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

Clostridium difficile 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 in vitro 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 Clostridium sordellii-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.