RESUMO
Current in vitro biofilm modelling of the opportunistic pathogen, Pseudomonas aeruginosa (PA) in people with cystic fibrosis (PwCF) is limited in its ability to mimic the complexities of the cystic fibrosis (CF) lung environment. Recent adaptations of the Microbial Identification after Passive CLARITY Technique (MiPACT) in CF research have allowed for the direct imaging of PA biofilm spatial organization and structure in expectorated sputum. Here, we performed a comparative analysis of in vitro and within patient (ex vivo) measures of PA biofilms using sputa from new onset infected children with CF. MiPACT-fluorescent in situ hybridization (FISH) and fluorescent anti-Psl monoclonal antibody (mAb) staining was performed to directly visualize PA and Psl (exopolysaccharide in PA biofilm matrix) in 11 CF sputum specimens. Corresponding PA isolates, recovered from the same sputum samples, were grown as biofilms in a glass slide chamber model, then visualized by fluorescent live-cell and anti-Psl mAb staining. We observed that PA biovolume, aggregation and Psl antibody binding (normalized per PA biovolume) in CF sputum did not correlate with the in vitro model, although a trend towards significance in the biovolume relationship was observed with the addition of sputum supernatant to the in vitro model.
