This study investigates the interaction between Pseudomonas aeruginosa and the immune response in cystic fibrosis zebrafish embryos. The effectiveness of phage therapy in reducing bacterial infection and inflammation is also examined.
Take anesthetized dechorionated cystic fibrosis zebrafish embryos susceptible to Pseudomonas aeruginosa infection.
Inject fluorescent protein-expressing Pseudomonas aeruginosa into a major blood vessel, the duct of Cuvier.
Transfer injected embryos to a plate with media and a pigmentation-blocking agent to inhibit embryo pigmentation and visualize bacteria. Incubate.
Injected bacteria move through the circulation and invade organs.
In response, the host immune system gets activated, releasing pro-inflammatory mediators and recruiting immune cells.
Following incubation, inject a cocktail of virulent phages against Pseudomonas aeruginosa into the duct of Cuvier and transfer the embryos to a plate containing media and the pigmentation-blocking agent.
Incubate. Phages infect the bacteria and use host machinery to produce new phage particles.
Bacteria lyse, releasing phages that infect neighboring bacteria, resulting in a reduced pro-inflammatory response.
Using a fluorescent microscope, observe the reduced bacterial fluorescence in the zebrafish embryos over time, indicating phage effectiveness against Pseudomonas aeruginosa.
At 26 hours post-fertilization, load a microinjection needle with approximately 5 microliters of P. aeruginosa inoculum, and insert the needle dorsally to the starting point of the duct of Cuvier, at which the duct starts spreading over the yolk sac. Inject 1 to 3 nanoliters of the PAO1 inoculum into the embryo, making sure that the volume expands directly within the duct, and enters into the circulation.
After the injection, transfer the embryo into one of two new Petri dishes containing fresh E3 medium supplemented with phenylthiourea for a 30-minute or 3-hour incubation at 28 degrees Celsius. Next, load a microinjection needle with approximately 5 microliters of the phage cocktail, and fix the needle to the microinjector. Then, inject 1 to 3 nanoliters of phage cocktail into the duct of Cuvier of each embryo previously injected with bacteria, and place the embryos into one of two new Petri dishes containing fresh E3 medium supplemented with phenylthiourea at 28 degrees Celsius.
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