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Kerr, Sarah (2023) Obtaining a lytic bacteriophage suitable for use in phage therapy against Clostridioides difficile infection. PhD thesis, University of Nottingham.

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Abstract

Clostridioides difficile is the leading cause of hospital-acquired and antibiotic-associated diarrhoea in the developed world. More effective countermeasures are required. Phage therapy, based on lytic bacteriophage, is a particularly attractive option. To date, however, all described C. difficile phages have proven to be temperate.

In the present study, two possible routes to obtaining the obligatory lytic phage on which a therapeutic treatment could be based were explored. In the one, a screening programme was instigated for naturally occurring lytic phage, while in parallel, rational genomic alterations were explored that could make an existing temperate phage lytic.

In the first approach, novel bacteriophages were isolated from sewage treatment plants and characterised. Genome sequencing revealed that one of the isolates (phiL14WAR): (i) lacked an obvious integrase gene; (ii) exhibited a much higher degree of antimicrobial activity against C. difficile than temperate phage, and; (iii) failed to convert its indicator strain to a lysogen. These data indicate that phiL14WAR may be a lytic phage, the first of its kind. Further work, however, is required before phiL14WAR can be definitively declared lytic, including determining whether it possesses other lysogeny-related genes.

In the second approach, genes involved in lysogeny were targeted for removal from the existing temperate phage, phiCD2301. CRISPR-mediated deletion of an integrase gene ablated the ability of the lysogen to form plagues, a phenotype entirely restored by its complementation. Further analysis was compromised by being unable to generate mutant phage particles. Clues as to the identity of a potential repressor gene(s) were sought through the identification of putative homologs to the phage ʎ cI repressor and by determining the highest expressed genes during lysogeny. Candidate genes were unsuccessfully targeted for knockout using CRIPSR/Cas9. Of these, structural predictions made using AlphaFold 2 suggested that the target identified through homology, rather than those identified by RNA seq, was more likely the repressor.

While it has not been possible to engineer a lytic phage within the time frame of this study, the work undertaken has provided a sound base for the future attainment of this goal. In the meantime, the increased antimicrobial action of the novel integrase-deficient phage makes it a promising candidate for further study while at the same time providing the basis of a phage suitable for therapeutic use against C. difficile infection.

Item Type:	Thesis (University of Nottingham only) (PhD)
Supervisors:	Minton, Nigel Griffin, Ruth
Keywords:	Bacteriophages; Clostridium difficile; Clostridium diseases
Subjects:	Q Science > QR Microbiology
Faculties/Schools:	UK Campuses > Faculty of Medicine and Health Sciences > School of Life Sciences
Item ID:	73695
Depositing User:	Kerr, Sarah
Date Deposited:	18 Jul 2023 04:40
Last Modified:	18 Jul 2023 04:40
URI:	https://eprints.nottingham.ac.uk/id/eprint/73695

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