Date of Award

2015

Document Type

Doctoral Thesis

Degree Name

Doctor of Philosophy

Department

Biological Sciences

First Advisor

Dr. Jim O'Mahony

Second Advisor

Dr. Aidan Coffey

Abstract

The work presented in this thesis describes the isolation and characterisation of novel bacteriophages, and the subsequent exploitation of their bactericidal properties against two opportunistic food related pathogens, Mycobacterium avium subsp. paratuberculosis (MAP) and Cronobacter sakazakii.

MAP is a proven animal pathogen known to cause Johne’s disease in cattle but has been implicated as a causative agent of Crohn’s disease in humans. Despite the fact that this association has yet to be proven, significant focus has been directed towards evaluating the consequences of consuming milk contaminated with MAP. Accordingly, six mycobacteriophages were isolated and characterised in terms of temperature and pH stability, and were subsequently demonstrated both individually and in combination to be active at inhibiting the growth of a variety of bovine MAP isolates, to varying degrees. In addition, the anti-mycobacterial efficacy of these phages was also demonstrated against the faster growing, non-pathogenic surrogate strain, M. smegmatis, in reconstituted skim milk.

Cronobacter sakazakii is an opportunistic foodborne pathogen that is responsible for causing necrotising enterocolitis, meningitis and bacteraemia in low birth weight infant and neonates with high mortality. In recent years, the microbiological safety of powdered infant formula has become the focus of much scientific research due to the identification of contaminating C. sakazakii and its epidemiological link with life threatening neonatal infections. In response to this, Cronobacter phage vB_CsaP_Ssl was isolated from soil and its genome was sequenced and annotated which resulted in the identification a putative endolysin that was subsequently cloned, expressed, purified and demonstrated to be effective at hydrolysing the peptidoglycan of C. sakazakii and other Gram-negative food related pathogens. Three additional phages were also isolated, characterised and demonstrated in combination, to be effective at controlling C. sakazakii in different brands of infant formula. Furthermore, the anti-biofilm properties of the combined phage preparation was also demonstrated. The remaining work in this thesis focused on harnessing a bacterial-derived peptidoglycan hydrolase from C. sakazakii for biocontrol of this infant formula pathogen and other related Gram-negative bacteria. In addition, the application of a novel strategy to overcome the limitations associated with the exogenous use of peptidoglycan hydrolases against Gram-negative bacteria was also investigated.

Access Level

info:eu-repo/semantics/openAccess

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