The isolation, identification and detection of Clostridium Difficile

Aspinall, Steven Thomas (1992) The isolation, identification and detection of Clostridium Difficile. Doctoral thesis, University of Central Lancashire.

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The aim of the study was to determine the feasibility of an enzyme linked immunosorbant assay (ELISA) for detection of Clostridium difficile in broth cultures and faeces, to develop a new selective isolation medium and rapid identification test for Cl.difficile, to investigate the use of SDS-PAGE for typing isolates and to determine the presence of antibodies against Cl.difficile antigens in the sera of infected patients.
Detection of C1.difficile antigens using ELJSA: Using a competitive ELISA, a methodology was developed to determine the feasibility of the test for detection of Cl.difficile in broth cultures and faeces. Negative broth cultures produced no reduction in absorbance, whilst positive cultures resulted in >84% reduction. Negative faecal extracts yielded reductions of (18%, whilst positive samples produced >64% reduction (with a coefficient of variation of 5%). ELISA was thus found to be a feasible method but highly specific antibodies are required.
Media development: Various culture media and supplements were investigated for optimal growth of Cl.difficile and minimum inhibitory concentration determinations were performed for several antibiotics against a range of faecal isolates. Using Cl.difficile agar base containing cysteine hydrochloride (0.5 gIl), moxalactam (32 pg/ml) and norfloxacin (12 pg/ml) as a new selective medium, the isolation rate of Cl.difficile from faeces was increased by 20% and the selectivity improved by 33%
compared with the commercially available cycloserine (500 pg/mi), cefoxitin (16 pg/mi), fructose agar.
Identification: By investigating several enzymic tests for various Clostridium spp., the use of prolyl aminopeptidase, p-galactosidase, leucine aminopeptidase, indole production and acid phosphatase allowed the rapid identification of 96.4% of strains of Cl.difficile (within 30 minutes). The remaining isolates required an additional test, whilst no other Clostridium spp. tested produced similar results. The incorporation of these tests into filter paper squares allowed all five tests to be combined onto a small plastic carrier strip for ease of use.
SDS-PAGE typing: A typing system using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) was developed for differentiation of strains of Cl.difficile. Although complex protein profiles were obtained, the method was found to be reproducible and produced ten different protein patterns from the isolates tested. There was no difference between the protein patterns of the isolates obtained from asymptomatic infants and those from symptomatic adults.
Antibody detection: Using a Western blotting technique, the antibodies raised in twelve patients with Cl.difficile infection against the individual protein bands of the isolates after SDS-PAGE were found to be highly variable. No common antibodies were detected and some individuals with no recent history of infection produced antibodies which reacted with certain Cl.difficile antigens.

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