to the signature reveals a wide range of functional processes with sporulation- and regulation-associated genes dominating.
Extended Data Figure 6 Validation and characterization of the sporulation signature. a, The signature accurately distinguishes spore-forming and non-spore-forming bacteria cultured from this study and from across different environments (known spore-formers n = 57, known non-spore-formers n = 50, cultured after ethanol treatment n = 69, cultured after no ethanol treatment n = 149). Refer to Supplementary Table 1 for signature scores of the bacteria tested. Mean ± s.d. b, Assignment of functional classes to the signature reveals a wide range of functional processes with sporulation- and regulation-associated genes dominating.CC BY · PubMed Central
Extended Data Figure 7 Spore-forming bacteria are more resilient than non-spore-forming bacteria to environmental stresses such as disinfectants. Pure bacterial cultures were immersed in ethanol for 4 h before being washed and inoculated onto YCFA growth medium with sodium taurocholate as a germinant. Only spore-forming bacteria survived. Taxonomic family names are shown in brackets. The dashed line indicates the culture detection limit of 50 c.f.u. ml−1. Mean ± s.d., n = 3 biological replicates for each species tested.
Extended Data Figure 7 Spore-forming bacteria are more resilient than non-spore-forming bacteria to environmental stresses such as disinfectants. Pure bacterial cultures were immersed in ethanol for 4 h before being washed and inoculated onto YCFA growth medium with sodium taurocholate as a germinant. Only spore-forming bacteria survived. Taxonomic family names are shown in brackets. The dashed line indicates the culture detection limit of 50 c.f.u. ml−1. Mean ± s.d., n = 3 biological replicates for each species tested.CC BY · PubMed Central
Extended Data Figure 8 Growth response of non-spore-forming bacteria to intestinal germinants. The number of c.f.u. present on plates in the presence of a particular germinant expressed as a fold change with respect to the number of c.f.u. present on plates in the absence of a germinant. No ethanol shock treatment was performed beforehand. A fold change of one (dashed line) would indicate that a germinant had no effect on the number of c.f.u. recovered from the bacteria. There was no statistically significant difference based on an unpaired t-test of each germinant condition against the no germinant condition. Mean and range, n = 3 biological replicates for both species.
Extended Data Figure 8 Growth response of non-spore-forming bacteria to intestinal germinants. The number of c.f.u. present on plates in the presence of a particular germinant expressed as a fold change with respect to the number of c.f.u. present on plates in the absence of a germinant. No ethanol shock treatment was performed beforehand. A fold change of one (dashed line) would indicate that a germinant had no effect on the number of c.f.u. recovered from the bacteria. There was no statistically significant difference based on an unpaired t-test of each germinant condition against the no germinant condition. Mean and range, n = 3 biological replicates for both species.CC BY · PubMed Central
Extended Data Figure 9 Validation of the estimation of the proportion of spore-formers in the intestinal microbiota. Full-length 16S rRNA gene amplicon sequencing was used to determine the taxonomic proportions of bacteria from the six donor faecal samples. Spore-forming bacteria were cultured from each donor and a taxonomic classification was assigned as described in the main text. The genus (circle) and family (square) taxonomic ranks were designated as the lower and upper limits for calculating the proportion of spore-formers at a taxonomic level. Specific genera and families were included if they contained a species that was cultured after ethanol shock treatment. Mean ± s.d.
Extended Data Figure 9 Validation of the estimation of the proportion of spore-formers in the intestinal microbiota. Full-length 16S rRNA gene amplicon sequencing was used to determine the taxonomic proportions of bacteria from the six donor faecal samples. Spore-forming bacteria were cultured from each donor and a taxonomic classification was assigned as described in the main text. The genus (circle) and family (square) taxonomic ranks were designated as the lower and upper limits for calculating the proportion of spore-formers at a taxonomic level. Specific genera and families were included if they contained a species that was cultured after ethanol shock treatment. Mean ± s.d.CC BY · PubMed Central

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Metadata

Authors
Browne, H. P., Forster, S. C., Anonye, B. O., Kumar, N., Neville, B. A., Stares, M. D., Goulding, D., & Lawley, T. D.
Affiliations
Wellcome Sanger Institute (GB)
Corresponding author
Lawley, Trevor D.
ORCID
Venue
Nature
Publisher
Springer Science and Business Media LLC
Published
2016-05-04
Volume
533
Issue
7604
Pages
543-546
ISSN
  • 0028-0836 (ISSN-L)
  • 1476-4687
DOI
10.1038/nature17645
PMC
PMC4890681
OpenAlex
W2345813655
Cited by
1,299
References
41
Citations by year
2026: 60 · 2025: 90 · 2024: 119 · 2023: 102 · 2022: 131 · 2021: 164 · 2020: 170 · 2019: 180 · 2018: 119 · 2017: 120 · 2016: 41
FWCI
79.72
Type
journal article
Language
en
Open access
Licence
Locations
  • Nature (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • CLOK (University of Central Lancashire) (acceptedVersion)
  • Monash University Research Portal (Monash University) (submittedVersion, other-oa)
  • RePEc: Research Papers in Economics (submittedVersion)
Funders
  • Wellcome Trust (098051)
  • State Government of Victoria
  • Medical Research Council (098051, PF451, MR/K000551/1)
  • National Health and Medical Research Council (1091097)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Gut microbiota and health
  • Physical Sciences › Environmental Science › Ecology › Microbial Community Ecology and Physiology
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Genomics and Phylogenetic Studies
Keywords
uncultured bacteria, human gut microbiome, bacterial sporulation, spore-forming bacteria, host-to-host transmission, novel bacterial taxa, Firmicutes, Bacteroidetes, anaerobic bacteria, metagenomics, whole genome sequencing
MeSH terms
Gastrointestinal Microbiome, Anaerobiosis, Bacteria, Feces, Health, Humans, Oxygen, Phenotype, Phylogeny, Species Specificity, Spores, Bacterial, Bacterial Typing Techniques, Genome, Bacterial, Sequence Analysis, DNA, Cell Culture Techniques, Metagenome, Metagenomics

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