Skip to main content
NCBI home page
Journal of Clinical Microbiology logoLink to Journal of Clinical Microbiology
. 1990 Aug;28(8):1808–1812. doi: 10.1128/jcm.28.8.1808-1812.1990

Identification of toxigenic Clostridium difficile strains by using a toxin A gene-specific probe.

B W Wren 1, C L Clayton 1, N B Castledine 1, S Tabaqchali 1
PMCID: PMC268051  PMID: 2118549

Abstract

A 4.5-kilobase PstI fragment encoding part of the toxin A gene was isolated and used as a DNA probe in colony hybridization studies with 58 toxigenic and 17 nontoxigenic Clostridium difficile strains. All 58 toxigenic strains showed positive hybridization, in contrast to the 17 nontoxigenic strains. Southern blot analysis with the toxin A gene probe showed hybridization to a single fragment of equal intensities for HindIII-digested genomic DNAs isolated from C. difficile strains of wide-ranging toxin production. The positive hybridization signals were due to fragments of heterogeneous lengths (9 to 13 kilobases) for toxigenic strains of different types but were absent for the nontoxigenic strains. These results suggest the presence of a single copy of the toxin A gene on the genome of C. difficile strains, and the wide variation of toxin expression is not a reflection of gene copy number. The lack of toxin activity for nontoxigenic strains can be explained by the absence of at least part of the toxin A gene. The toxin A gene probe was tested against clostridial strains from 18 other species, of which only toxigenic C. sordellii strains showed positive hybridization. The specificity of the toxin A gene probe for toxigenic strains may lead to improved methods for the specific identification of toxigenic C. difficile strains from clinical specimens.

Full text

PDF
1808

Images in this article

1810Image
on p.1810
1810Image
on p.1810

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Anderson D. G., McKay L. L. Simple and rapid method for isolating large plasmid DNA from lactic streptococci. Appl Environ Microbiol. 1983 Sep;46(3):549–552. doi: 10.1128/aem.46.3.549-552.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Dove C. H., Wang S. Z., Price S. B., Phelps C. J., Lyerly D. M., Wilkins T. D., Johnson J. L. Molecular characterization of the Clostridium difficile toxin A gene. Infect Immun. 1990 Feb;58(2):480–488. doi: 10.1128/iai.58.2.480-488.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Feinberg A. P., Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem. 1983 Jul 1;132(1):6–13. doi: 10.1016/0003-2697(83)90418-9. [DOI] [PubMed] [Google Scholar]
  4. George R. H., Symonds J. M., Dimock F., Brown J. D., Arabi Y., Shinagawa N., Keighley M. R., Alexander-Williams J., Burdon D. W. Identification of Clostridium difficile as a cause of pseudomembranous colitis. Br Med J. 1978 Mar 18;1(6114):695–695. doi: 10.1136/bmj.1.6114.695. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Grunstein M., Hogness D. S. Colony hybridization: a method for the isolation of cloned DNAs that contain a specific gene. Proc Natl Acad Sci U S A. 1975 Oct;72(10):3961–3965. doi: 10.1073/pnas.72.10.3961. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Heard S. R., Rasburn B., Matthews R. C., Tabaqchali S. Immunoblotting to demonstrate antigenic and immunogenic differences among nine standard strains of Clostridium difficile. J Clin Microbiol. 1986 Sep;24(3):384–387. doi: 10.1128/jcm.24.3.384-387.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Larson H. E., Price A. B., Honour P., Borriello S. P. Clostridium difficile and the aetiology of pseudomembranous colitis. Lancet. 1978 May 20;1(8073):1063–1066. doi: 10.1016/s0140-6736(78)90912-1. [DOI] [PubMed] [Google Scholar]
  8. Muldrow L. L., Ibeanu G. C., Lee N. I., Bose N. K., Johnson J. Molecular cloning of Clostridium difficile toxin A gene fragment in lambda gt11. FEBS Lett. 1987 Mar 23;213(2):249–253. doi: 10.1016/0014-5793(87)81500-4. [DOI] [PubMed] [Google Scholar]
  9. Pothoulakis C., Barone L. M., Ely R., Faris B., Clark M. E., Franzblau C., LaMont J. T. Purification and properties of Clostridium difficile cytotoxin B. J Biol Chem. 1986 Jan 25;261(3):1316–1321. [PubMed] [Google Scholar]
  10. Rihn B., Scheftel J. M., Girardot R., Monteil H. A new purification procedure for Clostridium difficile enterotoxin. Biochem Biophys Res Commun. 1984 Nov 14;124(3):690–695. doi: 10.1016/0006-291x(84)91013-1. [DOI] [PubMed] [Google Scholar]
  11. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  12. Strom M. S., Eklund M. W., Poysky F. T. Plasmids in Clostridium botulinum and related Clostridium species. Appl Environ Microbiol. 1984 Nov;48(5):956–963. doi: 10.1128/aem.48.5.956-963.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Tabaqchali S., O'Farrell S., Holland D., Silman R. Method for the typing of Clostridium difficile based on polyacrylamide gel electrophoresis of [35S]methionine-labeled proteins. J Clin Microbiol. 1986 Jan;23(1):197–198. doi: 10.1128/jcm.23.1.197-198.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Wilkins T., Krivan H., Stiles B., Carman R., Lyerly D. Clostridial toxins active locally in the gastrointestinal tract. Ciba Found Symp. 1985;112:230–241. doi: 10.1002/9780470720936.ch13. [DOI] [PubMed] [Google Scholar]
  15. Wren B. W., Clayton C. L., Mullany P. P., Tabaqchali S. Molecular cloning and expression of Clostridium difficile toxin A in Escherichia coli K12. FEBS Lett. 1987 Dec 10;225(1-2):82–86. doi: 10.1016/0014-5793(87)81135-3. [DOI] [PubMed] [Google Scholar]
  16. Wren B. W., Mullany P., Clayton C., Tabaqchali S. Molecular cloning and genetic analysis of a chloramphenicol acetyltransferase determinant from Clostridium difficile. Antimicrob Agents Chemother. 1988 Aug;32(8):1213–1217. doi: 10.1128/aac.32.8.1213. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Wren B. W., Tabaqchali S. Restriction endonuclease DNA analysis of Clostridium difficile. J Clin Microbiol. 1987 Dec;25(12):2402–2404. doi: 10.1128/jcm.25.12.2402-2404.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Wren B., Heard S. R., Tabaqchali S. Association between production of toxins A and B and types of Clostridium difficile. J Clin Pathol. 1987 Dec;40(12):1397–1401. doi: 10.1136/jcp.40.12.1397. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. von Eichel-Streiber C., Suckau D., Wachter M., Hadding U. Cloning and characterization of overlapping DNA fragments of the toxin A gene of clostridium difficile. J Gen Microbiol. 1989 Jan;135(1):55–64. doi: 10.1099/00221287-135-1-55. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Clinical Microbiology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES