Rapid detection of the Clostridium difficile ribotype 027 tcdC gene frame shift mutation at position 117 by real-time PCR and melt curve analysis

Article

Abstract

The emergence of the hypervirulent strain Clostridium difficile PCR ribotype 027 has increased the necessity for rapid C. difficile typing tests for clinical and epidemiological purposes. We developed a rapid real-time polymerase chain reaction (PCR) test for the detection of C. difficile. As the target, we chose the tcdC gene, which encodes for a negative regulator in toxin production. A deletion at position 117 of the tcdC gene, which is associated with severe tcdC truncation, is well conserved in all PCR ribotype 027 isolates. Probe sequences of the real-time PCR test were designed to result in distinct melt profiles for sequence variations at positions 117 to 120 of the tcdC gene. The tcdC gene deletion at position 117 was easily detected with real-time PCR and melt curve analysis in all C. difficile ribotype 027 isolates. In five non-027 strains and 46 hospitalised patient samples, melt curve analysis detected no deletion. PCR results were confirmed by DNA sequencing. The combination of real-time PCR and melt curve analysis is a rapid and accurate method for the detection of C. difficile DNA and simultaneous screening for the tcdC gene deletion at position 117, which is closely related to the C. difficile PCR ribotype 027 strain.

References

  1. 1.
    Lyerly DM, Krivan HC, Wilkins TD (1988) Clostridium difficile: its disease and toxins. Clin Microbiol Rev 1:1–18PubMedGoogle Scholar
  2. 2.
    Hammond GA, Johnson JL (1995) The toxigenic element of Clostridium difficile strain VPI 10463. Microb Pathog 19:203–213. doi:10.1016/S0882-4010(95)90263-5 PubMedCrossRefGoogle Scholar
  3. 3.
    Krausz S, Bessems M, Boermeester MA, Kuijper EJ, Visser CE, Speelman P (2005) Life-threatening infections with a new strain of Clostridium difficile. Ned Tijdschr Geneeskd 149:2081–2086. In DutchPubMedGoogle Scholar
  4. 4.
    Loo VG, Poirier L, Miller MA, Oughton M, Libman MD, Michaud S et al (2005) A predominantly clonal multi-institutional outbreak of Clostridium difficile-associated diarrhea with high morbidity and mortality. N Engl J Med 353:2442–2449. doi:10.1056/NEJMoa051639 PubMedCrossRefGoogle Scholar
  5. 5.
    Kuijper EJ, Coignard B, Tüll P; ESCMID Study Group for Clostridium difficile; EU Member States; European Centre for Disease Prevention and Control (2006) Emergence of Clostridium difficile-associated disease in North America and Europe. Clin Microbiol Infect 12:2–18. doi:10.1111/j.1469-0691.2006.01580.x PubMedCrossRefGoogle Scholar
  6. 6.
    Kleinkauf N, Weiss B, Jansen A, Eckmanns T, Bornhofen B, Küehnen E et al (2007) Confirmed cases and report of clusters of severe infections due to Clostridium difficile PCR ribotype 027 in Germany. Euro Surveill 12:E071115.2Google Scholar
  7. 7.
    Kuijper EJ, Coignard B, Brazier JS, Suetens C, Drudy D, Wiuff C et al (2007) Update of Clostridium difficile-associated disease due to PCR ribotype 027 in Europe. Euro Surveill 12:E1–E2PubMedGoogle Scholar
  8. 8.
    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. doi:10.1128/JCM.01484-07 PubMedCrossRefGoogle Scholar
  9. 9.
    Warny M, Pepin J, Fang A, Killgore G, Thompson A, Brazier J et al (2005) Toxin production by an emerging strain of Clostridium difficile associated with outbreaks of severe disease in North America and Europe. Lancet 366:1079–1084. doi:10.1016/S0140-6736(05)67420-X PubMedCrossRefGoogle Scholar
  10. 10.
    Spigaglia P, Mastrantonio P (2002) Molecular analysis of the pathogenicity locus and polymorphism in the putative negative regulator of toxin production (TcdC) among Clostridium difficile clinical isolates. J Clin Microbiol 40:3470–3475. doi:10.1128/JCM.40.9.3470-3475.2002 PubMedCrossRefGoogle Scholar
  11. 11.
    MacCannell DR, Louie TJ, Gregson DB, Laverdiere M, Labbe AC, Laing F et al (2006) Molecular analysis of Clostridium difficile PCR ribotype 027 isolates from Eastern and Western Canada. J Clin Microbiol 44:2147–2152. doi:10.1128/JCM.02563-05 PubMedCrossRefGoogle Scholar
  12. 12.
    Curry SR, Marsh JW, Muto CA, O’Leary MM, Pasculle AW, Harrison LH (2007) tcdC genotypes associated with severe TcdC truncation in an epidemic clone and other strains of Clostridium difficile. J Clin Microbiol 45:215–221. doi:10.1128/JCM.01599-06 PubMedCrossRefGoogle Scholar
  13. 13.
    Sloan LM, Duresko BJ, Gustafson DR, Rosenblatt JE (2008) Comparison of real-time PCR for detection of the tcdC gene with four toxin immunoassays and culture in diagnosis of Clostridium difficile infection. J Clin Microbiol 46:1996–2001. doi:10.1128/JCM.00032-08 PubMedCrossRefGoogle Scholar
  14. 14.
    Bidet P, Barbut F, Lalande V, Burghoffer B, Petit JC (1999) Development of a new PCR-ribotyping method for Clostridium difficile based on ribosomal RNA gene sequencing. FEMS Microbiol Lett 175:261–266. doi:10.1111/j.1574-6968.1999.tb13629.x PubMedCrossRefGoogle Scholar
  15. 15.
    Pituch H, Van Belkum A, Van Den Braak N, Obuch-Woszczatynski P, Verbrugh H, Meisel-Mikołajczyk F et al (2003) Recent emergence of an epidemic clindamycin-resistant clone of Clostridium difficile among Polish patients with C. difficile-associated diarrhea. J Clin Microbiol 41:4184–4187. doi:10.1128/JCM.41.9.4184-4187.2003 PubMedCrossRefGoogle Scholar
  16. 16.
    Pituch H, Kreft D, Obuch-Woszczatynski P, Wultańska D, Meisel-Mikołajczyk F, Łuczak M et al (2005) Clonal spread of a Clostridium difficile strain with a complete set of toxin A, toxin B, and binary toxin genes among Polish patients with Clostridium difficile-associated diarrhea. J Clin Microbiol 43:472–475. doi:10.1128/JCM.43.1.472-475.2005 PubMedCrossRefGoogle Scholar
  17. 17.
    Goorhuis A, Bakker D, Corver J, Debast SB, Harmanus C, Notermans DW, Bergwerff AA et al (2008) Emergence of Clostridium difficile infection due to a new hypervirulent strain, polymerase chain reaction ribotype 078. Clin Infect Dis 47:1162–1170. doi:10.1086/592257 PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2009

Authors and Affiliations

  1. 1.Medical Laboratory BremenHaferwende 12BremenGermany
  2. 2.Labor Nord-WestNordhornGermany

Personalised recommendations