Visualizing and quantifying Pseudomonas aeruginosa infection in the hindbrain ventricle of zebrafish using confocal laser scanning microscopy.

Pseudomonas aeruginosa colonizes surfaces using a stepwise process that involves several phases, including attachment, production of exopolysaccharides, formation of microcolonies and the eventual development of biofilms. This process has been extensively characterized in vitro using both light and electron microscopic techniques. However, our ability to visualize this process in situ at the site of infection has been limited by the nature of the vertebrate models available. The optically clear zebrafish (Danio rerio) is an emerging model well suited for imaging bacterial infections. In this study, we infected the hindbrain ventricle of 54 h post-fertilization zebrafish with P. aeruginosa PAO1 and visualized and quantified microcolony formation using confocal laser scanning microscopy and image analyses. In comparison to wildtype PAO1, infection with a P. aeruginosa mutant deficient in the ability to produce the exopolysaccharide Psl caused less zebrafish mortality and fewer, smaller microcolonies per zebrafish at both 18 h and 29 h post-infection. The work presented here demonstrates reproducible in situ visualization and quantification methods for determining the extent of P. aeruginosa infection in a vertebrate model. We demonstrate how this model system can be manipulated to understand the effect of virulence factors on pathogenicity. Furthermore, this model can be adapted to study biofilm formation in situ, thereby extending our understanding of how bacterial persistence leads to chronic infections.