Article | Published:

Endospores and other lysis-resistant bacteria comprise a widely shared core community within the human microbiota

The ISME Journalvolume 12pages24032416 (2018) | Download Citation

Abstract

Endospore-formers in the human microbiota are well adapted for host-to-host transmission, and an emerging consensus points to their role in determining health and disease states in the gut. The human gut, more than any other environment, encourages the maintenance of endospore formation, with recent culture-based work suggesting that over 50% of genera in the microbiome carry genes attributed to this trait. However, there has been limited work on the ecological role of endospores and other stress-resistant cellular states in the human gut. In fact, there is no data to indicate whether organisms with the genetic potential to form endospores actually form endospores in situ and how sporulation varies across individuals and over time. Here we applied a culture-independent protocol to enrich for endospores and other stress-resistant cells in human feces to identify variation in these states across people and within an individual over time. We see that cells with resistant states are more likely than those without to be shared among multiple individuals, which suggests that these resistant states are particularly adapted for cross-host dissemination. Furthermore, we use untargeted fecal metabolomics in 24 individuals and within a person over time to show that these organisms respond to shared environmental signals, and in particular, dietary fatty acids, that likely mediate colonization of recently disturbed human guts.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

References

  1. 1.

    Filippidou S, Junier T, Wunderlin T, Lo C-C, Li P-E, Chain PS, et al. Under-detection of endospore-forming Firmicutes in metagenomic data. Comput Struct Biotechnol J. 2015;13:299–306.

  2. 2.

    Ley RE, Hamady M, Lozupone C, Turnbaugh PJ, Ramey RR, Bircher JS, et al. Evolution of mammals and their gut microbes. Science. 2008;320:1647–51.

  3. 3.

    Browne HP, Forster SC, Anonye BO, Kumar N, Neville BA, Stares MD, et al. Culturing of ‘unculturable’ human microbiota reveals novel taxa and extensive sporulation. Nature. 2016;533:543–6.

  4. 4.

    Flint JF, Drzymalski D, Montgomery WL, Southam G, Angert ER. Nocturnal production of endospores in natural populations of epulopiscium-like surgeonfish symbionts. J Bacteriol. 2005;187:7460–70.

  5. 5.

    Angert ER, Losick RM. Propagation by sporulation in the guinea pig symbiont Metabacterium polyspora. Proc Natl Acad Sci USA. 1998;95:10218–23.

  6. 6.

    Alexander CJ, Citron DM, Brazier JS, Goldstein EJ. Identification and antimicrobial resistance patterns of clinical isolates of Clostridium clostridioforme, Clostridium innocuum, and Clostridium ramosum compared with those of clinical isolates of Clostridium perfringens. J Clin Microbiol. 1995;33:3209–15.

  7. 7.

    Paredes-Sabja D, Torres JA, Setlow P, Sarker MR. Clostridium perfringens Spore Germination: characterization of germinants and their receptors. J Bacteriol. 2008;190:1190–201.

  8. 8.

    Sokol H, Pigneur B, Watterlot L, Lakhdari O, Bermúdez-Humarán LG, Gratadoux J-J, et al. Faecalibacterium prausnitzii is an anti-inflammatory commensal bacterium identified by gut microbiota analysis of Crohn disease patients. Proc Natl Acad Sci USA. 2008;105:16731–6.

  9. 9.

    Png CW, Lindén SK, Gilshenan KS, Zoetendal EG, McSweeney CS, Sly LI, et al. Mucolytic bacteria with increased prevalence in IBD mucosa augment in vitro utilization of mucin by other bacteria. Am J Gastroenterol. 2010;105:2420–8.

  10. 10.

    Deakin LJ, Clare S, Fagan RP, Dawson LF, Pickard DJ, West MR, et al. The Clostridium difficile spo0A gene Is a persistence and transmission factor. Infect Immun. 2012;80:2704–11.

  11. 11.

    Atarashi K, Tanoue T, Oshima K, Suda W, Nagano Y, Nishikawa H, et al. Treg induction by a rationally selected mixture of Clostridia strains from the human microbiota. Nature. 2013;500:232–6.

  12. 12.

    Atarashi K, Tanoue T, Shima T, Imaoka A, Kuwahara T, Momose Y, et al. Induction of colonic regulatory T cells by indigenous clostridium species. Science. 2011;331:337–41.

  13. 13.

    Stefka AT, Feehley T, Tripathi P, Qiu J, McCoy K, Mazmanian SK, et al. Commensal bacteria protect against food allergen sensitization. Proc Natl Acad Sci USA. 2014;111:13145–50.

  14. 14.

    Kim Y-G, Sakamoto K, Seo S-U, Pickard JM, Gillilland MG, Pudlo NA, et al. Neonatal acquisition of em Clostridia/em species protects against colonization by bacterial pathogens. Science. 2017;356:315.

  15. 15.

    Ivanov II, Atarashi K, Manel N, Brodie EL, Shima T, Karaoz U, et al. Induction of intestinal Th17 cells by segmented filamentous bacteria. Cell. 2009;139:485–98.

  16. 16.

    Kuwahara T, Ogura Y, Oshima K, Kurokawa K, Ooka T, Hirakawa H, et al. The lifestyle of the segmented filamentous bacterium: a non-culturable gut-associated immunostimulating microbe inferred by whole-genome sequencing. DNA Res. 2011;18:291–303.

  17. 17.

    Sczesnak A, Segata N, Qin X, Gevers D, Petrosino JF, Huttenhower C, et al. The genome of Th17 cell-inducing segmented filamentous bacteria reveals extensive auxotrophy and adaptations to the intestinal environment. Cell Host Microbe. 2011;10:260–72.

  18. 18.

    Eeckhaut V, Van Immerseel F, Croubels S, De Baere S, Haesebrouck F, Ducatelle R, et al. Butyrate production in phylogenetically diverse Firmicutes isolated from the chicken caecum: butyrate-producing bacteria from the chicken caecum. Microb Biotechnol. 2011;4:503–12.

  19. 19.

    Furusawa Y, Obata Y, Fukuda S, Endo TA, Nakato G, Takahashi D, et al. Commensal microbe-derived butyrate induces the differentiation of colonic regulatory T cells. Nature. 2013;504:446–50.

  20. 20.

    Louis P, Young P, Holtrop G, Flint HJ. Diversity of human colonic butyrate-producing bacteria revealed by analysis of the butyryl-CoA:acetate CoA-transferase gene. Environ Microbiol. 2010;12:304–14.

  21. 21.

    Smith PM, Howitt MR, Panikov N, Michaud M, Gallini CA, Bohlooly-Y M, et al. The microbial metabolites, short-chain fatty acids, regulate colonic treg cell homeostasis. Science. 2013;341:569–73.

  22. 22.

    Van den Abbeele P, Belzer C, Goossens M, Kleerebezem M, De Vos WM, Thas O, et al. Butyrate-producing Clostridium cluster XIVa species specifically colonize mucins in an in vitro gut model. ISME J. 2013;7:949–61.

  23. 23.

    Yano JM, Yu K, Donaldson GP, Shastri GG, Ann P, Ma L, et al. Indigenous bacteria from the gut microbiota regulate host serotonin biosynthesis. Cell. 2015;161:264–76.

  24. 24.

    Makarova KS, Wolf YI, Koonin EV. Comprehensive comparative-genomic analysis of type 2 toxin-antitoxin systems and related mobile stress response systems in prokaryotes. Biol Direct. 2009;4:19.

  25. 25.

    Maurice CF, Haiser HJ, Turnbaugh PJ. Xenobiotics shape the physiology and gene expression of the active human gut microbiome. Cell. 2013;152:39–50.

  26. 26.

    Li L, Mendis N, Trigui H, Oliver JD, Faucher SP. The importance of the viable but non-culturable state in human bacterial pathogens. Front Microbiol. 2014;5:258.

  27. 27.

    Kaplan I, Williams JW. Spore formation among the anaerobic bacteria: I. The formation of spores by Clostridium sporogenes in nutrient agar media. J Bacteriol. 1941;42:265.

  28. 28.

    Dingman DW, Stahly DP. Medium promoting sporulation of Bacillus larvae and metabolism of medium components. Appl Environ Microbiol. 1983;46:860–9.

  29. 29.

    Wunderlin T, Junier T, Roussel-Delif L, Jeanneret N, Junier P. Endospore-enriched sequencing approach reveals unprecedented diversity of Firmicutes in sediments: endospore-forming enrichment. Environ Microbiol Rep. 2014;6:631–9.

  30. 30.

    Sekar R, Pernthaler A, Pernthaler J, Warnecke F, Posch T, Amann R. An improved protocol for quantification of freshwater actinobacteria by fluorescence in situ hybridization. Appl Environ Microbiol. 2003;69:2928–35.

  31. 31.

    Fahlgren A, Hammarström S, Danielsson Å, HAMMARSTRÖM M-L. Increased expression of antimicrobial peptides and lysozyme in colonic epithelial cells of patients with ulcerative colitis. Clin Exp Immunol. 2003;131:90–101.

  32. 32.

    Keshav S, Chung P, Milon G, Gordon S. Lysozyme is an inducible marker of macrophage activation in murine tissues as demonstrated by in situ hybridization. J Exp Med. 1991;174:1049.

  33. 33.

    Gueimonde M, Laitinen K, Salminen S, Isolauri E. Breast milk: a source of bifidobacteria for infant gut development and maturation? Neonatology. 2007;92:64–6.

  34. 34.

    Edgar RC. Search and clustering orders of magnitude faster than BLAST. Bioinformatics. 2010;26:2460–1.

  35. 35.

    Galperin MY, Mekhedov SL, Puigbo P, Smirnov S, Wolf YI, Rigden DJ. Genomic determinants of sporulation in Bacilli and Clostridia: towards the minimal set of sporulation-specific genes: distribution of sporulation genes in Bacilli and Clostridia. Environ Microbiol. 2012;14:2870–90.

  36. 36.

    Serra CR, Earl AM, Barbosa TM, Kolter R, Henriques AO. Sporulation during growth in a gut isolate of Bacillus subtilis. J Bacteriol. 2014;196:4184–96.

  37. 37.

    Francis MB, Allen CA, Shrestha R, Sorg JA. Bile acid recognition by the clostridium difficile germinant receptor, CspC, is important for establishing infection. PLoS Pathog. 2013;9:e1003356.

  38. 38.

    Devkota S, Wang Y, Musch MW, Leone V, Fehlner-Peach H, Nadimpalli A, et al. Dietary-fat-induced taurocholic acid promotes pathobiont expansion and colitis in Il10−/− mice. Nature. (2012). e-pub ahead of print, https://doi.org/10.1038/nature11225.

  39. 39.

    Sorg JA, Sonenshein AL. Inhibiting the initiation of clostridium difficile spore germination using analogs of chenodeoxycholic acid, a bile acid. J Bacteriol. 2010;192:4983–90.

  40. 40.

    Ceuppens S, Uyttendaele M, Drieskens K, Heyndrickx M, Rajkovic A, Boon N, et al. Survival and germination of bacillus cereus spores without outgrowth or enterotoxin production during in vitro simulation of gastrointestinal transit. Appl Environ Microbiol. 2012;78:7698–705.

  41. 41.

    Shah IM, Laaberki M-H, Popham DL, Dworkin J. A eukaryotic-like Ser/Thr kinase signals bacteria to exit dormancy in response to peptidoglycan fragments. Cell. 2008;135:486–96.

  42. 42.

    Koenig JE, Spor A, Scalfone N, Fricker AD, Stombaugh J, Knight R, et al. Succession of microbial consortia in the developing infant gut microbiome. Proc Natl Acad Sci USA. 2011;108:4578–85.

  43. 43.

    Chandan RC, Shahani KM, Holly RG. Lysozyme content of human milk. Nature. 1964;204:76.

  44. 44.

    Nayfach S, Rodriguez-Mueller B, Garud N, Pollard KS. An integrated metagenomics pipeline for strain profiling reveals novel patterns of bacterial transmission and biogeography. Genome Res. 2016;26:1612–25.

  45. 45.

    Allegretti JR, Kearney S, Li N, Bogart E, Bullock K, Gerber GK, et al. Recurrent Clostridium difficile infection associates with distinct bile acid and microbiome profiles. Aliment Pharmacol Ther. 2016;43:1142–53.

  46. 46.

    Youngster I, Sauk J, Pindar C, Wilson RG, Kaplan JL, Smith MB, et al. Fecal microbiota transplant for relapsing Clostridium difficile infection using a frozen inoculum from unrelated donors: a randomized, open-label, controlled pilot study. Clin Infect Dis. 2014;58:1515–22.

  47. 47.

    David LA, Materna AC, Friedman J, Campos-Baptista MI, Blackburn MC, Perrotta A, et al. Host lifestyle affects human microbiota on daily timescales. Genome Biol. 2014;15:R89.

  48. 48.

    Bueche M, Wunderlin T, Roussel-Delif L, Junier T, Sauvain L, Jeanneret N, et al. Quantification of endospore-forming firmicutes by quantitative PCR with the functional gene spo0A. Appl Environ Microbiol. 2013;79:5302–12.

  49. 49.

    Chomczynski P, Rymaszewski M. Alkaline polyethylene glycol-based method for direct PCR from bacteria, eukaryotic tissue samples, and whole blood. Biotechniques. 2006;40:454.

  50. 50.

    Preheim SP, Perrotta AR, Martin-Platero AM, Gupta A, Alm EJ. Distribution-based clustering: using ecology to refine the operational taxonomic unit. Appl Environ Microbiol. 2013;79:6593–603.

  51. 51.

    Fierer N, Jackson JA, Vilgalys R, Jackson RB. Assessment of soil microbial community structure by use of taxon-specific quantitative PCR assays. Appl Environ Microbiol. 2005;71:4117–20.

  52. 52.

    Zhang J, Kobert K, Flouri T, Stamatakis A. PEAR: a fast and accurate illumina paired-end read merger. Bioinformatics. 2014;30:614–20.

  53. 53.

    Callahan BJ, McMurdie PJ, Rosen MJ, Han AW, Johnson AJA, Holmes SP. DADA2: high-resolution sample inference from Illumina amplicon data. Nat Methods. 2016;13:581.

  54. 54.

    Maidak BL, Olsen GJ, Larsen N, Overbeek R, McCaughey MJ, Woese CR. The ribosomal database project (RDP). Nucleic Acids Res. 1996;24:82–85.

  55. 55.

    Wang Q, Garrity GM, Tiedje JM, Cole JR. Naive Bayesian classifier for rapid assignment of rRNA sequences into the new bacterial taxonomy. Appl Environ Microbiol. 2007;73:5261–7.

  56. 56.

    Paulson JN, Stine OC, Bravo HC, Pop M. Differential abundance analysis for microbial marker-gene surveys. Nat Methods. 2013;10:1200–2.

Download references

Acknowledgements

We thank Fatima Hussain and Mathieu Groussin for extensive discussion and experimental advice. We thank the MIT BioMicro Center for sequencing service. We thank the members of the Losick lab for helpful advice and feedback on assaying sporulation phenotypes. Special thanks to Claire Duvallet for feedback on figure design and editing and manuscript clarity as well as the MIT Biological Engineering Communication Lab. We thank the members of the Alm Lab in general for intellectual support, discussion of data, and design of experiments. We thank Hilary Browne for discussions involving the cultivation and revival of endospore-forming organisms in the gut.We thank the MIT BioMicro Center for sequencing service. We thank the Chisholm lab and the Weiss Lab at MIT for allowing us to use their microscopes and qPCR machines.

Funding

The funding was provided by the Broad Institute BN10 Training Grants. S.M.K. was funded by an NSF Graduate Research Fellowship.

Author information

Affiliations

  1. Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA

    • Sean M. Kearney
    • , Sean M. Gibbons
    • , Mathilde Poyet
    • , Thomas Gurry
    •  & Eric J. Alm
  2. The Broad Institute, Cambridge, MA, USA

    • Sean M. Kearney
    • , Sean M. Gibbons
    • , Mathilde Poyet
    • , Thomas Gurry
    • , Kevin Bullock
    • , Clary B. Clish
    •  & Eric J. Alm
  3. The Center for Microbiome Informatics and Therapeutics, Cambridge, MA, USA

    • Sean M. Kearney
    • , Sean M. Gibbons
    • , Mathilde Poyet
    • , Thomas Gurry
    •  & Eric J. Alm
  4. Division of Gastroenterology, Brigham and Women’s Hospital, Boston, MA, USA

    • Jessica R. Allegretti
  5. Harvard Medical School, Boston, MA, USA

    • Jessica R. Allegretti

Authors

  1. Search for Sean M. Kearney in:

  2. Search for Sean M. Gibbons in:

  3. Search for Mathilde Poyet in:

  4. Search for Thomas Gurry in:

  5. Search for Kevin Bullock in:

  6. Search for Jessica R. Allegretti in:

  7. Search for Clary B. Clish in:

  8. Search for Eric J. Alm in:

Conflict of interest

The authors declare that they have no conflict of interest.

Corresponding author

Correspondence to Eric J. Alm.

Electronic supplementary material

About this article

Publication history

Received

Revised

Accepted

Published

DOI

https://doi.org/10.1038/s41396-018-0192-z