Bacteriophage therapy for chronic rhinosinusitus: targeting Pseudomonas aeruginosa biofilms

Abstract

Chronic rhinosinusitis (CRS) is a chronic inflammatory condition involving the paranasal sinuses, affecting roughly 10.5% of the population. Current theories suggest that the aetiology of CRS is multifactorial, with evidence that microbial dysbiosis and bacterial biofilms may both play a role. Pseudomonas aeruginosa is a gram-negative bacterium, which is associated with poorer disease-specific quality of life and patients requiring revision sinus surgeries for CRS. P. aeruginosa is also a key pathogen in cystic fibrosis (CF), where it may cause both sinus and lower respiratory tract infections. Unfortunately, P. aeruginosa frequently displays intrinsic and acquired resistance to antibiotics. The use of lytic bacteriophages has been proposed as an alternative treatment for infections caused by antibiotic-resistant bacteria. Lytic bacteriophages are bacterial viruses that can infect, replicate within, and lyse bacteria, killing the host bacteria through lysis. This thesis examines the suitability of anti-P. aeruginosa lytic bacteriophages as a treatment for P. aeruginosa infections in CRS and CF-associated CRS. An in vitro study of the efficacy of a mixture of anti-P. aeruginosa bacteriophages (referred to as CT-PA) against a panel of 40 clinical P. aeruginosa respiratory isolates, from CRS and CF patients across 3 continents, was performed. CT-PA was found to have a broad host range, including activity against multidrug-resistant isolates. CT-PA was able to significantly reduce biofilm biomass after 24 and 48 hours exposure to biofilms. The sheep rhinosinusitis model was then adapted to simulate P. aeruginosa sinusitis. The growth of viable P. aeruginosa biofilms within sheep frontal sinuses was confirmed using fluorescence in situ hybridisation and LIVE/DEAD BacLight staining. This animal model was then used to assess the safety and efficacy of CT-PA sinus flushes in vivo. The Safety arm confirmed a good safety profile, with no signs of local or systemic toxicity observed after 3 weeks of twice-daily CT-PA sinus flushes. The Efficacy arm of the study showed a statistically significant reduction in sinus biofilm biomass in sheep treated with CT-PA sinus flushes, twice daily for 7 days, compared to sheep who received 0.9% saline flushes instead. The emergence of bacteriophage insensitive mutants (BIMs) in P. aeruginosa biofilms exposed to CT-PA was also explored in an in vitro biofilm model, using whole genome sequencing and antibiotic susceptibility testing. This revealed changes in antibiotic susceptibility in BIMs compared to their parent strains. Whole genome sequencing revealed several genetic variations between the BIM and phage-sensitive isolate of each strain, most notably in the prophage Pf1 region of the genome. In summary, bacteriophage therapy holds promise for treating P. aeruginosa infections in CRS and CF. Specifically, the CT-PA phage cocktail appears to be safe and effective in treating P. aeruginosa sinus infections in an in vivo animal model. Further investigations are required to determine whether the development of BIMs will hamper the efficacy of lytic bacteriophage therapy, or conversely whether the emergence of phage resistance may create evolutionary trade-offs that can be exploited to decrease resistance to conventional antibiotics.