Characterising the role and evolution of temperate bacteriophages in chronic respiratory infections including Cystic Fibrosis and Bronchiectasis

The aim of this research project was to determine whether temperate bacteriophages could be used as markers for bacterial evolution in chronic microbial infections and in the progression of respiratory diseases [Cystic Fibrosis (CF) and Bronchiectasis (BR)]. CF and BR have diverse clinical origins but similar pathophysiological burdens including inflammation and the production of a nutrient rich mucus. This thick, dehydrated mucus is an ideal colonisation site for opportunistic bacteria such as Pseudomonas aeruginosa (P. aeruginosa). Some strains of P. aeruginosa have been found to harbour multiple inducible temperate bacteriophages that are believed to have an effect on P. aeruginosa functionality. In order to try and answer the research question proposed, the research was split into three subgroups: (1) a cross-infection study utilising 94 clinical P. aeruginosa isolates and their associated mixed phage communities to determine changes in phage-host interactions alongside the progression of the clinical disease. We here illustrate that phages induced from the older CF patient isolates were the most infective, whilst the phages originating from the youngest CF patients or the patients with <10 years of BR diagnosis were the least infective. (2) Metagenome analysis of the total induced viral DNA from each of these 94 P. aeruginosa isolates was used to determine whether disease progression offered complexity or additional gene function that would offer a selective advantage for the bacterium or virus in these clinical backgrounds. This research importantly shows differences in phage metagenome complexity and an increase in gene function that correlates with the advancement of disease progression. Therefore, phage metagenomes originating from the older CF patients were the most enriched in functions relating to survival within the chronic lung. It also shows a snapshot of phage evolution and their impact on the bacteria colonising the lower lung. If higher numbers of phage genes with defined function is a marker of the levels of adaptation and evolution that has occurred, then this research determines that phages isolated from CF metagenomes had undergone more rounds of evolution compared to the BR metagenomes. (3) Changes in metabolite profiles in the bacterium when infected with a single phage may further show the involvement and subversion of host cell functionality by these phages. A panel of plaque-purified phages were used to create lysogens of lab strain PAO1. Changes in the metabolite profiles between naive and infected PAO1 were investigated throughout pellicle growth where distinct differences were seen and further show the impact of prophage formation on the core gene function of respiring bacteria. Both the E. coli metabolome database and the human metabolome database were utilised in order to identify potential metabolites that were both statistically significant and had a low CV score. We illustrate different metabolite profiles when comparing naive bacterial strain to lysogen and thus, we establish the intricacy of how the virus subverts host cell functionality. One metabolite was observed that was present in a lysogenic strain and not in the naive host cell, so potentially leading to the assumption that this metabolite is phage derived. Further LCMS work is required in order to confirm this preliminary finding.