(570) Visualization of Pseudomonas aeruginosa biofilms in sputum of children with cystic fibrosis who fail eradication therapy

Background: Pseudomonas aeruginosa (PA) is a common pathogen involved in chronic pulmonary infection in patients with CF. Early eradication of PA infection is key to preventing the lung function decline associated with chronic infection. The PA exopolysaccharide, Psl, is a key component of the biofilm matrix and may play a role in the establishment of persistent infection. We previously showed, in vitro, that PA isolates from new-onset infections that failed early antibiotic eradication treatment (AET) had greater binding of anti-Psl monoclonal antibodies (mAbs), leading to bacterial aggregation and tobramycin tolerance, compared to successfully eradicated PA isolates when grown as biofilms. The goal of this study was to measure PA and Psl directly ex vivo in sputum from patients with CF and correlate these findings with success/failure of eradication therapy.

Methods: This was a prospective, observational study of children with CF followed at SickKids (Toronto, Canada) who had a new onset PA infection (defined as a sputum culture positive for PA with at least 3 preceding negative cultures in the prior 12 months). Expectorated sputum specimens were collected from the patients prior to starting AET with inhaled tobramycin. If a subject failed AET by having a PA positive sputum culture in the 6 months following the initial positive culture, the initial PA isolate (obtained prior to commencing inhaled tobramycin) was defined as persistent; otherwise, the initial isolate was defined as eradicated. Sputum specimens were polymerized in a hydrogel mixture and cleared using our MiPACT (tissue clearing) technique. PA and the exopolysaccharide, Psl, within the sputum hydrogels were visualized using a fluorescent in situ hybridization (FISH) technique with the anti-Psl mAb Psl0096 (red), PsearA (green) PA probe and DAPI (blue) nucleic acid stain, followed by confocal microscopy and image analysis. The Volocity 6.3 software was used to quantify PA biovolume, maximum aggregate size and the total number of aggregates, along with anti-Psl mAb binding (per 100,000 μm³ of biovolume) and PA-Psl colocalization.

Results: Of the 11 patients enrolled, 7 were categorized as eradicated and 4 had persistent infection. Sputum from patients with persistent infection showed PA aggregates of greater biovolume, maximum aggregate size and number of aggregates compared to the eradicated PA sputum samples (p< 0.05). Anti-Psl mAb binding (per 100,000 µm³ of biovolume) was also greater in the sputum hydrogels containing persistent PA compared to eradicated PA (p< 0.05). The Manders (M2) coefficient representing the degree of Psl to PA colocalization was similar in both groups.

Conclusions: Children with CF who failed early AET were infected with PA that had greater biovolume and formed aggregates of greater size and number than the PA in children who cleared infection. The anti-Psl mAb binding in sputum (per 100,000 µm³ of biovolume) was greater in the persistent compared to eradicated infections, although the proportion of PA-associated and secreted Psl was similar in both groups. These results suggest that structural differences in PA colonization (mediated by Psl) within the CF lung may contribute to the failure of antibiotics to penetrate PA aggregates and eradicate infection.

Acknowledgements: The authors would like to acknowledge GlycoNet and the Cystic Fibrosis Foundation for funding this project and all patients and families who participated.

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