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

Denise Drudy, N. Harnedy, S. Fanning, R. O'Mahony, L. Kyne

Research output: Contribution to journalArticlepeer-review

Abstract

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.

Original languageEnglish
Pages (from-to)298-304
Number of pages7
JournalClinical Microbiology and Infection
Volume13
Issue number3
DOIs
Publication statusPublished - Mar 2007
Externally publishedYes

Keywords

  • Clostridium difficile
  • Cytotoxicity assay
  • ELISA
  • Molecular characterisation
  • PCR
  • Toxins

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