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Comparison of Molecular Typing Methods Applied to Clostridium difficile

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Molecular Epidemiology of Microorganisms

Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 551))

Abstract

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.

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References

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    PubMed  CAS  Google Scholar 

  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.

    PubMed  CAS  Google Scholar 

  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.

    PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    PubMed  CAS  Google Scholar 

  10. O’Neill, G. L., Beaman, M. H., and Riley, T. V. (1991). Relapse versus reinfection with Clostridium difficile. Epidemiol. Infect. 107, 627–635.

    Article  PubMed  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    PubMed  CAS  Google Scholar 

  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.

    PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  23. Gurtler, V., and Mayall, B. C. (1994). Genotyping of Clostridium difficile isolates. J. Clin. Microbiol. 32, 3095.

    PubMed  CAS  Google Scholar 

  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.

    PubMed  CAS  Google Scholar 

  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.

    PubMed  CAS  Google Scholar 

  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.

    Article  Google Scholar 

  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.

    PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  Google Scholar 

  31. Brazier, J. S. (2001). Typing of Clostridium difficile. Clin. Microbiol. Infect. 7, 428–431.

    Article  PubMed  CAS  Google Scholar 

  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.

    PubMed  CAS  Google Scholar 

  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.

    PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  47. Lindstedt, B.-A. (2005). Multiple-locus variable number tandem repeats analysis for genetic fingerprinting of pathogenic bacteria. Electrophoresis 26, 2567–2582.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  PubMed  Google Scholar 

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Authors and Affiliations

  1. Reference Laboratory for Clostridium difficile, Medical Microbiology Department, LUMC, Leiden, The National Institute for Public Health and Environment, Bangkok, Bilthoven, The Netherlands

    Ed J. Kuijper, Renate J. van den Berg & Jon S. Brazier

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  1. Ed J. Kuijper
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  2. Renate J. van den Berg
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  3. Jon S. Brazier
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    © 2009 Humana Press, a part of Springer Science+Business Media, LLC

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    Kuijper, E.J., van den Berg, R.J., Brazier, J.S. (2009). Comparison of Molecular Typing Methods Applied to Clostridium difficile . In: Caugant, D. (eds) Molecular Epidemiology of Microorganisms. Methods in Molecular Biology™, vol 551. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60327-999-4_13

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    • DOI: https://doi.org/10.1007/978-1-60327-999-4_13

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