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Identification and characterisation of lysin enzymes as potential therapeutics for the treatment of Clostridium difficile

Alyousef, Abdullah 2013. Identification and characterisation of lysin enzymes as potential therapeutics for the treatment of Clostridium difficile. PhD Thesis, Cardiff University.
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Clostridium difficile is the most common cause of hospital acquired infections. While the current treatments of choice, antibiotics, are generally effective in promoting recovery ,the increased incidence of C. difficile infections and treatment failure associated with antibiotic resistance combined with the emergence of hypervirulent strains highlights the need to develop therapeutic approaches that specifically target the pathogen without causing collateral damage to the protective microbiota. Several non-antibiotic approaches are currently being investigated, such as bacteriophage therapy. For this reason, we attempted to isolate C. difficile specific lytic bacteriophages which could form the basis of a treatment for C. difficile. While we were unable to isolate lytic phages, we were able to isolate twelve temperate bacteriophages from twenty-three clinical isolates of C. difficile using mitomycin C. Unfortunately we failed to identify a susceptible host strain capable of supporting the replication of these phages. This failure may in part be due to repeated episodes of phage infection over time, which have resulted in the emergence of “phage resistant” species mediated by systems such as Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR). Employing a PCR-based approach using primers specific for the lysin genes of five previously isolated C. difficile phages, we found evidence to suggest that repeated bacteriophage infection is a common event for clinical isolates of C. difficile. Our inability to isolate a lytic bacteriophage prompted us to adopt an alternative approach in which we used endolysin enzyme of five previously identified C. difficile phages as recombinant protein. These lysins showed broad spectrum activity against the vegetative forms of a large collection of C. difficile ribotypes with little or no activity against other species, supporting their potential as therapeutic agents. We also identified a genome associated lysin (CD630, YP_001088405), which lysed vegetative C. difficile in a similar manner to the phage derived lysins. We also cloned and expressed a spore cortex lytic enzyme (SleC) which targeted the cortex of C. difficile spores. Unfortunately this enzyme was inactive against intact spores, suggesting that the outer layers of the spore act as a permeability barrier. The results of this study showed in vitro the applicability of endolysins against the vegetative form of C. difficile and the activity of spore cortex lytic enzyme against coatless spores, offering interesting perspectives for evaluation of the antibacterial activity of a mixture of endolysin and spore cortex lytic enzyme.

Item Type: Thesis (PhD)
Status: Unpublished
Schools: Pharmacy
Subjects: Q Science > Q Science (General)
R Medicine > RM Therapeutics. Pharmacology
Uncontrolled Keywords: Clostridium difficile lysin pathogen in vitro bacteriophages
Last Modified: 19 Mar 2016 23:23

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