The extensive use of antibiotics has led to the emergence of methicillin-resistant Staphylococcus pseudintermedius, a bacterium causing difficult-to-treat canine skin infection (pyoderma). The administration of bacteriophages (phage therapy) can be an alternative to antibiotic therapy. Lytic phages, which lyse their host, are considered the only appropriate type of phages for phage therapy as opposed to temperate phages, which can survive within their host (lysogeny). However, it is possible to mutate temperate phages so that they cannot establish lysogeny anymore. Phage λ virulent (Vir) mutants have lost the operator to which the CI repressor binds to inhibit the expression of lytic genes. As a result, Vir mutants are strictly lytic. The work presented in this thesis was undertaken to isolate S. pseudintermedius phages and gain knowledge about their biology with the aim to develop phage therapy to treat pyoderma. The work was novel; very few data were available on S. pseudintermedius phages and no data have been published on phage therapy to treat canine skin infection. Four temperate phage candidates were selected after phenotypic and genotypic characterisation. No lytic phages were found. Random mutagenesis approaches were unsuccessful for the isolation of Vir mutants. An operator and three point mutations leading to the absence of CI repressor binding to this operator were identified through gel shift assay. These mutations should lead to a virulent phenotype if introduced in the relevant phage genome through site-directed mutagenesis. A PCR-based assay was performed to explore how widespread lysogeny was in S. pseudintermedius: 11 out of 45 tested strains were positive for the presence of prophage genes. Bioinformatic analyses revealed some of the genetic characteristics of S. pseudintermedius phages: genomic circular permutation and the presence of a genetic switch similar to that of phage λ. The work reported in this thesis represents a first step towards understanding the biology of S. pseudintermedius phages and developing phage therapy.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:704106 |
| Date | January 2016 |
| Creators | Breteau, Muriel |
| Publisher | University of Warwick |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://wrap.warwick.ac.uk/86043/ |
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