1887

Abstract

We have developed a PCR ribotyping method based on capillary gel electrophoresis and have compared it with conventional PCR ribotyping. A total of 146 isolates were studied: five isolates were reference strains (PCR ribotypes 001, 014, 017, 027 and 053); 141 were clinical isolates comprising 39 Austrian PCR ribotypes collected in the period 2006–2007 at 25 Austrian healthcare facilities. Capillary gel electrophoresis yielded up to 11 fragments per isolate and 47 ribotype patterns. All but one of the five PCR ribotypes of reference strains were clearly reflected in the chromatograms of capillary-based typing. Capillary gel electrophoresis divided 24 isolates belonging to PCR ribotype type 014 into seven subgroups, whereas subtyping the same isolates using multiple-locus variable-number tandem-repeat analysis yielded three unrelated subgroups, without obvious correlation to sr subgroups. Using a web-based software program (http://webribo.ages.at), we were able to correctly identify these 014 isolates by simply allocating the seven subgroup patterns to one ribotype, i.e. to PCR ribotype 014. We consider capillary gel electrophoresis-based PCR ribotyping to be a way of overcoming the problems associated with inter-laboratory comparisons of typing results, while at the same time substantially diminishing the hands-on time for PCR ribotyping.

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2008-11-01
2024-12-12
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References

  1. Aspevall O., Lundberg A., Burman L. G., Akerlund T., Svenungsson B. 2006; Antimicrobial susceptibility pattern of Clostridium difficile and its relation to PCR ribotypes in a Swedish university hospital. Antimicrob Agents Chemother 50:1890–1892 [CrossRef]
    [Google Scholar]
  2. Barbut F., Mastrantonio P., Delmée M., Brazier J., Kuijper E., Poxton I. 2007; Prospective study of Clostridium difficile infections in Europe with phenotypic and genotypic characterisation of the isolates. Clin Microbiol Infect 13:1048–1057 [CrossRef]
    [Google Scholar]
  3. Bidet P., Barbut F., Lalande V., Burghoffer B., 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 [CrossRef]
    [Google Scholar]
  4. Bidet P., Lalande V., Salauze B., Burghoffer B., Avesani V., Delmee M., Rossier A., Barbut F., Petit J. C. 2000; Comparison of PCR-ribotyping, arbitrarily primed PCR, and pulsed-field gel electrophoresis for typing Clostridium difficile . J Clin Microbiol 38:2484–2487
    [Google Scholar]
  5. Gal M., Northey G., Brazier J. S. 2005; A modified pulsed-field gel electrophoresis (PFGE) protocol for subtyping previously non-PFGE typeable isolates of Clostridium difficile polymerase chain reaction ribotype 001. J Hosp Infect 61:231–236 [CrossRef]
    [Google Scholar]
  6. Indra A., Schmid D., Huhulescu H., Hell M., Gattringer R., Hasenberger P., Fiedler A., Wewalka G., Allerberger F. 2008; Characterization of clinical Clostridium difficile isolates by PCR ribotyping and detection of toxin genes in Austria, 2006–2007. J Med Microbiol 57:702–708 [CrossRef]
    [Google Scholar]
  7. Kato N., Ou C. Y., Kato H., Bartley S. L., Brown V. K., Dowell V. R. J., Ueno K. 1991; Identification of toxigenic Clostridium difficile by the polymerase chain reaction. J Clin Microbiol 29:33–37
    [Google Scholar]
  8. Kato H., Kato N., Katow S., Maegawa T., Nakamura S., Lyerly D. M. 1999; Deletions in the repeating sequences of the toxin A gene of toxin A-negative, toxin B-positive Clostridium difficile strains. FEMS Microbiol Lett 175:197–203 [CrossRef]
    [Google Scholar]
  9. Kato H., Kato N., Watanabe K., Yamamoto T., Suzuki K., Ishigo S., Kunihiro S., Nakamura I., Killgore G. E. other authors 2001; Analysis of Clostridium difficile isolates from nosocomial outbreaks at three hospitals in diverse areas of Japan. J Clin Microbiol 39:1391–1395 [CrossRef]
    [Google Scholar]
  10. Kuijper E. J., Oudbier J. H., Stuifbergen W. N., Jansz A., 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
    [Google Scholar]
  11. Kuijper E. J., Coignard B., Tull P. 2006; Emergence of Clostridium difficile -associated disease in North America and Europe. Clin Microbiol Infect 12 (Suppl. 6):2–18
    [Google Scholar]
  12. Lemee L., Dhalluin A., Pestel-Caron M., Lemeland J., Pons J. 2004; Multilocus sequence typing analysis of human and animal Clostridium difficile isolates of various toxigenic types. J Clin Microbiol 42:2609–2617 [CrossRef]
    [Google Scholar]
  13. McDonald L. C., Owings M., Jernigan D. B. 2006; Clostridium difficile infection in patients discharged from US short-stay hospitals, 1996–2003. Emerg Infect Dis 12:409–415 [CrossRef]
    [Google Scholar]
  14. Northey G., Gal M., Rahmati A., Brazier J. S. 2005; Subtyping of Clostridium difficile PCR ribotype 001 by REP-PCR and PFGE. J Med Microbiol 54:543–547 [CrossRef]
    [Google Scholar]
  15. Pituch H., Brazier J. S., Obuch-Woszczatynski P., Wultanska D., Meisel-Mikolajczyk F., Luczak M. 2006; Prevalence and association of PCR ribotypes of Clostridium difficile isolated from symptomatic patients from Warsaw with macrolide-lincosamide-streptogramin B (MLSB) type resistance. J Med Microbiol 55:207–213 [CrossRef]
    [Google Scholar]
  16. Rupnik M., Brazier J. S., Duerden B. I., Grabnar M., Stubbs S. L. 2001; Comparison of toxinotyping and PCR ribotyping of Clostridium difficile strains and description of novel toxinotypes. Microbiology 147:439–447
    [Google Scholar]
  17. Sadeghifard N., Gurtler V., Beer M., Seviour R. J. 2006; The mosaic nature of intergenic 16S–23S rRNA spacer regions suggests rRNA operon copy number variation in Clostridium difficile strains. Appl Environ Microbiol 72:7311–7323 [CrossRef]
    [Google Scholar]
  18. Sebaihia M., Wren B. W., Mullany P., Fairweather N. F., Minton N., Stabler R., Thomson N. R., Roberts A. P., Cerdeno-Tarraga A. M. other authors 2006; The multidrug-resistant human pathogen Clostridium difficile has a highly mobile, mosaic genome. Nat Genet 38:779–786 [CrossRef]
    [Google Scholar]
  19. Spigaglia P., Mastrantonio P. 2004; Comparative analysis of Clostridium difficile clinical isolates belonging to different genetic lineages and time periods. J Med Microbiol 53:1129–1136 [CrossRef]
    [Google Scholar]
  20. Stubbs S. L., Brazier J. S., O'Neill G. L., 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
    [Google Scholar]
  21. Stubbs S., Rupnik M., Gibert M., Brazier J., Duerden B., Popoff M. 2000; Production of actin-specific ADP-ribosyltransferase (binary toxin) by strains of Clostridium difficile . FEMS Microbiol Lett 186:307–312 [CrossRef]
    [Google Scholar]
  22. van den Berg R. J., Claas E. C. J., Oyib D. H., Klaassen C. H. W., Dijkshoorn L., Brazier J. S., Kuijper E. J. 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 [CrossRef]
    [Google Scholar]
  23. van den Berg R. J., Schaap I., Templeton K. E., Klaassen C. H. W., 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 [CrossRef]
    [Google Scholar]
  24. Vonberg R. P., Schwab F., Gastmeier P. 2007; Clostridium difficile in discharged inpatients, Germany. Emerg Infect Dis 13:179–180 [CrossRef]
    [Google Scholar]
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