A Phage Therapy against Pseudomonas aeruginosa Infection in Zebrafish Embryos

Abstract

Source: Cafora, M., et al., A. Phage Therapy Application to Counteract Pseudomonas aeruginosa Infection in Cystic Fibrosis Zebrafish Embryos. J. Vis. Exp. (2020)

The video demonstrates the phage therapy technique targeting Pseudomonas aeruginosa infection in cystic fibrosis zebrafish embryos. Phage therapy effectively diminishes bacterial load and mitigates proinflammatory immune responses in CF zebrafish embryos infected with bacteria, leading to reduced CF embryo lethality.

Protocol

1. 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, 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.
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 (Table 1) 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 (mix of four phages that infect the strain PAO1) with a fine gel loading tip and fix it to the microinjector.
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.

Table 1: Preparation of solutions

Solutions	Preparation
Anaesthetic stock solution 25X	4 mg/mL of Tricaine in distilled H2O.
Anaesthetic working solution 1X	dilute in distilled H2O the Tricaine stock solution 25X preparation to reach the 1X concentration (0.16 mg/mL) Tricaine of distilled H2O.
CsCl d=1.3	20.49 g in 50 mL TN
CsCl d=1.4	20.28 g in 50 mL TN
CsCl d=1.5	34.13 g in 50 mL TN
CsCl d=1.6	41.2 g in 50 mL of TN
E3 embryo medium for zebrafish embryo	1 L 1of E3 (dilute the 50X stock with distilled H2O) + 200 μl of 0.05% methyl blue . Store at RT.
E3 embryo medium stock solution (50X)	73.0 g NaCl, 3.15 g KCl , 9.15 g CaCl2 , and 9.95 g MgSO4 in 5 L of distilled H2O. Store at RT.
LB agar	10 g/L tryptone, 5 g/L yeast extract, 5 g/L NaCl, 10 g/L agar
LB broth	for 1L: 950 mL H2O, 10 g Tryptone, 10 g NaCl, 5 g Yeast extract
PBST	PBS 1X + Triton X 1%
Physiological solution	0.9% NaCl
Pigmentation blocking stock solution 10X	0.3 mg/mL phenyl thiourea (PTU) powder in E3 embryo medium for zebrafish embryo
Pronase stock solution 5X	5 mg/mL pronase powder in E3 embryo medium for zebrafish embryo
TN buffer	10 mM Tris HCl pH 8.0, 150 mM NaCl

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Bacto Agar	BD	214010
Dulbecco's phospate buffered saline PBS	Sigma-Aldrich	D8537
Ethyl 3-aminobenzoate methanesulfonate	Sigma-Aldrich	886-86-2	Common name tricaine
Femtojet Micromanipulator	Eppendorf	5247
Microinjection needles	Harvard apparatus
N-Phenylthiourea >=98%	Aldrich-P7629	103-85-5
Pronase	Sigma-Aldrich	9036-06-0
Z-MOLDS Microinjection	Word Precision Instruments