The role of Psl in the failure to eradicate Pseudomonas aeruginosa biofilms in children with cystic fibrosis

Abstract

The exopolysaccharide Psl contributes to biofilm structure and antibiotic tolerance and may play a role in the failure to eradicate Pseudomonas aeruginosa from cystic fibrosis (CF) airways. The study objective was to determine whether there were any differences in Psl in P. aeruginosa isolates that were successfully eradicated compared to those that persisted, despite inhaled tobramycin treatment, in children with CF. Initial P. aeruginosa isolates were collected from children with CF undergoing eradication treatment, grown as biofilms and labeled with 3 anti-Psl monoclonal antibodies (Cam003/Psl0096, WapR001, WapR016) before confocal microscopy visualization. When grown as biofilms, P. aeruginosa isolates from children who failed antibiotic eradication therapy, had significantly increased Psl0096 binding compared to isolates from those who cleared P. aeruginosa. This was confirmed in P. aeruginosa isolates from the SickKids Eradication Cohort as well as the Early Pseudomonas Infection Control (EPIC) trial. Increased anti-Psl antibody binding was associated with bacterial aggregation and tobramycin tolerance. The biofilm matrix represents a potential therapeutic target to improve P. aeruginosa eradication treatment.

Fig. 1. Dot blot analysis of Psl levels in persistent (N = 29, black triangles) versus eradicated (N = 63, white triangles) P.aeruginosa isolates from SickKids cohort.

SickKids P. aeruginosa isolates, PAO1 and ΔPsl (Psl deficient P. aeruginosa) were grown as biofilms. The supernatant and lysate were spotted on nitrocellulose membrane and incubated with Cam003 (A), WapR001 (B) or WapR016 (C) anti-Psl antibodies. Bound antibody was detected with a chemiluminescent substrate after secondary antibody incubation, and signal intensities were analyzed using the ChemiDoc Imaging System. The mean intensity values for each isolate (done in triplicate) were plotted as arbitrary units (a.u.) with standard error of the mean (SEM). Statistics were performed using non-parametric Mann–Whitney test ***p < 0.001, *p < 0.05, ns not significant.

Fig. 2. P. aeruginosa isolates were grown as biofilms for 48 h then incubated for 90 min with fluorescently labeled anti-Psl antibodies (IgG control, Psl0096, WapR01, and WapR016).

Representative images are shown for a SickKids eradicated (Pa50) and persistent (Pa565) isolate, where biofilms are stained green and the antibody binding is magenta.

Fig. 3. Anti-Psl antibody binding.

SickKids P. aeruginosa isolates (N = 7 Persistent; N = 7 eradicated), EPIC P. aeruginosa isolates (N = 7 Persistent; N = 7 eradicated), laboratory P. aeruginosa strain PAO1 and Δpsl (Psl deficient PAO1) were grown as biofilms for 48 h then incubated for 90 min with fluorescently labeled monoclonal anti-Psl antibodies, A Psl0096, B WapR001, and C WapR016. The mean antibody fluorescence volume (µm³/100,000 µm³ biofilm) for each isolate (done in triplicate) was calculated and the mean of all isolates was plotted with standard error of the mean (SEM). Statistics were performed using non-parametric Mann–Whitney test ***p < 0.001, **p < 0.01, *p < 0.05, ns not significant.

Fig. 4. SickKids P. aeruginosa isolates (N = 7 persistent; N = 7 eradicated) were grown as biofilms for 24 h after which antibodies (LB control, IgG or anti-Psl 0096) and tobramycin 1000 µg/ml (or LB alone) were added for the following 24 h before imaging was performed.

A The mean biofilm volume (measured in µm³) for each isolate (done in triplicate) was calculated and the mean of all isolates was plotted with standard error of the mean (SEM). Comparisons with and without tobramycin treatment were performed for each condition using non-parametric Mann–Whitney test ***p < 0.001, **p < 0.01, *p < 0.05, ns not significant. B In the presence of tobramycin, aggregation was measured using biofilm surface to volume ratio. The mean biofilm surface to volume ratio (µm²/µm³) was calculated for each isolate (done in triplicate) and the mean of all isolates was plotted with standard error of the mean (SEM). Comparisons to IgG control were performed for each condition using non-parametric Mann–Whitney test ***p < 0.001, **p < 0.01, *p < 0.05, ns: not significant. C Representative images are shown for a persistent (Pa375) and eradicated (Pa558) isolate.

Fig. 5. SickKids P. aeruginosa isolates (N = 7 persistent; N = 7 eradicated) were grown as biofilms for 24 h after which antibodies (LB control, IgG or anti-Psl 0096) and tobramycin (or LB alone) were added for the following 24 h before ATP was measured.

The mean ATP activity (measured in Relative Light Units (RLU) ×10⁷) for each isolate (done in triplicate) was calculated and the mean of all isolates was plotted with standard error of the mean (SEM). Comparisons with and without tobramycin treatment were performed for each condition using non-parametric Mann–Whitney test ***p < 0.001, **p < 0.01, *p < 0.05, ns not significant.