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Similar to Journal Club Presentation on "Culturing of ‘unculturable’ human microbiota reveals novel taxa and extensive sporulation"
Similar to Journal Club Presentation on "Culturing of ‘unculturable’ human microbiota reveals novel taxa and extensive sporulation" (20)
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Journal Club Presentation on "Culturing of ‘unculturable’ human microbiota reveals novel taxa and extensive sporulation"
- 2. Detection of microbial species in an environmental sample 1. Microscopy 2. Culturing • Allows for in-depth analysis, genetics, etc. • Requires that you know how to culture it 3. Sequencing-based approaches: • 16S sequencing (Sanger or NGS) • Metagenomic shotgun sequencing • Transcriptomics • Single-cell WGS Successfully culturing a microbe is critical for understanding the function of its genes
- 3. Detection of microbial species in a sample Method Advantages Disadvantages Culturing In-depth analysis of species (e.g. characterization of hypothetical genes) Have to know how to culture the species. Low-throughput 16S Sequencing High-throughput Limited to taxonomic information Metagenomic Shotgun Sequencing High-throughput. Information about all genes (e.g. metabolism) Follow-up work is difficult without culturing Single-cell WGS No culturing required for WGS Follow-up work is difficult without culturing
- 4. Novel culturing methods • “Microbial Culturomics” • e.g. culturing fecal samples in 212 conditions. (Lagier et al. Clin Microbiol Infect. 2012 18(12):1185-93) • Rational design of culturing conditions • e.g. culture of oligotrophs in low nutrient media (Cho & Giovannoni. Appl Environ Microbiol. 2004. 70(1): 432-440 • Community culturing • e.g. iChip to culture soil bacteria (Ling et al. Nature. 2015. 517(7535):455-9)
- 5. Culturing of gut microbiota members using the rich medium YCFA • Casitone • Yeast extract • NaHCO3 • Cysteine • K2HPO4 • KH2PO4 • NaCl • MgSO4 • CaCl2 • Resazurin • Haemin • Biotin • Cobalamin • P-aminobenzoic acid • Folic acid • Pyridoxamine • SCFAs: acetate, propionate, isobutyrate, isovalerate, valerate • Thiamine • Riobflavin Duncan et al. Int J Syst Evol Microbiol. 2002 52(6):2141-6
- 6. Overview of Workflow Fecal Samples (n=6) EtOH treatment + culturing with germination culturing Total Microbiota Spore-Formers Metagenomic and 16S sequencing Full-length 16S sequencing (Sanger)
- 7. Sporulation and Germination Al-Hinai et al. 2015. Microbiol Mol Biol Rev. 79(1): 19-37
- 8. “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.”
- 9. The relative abundances of bacterial species correlate between fecal samples and total microbiota culture “…73.5% of the 741 computationally derived metagenomic species…were also detectable in the cultured samples”
- 10. 16S sequencing profiles show similarity between fecal and cultured samples, dissimilarity with spore-forming cultures
- 11. Phylogenetic tree of isolates based on full- length 16S sequencing Pure culture of ~2000 colonies results in 137 unique species Represent 90%+ of total abundance 67/137 isolates are novel
- 12. Phylogeny of spore- forming isolates shows that sporulation is spread across many families of the Firmicutes phylum
- 13. Novel spore-formers germinate in response to bile acids (just like C. difficile)
- 14. Spore-formers are more aero- tolerant (just like C. difficile) Dashed lines: non-spore-formers Solid lines: spore-formers
- 15. Identification of an ethanol- resistance gene signature “Support vector, machine- based, contrast set association mining was applied to identify the optimal, weighted gene signature consisting of 66 genes.” Training Dataset
- 16. Application of the gene signature demonstrates that ~1/3 of the microbiota is capable of spore formation
- 17. The spore-former population is more diverse and more dynamic Diversity Index Change in abundance over 1 y
- 18. Conclusions (1) A large number of species can be cultivated using one protocol (2) Sporulation is a common phenotype of the gut microbiota Strengths: • Applies “culturomics” to a specific hypothesis • Helpful machine learning model for predicting sporulation ability • Establishes a general model for gut-to-gut transmission • Opens the door for more detailed studies of the cultured species Weaknesses: • Are only able to culture a few phyla (Bacteroidetes, Actinobacteria, and Firmicutes)
Editor's Notes
- Data: https://www.ebi.ac.uk/ena/data/view/PRJEB10915
- Fig 1A: Metagenomic sequencing of fecal samples and colonies scraped from YCFA plates grown anaerobically What did they do with zeros? I.E. those species detected in the fecal sample but which didn’t grow when cultured, or vice versa.
- 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).
- 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.
- Novel genus member: they were able to characterize the isolate taxonomically to the family level, and then the genus was “unclassified” Novel family member: they were able to characterize the isolate taxonomically to the order level, and then the family was “unclassified” Novel: Species=45 Genera=19 Families=3 Total=67
- Streak colonies to singles (from ethanol treated and untreated samples), full-length 16S sequencing to identify species. Here showing what was isolated and what genera or species they belong to, overlaying the relative abundances of these groups as determined by metagenomic sequencing of fecal samples.
- “species” here, means novel species, genera, and families (as worded in Fig 1C)
- Full-length 16S used to make tree Branch colors are families Shaded text are isolated from the ethanol treatment, unshaded from an untreated sample
- Grow in broth, treat with EtOH, plate onto YCFA +/- bile acid germinant. Non-spore-formers did not survive the EtOH treatment.
- Grow O/N anaerobically in YCFA broth. Dilute and spot onto YCFA agar + taurocholate anaerobically. Move plates to aerobic conditions. At time points, transfer back to anaerobic conditions and incubate for 72 h, then count colonies.
- 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.