Comparison of Molecular Typing Methods Applied to Clostridium difficile

  • Ed J. Kuijper
  • Renate J. van den Berg
  • Jon S. Brazier
Part of the Methods in Molecular Biology™ book series (MIMB, volume 551)


Since the 1980s the epidemiology of Clostridium difficile infection (CDI) has been investigated by the application of many different typing or fingerprinting methods. To study the epidemiology of CDI, a typing method with a high discriminatory power, typeability, and reproducibility is required. Molecular typing methods are generally regarded as having advantages over phenotypic methods in terms of the stability of genomic markers and providing greater levels of typeability. A growing number of molecular methods have been applied to C. difficile. For the early and rapid detection of outbreak situations, methods such as restriction enzyme analysis, arbitrary primed polymerase chain reaction (PCR), and PCR ribotyping are commonly used. For long-term epidemiology, multilocus sequence typing, multilocus variable number of tandem repeats analysis, and amplified fragment length polymorphism are of interest. Currently, the PCR-ribotyping method and the library of PCR ribotypes in Cardiff are the benchmarks to which most typing studies around the world are compared. Multilocus variable number of tandem repeats analysis is the most discriminative typing method and will contribute significantly to our understanding of the epidemiology of this important nosocomial pathogen.

Key words

Clostridium difficile MLVA PCR ribotyping REA subtyping 


  1. 1.
    Kuijper, E. J., Coignard, B., and Tull, P. (2006). Emergence of Clostridium difficile-associated disease in North America and Europe. Clin. Microbiol. Infect. 12(Suppl. 6), 2–18.PubMedCrossRefGoogle Scholar
  2. 2.
    Arai, T., Kusakabe, A., Nakashio, S., and Nakamura, M. (1984). A survey of plasmids in Clostridium difficile strains. Kitasato Arch. Exp. Med. 57, 285–288.PubMedGoogle Scholar
  3. 3.
    Steinberg, J. P., Beckerdite, M. E., and Westenfelder, G. O. (1987). Plasmid profiles of Clostridium difficile isolates from patients with antibiotic-associated colitis in two community hospitals. J. Infect. Dis. 156, 1036–1038.PubMedCrossRefGoogle Scholar
  4. 4.
    Clabots, C., Lee, S., Gerding, D., Mulligan, M., Kwok, R., Schaberg, D., et al (1988). Clostridium difficile plasmid isolation as an epidemiologic tool. Eur. J. Clin. Microbiol. Infect. Dis. 7, 312–315.PubMedCrossRefGoogle Scholar
  5. 5.
    Kuijper, E. J., Oudbier, J. H., Stuifbergen, W. N., Jansz, A., and Zanen, H. C. (1987). Application of whole-cell DNA restriction endonuclease profiles to the epidemiology of Clostridium difficile-induced diarrhea. J. Clin. Microbiol. 25, 751–753.PubMedGoogle Scholar
  6. 6.
    Devlin, H. R., Au, W., Foux, L., and Bradbury, W. C. (1987). Restriction endonuclease analysis of nosocomial isolates of Clostridium difficile. J. Clin. Microbiol. 25, 2168–2172.PubMedGoogle Scholar
  7. 7.
    Clabots, C. R., Johnson, S., Bettin, K. M., Mathie, P. A., Mulligan, M. E., Schaberg, D. R., et al. (1993). Development of a rapid and efficient restriction endonuclease analysis typing system for Clostridium difficile and correlation with other typing systems. J. Clin. Microbiol. 31, 1870–1875.PubMedGoogle Scholar
  8. 8.
    Bowman, R. A., O’Neill, G. L., and Riley, T. V. (1991). Non-radioactive restriction fragment length polymorphism (RFLP) typing of Clostridium difficile. FEMS Microbiol. Lett. 63, 269–272.PubMedCrossRefGoogle Scholar
  9. 9.
    Wolfhagen, M. J., Fluit, A. C., Torensma, R., Jansze, M., Kuypers, A. F., Verhage, E. A., et al. (1993). Comparison of typing ­methods for Clostridium difficile isolates. J. Clin. Microbiol. 31, 2208–2211.PubMedGoogle Scholar
  10. 10.
    O’Neill, G. L., Beaman, M. H., and Riley, T. V. (1991). Relapse versus reinfection with Clostridium difficile. Epidemiol. Infect. 107, 627–635.PubMedCrossRefGoogle Scholar
  11. 11.
    McMillin, D. E., and Muldrow, L. L. (1992). Typing of toxic strains of Clostridium difficile using DNA fingerprints generated with arbitrary polymerase chain reaction primers. FEMS Microbiol. Lett. 71, 5–9.PubMedCrossRefGoogle Scholar
  12. 12.
    Barbut, F., Mario, N., Frottier, J., and Petit, J. C. (1993). Use of the arbitrary primer polymerase chain reaction for investigating an outbreak of Clostridium difficile-associated diarrhea in AIDS patients. Eur. J. Clin. Microbiol. Infect. Dis. 12, 794–795.PubMedCrossRefGoogle Scholar
  13. 13.
    Wilks, M., and Tabaqchali, S. (1994). Typing of Clostridium difficile by polymerase chain reaction with an arbitrary primer. J. Hosp. Infect. 28, 231–234.PubMedCrossRefGoogle Scholar
  14. 14.
    Killgore, G. E., and Kato, H. (1994). Use of arbitrary primer PCR to type Clostridium difficile and comparison of results with those by immunoblot typing. J. Clin. Microbiol. 32, 1591–1593.PubMedGoogle Scholar
  15. 15.
    Tang, Y. J., Houston, S. T., Gumerlock, P. H., Mulligan, M. E., Gerding, D. N., Johnson, S., et al (1995). Comparison of arbitrarily primed PCR with restriction endonuclease and immunoblot analyses for typing Clostridium difficile isolates. J. Clin. Microbiol. 33, 3169–3173.PubMedGoogle Scholar
  16. 16.
    Samore, M., Killgore, G., Johnson, S., Goodman, R., Shim, J., Venkataraman, L., et al (1997). Multicenter typing comparison of sporadic and outbreak Clostridium difficile isolates from geographically diverse hospitals. J. Infect. Dis. 176, 1233–1238.PubMedCrossRefGoogle Scholar
  17. 17.
    Rafferty, M. E., Baltch, A. L., Smith, R. P., Bopp, L. H., Rheal, C., Tenover, F. C., et al. (1998). Comparison of restriction enzyme analysis, arbitrarily primed PCR, and protein profile analysis typing for epidemiologic investigation of an ongoing Clostridium difficile outbreak. J. Clin. Microbiol. 36, 2957–2963.PubMedGoogle Scholar
  18. 18.
    Cohen, S. H., Tang, Y. J., and Silva, J., Jr. (2001). Molecular typing methods for the epidemiological identification of Clostridium difficile strains. Expert. Rev. Mol. Diagn. 1, 61–70.PubMedCrossRefGoogle Scholar
  19. 19.
    Barbut, F., Mario, N., Delmee, M., Gozian, J., and Petit, J. C. (1993). Genomic fingerprinting of Clostridium difficile isolates by using a random amplified polymorphic DNA (RAPD) assay. FEMS Microbiol. Lett. 114, 161–166.PubMedCrossRefGoogle Scholar
  20. 20.
    Chachaty, E., Saulnier, P., Martin, A., Mario, N., and Andremont, A. (1994). Comparison of ribotyping, pulsed-field gel electrophoresis and random amplified polymorphic DNA for typing Clostridium difficile strains. FEMS Microbiol. Lett. 122, 61–68.PubMedCrossRefGoogle Scholar
  21. 21.
    Alonso, R., Gros, S., Pelaez, T., Garcia-de-Viedma, D., Rodriguez-Creixems, M., and Bouza, E. (2001). Molecular analysis of relapse vs re-infection in HIV-positive patients suffering from recurrent Clostridium difficile associated diarrhoea. J. Hosp. Infect. 48, 86–92.PubMedCrossRefGoogle Scholar
  22. 22.
    Gurtler, V. (1993). Typing of Clostridium difficile strains by PCR-amplification of variable length 16S-23S rDNA spacer regions. J. Gen. Microbiol. 139, 3089–3097.PubMedCrossRefGoogle Scholar
  23. 23.
    Gurtler, V., and Mayall, B. C. (1994). Genotyping of Clostridium difficile isolates. J. Clin. Microbiol. 32, 3095.PubMedGoogle Scholar
  24. 24.
    Cartwright, C. P., Stock, F., Beekmann, S. E., Williams, E. C., and Gill, V. J. (1995). PCR amplification of rRNA intergenic spacer regions as a method for epidemiologic typing of Clostridium difficile. J. Clin. Microbiol. 33, 184–187.PubMedGoogle Scholar
  25. 25.
    Collier, M. C., Stock, F., DeGirolami, P. C., Samore, M. H., and Cartwright, C. P. (1996). Comparison of PCR-based approaches to molecular epidemiologic analysis of Clostridium difficile. J. Clin. Microbiol. 34, 1153–1157.PubMedGoogle Scholar
  26. 26.
    O’Neill, G. L., Ogunsola, F. T., Brazier, J. S., and Duerden, B. I. (1996). Modification of a PCR ribotyping method for application as a routine typing scheme for Clostridium difficile. Anaerobe 2, 205–209.CrossRefGoogle Scholar
  27. 27.
    Stubbs, S. L., Brazier, J. S., O’Neill, G. L., and Duerden, B. I. (1999). PCR targeted to the 16S-23S rRNA gene intergenic spacer region of Clostridium difficile and construction of a library consisting of 116 different PCR ribotypes. J. Clin. Microbiol. 37, 461–463.PubMedGoogle Scholar
  28. 28.
    Bidet, P., Barbut, F., Lalande, V., Burghoffer, B., and Petit, J. C. (1999). Development of a new PCR ribotyping method for Clostridium difficile based on ribosomal RNA gene sequencing. FEMS Microbiol. Lett. 175, 261–266.PubMedCrossRefGoogle Scholar
  29. 29.
    Barbut, F., Richard, A., Hamadi, K., Chomette, V., Burghoffer, B., and Petit, J. C. (2000). Epidemiology of recurrences or reinfections of Clostridium difficile-associated diarrhea. J. Clin. Microbiol. 38, 2386–2388.PubMedGoogle Scholar
  30. 30.
    Brazier, J. S., Mulligan, M. E., Delmee, M., and Tabaqchali, S. (1997). Preliminary findings of the international typing study on Clostridium difficile. International Clostridium Difficile Study Group. Clin. Infect. Dis. 25(Suppl. 2), S199–S201.PubMedCrossRefGoogle Scholar
  31. 31.
    Brazier, J. S. (2001). Typing of Clostridium difficile. Clin. Microbiol. Infect. 7, 428–431.PubMedCrossRefGoogle Scholar
  32. 32.
    Rupnik, M., Avesani, V., Janc, M., von Eichel-Streiber, C., and Delmee, M. (1998). A novel toxinotyping scheme and correlation of toxinotypes with serogroups of Clostridium difficile isolates. J. Clin. Microbiol. 36, 2240–2247.PubMedGoogle Scholar
  33. 33.
    Rupnik, M., Brazier, J. S., Duerden, B. I., Grabnar, M., and Stubbs, S. L. (2001). Comparison of toxinotyping and PCR ribotyping of Clostridium difficile strains and description of novel toxinotypes. Microbiology 147, 439–447.PubMedGoogle Scholar
  34. 34.
    Rupnik, M., Kato, N., Grabnar, M., and Kato, H. (2003). New types of toxin A-negative, toxin B-positive strains among Clostridium difficile isolates from Asia. J. Clin. Microbiol. 41, 1118–1125.PubMedCrossRefGoogle Scholar
  35. 35.
    Barbut, F., Lalande, V., Burghoffer, B., Thien, H. V., Grimprel, E., and Petit, J. C. (2002). Prevalence and genetic characterization of toxin A variant strains of Clostridium difficile among adults and children with diarrhea in France. J. Clin. Microbiol. 40, 2079–2083.PubMedCrossRefGoogle Scholar
  36. 36.
    Geric, B., Rupnik, M., Gerding, D. N., Grabnar, M., and Johnson, S. (2004). Distribution of Clostridium difficile variant toxinotypes and strains with binary toxin genes among clinical isolates in an American hospital. J. Med. Microbiol. 53, 887–894.PubMedCrossRefGoogle Scholar
  37. 37.
    Tasteyre, A., Karjalainen, T., Avesani, V., Delmee, M., Collignon, A., Bourlioux, P., et al. (2000). Phenotypic and genotypic diversity of the flagellin gene (fliC) among Clostridium difficile isolates from different serogroups. J. Clin. Microbiol. 38, 3179–3186.PubMedGoogle Scholar
  38. 38.
    Pituch, H., Obuch-Woszczatynski, P., van den Braak, N., van Belkum, A., Kujawa, M., Luczak, M., et al. (2002). Variable flagella expression among clonal toxin A−/B+ Clostridium difficile strains with highly homogeneous flagellin genes. Clin. Microbiol. Infect. 8, 187–188.PubMedCrossRefGoogle Scholar
  39. 39.
    McCoubrey, J., Starr, J., Martin, H., and Poxton, I. R. (2003). Clostridium difficile in a geriatric unit: a prospective epidemiological study employing a novel S-layer typing method. J. Med. Microbiol. 52, 573–578.PubMedCrossRefGoogle Scholar
  40. 40.
    Karjalainen, T., Saumier, N., Barc, M. C., Delmee, M., and Collignon, A. (2002). Clostridium difficile genotyping based on slpA variable region in S-layer gene sequence: an alternative to serotyping. J. Clin. Microbiol. 40, 2452–2458.PubMedCrossRefGoogle Scholar
  41. 41.
    Kato, H., Yokoyama, T., and Arakawa, Y. (2005). Typing by sequencing the slpA gene of Clostridium difficile strains causing multiple outbreaks in Japan. J. Med. Microbiol. 54, 167–171.PubMedCrossRefGoogle Scholar
  42. 42.
    Eidhin, D. N., Ryan, A. W., Doyle, R. M., Walsh, J. B., and Kelleher, D. (2006). Sequence and phylogenetic analysis of the gene for surface layer protein, slpA, from 14 PCR ribotypes of Clostridium difficile. J. Med. Microbiol. 55, 69–83.PubMedCrossRefGoogle Scholar
  43. 43.
    Klaassen, C. H., van Haren, H. A., and Horrevorts, A. M. (2002). Molecular fingerprinting of Clostridium difficile isolates: pulsed-field gel electrophoresis versus amplified fragment length polymorphism. J. Clin. Microbiol. 40, 101–104.PubMedCrossRefGoogle Scholar
  44. 44.
    van den Berg, R. J., Claas, E. C., Oyib, D. H., Klaassen, C. H., Dijkshoorn, L., Brazier, J. S., et al (2004). Characterization of toxin A-negative, toxin B-positive Clostridium difficile isolates from outbreaks in different countries by amplified fragment length polymorphism and PCR ribotyping. J. Clin. Microbiol. 42, 1035–1041.PubMedCrossRefGoogle Scholar
  45. 45.
    Lemee, L., Dhalluin, A., Pestel-Caron, M., Lemeland, J. F., and Pons, J. L. (2004). Multilocus sequence typing analysis of human and animal Clostridium difficile isolates of various toxigenic types. J. Clin. Microbiol. 42, 2609–2617.PubMedCrossRefGoogle Scholar
  46. 46.
    Lemee, L., Bourgeois, I., Ruffin, E., Collignon, A., Lemeland, J. F., and Pons, J. L. (2005). Multilocus sequence analysis and comparative evolution of virulence-associated genes and housekeeping genes of Clostridium difficile. Microbiology 151, 3171–3180.PubMedCrossRefGoogle Scholar
  47. 47.
    Lindstedt, B.-A. (2005). Multiple-locus variable number tandem repeats analysis for genetic fingerprinting of pathogenic bacteria. Electrophoresis 26, 2567–2582.PubMedCrossRefGoogle Scholar
  48. 48.
    Sebaihia, M., Wren, B. W., Mullany, P., Fairweather, N. F., Minton, N., Stabler, R., et al (2006). The multidrug-resistant human pathogen Clostridium difficile has a highly mobile, mosaic genome. Nat. Genet. 38, 779–786.PubMedCrossRefGoogle Scholar
  49. 49.
    Marsh, J. W., O’Leary, M. M., Shutt, K. A., Pasculle, A. W., Johnson, S., Gerding, D. N., et al. (2006). Multilocus variable-number tandem-repeat analysis for investigation of Clostridium difficile transmission in Hospitals. J. Clin. Microbiol. 44, 2558–2566.PubMedCrossRefGoogle Scholar
  50. 50.
    van den Berg, R. J., Schaap, I., Templeton, K. E., Klaassen, C. H., and Kuijper, E. J. (2007). Typing and subtyping of Clostridium difficile isolates by using multiple-locus variable-number tandem-repeat analysis. J. Clin. Microbiol. 45, 1024–1028.PubMedCrossRefGoogle Scholar
  51. 51.
    Fawley, W. N., Freeman, J., Smith, C., Harmanus, C., van den Berg, R. J., Kuijper, E. J., et al (2008). Use of highly discriminatory fingerprinting to analyze clusters of Clostridium difficile infection cases due to epidemic ribotype 027 strains. J. Clin. Microbiol. 46, 954–960.PubMedCrossRefGoogle Scholar
  52. 52.
    Killgore, G., Thompson, A., Johnson, S., Brazier, J., Kuijper, E., Pepin, J., et al (2008). Comparison of seven techniques for typing international epidemic strains of Clostridium difficile: restriction endonuclease analysis, pulsed-field gel electrophoresis, PCR-ribotyping, multilocus sequence typing, multilocus variable-number tandem-repeat analysis, amplified fragment length polymorphism, and surface layer protein A gene sequence typing. J. Clin. Microbiol. 46, 431–437.PubMedCrossRefGoogle Scholar
  53. 53.
    Fenner, L., Widmer, A. F., Stranden, A., Conzelmann, M., Goorhuis, A., Harmanus, C., et al (2008). First cluster of clindamycin-resistant Clostridium difficile PCR-ribotype 027 in Switzerland. Clin. Microbiol. 14, 514–515.CrossRefGoogle Scholar
  54. 54.
    Drudy, D., Goorhuis, B., Bakker, D., Kyne, L., van den Berg, R., Fanning, S., et al (2008). Clindamycin-resistant clone of Clostridium difficile PCR ribotype 027, Europe. Emerg. Infect. Dis. 14, 1485–1487.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2009

Authors and Affiliations

  • Ed J. Kuijper
    • 1
  • Renate J. van den Berg
    • 1
  • Jon S. Brazier
    • 1
  1. 1.Reference Laboratory for Clostridium difficile, Medical Microbiology Department, LUMC, LeidenThe National Institute for Public Health and EnvironmentBangkokThe Netherlands

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