Isolation and characterisation of toxin A-negative, toxin B-positive Clostridium difficile in Dublin, Ireland

Clin Microbiol Infect. 2007 Mar;13(3):298-304. doi: 10.1111/j.1469-0691.2006.01634.x.


Clostridium difficile is a major cause of infectious diarrhoea in hospitalised patients. Most pathogenic C. difficile strains produce two toxins, A and B; however, clinically relevant toxin A-negative, toxin B-positive (A- B+) strains of C. difficile that cause diarrhoea and colitis in humans have been isolated worldwide. The aims of this study were to isolate and characterise A- B+ strains from two university hospitals in Dublin, Ireland. Samples positive for C. difficile were identified daily by review of ELISA results and were cultured on selective media. Following culture, toxin-specific immunoassays, IMR-90 cytotoxicity assays and PCR were used to analyse consecutive C. difficile isolates from 93 patients. Using a toxin A-specific ELISA, 52 samples produced detectable toxin. All isolates were positive using a toxin A/B ELISA. Similarly, all isolates were positive with the cytoxicity assay, although variant cytopathic effects were observed in 41 cases. PCR amplification of the toxin A and toxin B genes revealed that 41 of the previous A- B+ strains had a c. 1.7-kb deletion in the 3'-end of the tcdA gene. Restriction enzyme analysis of these amplicons revealed the loss of polymorphic restriction sites. These 41 A- B+ isolates were designated toxinotype VIII by comparison with C. difficile strain 1470. PCR ribotyping revealed that all A- B+ isolates belonged to PCR-ribotype 017. A- B+ C. difficile isolates accounted for 44% of the isolates examined in this study, and appeared to be isolated more frequently in Dublin, Ireland, than reported rates for other countries.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacterial Proteins / analysis*
  • Bacterial Proteins / genetics
  • Bacterial Toxins / analysis*
  • Bacterial Toxins / genetics
  • Clostridium difficile / classification
  • Clostridium difficile / genetics
  • Clostridium difficile / isolation & purification*
  • Enterotoxins / analysis*
  • Enterotoxins / genetics
  • Polymerase Chain Reaction
  • Ribotyping


  • Bacterial Proteins
  • Bacterial Toxins
  • Enterotoxins
  • tcdA protein, Clostridium difficile
  • toxB protein, Clostridium difficile