Date of Award
Doctor of Philosophy
Prof. Aidan Coffey
This thesis focussed on Gram positive phages and their endolysins. Here, two similar kay-like staphylococcal phages B1 (vB_SauM_B1) and JA1 (vB_SauM_JA1) were isolated from a commercial therapeutic phage mix. Their host range was established on the Irish National MRSA bank, which included twenty one sequence types in addition relevant control strains. Based on this, distinct phages were identified and subjected to genome sequencing. The sequences were compared with the sequence of phage K (vB_SauM_K), which was also determined in this work. All three phages had a genome size of at least 139 kb, although some key differences were identified between each. The new phages B1 and JA1 possessed double stranded DNA and generally had a broader host range than phage K. A comparative genomic analysis on the phage genomes identified several (open reading frames) ORFs that were absent in the genome of phage K but present in genomes of phages B1 and JA1. One of the cloned genes from phage K was shown to encode a protein for the receptor-binding-protein and this protein was demonstrated to slightly inhibit phage adsorption. The other cloned gene encoded the phage endolysin and this peptidoglycan hydrolase were identical across all three phages and thus, the CHAPk endolysin of phage K was chosen to demonstrate the application of the endolysin for the control of staphylococci in milk. A two-log reduction in staphylococcal numbers in milk was observed. When the endolysin was introduced into a lactococcal secretion system using the pNZ8048 vector, detectable secretion was successfully demonstrated. Simultaneously, a Clostridium difficile phage endolysin, an amidase, was also cloned into the same secretion system with successful secretion also being demonstrated. In addition, this latter endolysin was also secreted from a recombinant E. coli strain, suggesting potential applications for delivery of the endolysin to the intestine from a hypothetical probiotic E. coli strain.
Ajuebor, Jude, "Phage Therapy and Development of Delivery Systems for Gram-Positive Phage Endolysins" (2018). PhDs [online].
Available at: https://sword.cit.ie/scidiss/4
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