Abstract
Pseudomonas aeruginosa evolves during chronic pulmonary infections of cystic fibrosis (CF) patients, forming pathoadapted variants that are persistent. Mucoid and rugose small-colony variants (RSCVs) are typically isolated from sputum of CF patients. These variants overproduce exopolysaccharides in the biofilm extracellular polymeric substance (EPS). Currently, changes to the biophysical properties of RSCV and mucoid biofilms due to variations in EPS are not well understood. This knowledge may reveal how lung infections resist host clearance mechanisms. Here, we used mechanical indentation and shear rheometry to analyse the viscoelasticity of RSCV and mucoid colony-biofilms compared to their isogenic parent at 2-, 4-, and 6-d. While the viscoelasticity of parental colony-biofilms underwent fluctuating temporal changes, in contrast, RSCV and mucoid colony-biofilms showed a gradual progression to more elastic-solid behaviour. Theoretical indices of mucociliary and cough clearance predict that mature 6-d parental and RSCV biofilms may show reduced cough clearance from the lung, while early mucoid biofilms may show reduced clearance by both mechanisms. We propose that viscoelasticity be considered a virulence property of biofilms.
Highlights
In the cystic fibrosis (CF) lung mucus accumulates due to impaired clearance forming a niche that is readily colonised by bacteria
To determine if the viscoelasticity of P. aeruginosa pathoadapted variants differ from the parent strain due to changes to the extracellular polymeric substance (EPS), mechanical tests were performed on representative rugose small-colony variants (RSCVs) and mucoid colony-biofilms and compared to the isogenic parent WT
RSCV colony-biofilms were macroscopically similar to WT on 2-d
Summary
In the cystic fibrosis (CF) lung mucus accumulates due to impaired clearance forming a niche that is readily colonised by bacteria. Compounding the chronicity of these infections is the ability of P. aeruginosa to pathoadapt, resulting in the evolution and selection of variants that are more fit Emergence of these variants is associated with increased treatment difficulties and worsening patient prognosis[5]. Two unique classes of pathoadapted variants isolated from the lungs of CF patients that we are interested in are mucoid and rugose small-colony variants (RSCVs)[7,8] These variants show phenotypic differences compared to the isogenic parental strain due to overproduction of different exopolysaccharides in the biofilm extracellular polymeric substance (EPS). Theoretical mucociliary- and cough-clearance indices (MCI and CCI respectively) have been determined for sputum (expectorated mucus), derived from in vitro lung clearance models[22,23] These indices correlate sputum viscoelasticity to predicted levels of clearance from the lung. This indicates that the viscoelasticity of healthy mucus maintains a balance that promotes clearance by both mechanisms[24,25]
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