Phage Therapy Application to Counteract Pseudomonas aeruginosa Infection in Cystic Fibrosis Zebrafish Embryos

Presented here is a protocol for Pseudomonas aeruginosa infection and phage therapy application in cystic fibrosis (CF) zebrafish embryos.

This protocol facilitates the preparation and administration of a viral and phage cocktail against pseudomonas aeruginosa in zebrafish embryos. The main advantage of this technique is that it allows an in vivo assessment of the phage efficacy in counteracting the bacterial infection. The phage cocktail is efficient in both wild type and cystic fibrosis zebrafish models.

Opening the possibility of applying the phage therapy to bacterial infections in cystic fibrosis patients. Phage therapy can also be applied to counteract specific bacterial infections in other model systems. To prepare a phage stock for the experiment.

Add 1.25 times 10 to the seventh phages to an OD 600 of 0.05 P.aeruginosa culture to a multiplicity of infection of one times 10 to the negative three and incubate the culture for three to four hours with shaking until the OD 600 drops to 0.1 to 0.3. At the end of the incubation incubate the lysate with one microgram per milliliter of DNAse and RNAse for 30 minutes at 37 degrees Celsius. And pellet the cells by centrifugation.

At the end of the centrifugation filter the supernatant through a 0.8 micro meter pore diameter and add 58 grams per liter of sodium chloride and 105 grams per liter of PEG 6000. Incubate the solution at four degrees Celsius overnight. The next morning, precipitate the phage by centrifugation.

At the end of the centrofugation remove the supernatant and carefully dissolve the phage pellet in 15 milliliters of Tris sodium chloride buffer. To purify the phages by cesium chloride density gradient, first stratify two milliliters of four different cesium chloride solutions in polyallomer ultracentrifuge tubes for an SW 41 Rotor and add 3.5 milliliters of the phage suspension to each tube. Cesium chloride is toxic.

Always adopt proper safety procedures when handling and discarding this compound. When all of the tubes have been loaded, place the tubes into the rotor taking care that the tubes are balanced. And centrifuge the samples for two hours at 100, 000 times g and four degrees Celsius.

At the end of the centrifugation, use a syringe with a 19 gauge needle to aspirate the white layer between the D equals 1.5 and the D equals 1.4 density regions. And transfer the suspensions into new SW 60 size polyallomer tubes. Centrifuge the suspensions for at least 16 hours at 150, 000 times g and four degrees Celsius.

And transfer the visible bands into dialysis tubes with a 6, 000 Dalton cutoff. Then dialyze the collected samples two times against 500 milliliters of water for 20 minutes per dialysis and overnight against 500 milliliters of Tris sodium chloride buffer. The next morning, filter the resulting phage stock with a 0.22 micro meter pore filter and store the stock at four degrees Celsius.

On the day of the micro injection dilute two one micromolar morpholinos stock solutions in sterile water to a final concentration of 0.25 picomolar per embryo to obtain a five microliter morpholino injection solution. And add 0.5 microliters of phenol red to each solution. Next use a 20 microliter micropipette with a fine gel loading tip to load a micro injection needle with the entire volume of morpholino mixed solution and secure the needle to a micro manipulator connected to a stand under a stereo microscope.

Then with one or two cells, zebra fish embryos arranged on a glass slide placed within a 96 millimeter diameter Petri dish, penetrate the chorion and the yolk with the micro injection needle tip and inject two nanoliters of the morphlino mixture into the embryo. At 26 hours post fertilization, load a micro injection needle with approximately five 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 one to three 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 three hour incubation at 28 degrees Celsius. Next, load a micro injection needle with approximately five microliters of the phage cocktail and fix the needle to the micro injector. Then inject one to three 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.

At four hours post-infection, use a plastic pipette to transfer an anesthetized embryo into a glass bottom dish. And fill the dish with warm low melting point agarose solution. When the agarose is cold, gently fill the dish with E3 medium supplemented with anesthetic solution.

Place the dish under the stereo microscope and use a pipette tip to position the embryo in the desired orientation. Then place the dish under a fluorescent stereo microscope with a fluorescent filter for GFP positive bacteria for up to 18 hours post-infection and image the embryo for the progression of GFP expression at nine, 14 and 18 hours post-infection. To determine the bacterial burden at eight hours post-infection, transfer 15 anesthetized micro-injected embryos into a 1.5 milliliter centrifuge tube and replace the anesthetic solution with 300 microliters of 1%Triton X-100 in PBS.

Pass the needles through the sterile 27 gauge needle of an insulin syringe at least 15 times to homogenize the embryos. And prepare serial dilutions of the homogenate by transferring 100 microliters of the resulting mixture into 900 microliters of sterile PBS per dilution. To select for the naturally ampicillin resistant P.aeruginosa strain, plate 10 microliters of the dilutions onto LB agar supplemented with ampicillin and incubate them overnight at 37 degrees Celsius.

The next day count the number of colonies. To allow calculation of the total number of colony forming units and the average number of colony forming units per infected embryo. To evaluate the lethality of the P.aeruginosa infection score the injected embryos at 20 hours post-infection under a stereo microscope by counting the number of dead white opaque embryos.

Then calculate the half maximal lethal concentration 50 dose of P.aeruginosa that resulted in the death of 50%of the injected embryos at 20 hours post-infection. To validate the cystic fibrosis phenotype the impaired position of internal organs such as the heart liver and pancreas can be evaluated. The bacterial burden is reduced by phage therapy in cystic fibrosis embryos infected with P.Aeruginosa.

In 48 hours post fertilization cystic fibrosis embryos infected with GFP positive P.Aeroginosa bacteria a 30 colony forming units per embryo dose demonstrates a 50%lethality at 20 hours post-infection. Phage therapy is equally effective at 20 hours post-infection when delivered 30 minutes or seven hours after bacterial injection. Here, a cystic fibrosis and GFP positive P.Aeruginosa injected embryo at four, nine, 14 and 18 hours post-infection is shown.

In contrast the cystic fibrosis plus P.Aeruginosa plus phage injected embryo demonstrates a reduced fluorescence due to the phage action against the bacteria. The inflammatory response generated by P.aeruginosa infection in cystic fibrosis embryos is significantly increased as revealed by the expression of the pro-inflammatory cytokines, TNF alpha and interleukin 1 beta following P.aeruginosa injection compared to control injected zebrafish. The expression of both inflammatory cytokines is reduced in phage cocktail co-injected animals however.

The phages must be carefully purified to remove any contaminating endotoxin that may be retained during the preparation. The phage preparations can be analyzed by electron microscopy to assess the virion morphology. It would be interesting to test whether the infection in human patients by bacteria other than pseudomonas aeruginosa can be cured with phage therapy in the cystic fibrosis zebrafish model.