Isolating and Purifying Clostridium difficile Spores

  • Adrianne N. Edwards
  • Shonna M. McBrideEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1476)


The ability for the obligate anaerobe, Clostridium difficile to form a metabolically dormant spore is critical for the survival of this organism outside of the host. This spore form is resistant to a myriad of environmental stresses, including heat, desiccation, and exposure to disinfectants and antimicrobials. These intrinsic properties of spores allow C. difficile to survive long-term in an oxygenated environment, to be easily transmitted from host-to-host, and to persist within the host following antibiotic treatment. Because of the importance of the spore form to the C. difficile life cycle and treatment and prevention of C. difficile infection (CDI), the isolation and purification of spores are necessary to study the mechanisms of sporulation and germination, investigate spore properties and resistances, and for use in animal models of CDI. Here we provide basic protocols, in vitro growth conditions, and additional considerations for purifying C. difficile spores for a variety of downstream applications.

Key words

Clostridium difficile Sporulation Endospore Anaerobe Anaerobic chamber Antibiotic-associated diarrhea 


  1. 1.
    CDC (2013) Antibiotic resistance threats in the United States. In: Control CfD (ed)
  2. 2.
    Dubberke ER, Olsen MA (2012) Burden of Clostridium difficile on the healthcare system. Clin Infect Dis 55(Suppl 2):S88–S92, PMCID: 3388018CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Sorg JA, Sonenshein AL (2008) Bile salts and glycine as cogerminants for Clostridium difficile spores. J Bacteriol 190(7):2505–2512, PMCID: 2293200CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Chen X, Katchar K, Goldsmith JD, Nanthakumar N, Cheknis A, Gerding DN, Kelly CP (2008) A mouse model of Clostridium difficile-associated disease. Gastroenterology 135(6):1984–1992CrossRefPubMedGoogle Scholar
  5. 5.
    Goulding D, Thompson H, Emerson J, Fairweather NF, Dougan G, Douce GR (2009) Distinctive profiles of infection and pathology in hamsters infected with Clostridium difficile strains 630 and B1. Infect Immun 77(12):5478–5485, PMCID: 2786451CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Douce G, Goulding D (2010) Refinement of the hamster model of Clostridium difficile disease. Methods Mol Biol 646:215–227CrossRefPubMedGoogle Scholar
  7. 7.
    Sorg JA, Sonenshein AL (2010) Inhibiting the initiation of Clostridium difficile spore germination using analogs of chenodeoxycholic acid, a bile acid. J Bacteriol 192(19):4983–4990, PMCID: 2944524CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Francis MB, Allen CA, Shrestha R, Sorg JA (2013) Bile acid recognition by the Clostridium difficile germinant receptor, CspC, is important for establishing infection. PLoS Pathog 9(5):e1003356, PMCID: 3649964CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Liu R, Suarez JM, Weisblum B, Gellman SH, McBride SM (2014) Synthetic polymers active against Clostridium difficile vegetative cell growth and spore outgrowth. J Am Chem Soc. PMCID: 4210120Google Scholar
  10. 10.
    Kuijper EJ, Coignard B, Tull P (2006) Emergence of Clostridium difficile-associated disease in North America and Europe. Clin Microbiol Infect 12(Suppl 6):2–18CrossRefPubMedGoogle Scholar
  11. 11.
    Berg JM, Tymoczko JL, Stryer L, Stryer L (2002) Biochemistry, 5th edn. W.H. Freeman, New YorkGoogle Scholar
  12. 12.
    Putnam EE, Nock AM, Lawley TD, Shen A (2013) SpoIVA and SipL are Clostridium difficile spore morphogenetic proteins. J Bacteriol 195(6):1214–1225CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    George WL, Sutter VL, Citron D, Finegold SM (1979) Selective and differential medium for isolation of Clostridium difficile. J Clin Microbiol 9(2):214–219, PMCID: 272994PubMedPubMedCentralGoogle Scholar
  14. 14.
    Wilson KH, Kennedy MJ, Fekety FR (1982) Use of sodium taurocholate to enhance spore recovery on a medium selective for Clostridium difficile. J Clin Microbiol 15(3):443–446, PMCID: 272115PubMedPubMedCentralGoogle Scholar
  15. 15.
    Edwards AN, Nawrocki KL, McBride SM (2014) Conserved oligopeptide permeases modulate sporulation initiation in Clostridium difficile. Infect Immun 82(10):4276–4291CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Bouillaut L, McBride SM, Sorg JA (2011) Genetic manipulation of Clostridium difficile. Curr Protoc Microbiol; Chapter 9: Unit 9A 2. PMCID: 3615975Google Scholar
  17. 17.
    Kuehne SA, Heap JT, Cooksley CM, Cartman ST, Minton NP (2011) ClosTron-mediated engineering of Clostridium. Methods Mol Biol 765:389–407CrossRefPubMedGoogle Scholar
  18. 18.
    Francis MB, Sorg JA (2013) Virulence studies of Clostridium difficile. Bio-protocol 3(24):e1002Google Scholar
  19. 19.
    Edwards AN, Suarez JM, McBride SM (2013) Culturing and maintaining Clostridium difficile in an anaerobic environment. J Vis Exp (79)Google Scholar
  20. 20.
    Sorg JA, Dineen SS (2009) Laboratory maintenance of Clostridium difficile. Curr Protoc Microbiol; Chapter 9: Unit 9A.1Google Scholar
  21. 21.
    Lawley TD, Croucher NJ, Yu L, Clare S, Sebaihia M, Goulding D, Pickard DJ, Parkhill J, Choudhary J, Dougan G (2009) Proteomic and genomic characterization of highly infectious Clostridium difficile 630 spores. J Bacteriol 191(17):5377–5386, PMCID: 2725610CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Sorg JA, Sonenshein AL (2009) Chenodeoxycholate is an inhibitor of Clostridium difficile spore germination. J Bacteriol 191(3):1115–1117CrossRefPubMedGoogle Scholar
  23. 23.
    Francis MB, Allen CA, Sorg JA (2015) Spore cortex hydrolysis precedes DPA release during Clostridium difficile spore germination. J Bacteriol 197(14):2276–2283CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Paredes-Sabja D, Bond C, Carman RJ, Setlow P, Sarker MR (2008) Germination of spores of Clostridium difficile strains, including isolates from a hospital outbreak of Clostridium difficile-associated disease (CDAD). Microbiology 154(Pt 8):2241–2250CrossRefPubMedGoogle Scholar
  25. 25.
    Permpoonpattana P, Tolls EH, Nadem R, Tan S, Brisson A, Cutting SM (2011) Surface layers of Clostridium difficile endospores. J Bacteriol 193(23):6461–6470, PMCID: 3232898CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Hashimoto T, Black SH, Gerhardt P (1960) Development of fine structure, thermostability, and dipicolinate during sporogenesis in a bacillus. Can J Microbiol 6:203–212CrossRefPubMedGoogle Scholar
  27. 27.
    Hitchins AD, Kahn AJ, Slepecky RA (1968) Interference contrast and phase contrast microscopy of sporulation and germination of Bacillus megaterium. J Bacteriol 96(5):1811–1817, PMCID: 315245PubMedPubMedCentralGoogle Scholar
  28. 28.
    Burns DA, Minton NP (2011) Sporulation studies in Clostridium difficile. J Microbiol Methods 87(2):133–138CrossRefPubMedGoogle Scholar
  29. 29.
    Pereira FC, Saujet L, Tome AR, Serrano M, Monot M, Couture-Tosi E, Martin-Verstraete I, Dupuy B, Henriques AO (2013) The spore differentiation pathway in the enteric pathogen Clostridium difficile. PLoS Genet 9(10):e1003782, PMCID: 3789829CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Fimlaid KA, Bond JP, Schutz KC, Putnam EE, Leung JM, Lawley TD, Shen A (2013) Global analysis of the sporulation pathway of Clostridium difficile. PLoS Genet 9(8):e1003660, PMCID: 3738446CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Saujet L, Pereira FC, Serrano M, Soutourina O, Monot M, Shelyakin PV, Gelfand MS, Dupuy B, Henriques AO, Martin-Verstraete I (2013) Genome-wide analysis of cell type-specific gene transcription during spore formation in Clostridium difficile. PLoS Genet 9(10):e1003756, PMCID: 3789822CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    Sebaihia M, Wren BW, Mullany P, Fairweather NF, Minton N, Stabler R, Thomson NR, Roberts AP, Cerdeno-Tarraga AM, Wang H, Holden MT, Wright A, Churcher C, Quail MA, Baker S, Bason N, Brooks K, Chillingworth T, Cronin A, Davis P, Dowd L, Fraser A, Feltwell T, Hance Z, Holroyd S, Jagels K, Moule S, Mungall K, Price C, Rabbinowitsch E, Sharp S, Simmonds M, Stevens K, Unwin L, Whithead S, Dupuy B, Dougan G, Barrell B, Parkhill J (2006) The multidrug-resistant human pathogen Clostridium difficile has a highly mobile, mosaic genome. Nat Genet 38(7):779–786CrossRefPubMedGoogle Scholar
  33. 33.
    Monot M, Boursaux-Eude C, Thibonnier M, Vallenet D, Moszer I, Medigue C, Martin-Verstraete I, Dupuy B (2011) Reannotation of the genome sequence of Clostridium difficile strain 630. J Med Microbiol 60(Pt 8):1193–1199CrossRefPubMedGoogle Scholar
  34. 34.
    Stabler RA, He M, Dawson L, Martin M, Valiente E, Corton C, Lawley TD, Sebaihia M, Quail MA, Rose G, Gerding DN, Gibert M, Popoff MR, Parkhill J, Dougan G, Wren BW (2009) Comparative genome and phenotypic analysis of Clostridium difficile 027 strains provides insight into the evolution of a hypervirulent bacterium. Genome Biol 10(9):R102, PMCID: 2768977CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Department of Microbiology and ImmunologyEmory University School of MedicineAtlantaUSA

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