Phenotypic heterogeneity in Pseudomonas aeruginosa virulence revealed through passive exposure in zebrafish (Danio rerio) and nematodes (Caenorhabditis elegans).

The rapid emergence of diseases and parasites in aquatic wildlife requires improved methodologies to identify and characterize new and future pathogens. While microinjection of pathogens directly into a model organism, such as zebrafish, enables the exploration of infection and immune response in the host, such methodology focuses primarily on identifying causative agents in events of aquatic wildlife mortality due to acute infection. Here, we present an updated protocol of infection by static immersion in larval zebrafish to investigate the possible effect of prolonged environmental exposure to an opportunistic pathogen. By controlling microbial growth and monitoring mortality over 72 h, we show that static immersion can detect minute differences in virulence profiles between and within different strains of Pseudomonas aeruginosa, an important opportunistic pathogen of animals and humans. We then conducted two sets of passive exposure virulence assays in Caenorhabditis elegans, an alternative model. We demonstrated the virulence phenotype, while showing slight differences between experimental models, and showed similar trends. We believe that passive exposure thus offers a practical host-pathogen model that simulates opportunistic infection occurring in the environment and enables the detection of minute changes in virulence between and within bacterial strains.