Clostridium difficile: expression of virulence factors, resistance to disinfectants and interactions with human cells
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Date
22/06/2012Author
Vohra, Prerna
Metadata
Abstract
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.