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

Adult zebrafish (Danio rerio) from the AB strain (European Zebrafish Resource Center EZRC) are maintained according to international (EU Directive 2010/63/EU) and national guidelines (Italian decree 4^th March 2014, n. 26) on the protection of animals used for scientific purposes. Standard conditions are set in the fish facility with a 14 h of light/10 h dark cycle and tank water temperature at 28° C.

1. Preparation of solutions and tools

1. Prepare a 50x stock solution and 1x working solutions for E3 embryo medium for growing zebrafish embryos (see Table 1).
2. Make pigmentation blocking solution, to block the embryo pigmentation from 24 hours post fertilization (24 hpf) (see Table 1).
3. Prepare 25x stock and 1x working Tricane anesthetic solution as described in Table 1.
   NOTE: To avoid the repeated freezing and thawing of the solution that could damage the stock solution, make aliquots of 2 mL of 25x tricaine and store them at -20 °C.
4. Prepare tracks for embryo microinjection by dissolving 1 g of agarose in 100 mL of distilled H₂O in a microwavable flask or bottle. Microwave for 1-3 min until the agarose is completely dissolved.
5. Position zebrafish microinjection molds (see Table of Materials) turned upside down in a Petri dish (90 mm x 15 mm) and cover the entire surface by pouring the liquid agarose gel with a width of approximately 10 mm.
6. Remove the mold once the agarose has solidified. Fill the Petri dish with 1x E3 embryo medium. This can be stored at 4 °C for approximately one week. Before use, replace with the fresh E3 medium containing Tricaine.
   NOTE: Older molds might develop fungi or bacterial contaminations.
7. Use fire polished 10 cm borosilicate capillaries to prepare needles for microinjection and secure them to the micropipette puller by tightening the handles. Set the puller with the following settings: heat 500, velocity 100, time 150, pull 150.

2. P. aeruginosa (PAO1) inoculum preparation

1. Inoculate 1 colony of GFP⁺ P. aeruginosa strain PAO1¹⁹ in 5 mL of LB broth (see Table 1) and grow overnight at 37 °C with shaking (200 rpm).
2. Dilute the above overnight culture to OD₆₀₀ = 0.1 in 10 mL of LB broth and grow to OD₆₀₀ = 0.5 at 37 °C with shaking (200 rpm).
3. Centrifuge 2 mL of the culture for 2 min at 4 °C at 16,100 x g and resuspend the cell pellet to OD₆₀₀ = 1, equivalent to about 1 x 10⁹ cfu/mL, in 1 mL of the physiological solution (see Table 1).
4. Dilute the cell suspension to about 5 x 10⁷cfu/mL in physiological solution and store at 4 °C.

3. Phage stock preparation

1. Inoculate 1 colony of P. aeruginosa strain PAO1 in 5 mL of LB broth and incubate overnight at 37 °C with shaking. Dilute the overnight culture to OD₆₀₀ = 0.01 in 500 mL of LB broth and grow at 37 °C with shaking to OD₆₀₀ = 0.05, equivalent to about 2.5 x 10⁷ cfu/mL.
2. To the diluted culture of P. aeruginosa, add about 1.25 x 10⁷ phages, to get the multiplicity of infection (M.O.I.) of 10^-3. Incubate at 37 °C with shaking until the OD₆₀₀ drops to about 0.1-0.3 (in about 3 to 5 h, depending on the phage used).
   NOTE: Four phages were selected for this experiment that infect PAO1 strain: Two Podoviridae, GenBank accession numbers vB_PaeP_PYO2, MF490236, and vB_PaeP_DEV, MF490238, and two Myoviridae, vB_PaeM_E215, MF490241, and vB_PaeM_E217, MF490240. Perform the steps below for each phage individually.
3. Incubate the lysate with 1 mg/mL of DNase and RNase for 30 min at 37 °C. Centrifuge at 5,000 x g for 30 min at 4 °C and carefully recover the supernatant (SN). Filter the SN with a filter having 0.8 μm pore diameter.
4. To the SN, add 58 g/L of NaCl and 105 g/L of PEG6000. Dissolve with stirring at RT 20 min and then keep it overnight at 4 °C. Centrifuge at 20, 000 x g for 30 min at 4 °C for phage precipitation. Remove the SN and carefully dissolve the phage pellet in 15 mL of TN buffer (see Table 1).
5. Purify the phages with CsCl density gradient as described in steps below.
   1. In a polyallomer ultracentrifuge tubes for SW41 rotor, prepare a CsCl step density gradient by stratifying 2 mL of the four CsCl solutions described in Table 1.  Add 3.5 mL of phage suspension in each of the 4 tubes.
      NOTE: CsCl is toxic, adopt proper safety procedures to handle and discard it.
   2. Introduce the tubes into the rotor and ensure that the tubes are balanced (the weight difference between the two facing tubes must be ≤ 0.01g).
   3. Centrifuge for 2 h at 4 °C and 100, 000 x g (25,000 rpm for SW41 rotor). After centrifugation, slowly remove the tubes and carefully immobilize them on a support. Using a syringe with 19 G needle, draw up the white/opalescent band that is located usually between the d=1.5 and the d=1.4 density region.
   4. Transfer the suspensions from the syringe into new polyallomer tubes for SW60 rotor and use the d=1.4 solution to precisely balance the tubes.
   5. Centrifuge the suspension at 4 °C and 150,0000 x g (38,000 rpm for the SW60 rotor) for at least 16 h.
   6. Collect the visible band as above (see step 3.3.3) and transfer them into dialysis tubes with 6,000 Da cut-off. Dialyze the obtained visible band 2x for 20 min against 500 mL of water and overnight against 500 mL of TN buffer. Filter with a 0.22 µm pore filter and store at 4 °C.

4. Phage cocktail preparation

1. Make a mix of four phages that infect the strain PAO1, previously isolated and characterized⁸. Before mixing, estimate the titer of each phage stock by plaque assay²⁰.
2. Assemble the phage cocktail, with an overall titer of 5 × 10⁸ pfu/mL, by equally mixing four phage preparations at the same pfu/mL immediately before each experiment, to ensure accurate phage titers. 

5. Collection and preparation of zebrafish embryos for cftr morpholinos microinjection

NOTE: Collect 1-2 cell embryos from wild type zebrafish for cftr morpholinos (cftr-MOs) microinjection.

1. Two days before the bacterial microinjection experiment, set up breeding pairs and collect embryos as described previously²¹. Adult zebrafish (Danio rerio) of the AB strain were purchased from the European Zebrafish Resource Center, EZRC.
2. The day after, collect the embryos immediately after fertilization with a plastic pipette or using a fine-mesh strainer. Place the collected embryos in a Petri dish containing E3 embryo medium and carefully remove the debris with a plastic pipette to avoid contamination that might damage the embryo development.
3. Prepare 5 µL of morpholino injection solution by diluting the two morpholino stock solutions (1 mM) in sterile water to a final concentration of 0.25 pmol/embryo ATG-MO + 0.25 pmol/embryo splice-MO. To trace the embryo injection, add 0.5 µL of phenol red to the morpholino injection solution mix.
4. Load a microinjection needle with approximately 5 μL of the morpholino mix solution using a 20 μL micropipette with a fine gel loading tip. Secure the needle to the micromanipulator connected to a stand and position it under a stereomicroscope.
   NOTE: It is not necessary that the solution reaches the tip of the needle as the pressure of the microinjector pump will push it.
5. Clip off the tip of the needle by cutting the needle tip with fine sterile tweezers. Measure the diameter of the drop using a scale bar in the ocular of the microscope. Alternatively, create a drop of mineral oil onto a micrometer slide and set the microinjection volume by injecting the solution into the oil-drop to evaluate the droplet size. Use a drop with a 156 μm diameter to inject a volume of 2 nL.
6. Set the microinjector by adjusting the compensation pressure to 15 hPa, injection time to 0.5 s, and injection pressure between 300 and 600 hPa to obtain the correct injection volume depending on the needle used.
7. With a plastic pipette arrange the embryos from step 5.2 onto a glass positioned in a 96 mm diameter Petri dish.
   NOTE: Too much E3 medium will prevent the proper penetration of the microinjection needle into the chorion of the embryos.
8. Penetrate the chorion and then the yolk with the needle to inject 2 nL into the embryo as described previously²².
9. Place injected embryos into a Petri dish with E3 medium and put them in an incubator at 28 °C to let them develop until the day after.

6. Microinjection of zebrafish embryos with bacteria and phage cocktail

NOTE: To perform a systemic infection, the embryo must have blood circulation that usually starts after 26 hpf.

1. After 26 hpf (for zebrafish developmental stages refer to Kimmel²¹) dechorionate embryos with pronase solution prepared by dissolving pronase powder in E3 medium at a concentration of 2 mg/mL. Gently pipette the embryos to break the chorion. Discard the pronase/E3 medium and rinse the dish several times with fresh E3 to remove all pronase.
2. Anesthetize zebrafish embryos in E3 medium containing Tricaine approximately 5 min prior to injections.
3. Pipette the anesthesized embryos into the track prepared in step 1. Use a fine gel loading tip to line the embryos in the lateral position.
4. Load a microinjection needle with approximately 5 μL of the P. aeruginosa preparation as described in part 5.
5. Insert the needle dorsally to the starting point of the duct of Cuvier where it starts spreading over the yolk sac. Inject a volume of 1-3 nL and ensure that the volume expands directly within the duct and enters into the circulation.
6. Approximately after every 50 injected-embryo, inject a drop of the bacteria into a 1.5 mL centrifuge tube with 100 μL of sterile PBS to check if the injection volume remains the same. Plate the inoculum on LB agar (see Table 1) at 37 °C overnight to assess the CFU.
7. Transfer the microinjected embryos in two clean Petri dishes with fresh E3 medium + PTU and incubate them at 28 °C.  
8. At two selected time points: 30 min or 3 h after the bacterial injection, take out one of the Petri dishes with bacterial-injected embryos from the incubator and align them in the track as described in 6.3 for the phage cocktail injection.
9. Load a microinjection needle with approximately 5 μL of the phage cocktail (prepared in step 4) with a fine gel loading tip and fix it to the microinjector (see step 5).
10. Inject the phage cocktail in the duct of Cuvier of the embryos previously injected with bacteria.
11. Transfer the microinjected embryos in two clean Petri dishes with fresh E3 medium + PTU and incubate them at 28 °C.

7. Evaluation of the bacterial burden of embryos injected with PAO1 and phages

1. At 8 hpi, pipette 15 anesthetized embryos from the Petri dish to a 1.5 mL centrifuge tube.
2. Replace the anesthetic solution with 300 μL of 1% Triton X-100 in PBS (PBSTritonX) and homogenize the embryos by passing them at least 15x through an insulin syringe with a sterile 27-G needle.
3. Prepare serial dilutions of homogenates in sterile PBS by transferring 100 μL of the diluted homogenate into 900 μL of sterile PBS.
4. To select for the naturally amp-resistant PAO1 strain, plate 100 μL of the dilutions on LB agar added with ampicillin (100 μg/mL), and incubate at 37 °C over-night.
5. The day after, count the number of colonies, multiply by the dilution factor to determine the total number of CFU, and divide by the number of embryos homogenized to obtain the average number of CFU/infected embryo.

8. Evaluation of the lethality of embryos injected with PAO1 and phages

1. To evaluate the lethality of the PAO1 infection, score the injected embryos at 20 hpi under a stereomicroscope and count for the dead embryos (white/not transparent).
2. Calculate the half-maximal lethal concentration 50 (LD50) dose of PAO1 that determines the death of 50% of injected embryos at 20 hpf.

9. Embryo preparation for stereomicroscope time-lapse imaging of GFP⁺ PAO1 infection

1. Preparate the mold for live-embryo imaging by dissolving 1.5% low melting agarose in 100 mL of E3 solution.
2. Add 1% Tricaine (see Table 1) to anesthetize the embryos.
3. At 4 hpi transfer one embryo with a plastic pipette in a glass bottom dish and add the warm low-melting-point agarose solution.
4. Position the glass bottom dish under a stereomicroscope and with a tip position the embryo in the desired orientation. Use a plastic pipette to carefully add a drop of agarose on the embryo.
5. Let the agarose cool for 5-10 min and gently fill the glass bottom dish with E3 containing the anesthetic solution to keep the embryo moist.
6. Place the glass bottom dish with the embryo under the fluorescent stereomicroscope with a fluorescent filter (channel 488 nm) for GFP⁺ bacteria. Do not remove the Petri dish until further acquisitions with the same parameters at 9, 14 and 18 hpi.

10. Expression analyses of pro-inflammatory cytokines

1. At 20 hpi anesthetize the embryos with tricaine 1x solution and transfer them from the Petri dish to a 1.5 mL centrifuge tube.
2. Under a fume hood remove the anesthetic solution and replace with 200 μL of guanidium hydrochloride reagent.
3. Homogenize embryos by pipetting them repetitively through a 200 μL pipette first and then at least 15x with an insulin syringe with a sterile 27 G needle.
4. Extract total RNA from homogenized zebrafish embryos using a commercially available guanidium hydrochloride reagents as per the manufacturer’s instructions.
5. Treat 1 μg of each sample of RNA with 2 μL of 1U/μL of DNase I  (RNase-free), following the manufacturer’s instructions.
6. Use 1 μg of DNase I treated RNA for reverse-transcription reaction (RT) using commercially available kit (see Table of Materials), in a total volume of 20 μL according to manufacturer’s instructions.
7. Perform RT qPCR reaction in a total volume of 10 μL containing 1x SYBR Green mastermix using 1.5 μL of a 1:6 dilution of RT reaction. For normalization purposes, use the primers for the house-keeping genes rpl8 and for pro-inflammatory cytokines, use primers for TNF-α and IL-1β (see Table 2). qPCR protocol was: cycle 1 step 1: 95 °C, 2 min, repeat once; cycle 2: step 1 95 °C, 10 s, step 2 55 °C, 30 s, repeat 40x; cycle 3: step 1 72 °C, 15 min.