Culture, epidemiology and virulence factors of Clostridium difficile

Abstract

The literature on the culture, epidemiology and possible virulence factors of Clostridium difficile is reviewed with reference to the association of the organism with pseudomembranous colitis and other bowel disorders.

Studies were done to assess the use of enrichment broths for increasing isolation of C. difficile from faeces. Enrichment culture to determine the level of carriage of the organism in healthy adults resulted in isolation of the organism from 11% of such individuals. No correlation was found with previous antimicrobial therapy or the sex of individuals.

Recommendations are made for routine culture of the organism in diagnostic laboratories. It is suggested that about lg of faeces be mixed with 1ml of industrial methylated spirits, left on the bench at room temperature for one to two minutes and then plated onto CCFA medium containing 250μg/ml cycloserine and 8μg/ml cefoxitin.

Plates should be incubated for 48h.

SDS-PAGE and immunoblotting were used to characterize isolates from various outbreaks of C. difficile-associated disease where cross infection was suspected. Although cross infection was shown to be a possible mechanism of transmission in some cases, it was not the only factor involved in developing disease.

Cell surface components were extracted from strains of C. difficile for immunochemical analysis. Two antigens were extracted from crude cell membranes. The lipoteichoic acid (LTA) moiety was shown to form a ladder pattern, reminiscent of that seen with the lipopolysaccharide from smooth Gram-negative organisms, when analysed by SDS-PAGE. This was a common antigen in four strains studied. Similar molecules were not demonstrated in C. sordelli or C. bifermentans. The identity of the second antigen is uncertain. It may be a deacylated form of the LTA-type molecule. Cell wall carbohydrate antigen, extracted from three strains, was shown to cross-react with some, but not all, heterologous antisera.

Cell wall proteins, extracted with 6M urea, were found to be different in each of five C. difficile strains studied. This indicates greater diversity in such proteins than previously described. Each strain had one to three major proteins, with molecular masses (Mᵣₛ) of between 28.3kDa and 54kDa. These were always antigenic when probed with homologous antiserum (by immunoblotting) but were not detected with heterologous antisera. There was one antigen with Mᵣₛ of about 73kDa that was common to all strains.

Most strains of the organism tested were found to be relatively hydrophilic in nature. However it was demonstrated that different culture techniques could alter the results obtained.

Flagella isolated from two C. difficile strains were shown to have an Mᵣₛ of about 38kDa. They were antigenic, showing cross-reaction with antiserum raised against different strains of the organism.

Studies were performed to assess the ability of the organism to adhere to mouse ileum. It appeared that viable C. difficile cells associated with this tissue and that the association increased with time. Bacteroides fragilis was also shown to associate with the ileum but not in the same manner as C. difficile. AL