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fbtwitterlinkedinvimeoflicker grey 14rssslideshare1
Languages: English
Types: Doctoral thesis
Subjects: RM
A rise in antibiotic resistance has prompted renewed interest in the use of phages to treat bacterial infections. This project explored the use of phages to treat Ps. aeruginosa infections in the context of potential treatment for Cystic Fibrosis patients. Initial characterisation of phage activity, by both a conventional plaque based assay and a novel Bioscreen C assay, revealed that 4 Ps. aeruginosa phages could infect multiple strains of Ps. aeruginosa including some clinically prevalent strains within the UK. This initial activity was increased in some strains following selection with a selective virucide, and a cocktail was designed. The cocktail showed a >2 Log10 reduction after 20 h in 10 of 14 strains. When the cocktail was tested against 4 Ps. aeruginosa strain biofilms, only a modest level of activity (<1 Log10) was measured. This was increased (>5 Log10) when combined with antibiotics in one bacterial strain in particular i.e. the strain against which the phage were initially propagated. Assessment of the endotoxin content of the cocktail showed a level far in excess of acceptable levels (approximately 6 Log10 EU/mL) that were not reduced sufficiently following purification by affinity chromatography (5.81 0.37 Log10 EU/mL). Delivery of the phage cocktail showed acceptable levels of recovery following nebulisation (approximately 99%). The methods developed here, particularly the Bioscrecn C assay for the assessment of phage lytic activity is applicable to a variety of different arenas by the application of standard acceptance criteria on activity levels. The direct quantification of the bacterial content of biofilms following the addition of phage and virucide treatment is novel and allows a cidal activity to be measured. Results have also highlighted the need for proper quality control within phage preparations for chronic infections. This investigation also highlighted the need for a standardised in vitro model that is more representative of the conditions found within a CF lung.

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