Study of Clostridium difficile isolates from the Singapore General Hospital

Abstract

The pathogenecity locus (PaLoc) of C. difficile, comprising the genes tcd A-E, is its main virulence factor. 110 samples obtained from the Singapore General Hospital (SGH) were studied for the presence or absence of these genes by PCR. The strains were divided into 10 groups based on their genotype. 8.1% of the strains had a truncated tcdA which encodes for toxin A. The correlation between the PaLoc components present and pathogenecity in clinical isolates was studied using an ELISA for toxin A and a cytotoxicity assay for the activity of toxin B. Truncated tcdA lacks the repeating units required for substrate binding, hence gives a negative result in the ELISA. A RT-PCR revealed that the tcdA binding domain is affected at the transcription level. The cytotoxicity assay, followed by a PCR targeting the entire tcdB gene at 6 smaller fragments showed that the presence of full length tcdB is necessary for the cytotoxic activity of C. difficile from the SGH. Strain CD19, which has tcdB but lacks all other PaLoc genes has a very low level of toxin B expression compared to other strains.