Characterising temperate bacteriophages isolated from the microbiota of chronic respiratory disease

Tariq, Mohammad (2016) Characterising temperate bacteriophages isolated from the microbiota of chronic respiratory disease. Doctoral thesis, Northumbria University.

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Abstract

Cystic Fibrosis (CF) is the most common autosomal recessive genetic disorder in the UK. A mutation in the CFTR gene, alters a Cl- transporter protein resulting in dehydration at epithelial surface and a thick mucus layer that provides a nutrient rich environment ideal for opportunistic bacteria to colonise. Bronchiectasis (BR) is similar symptomatically to CF, with localised dilations and inflammation events of the bronchial tree that can be linked to lung trauma or allergy. Pseudomonas aeruginosa (Pa) is the most common opportunistic pathogen in CF that correlates to lowered lung function. Burkholderia cepacia complex (Bcc) species have been shown to be more problematic to clear than Pa, due to increased antimicrobial resistance and progression to Cepacia syndrome that can be fatal.
These opportunistic bacteria and their genome plasticity allows adaptation to the lung and can correlate to pathogenicity of chronic infection which is associated with poor clinical outcomes. As adaptation to the lung environment is such a key aspect of chronic bacterial infection in the lung this study focuses on the temperate phages infecting Pa and Bcc. Temperate phages once integrated into the bacterial chromosome have been shown in other bacterial backgrounds to aid bacterial adaptation through increasing rates of recombination. They have also been previously characterised to aid positive selection by carrying genes that aid bacterial survival, aiding evolution of both the bacterium and phage. The aim of this project was to characterise temperate bacteriophages chemically induced from 94 Pa (47 associated with CF and 47 with BR patients), 47 Bcc isolates (associated with CF patients).
This study focuses on 3 key areas. The first studies how phages induced from Pa and Bcc infect isolates from patients at different stages of chronic infection. All the bacterial isolates used in this study had at least one inducible lysogenic phage, in some instances polylysogens.
Infectivity through Pa phage cross infection showed that adult CF phage were more infective across the Pa panel. Paediatric CF phages showed an infection profile similar to that of Pa phage induced from BR patients >10 years after clinical diagnosis. Pa phage associated with BR patients <10 years after clinical diagnosis showed the least infection across the Pa panel.
Secondly, isolating and purifying temperate phages can be difficult and time consuming as phages that induce from the bacterial host in high numbers can mask secondary or tertiary phages. Importantly identifying individual phages can be complex, as a sensitive bacterial host is needed to isolate and propagate. This study uses novel genomic approaches to overcome this problem and separate mixed phage communities using k-mer abundance. Stratified phages have been annotated and compared for similarities that link to the clinical aetiology of the bacteria that carried that phage.
Finally, this study begins to map genetic traits that may aid phage longevity in a microbial system. A model has been previously characterised with lytic phages where Ig-like domains or BAM motifs may explain how phages bind to complex carbohydrates in mucus. When evaluating the incidence of BAM, Ig-like domains in temperate phage DNA sequences isolated in this study we identify an increase in BAM motifs, that may correlating to the evolution of disease in both CF and BR. This aids the proposed BAM model and the evolution of temperate phages interacting at mucosal surfaces.

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