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Published on: July 30, 2012
Visualizing and quantifying Pseudomonas aeruginosa infection in the hindbrain ventricle of zebrafish using confocal
Adam J Rocker1, Alexander R E Weiss1, Joseph S Lam1
1Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON N1G 2W1, Canada.
Abstract:
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.
Insights
Pseudomonas aeruginosa biofilm formation in zebrafish was visualized in situ. A mutant lacking the Psl exopolysaccharide showed reduced virulence and microcolony development, highlighting Psl
Area of Science:
- Microbiology
- Infectious Diseases
- Zebrafish Models
Background:
- Pseudomonas aeruginosa biofilm formation is a complex, multi-phase process crucial for chronic infections.
- In vitro studies are well-established, but in situ visualization in vertebrate models remains challenging.
- The zebrafish (Danio rerio) offers optical clarity, making it a promising model for imaging bacterial infections.
Purpose of the Study:
- To develop and validate reproducible in situ methods for visualizing and quantifying Pseudomonas aeruginosa infection in zebrafish.
- To investigate the role of the Psl exopolysaccharide in P. aeruginosa pathogenicity and biofilm development in a vertebrate model.
- To establish the zebrafish model for studying bacterial virulence factors and biofilm formation in vivo.
Main Methods:
- Infection of zebrafish larvae (54 hpf) with Pseudomonas aeruginosa PAO1.
- In situ visualization and quantification of bacterial microcolonies using confocal laser scanning microscopy.
- Comparison of wildtype PAO1 with a Psl-deficient mutant.
Main Results:
- Successful reproducible in situ visualization and quantification of P. aeruginosa infection in zebrafish.
- P. aeruginosa mutants deficient in Psl production exhibited reduced zebrafish mortality.
- Fewer and smaller microcolonies were observed in zebrafish infected with the Psl-deficient mutant compared to wildtype.
Conclusions:
- The zebrafish model provides a robust system for in situ study of P. aeruginosa infection dynamics.
- The Psl exopolysaccharide plays a significant role in P. aeruginosa virulence and microcolony formation in vivo.
- This model facilitates the study of bacterial virulence factors and biofilm development, advancing understanding of chronic infections.
