1887

Journal of Medical Microbiology logo

Volume 53, Issue 3

Immunological detection and cytotoxic properties of toxins from toxin A-positive, toxin B-positive variants Free

Abstract

is a major nosocomial pathogen and a causative agent of antibiotic-associated diarrhoea and pseudomembranous colitis. PCR analysis of the toxin A and B genes of this bacterium has revealed 20 variant types (toxinotypes I–XX), many of which can cause human disease. Strains comprising the 15 toxin A-positive, toxin B-positive toxinotypes are not usually differentiated from non-variant strains by routine laboratories that do not utilize PCR tests. Consequently, the toxins from these variant strains have not been investigated thoroughly. The present studies revealed that toxin A-positive (A+B+) strains representing 12 variant toxinotypes all express considerably lower levels of toxin A and are less cytotoxic than non-variant strain VPI 10463. Truncated forms of toxin A were detected by immunoblotting in toxinotype VI and VII strains and these toxins were differentiated from each other and from toxin A of the non-variant strain. A further novel finding was the ability of toxin A-positive (A+B+) strains of toxinotypes IX, XIV and XV to exhibit an alternative -like cytopathic effect on Vero cells, characterized by marked cell clumping. A rapid and simple method for toxin A removal from culture filtrates was developed. This enabled confirmation that the abnormal cytotoxicity observed for these strains is due to an altered toxin B, as has been found in toxin A-negative (A−B+) strains. These findings indicate the potential for differentiation of certain toxin A-positive (A+B+) toxinotypes without the need for PCR techniques.

  • Received:
  • Accepted:
  • Published Online:
© Microbiology Society
Loading

Article metrics loading...

/content/journal/jmm/10.1099/jmm.0.05404-0
2004-03-01
2024-04-19
Loading full text...

Full text loading...

/deliver/fulltext/jmm/53/3/JM530304.html?itemId=/content/journal/jmm/10.1099/jmm.0.05404-0&mimeType=html&fmt=ahah

References

  1. Alfa M. J., Kabani A., Lyerly D. & 7 other authors; 2000; Characterisation of a toxin A-negative, toxin B-positive strain of Clostridium difficile responsible for a nosocomial outbreak of Clostridium difficile- associated diarrhoea. J Clin Microbiol 38:2706–2714
     [Google Scholar]
  2. Barbut F., Lalande V., Burghoffer B., Thien H. V., Grimprel E., Petit J. C. 2002; Prevalence and genetic characterisation of toxin A variant strains of Clostridium difficile among adults and children with diarrhea in France. J Clin Microbiol 40:2079–2083 [CrossRef]
     [Google Scholar]
  3. Bentley A. H., Patel N. B., Sidorczuk P. & 7 other authors; 1998; Multicentre evaluation of a commercial test for the rapid diagnosis of Clostridium difficile -mediated antibiotic-associated diarrhoea. Eur J Clin Microbiol Infect Dis 17:788–790 [CrossRef]
     [Google Scholar]
  4. Borriello S. P., Wren B. W., Hyde S., Seddon S. V., Sibbons P., Krishna M. M., Tabaqchali S. S., Manek S., Price A. B. 1992; Molecular, immunological, and biological characterisation of a toxin A-negative, toxin B-positive strain of Clostridium difficile . Infect Immun 60:4192–4199
     [Google Scholar]
  5. Chaves-Olarte E., Weidmann M., von Eichel-Streiber C., Thelestam M. 1997; Toxins A and B from Clostridium difficile differ with respect to enzymatic potencies, cellular substrate specificities, and surface binding to cultured cells. J Clin Invest 100:1734–1741 [CrossRef]
     [Google Scholar]
  6. Chaves-Olarte E., Low P., Freer E., Norlin T., Weidmann M., von Eichel-Streiber C., Thelestam M. 1999; A novel cytotoxin from Clostridium difficile serogroup F is a functional hybrid between two other large clostridial cytotoxins. J Biol Chem 274:11046–11052 [CrossRef]
     [Google Scholar]
  7. Depitre C., Delmee M., Avesani V., L'Haridon R., Roels A., Popoff M., Corthier G. 1993; Serogroup F strains of Clostridium difficile produce toxin B but not toxin A. J Med Microbiol 38:434–441 [CrossRef]
     [Google Scholar]
  8. Frey S. M., Wilkins T. D. 1992; Localisation of two epitopes recognised by monoclonal antibody PCG-4 on Clostridium difficile toxin A. Infect Immun 60:2488–2492
     [Google Scholar]
  9. Hundsberger T., Braun V., Weidmann M., Leukel P., Sauerborn M., von Eichel-Streiber C. 1997; Transcription analysis of the genes tcdA-E of the pathogenicity locus of Clostridium difficile . Eur J Biochem 244:735–742 [CrossRef]
     [Google Scholar]
  10. Just I., Selzer J., Wilm M., von Eichel-Streiber C., Mann M., Aktories K. 1995; Glucosylation of Rho proteins by Clostridium difficile toxin B. Nature 375:500–503 [CrossRef]
     [Google Scholar]
  11. Kato H., Kato N., Watanabe K. & 7 other authors; 1998; Identification of toxin A-negative, toxin B-positive Clostridium difficile by PCR. J Clin Microbiol 36:2178–2182
     [Google Scholar]
  12. Kuijper E. J., de Weerdt J., Kato H., Kato N., van Dam A. P., van der Vorm E. R., Weel J., van Rheenen C., Dankert J. 2001; Nosocomial outbreak of Clostridium difficile -associated diarrhoea due to a clindamycin-resistant enterotoxin A-negative strain. Eur J Clin Microbiol Infect Dis 20:528–534 [CrossRef]
     [Google Scholar]
  13. Limaye A. P., Turgeon D. K., Cookson B. T., Fritsche T. R. 2000; Pseudomembranous colitis caused by a toxin A−B+ strain of Clostridium difficile . J Clin Microbiol 38:1696–1697
     [Google Scholar]
  14. Lyerly D. M., Saum K. E., MacDonald D. K., Wilkins T. D. 1985; Effects of Clostridium difficile toxins given intragastrically to animals. Infect Immun 47:349–352
     [Google Scholar]
  15. Lyerly D. M., Phelps C. J., Toth J., Wilkins T. D. 1986; Characterisation of toxins A and B of Clostridium difficile with monoclonal antibodies. Infect Immun 54:70–76
     [Google Scholar]
  16. Lyerly D. M., Barroso L. A., Wilkins T. D., Depitre C., Corthier G. 1992; Characterisation of a toxin A-negative, toxin B-positive strain of Clostridium difficile . Infect Immun 60:4633–4639
     [Google Scholar]
  17. Mani N., Dupuy B. 2001; Regulation of toxin synthesis in Clostridium difficile by an alternative RNA polymerase sigma factor. Proc Natl Acad Sci U S A 98:5844–5849 [CrossRef]
     [Google Scholar]
  18. McMillin D. E., Muldrow L. L., Leggette S. J., Abdulahi Y., Ekanemesang U. M. 1991; Molecular screening of Clostridium difficile toxins A and B genetic determinants and identification of mutant strains. FEMS Microbiol Lett 78:75–80 [CrossRef]
     [Google Scholar]
  19. Perelle S., Gibert M., Bourlioux P., Corthier G., Popoff M. 1997; Production of a complete binary toxin (actin-specific ADP- ribosyltransferase) by Clostridium difficile CD196. Infect Immun 65:1402–1407
     [Google Scholar]
  20. Popoff M. R., Chaves-Olarte E., Lemichez E. & 10 other authors; 1996; Ras, Rap, and Rac small GTP-binding proteins are targets for Clostridium sordellii lethal toxin glucosylation. J Biol Chem 271:10217–10224 [CrossRef]
     [Google Scholar]
  21. Riley T. V. 1998; Clostridium difficile ; a pathogen of the nineties. Eur J Clin Microbiol Infect Dis 17:137–141
     [Google Scholar]
  22. Rupnik M. 2001; How to detect Clostridium difficile variant strains in a routine laboratory. Clin Microbiol Infect 7:417–420 [CrossRef]
     [Google Scholar]
  23. Rupnik M., Avesani V., Janc M., von Eichel-Streiber C., Delmee M. 1998; A novel toxinotyping scheme and correlation of toxinotypes with serogroups of Clostridium difficile isolates. J Clin Microbiol 36:2240–2247
     [Google Scholar]
  24. Rupnik M., Brazier J. S., Duerden B. I., Grabnar M., Stubbs S. L. J. 2001; Comparison of toxinotyping and PCR ribotyping of Clostridium difficile strains and description of novel toxinotypes. Microbiology 147:439–447
     [Google Scholar]
  25. 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 [CrossRef]
     [Google Scholar]
  26. Sambol S. P., Merrigan M. M., Lyerly D., Gerding D. N., Johnson S. 2000; Toxin gene analysis of a variant strain of Clostridium difficile that causes human clinical disease. Infect Immun 68:5480–5487 [CrossRef]
     [Google Scholar]
  27. Samra Z., Talmor S., Bahar J. 2002; High prevalence of toxin A-negative toxin B-positive Clostridium difficile in hospitalised patients with gastrointestinal disease. Diagn Microbiol Infect Dis 43:189–192 [CrossRef]
     [Google Scholar]
  28. Soehn F., Wagenknecht-Wiesner A., Leukel P., Kohl M., Weidmann M., von Eichel-Streiber C., Braun V. 1998; Genetic rearrangements in the pathogenicity locus of Clostridium difficile strain 8864 – implications for transcription, expression and enzymatic activity of toxins A and B. Mol Gen Genet 258:222–232 [CrossRef]
     [Google Scholar]
  29. 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 [CrossRef]
     [Google Scholar]
  30. 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]
  31. von Eichel-Streiber C., zu Heringdorf D. M., Habermann E., Sartingen S. 1995; Closing in on the toxic domain through analysis of a variant Clostridium difficile cytotoxin B. Mol Microbiol 17:313–321 [CrossRef]
     [Google Scholar]
  32. Wozniak G., Trontelj P., Rupnik M. 2000; Genomic relatedness of Clostridium difficile strains from different toxinotypes and serogroups. Anaerobe 6:261–267 [CrossRef]
     [Google Scholar]
  33. Yassin S. F., Young-Fadok T. M., Zein N. N., Pardi D. S. 2001; Clostridium difficile -associated diarrhea and colitis. Mayo Clin Proc 76:725–730 [CrossRef]
     [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jmm/10.1099/jmm.0.05404-0
Loading
Immunological detection and cytotoxic properties of toxins from toxin A-positive, toxin B-positive Clostridium difficile variants
J. Med. Microbiol. 53, 197 (2004); https://doi.org/10.1099/jmm.0.05404-0
/content/journal/jmm/10.1099/jmm.0.05404-0
/content/journal/jmm/10.1099/jmm.0.05404-0
Loading

Data & Media loading...

Most cited Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error