Bacteriophage therapy for application against Staphylococcus aureus infection and biofilm in chronic rhinosinusitis

Abstract

Chronic rhinosinusitis (CRS) is a debilitating condition characterised by critical inflammation of the mucosa of the nose and paranasal sinuses. Effecting up to 14% of the world’s population CRS severely impacts a patient’s quality of life. The aetiology of CRS is complex and relatively undefined encompassing a multitude of contributing factors. Bacterial infection is one factor thought to play a role in the pathogenesis of CRS. More specifically biofilm forms of the bacterial species Staphylococcus aureus have been shown to negatively influence post-operative progression. Current practice treatment strategies often fail to remove biofilms from the mucosa of the nose. It is therefore of import to develop novel anti-biofilm therapeutics. Our understanding of the epidemiology of S. aureus infections and biofilms in CRS is also limited. Increasing our epidemiological knowledge would help in the development of effective treatment strategies against recurrent infections. Investigation into the epidemiology of S. aureus infections was undertaken by collecting S. aureus isolates from mucous and biofilm structures of CRS patients. The clonal type of each isolate was then compared to the other isolates using pulse field gel-electrophoresis. Results of this study indicated that the majority of patients experiencing recurrent infections maintained the same clonal type. Furthermore the study suggested that long-term antibiotic therapy in some patients can lead to the development of bacterial antibiotic resistance. Therefore development of a novel antibacterial therapy outside of antibiotics is required. A potential anti-biofilm therapy both eliminating and preventative in nature is the application of bacteriophage. Bacteriophage (phage) are viruses that specifically target, infect and destroy bacterial cells. Initially in vitro study was undertaken to assess the anti-biofilm activity of a phage cocktail specific for S. aureus (CT-SA) using a minimal biofilm eradication assay plate. S. aureus isolates from CRS patients were grown to mature biofilm form and treated with CT-SA for 48hrs. Following treatment biofilm biomass was determined by staining bacteria with a Live/Dead BacLight stain, imaging the biofilm using confocal scanning laser microscopy and determining biofilm biomass using software COMSTAT2. Results showed CT-SA significantly reduced S. aureus biofilms of susceptible strains. Results also indicated that a cocktail of phage was superior to use of a single phage as it reduced the frequency of bacterial resistant to the phage treatment. Following on from in vitro work, the safety and efficacy of CT-SA was assessed in vivo using a sheep model of frontal sinusitis associated with S. aureus infections. CT-SA was also combined with ethylenediaminetetraaceticacid (EDTA) to observe if these therapies would synergise. Results indicated both CT-SA and EDTA were safe for short term topical application to the sinus regions. Furthermore both CT-SA and EDTA individually significantly reduced S. aureus biofilm levels in the frontal sinus, but were not seen to synergise. Work conducted in this thesis has helped lead towards development of a novel anti-S. aureus biofilm agent. Future translation of CT-SA to a clinical trial setting may not only reduce or remove S. aureus biofilm from CRS patient noses but also improve their symptomatology and quality of life.