Pseudomonas aeruginosa Exoprotein-Induced Barrier Disruption Correlates With Elastase Activity and Marks Chronic Rhinosinusitis Severity.

Background:Pseudomonas aeruginosa causes severe chronic respiratory diseases and is associated with recalcitrant chronic rhinosinusitis (CRS). P. aeruginosa exoproteins contain virulence factors and play important roles in the pathogenicity of P. aeruginosa, however their role in CRS pathophysiology remains unknown. Methods: We isolated P. aeruginosa clinical isolates (CIs) and obtained clinical information from 21 CRS patients. Elastase activity of the CIs was measured at different phases of growth. Primary human nasal epithelial cells (HNECs) were cultured at air-liquid interface (ALI) and challenged with P. aeruginosa exoproteins or purified elastase, followed by measuring Transepithelial Electrical Resistance (TEER), permeability of FITC-dextrans, western blot, and immunofluorescence. Results: 14/21 CIs had a significant increase in elastase activity in stationary phase of growth. There was a highly significant strong correlation between the in vitro elastase activity of P. aeruginosa CIs with mucosal barrier disruption evidenced by increased permeability of FITC-dextrans (r = 0.95, p = 0.0004) and decreased TEER (r = -0.9333, P < 0.01) after 4 h of challenge. Western blot showed a significant degradation of ZO-1, Occludin and ß-actin in relation to the elastase activity of the exoproteins. There was a highly significant correlation between the in vitro elastase activity of P. aeruginosa CIs and CRS disease severity (for log phase, r = 0.5631, p = 0.0097; for stationary phase, r = 0.66, p = 0.0013) assessed by CT imaging of the paranasal sinuses. Conclusion: Our results implicate P. aeruginosa exoproteins as playing a major role in the pathophysiology of P. aeruginosa associated CRS by severely compromising mucosal barrier structure and function.

Safety and efficacy of a bacteriophage cocktail in an in vivo model of Pseudomonas aeruginosa sinusitis.

Pseudomonas aeruginosa (PA) is a bacterial pathogen that frequently displays antibiotic resistance. Its presence within the sinuses of chronic rhinosinusitis sufferers is associated with poorer quality of life. Obligately lytic bacteriophages (phages) are viruses that infect, replicate within, and lyse bacteria, causing bacterial death. The aims of this study were to assess the safety and efficacy of a PA phage cocktail (CT-PA) in a sheep model of rhinosinusitis. The sheep rhinosinusitis model was adapted to simulate PA infection in sheep frontal sinuses. To assess efficacy, after a 7-day biofilm formation period, sheep received twice-daily frontal trephine flushes of CT-PA or saline for 1 week. Biofilm quantitation on frontal sinus mucosa was performed using LIVE/DEAD BacLight staining. To assess safety, sheep received twice-daily frontal trephine flushes of CT-PA or vehicle control for 3 weeks. Blood and fecal samples were collected throughout treatment. Histopathology of frontal sinus, lung, heart, liver, spleen, and kidney tissue was performed. Sinus cilia were visualized using scanning electron microscopy (SEM). The Efficacy arm showed a statistically significant reduction in biofilm biomass with all concentrations of CT-PA tested (P < 0.05). Phage presence in sinuses was maintained for at least 16hours after the final flush. All Safety arm sheep completed 3 weeks of treatment. Phage was detected consistently in feces and sporadically in blood and organ samples. Histology and SEM of tissues revealed no treatment-related damage. In conclusion, CT-PA was able to decrease sinus PA biofilm at concentrations of 108-1010 PFU/mL. No safety concerns were noted.

Activity of Bacteriophages in Removing Biofilms of Pseudomonas aeruginosa Isolates from Chronic Rhinosinusitis Patients.

Introduction:Pseudomonas aeruginosa infections are prevalent amongst chronic rhinosinusitis (CRS) sufferers. Many P. aeruginosa strains form biofilms, leading to treatment failure. Lytic bacteriophages (phages) are viruses that infect, replicate within, and lyse bacteria, causing bacterial death. Aim: To assess the activity of a phage cocktail in eradicating biofilms of ex vivo P.aeruginosa isolates from CRS patients. Methods: P. aeruginosa isolates from CRS patients with and without cystic fibrosis (CF) across three continents were multi-locus sequence typed and tested for antibiotic resistance. Biofilms grown in vitro were treated with a cocktail of four phages (CT-PA). Biofilm biomass was measured after 24 and 48 h, using a crystal violet assay. Phage titrations were performed to confirm replication of the phages. A linear mixed effects model was applied to assess the effects of treatment, time, CF status, and multidrug resistance on the biomass of the biofilm. Results: The isolates included 44 strain types. CT-PA treatment significantly reduced biofilm biomass at both 24 and 48 h post-treatment (p < 0.0001), regardless of CF status or antibiotic resistance. Biomass was decreased by a median of 76% at 48 h. Decrease in biofilm was accompanied by a rise in phage titres for all except one strain. Conclusion: A single dose of phages is able to significantly reduce biofilms formed in vitro by a range of P.aeruginosa isolates from CRS patients. This represents an exciting potential and novel targeted treatment for P. aeruginosa biofilm infections and multidrug resistant bacteria.