Clostridium difficile: expression of virulence factors, resistance to disinfectants and interactions with human cells
Clostridium difficile is the most common cause of nosocomial diarrhoea today. Through the changing epidemiology of C. difficile infection, the emergence and decline of different strains of varying virulence and a broad spectrum of disease from asymptomatic carriage and mild infection to severe pseudomembranous colitis have been observed. The main aim of this three-part thesis was to identify bacterial factors that might explain these variations by comparing five C. difficile strains - strain 630, an historic strain, strain VPI 10463, a reference strain, the hypervirulent ribotype 027 and the current locally endemic ribotypes 001 and 106. The first study focussed on the growth-related phenotypic and genotypic expression of virulence factors in C. difficile. Growth was studied over twenty-four hours, with simultaneous assessment of toxin and spore production. Total toxin production was measured by a commercial ELISA, while a quantitative ELISA for toxin A and a quantitative cytotoxicity assay for toxin B were developed for individual toxin levels, and spores were enumerated by viable counts. Ribotype 027 produced large amounts of toxin A and toxin B and was the second highest spore producer after ribotype 106. Growth may not affect virulence, but the ability to produce more toxins and spores could. To study the transcription of the genes involved in these processes, a real-time RT-PCR was developed. The transcription of the pathogenicity locus (tcdA-E) that regulates toxin production in C. difficile, and of spo0A, the initiator of sporulation, was studied. There were three key observations: firstly, the transcription of tcdC, the negative regulator of toxin production, did not decrease over time, suggesting it has a modulatory rather than repressive effect on the process. Secondly, tcdE expression was highest in ribotype 027, which might explain its hypertoxicity by greater toxin release. Thirdly, there was almost steady state expression of spo0A during the exponential growth phase in ribotypes 106 and 027, the highest spore producers, suggesting prolonged activation of sporulation. Thus, distinct inter-strain differences exist between C. difficile strains in vitro, which could mirror their virulence in vivo, and several traits contribute synergistically to the hypervirulence of ribotype 027. The second study aimed to identify suitable laboratory disinfectants against C. difficile. The efficacy of four commonly-used disinfectants and one decontaminant was tested; one disinfectant was a chlorine-based agent commonly used in hospitals. In conventional susceptibility tests, all five agents were effective against vegetative cells and spores of C. difficile. However, only the chlorine-based disinfectant was effective against spores dried onto surfaces, but this too required more than two minutes of treatment. The presence of organic matter significantly impaired the efficacy of the non-chlorine agents. The spores of epidemic strains were destroyed less effectively and exposure to sub-MIC levels of disinfectant increased sporulation, especially in ribotype 001, a common outbreak strain. Environmental sampling of the laboratory and surrounding areas showed considerable dissemination of C. difficile, highlighting the need for effective decontamination in conjunction with basic hygiene methods like hand-washing. The third study examined the biological activity of C. difficile. Macrophages were challenged in vitro with S-layer proteins, flagella, heat-shock proteins and culture supernatants of the five strains and cytokine production was measured by specially developed ELISAs. No significant inter-strain differences were observed, although the epidemic strains generally elicited a slightly greater cytokine response. Using epithelial cell lines it was observed that epidemic strains showed greater adherence; from inhibition assays, flagella and S-layer proteins were found to contribute equally to this. Through these studies, inter-strain differences between epidemic and historic isolates were identified with respect to virulence factors, survival in the environment and possible behaviour within the host. A sum of these observations suggests increased virulence in contemporary versus historical C. difficile strains. Finally, a supplementary study characterising a collection of ribotype 027 strains isolated in Scotland and the Netherlands by typing schemes, gene sequencing, susceptibility testing and phenotypic studies was performed. In agreement with other studies, the clonality of these hypervirulent strains was observed.