Human Microbiota Reveals Novel Taxa and Extensive Sporulation Hilary P
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OPEN LETTER doi:10.1038/nature17645 Culturing of ‘unculturable’ human microbiota reveals novel taxa and extensive sporulation Hilary P. Browne1*, Samuel C. Forster1,2,3*, Blessing O. Anonye1, Nitin Kumar1, B. Anne Neville1, Mark D. Stares1, David Goulding4 & Trevor D. Lawley1 Our intestinal microbiota harbours a diverse bacterial community original faecal sample and the cultured bacterial community shared required for our health, sustenance and wellbeing1,2. Intestinal an average of 93% of raw reads across the six donors. This overlap was colonization begins at birth and climaxes with the acquisition of 72% after de novo assembly (Extended Data Fig. 2). Comparison to a two dominant groups of strict anaerobic bacteria belonging to the comprehensive gene catalogue that was derived by culture-independent Firmicutes and Bacteroidetes phyla2. Culture-independent, genomic means from the intestinal microbiota of 318 individuals4 found that approaches have transformed our understanding of the role of the 39.4% of the genes in the larger database were represented in our cohort human microbiome in health and many diseases1. However, owing and 73.5% of the 741 computationally derived metagenomic species to the prevailing perception that our indigenous bacteria are largely identified through this analysis were also detectable in the cultured recalcitrant to culture, many of their functions and phenotypes samples. remain unknown3. Here we describe a novel workflow based on Together, these results demonstrate that a considerable proportion of targeted phenotypic culturing linked to large-scale whole-genome the bacteria within the faecal microbiota can be cultured with a single sequencing, phylogenetic analysis and computational modelling that growth medium. However, more than 8 × 106 distinct colonies would demonstrates that a substantial proportion of the intestinal bacteria need to be picked from YCFA agar plates to match the species detection are culturable. Applying this approach to healthy individuals, sensitivity of metagenomic sequencing. Thus, we established a broad- we isolated 137 bacterial species from characterized and candidate range culturing method that, when combined with high-throughput novel families, genera and species that were archived as pure archiving or specific phenotypic selection, can be used to isolate and cultures. Whole-genome and metagenomic sequencing, combined identify novel bacteria from the gastrointestinal tract. with computational and phenotypic analysis, suggests that at least The human intestinal microbiota is dominated by strict anaerobic 50–60% of the bacterial genera from the intestinal microbiota bacteria that are extremely sensitive to ambient oxygen, so it is not of a healthy individual produce resilient spores, specialized for known how these bacteria survive environmental exposure to be trans- host-to-host transmission. Our approach unlocks the human mitted between individuals. Certain members of the Firmicutes phylum, intestinal microbiota for phenotypic analysis and reveals how a including the diarrhoeal pathogen Clostridium difficile, produce met- marked proportion of oxygen-sensitive intestinal bacteria can be abolically dormant and highly resistant spores during colonization transmitted between individuals, affecting microbiota heritability. that facilitate both persistence within the host and environmental A typical human intestinal microbiota contains 100–1,000 bacterial transmission8–10. Relatively few intestinal spore-forming bacteria have species with tremendous compositional diversity between individuals, been cultured to date, and while metagenomic studies suggest that such that each individual’s microbiota is as unique as a fingerprint1,4. other unexpected members of the intestinal microbiota possess poten- Despite the taxonomic diversity, metagenomic sequencing has high- tial sporulation genes, these bacteria remain poorly characterized11–14. lighted that a health-associated intestinal microbiome codes for highly We hypothesized that sporulation is an unappreciated basic phenotype conserved gene families and pathways associated with basic bacterial of the human intestinal microbiota that may have a profound impact physiology and growth2. However, many basic microbiota functions on microbiota persistence and spread between humans. Spores from related to homeostasis, immune system development, digestion, path- C. difficile are resistant to ethanol and this phenotype can be used to ogen resistance and microbiota inheritance have yet to be discovered5. select for spores from a mixed population of spores and ethanol-sensitive This formidable challenge to validate and decipher the functional vegetative cells15. Faecal samples with or without ethanol treatment were attributes of the microbiota has been hindered because the majority of processed using our combined culture and metagenomics workflow intestinal bacteria are widely considered to be ‘unculturable’ and have (Extended Data Fig. 1). Principle component analysis demonstrated never been isolated in the laboratory3,6. that ethanol treatment profoundly altered the culturable bacterial com- We sought to establish a genomic-based workflow that could be used position and, when compared to the original profile, efficiently enriched as a platform for targeted culturing of specific bacterial phenotypes for ethanol-resistant bacteria, facilitating their isolation (Fig. 1b). (Extended Data Fig. 1). Accordingly, we collected fresh faecal samples We picked ~2,000 individual bacterial colonies from both ethanol- from six healthy humans and defined the resident bacterial communi- treated and non-ethanol-treated conditions, re-streaked them to purity, ties with a combined metagenomic sequencing and bacterial culturing and performed full-length 16S ribosomal RNA gene sequencing to approach. Applying shotgun metagenomic sequencing, we profiled and enable taxonomic characterization. Unique taxa were then archived as compared the bacterial species present in the original faecal samples frozen stocks for future phenotypic analysis. to those that grew as distinct colonies on agar plates containing the In total, we archived bacteria representing 96% of the bacterial abun- complex, broad-range bacteriological medium, YCFA7. Importantly, we dance at the genus level and 90% of the bacterial abundance at the observed a strong correlation between the two samples at the species species level based on average relative abundance across the six donors level (Spearman’s ρ = 0.75, P < 0.01) (Fig. 1a). When sequenced, the (Extended Data Fig. 3a, b). Even genera that were present at low average 1Host-Microbiota Interactions Laboratory, Wellcome Trust Sanger Institute, Hinxton CB10 1SA, UK. 2Centre for Innate Immunity and Infectious Diseases, Hudson Institute of Medical Research, Clayton, Victoria 3168, Australia. 3Department of Molecular and Translational Sciences, Monash University, Clayton, Victoria 3800, Australia. 4Microbial Pathogenesis Laboratory, Wellcome Trust Sanger Institute, Hinxton CB10 1SA, UK. *These authors contributed equally to this work. 26 MAY 2016 | VOL 533 | NATURE | 543 © 2016 Macmillan Publishers Limited. All rights reserved RESEARCH LETTER a b Ethanol-treated culture Complete faecal sample 2 newly identified candidate families (Extended Data Fig. 3d and –1 Complete culture Bacterial species 6 Extended Data Fig. 4). The identification of these new and unex- –2 pected spore-formers highlights the broad taxonomic distribution of 4 this phenotype among the enteric species of the Firmicutes. To define –3 2 the conserved genetic pathways underlying sporulation and germina- –4 tion within the intestinal microbiota, we sequenced, assembled and 0 Axis 2 (13.5%) annotated the whole genomes of 234 archived ethanol-resistant and –5 −2 ethanol-sensitive bacteria. Previously, the gene markers used to identify –6 spore-forming bacterial species have been based on underlying genetic −4 Spearman U = 0.75 13,17,18 Bacterial growth on culture plate (log10) assumptions ; here we applied an unbiased computational –6 –5 –4 –3 –2 –1 –2 024 6 approach to define 66 conserved genes linked to an ethanol-resistance Bacterial abundance in faecal samples (log10) Axis 1 (19.5%) phenotype (Extended Data Fig. 5 and Supplementary Table 1). This gene set allows for the prediction of the sporulation capabilities of bac- c Bacteroidetes terial species isolated from diverse environments with a high degree Characterized species Novel species of accuracy (Extended Data Fig. 6a and Supplementary Table 1) and Novel genus members consists of genes from a wide range of functional classes (Extended Novel family members Data Fig. 6b and Supplementary Table 1). To test whether commensal spore formation facilitates long-term Erysipelotrichaceae environmental survival, we exposed a phylogenetically diverse selec- Actinobacteria Peptostreptococcaceae tion of commensal spore-forming and non-spore-forming bacteria and Clostridiaceae C. difficile to ambient oxygen for increasing periods of time. Under these Proteobacteria 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 sur- vive 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 Ruminococcaceae ethanol (Extended Data Fig. 7).