1887

Abstract

The increasing incidence of infection (CDI) in Bulgaria has indicated the need to implement better surveillance approaches. The aim of the present work was to improve the current surveillance of CDI in Bulgaria by introducing innovative methods for identification and typing. One hundred and twenty stool samples obtained from 108 patients were studied over 4 years from which 32 isolates were obtained. An innovative duplex EvaGreen real-time PCR assay based on simultaneous detection of the and genes was developed for rapid identification. Four toxigenic profiles were distinguished by PCR: ABCDT (53.1 %, 17/32), ABCDT (28.1 %, 9/32), ABCDT (9.4 %, 3/32) and ABCDT (9.4 %, 3/32). PCR ribotyping and multilocus variable number of tandem repeat analysis (MLVA7) were used for molecular characterization of the isolates. In total, nine distinct ribotypes were confirmed and the most prevalent for Bulgarian hospitals was 017 followed by 014/020, together accounting for 44 % of all isolates. Eighteen per cent of the isolates (6/32) did not match any of the 25 reference ribotypes available in this study. Twenty-four MLVA7 genotypes were detected among the clinical isolates, distributed as follows: five for 017 ribotype, two for 014/020, 001, 002, 012 and 046 each, and one each for ribotypes 023, 070 and 078. The correlation between the typing methods was significant and allowed the identification of several clonal complexes. These results suggest that most cases in the eight Bulgarian hospitals studied were associated with isolates belonging to the outbreak ribotypes 017 and 014/20, which are widely distributed in Europe. The real-time PCR protocol for simultaneous detection of and proved to be very effective and improved identification and confirmation of clinical isolates.

  • This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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2013-09-01
2024-03-28
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References

  1. Bakker D., Corver J., Harmanus C., Goorhuis A., Keessen E. C., Fawley W. N., Wilcox M. H., Kuijper E. J. 2010; Relatedness of human and animal Clostridium difficile PCR ribotype 078 isolates determined on the basis of multilocus variable-number tandem-repeat analysis and tetracycline resistance. J Clin Microbiol 48:3744–3749 [View Article][PubMed]
    [Google Scholar]
  2. Baldan R., Cavallerio P., Tuscano A., Parlato C., Fossati L., Moro M., Serra R., Cirillo D. M. 2010; First report of hypervirulent strains polymerase chain reaction ribotypes 027 and 078 causing severe Clostridium difficile infection in Italy. Clin Infect Dis 50:126–127 [View Article][PubMed]
    [Google Scholar]
  3. Barbut F., Mastrantonio P., Delmée M., Brazier J., Kuijper E., Poxton I.European Study Group on Clostridium difficile (ESGCD) 2007; Prospective study of Clostridium difficile infections in Europe with phenotypic and genotypic characterisation of the isolates. Clin Microbiol Infect 13:1048–1057 [View Article][PubMed]
    [Google Scholar]
  4. Bartlett J. G., Perl T. M. 2005; The new Clostridium difficile – what does it mean?. N Engl J Med 353:2503–2505 [View Article][PubMed]
    [Google Scholar]
  5. Bauer M. P., Notermans D. W., van Benthem B. H., Brazier J. S., Wilcox M. H., Rupnik M., Monnet D. L., van Dissel J. T., Kuijper E. J.ECDIS Study Group 2011; Clostridium difficile infection in Europe: a hospital-based survey. Lancet 377:63–73 [View Article][PubMed]
    [Google Scholar]
  6. Bidet P., Lalande V., Salauze B., Burghoffer B., Avesani V., Delmée 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[PubMed]
    [Google Scholar]
  7. Dawson L. F., Valiente E., Donahue E. H., Birchenough G., Wren B. W. 2011; Hypervirulent Clostridium difficile PCR-ribotypes exhibit resistance to widely used disinfectants. PLoS ONE 6:e25754 [View Article][PubMed]
    [Google Scholar]
  8. Gonçalves C., Decré D., Barbut F., Burghoffer B., Petit J. C. 2004; Prevalence and characterization of a binary toxin (actin-specific ADP-ribosyltransferase) from Clostridium difficile. J Clin Microbiol 42:1933–1939 [View Article][PubMed]
    [Google Scholar]
  9. Huovinen P., Räihä I., Vuento R., Eerola E., Lehtonen A. 1990; False-positive Clostridium difficile latex agglutination tests. Lancet 335:1467–1468 [View Article][PubMed]
    [Google Scholar]
  10. Janezic S., Ocepek M., Zidaric V., Rupnik M. 2012; Clostridium difficile genotypes other than ribotype 078 that are prevalent among human, animal and environmental isolates. BMC Microbiol 12:48 [View Article][PubMed]
    [Google Scholar]
  11. Kato H., Kato N., Watanabe K., Iwai N., Nakamura H., Yamamoto T., Suzuki K., Kim S. M., Chong Y., Wasito E. B. 1998; Identification of toxin A-negative, toxin B-positive Clostridium difficile by PCR. J Clin Microbiol 36:2178–2182[PubMed]
    [Google Scholar]
  12. 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 [View Article][PubMed]
    [Google Scholar]
  13. Marsden G. L., Davis I. J., Wright V. J., Sebaihia M., Kuijper E. J., Minton N. P. 2010; Array comparative hybridisation reveals a high degree of similarity between UK and European clinical isolates of hypervirulent Clostridium difficile. BMC Genomics 11:389 [View Article][PubMed]
    [Google Scholar]
  14. McFarland L. V. 1998; Epidemiology, risk factors and treatments for antibiotic-associated diarrhea. Dig Dis 16:292–307 [View Article][PubMed]
    [Google Scholar]
  15. McFarland L. V., Mulligan M. E., Kwok R. Y. Y., Stamm W. E. 1989; Nosocomial acquisition of Clostridium difficile infection. N Engl J Med 320:204–210 [View Article][PubMed]
    [Google Scholar]
  16. Paltansing S., van den Berg R. J., Guseinova R. A., Visser C. E., van der Vorm E. R., Kuijper E. J. 2007; Characteristics and incidence of Clostridium difficile-associated disease in The Netherlands, 2005. Clin Microbiol Infect 13:1058–1064 [View Article][PubMed]
    [Google Scholar]
  17. 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 [View Article][PubMed]
    [Google Scholar]
  18. Rupnik M., Kato N., Grabnar M., 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 [View Article][PubMed]
    [Google Scholar]
  19. Rupnik M., Dupuy B., Fairweather N. F., Gerding D. N., Johnson S., Just I., Lyerly D. M., Popoff M. R., Rood J. I. other authors 2005; Revised nomenclature of Clostridium difficile toxins and associated genes. J Med Microbiol 54:113–117 [View Article][PubMed]
    [Google Scholar]
  20. Tanner H. E., Hardy K. J., Hawkey P. M. 2010; Coexistence of multiple multilocus variable-number tandem-repeat analysis subtypes of Clostridium difficile PCR ribotype 027 strains within fecal specimens. J Clin Microbiol 48:985–987 [View Article][PubMed]
    [Google Scholar]
  21. 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 [View Article][PubMed]
    [Google Scholar]
  22. Wei H. L., Kao C. W., Wei S. H., Tzen J. T., Chiou C. S. 2011; Comparison of PCR ribotyping and multilocus variable-number tandem-repeat analysis (MLVA) for improved detection of Clostridium difficile. BMC Microbiol 11:217 [View Article][PubMed]
    [Google Scholar]
  23. Xiao M., Kong F., Jin P., Wang Q., Xiao K., Jeoffreys N., James G., Gilbert G. L. 2012; Comparison of two capillary gel electrophoresis systems for Clostridium difficile ribotyping, using a panel of ribotype 027 isolates and whole-genome sequences as a reference standard. J Clin Microbiol 50:2755–2760 [View Article][PubMed]
    [Google Scholar]
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