Modeling Persistent Pseudomonas aeruginosa Infection in Wounded Zebrafish Larvae

Summary

There is currently a lack of in vivo models amenable to drug screening against chronic bacterial infections. Here, we describe a protocol for wound infection by a Pseudomonas aeruginosa clinical isolate to generate a persistent infection in zebrafish larvae.

Abstract

Pseudomonas aeruginosa is a major human pathogen, particularly in chronic wound infections and chronic pulmonary infections (especially in patients with cystic fibrosis (CF)). Chronic bacterial infections are refractory to antibiotic treatments and there is an urgent need for in vivo chronic infection models amenable to drug screening to develop efficient therapies. Here, we describe a protocol of infection by a P. aeruginosa clinical isolate from a CF patient, expressing constitutively the Green Fluorescent Protein (GFP), for generating a persistent wound infection in zebrafish larvae. Tail fin-injured embryos are immersed in a bacterial suspension for 1.5 h, washed, and monitored for bacterial load for 3 days. The bacterial burden was quantified daily by counting fluorescent colony-forming units (CFU) from lysed infected larvae, which allowed a persistent infection. Moreover, persistent P. aeruginosa bacteria were refractory to antibiotic treatment. This novel in vivo model of persistent P. aeruginosa infection offers opportunities to evaluate the efficacy of innovative treatments against chronic infections.

Introduction

Pseudomonas aeruginosa is a Gram-negative pathogenic bacterium responsible for chronic colonization in patients with cystic fibrosis (CF) and wounds^1,2. P. aeruginosa belongs to the group of ESKAPEE pathogens and is recognized by the World Health Organization as a critical priority for new therapeutics³. Chronic bacterial infections are difficult to treat with antibiotics due to adaptive drug resistance, which is related to multiple factors, including biofilm lifestyle, reduced growth, low metabolic activity⁴, as well as intracellular life cycle⁵. In vivo models are essential to better understand and treat P. aeruginosa chronic infection.

While several in vivo models have been used to assess P. aeruginosa virulence, very few models allowed to mimic persistent colonization and test the efficacy of treatments on chronic infection⁶. Animal models to study P. aeruginosa chronic pathogenesis mainly rely on the administration of the bacteria embedded in agar beads into the lungs⁷. A murine chronic cutaneous infection model has also been developed⁸. Zebrafish (Danio rerio), which has numerous advantages (moderate ethical issues, low cost, high egg production), is an appealing in vivo vertebrate model for drug testing, which also allows high-resolution real-time visualization of P. aeruginosa and host cells thanks to embryo transparency⁹.

As reported in a review, in previous studies P. aeruginosa laboratory strains (PAO1, PA14, and PAK) were mainly used in the zebrafish infection model, where they caused an acute infection⁹. We recently developed a wound infection protocol based on the immersion of tail fin-amputated embryos with P. aeruginosa PAO1 strain, which caused an acute infection¹⁰. The infection by immersion of injured embryos, which reflects a natural infection mode of P. aeruginosa, is a mode of infection that is reproducible and easier than microinjection.

Our goal was to establish a protocol of persistent P. aeruginosa infection in zebrafish. For this purpose, our strategy was to use P. aeruginosa clinical strains, which have been rarely considered in the zebrafish model^11,12, combined with the wound route of infection. Here, we describe the protocol that we have developed to model the persistent colonization of zebrafish embryos based on a wound infection by P. aeruginosa CF clinical isolates. This novel in vivo model meets our expectations and offers new opportunities to assess the efficacy of therapeutics in the context of persistent colonization, as described here and in a companion article¹³.

Protocol

All zebrafish experiments described in the present study were conducted at the University of Montpellier by following the 3Rs -Replacement, Reduction and Refinement- principles according to the European Union guidelines for handling of laboratory animals and were approved by the Direction Sanitaire et Vétérinaire de l'Hérault and the Comité d'Ethique pour l'utilisation d'animaux à des fins scientifiques under reference CEEA-LR-B4-172-37. All infection experiments were carried out on embryos up to 5 days post-fertilization. The breeding of adult zebrafish (Danio rerio) from the AB or Golden Lines fish adhered to the international guidelines specified by the EU Animal Protection Directive 2010/63/EU. Zebrafish facility of the University of Montpellier with standard conditions set as a 12 h:12 h light: dark cycle, 3.5 L polycarbonate tank (maximum 22 fish per tank) connected to a recirculating system with 4% salinity/400 µS conductivity and water temperature of 28 °C. The fish were fed 2x per day with fish food.

1. Preparation of solutions

1. Prepare a 1x working solution for fish water with or without methylene blue for growing zebrafish embryos (see Table 1).
2. Prepare 20x stock (0.4%) and 1x working Tricaine anesthetic solution as described in Table 1. To avoid repeated freezing and thawing of the stock solution, prepare 10 mL aliquots of 20x Tricaine and store at -20 °C.
3. Prepare 2% Triton solution diluted in 1x Phosphate Buffered Saline (PBS; Table 1).

2. Preparation of inoculum of fluorescent  P. aeruginosa expressing GFP constitutively

NOTE: All work with P. aeruginosa is done with BSL-2 precautions in a biosafety cabinet. P. aeruginosa strains should be genetically modified to express constitutively a fluorescent reporter, such as GFP (as used here and described in¹³). The use of strains with a chromosomally encoded fluorescent reporter is strongly recommended to avoid the loss of plasmid in the case of a plasmid-encoded fluorescent reporter.

1. At 2 days before infection, streak out from -80 °C a stock of genetically modified P. aeruginosa expressing GFP on LB agar plates (Table 1) and incubate overnight at 37 °C.
2. Check the fluorescence of the colonies using a fluorescent microscope (Magnification 6x) to inoculate a GFP⁺P. aeruginosa colony in 1 mL of LB broth (Table 1) and grow overnight at 37 °C with shaking (180 rpm).
3. On the day of the infection, dilute the above overnight culture of P. aeruginosa at 1:20 into 5 mL of fresh LB broth. Grow the bacteria at 37 °C with shaking (180 rpm) until the culture reaches an OD₆₀₀ = 0.8. This should take approximately 2-3 h.
4. Centrifuge the bacterial culture for 10 min at room temperature (RT) at 3584 x g. Remove the supernatant and resuspend the pellet in 4 mL of fish water without methylene blue.
5. Adapt the volume of culture with fish water to have 7 x 10⁷ to 1 x 10⁸ CFU/mL. This corresponds to an OD₆₀₀ of approximately 0.15 (to be adapted for each strain).
6. To quantify the inoculum, do a serial dilution of the bacterial suspension, plate 20 µL of the 10^-4 dilution on an LB agar plate, and incubate overnight at 37 °C.
7. Count GFP⁺ colonies under a fluorescent microscope to determine the CFUs of the inoculum.

3. Collection and preparation of zebrafish embryos

1. At 3 days before the bacterial immersion experiment, set up zebrafish breeding pairs. Place a breeding male and female in a crossbreeding tank.
2. The next morning, collect the eggs after fertilization using a fine-mesh strainer. Place the collected eggs in a Petri dish containing fish water with methylene blue.
3. To avoid contamination, add 1 drop of bleach to the Petri dish. After 5 min, change the fish water with methylene blue to remove the bleach.
4. Discard eggs with abnormal embryo development (unfertilized eggs are similar to a 1-cell egg stage) or dead ones (white appearance).
5. On the morning of the infection (at 2 dpf), dechorionate the embryos with two very thin forceps under the stereo microscope (magnification 11x). Tear the chorion using forceps without pinching the embryo in order to release it.
6. Place the dechorionated embryos in a Petri dish containing fish with methylene blue. Incubate the dishes at 28 °C until the infection.

4. Embryo injury

1. Anesthetize zebrafish embryos in fish water without methylene blue, containing 0.02% Tricaine, approximately 5 min before injury in a cell culture dish (35 mm x 10 mm). The embryos no longer move and do not respond to stimuli.
2. Place 25G needles on the top of two chopsticks to facilitate the handling of needles.
3. Under a stereo microscope, eliminate embryos with abnormal development or dead ones. Using a circular movement of the dish, gather all the embryos in the center.
4. Isolate embryos one by one using two needles in a vertical position (head down - tail up) and place the left needle at the tail to keep it straight. Cut the fin at the edge of the notochord with the right needle. Make the cut in one go. Do not exceed 10 min between cutting and immersion in the bacterial solution.

5. Infection of wounded embryos

1. Under the biological safety cabinet type II, vortex and add the P. aeruginosa solution at approximately 1 x 10⁷ CFU/mL to a 6-well plate.
2. Collect the injured embryos with a disposable Pasteur glass pipette and add them to the bacterial solution under the microbiological safety post. Incubate the plate at 28 °C for 1.5 h.
   NOTE: If a large number of infected larvae are needed, make groups of about 50 larvae, make the wound, immerse them, and repeat for the following group.
3. After infection, wash the embryos under the microbiological safety post as described below.
   1. Transfer the embryos with a glass pipette for a wash into 10 mL of fish water without methylene blue, adding as little liquid as possible, for 30 min at room temperature.
   2. Transfer the infected embryos again using a glass pipette for a second wash into 4 mL of fish water without methylene blue for a few minutes.
   3. Transfer 24 infected embryos individually into a multi-well plate with 1 mL of fish water without methylene blue.
4. Place the plates in a plastic box in the incubator at 28 °C. Humidify the box (with damp paper towels, for example) to limit evaporation.

6. Survival of infected embryos

1. For embryo survival studies, check viability under a stereo microscope at 18 h, 24 h, and 48 h post-infection (hpi). An embryo is considered dead when the heart and blood circulation stops.
2. Represent mortality/survival rates using Kaplan-Meier staircase graphic representations (GraphPad Prism 8.3.0) and analyze the data with a log-rank or other appropriate statistical tests.

7. Counting bacterial burden in infected embryos (Figure 1)

1. Perform CFU determination from infected embryos at four different time-points: 1.5 hpi, i.e., directly after infection, and after each wash at 24, 48, and 72 hpi with 5 embryos/time-point. Perform all steps under the microbiological safety post.
2. Prepare for each infected larvae 1.5 mL microcentrifuge tubes containing 95 µL of 1x PBS. The volume of the larvae is around 5 µL.
3. Transfer the larvae to a 6-well plate with 4 mL of fish water without methylene blue to perform a wash and eliminate planktonic bacteria.
4. Place each embryo in a microcentrifuge tube containing PBS, adding as little liquid as possible. Just touch the glass pipette containing the larvae on the surface of the PBS to allow the larvae to sink into the microcentrifuge tube without adding liquid.
5. Crush the embryos individually using a pestle on the side of the microcentrifuge tube. Keep the pestle inside the tube. Determine the endpoint when there is no intact embryo left.
6. Raise the pestle and add 100 µL of PBS-Triton 2% to rinse any residual bacteria from the pestle (the final concentration is 1%). Vortex and wait 10 min. Do not process more than five embryos at the same time.
7. Drop 3x, 10 µL of non-diluted lysates on LB agar plates for each embryo. Do a serial dilution of the lysates in a 96-well plate until a 10^-3 dilution is reached using a multichannel pipette. Drop 3x, 10 µL of dilution 10^-1, 10^-2, and 10^-3 next to the undiluted ones on LB agar plates and incubate the plates overnight at 37 °C.
8. Count fluorescent colonies of each dilution under the microscope and calculate the CFU per embryo infected.

Figure 1: Experimental timeline to assess bacterial virulence and persistence in zebrafish embryo. Abbreviations: CFU = Colony forming units, hpi = hours post-infection. The drawing was created with BioRender.com. The figure has been modified from¹³. Please click here to view a larger version of this figure.

8. Testing treatment against persistent infection (Figure 2)

1. Prepare antibiotic solutions at 40x the minimum inhibitory concentration (MIC) for the P. aeruginosa strain. Before the experiment, check that the chosen concentration of antibiotic is not toxic to the embryo.
2. Follow steps 7.1 and 7.2. Place 1 mL of fish water with antibiotics in a 24-well plate and do a control without antibiotics.
3. Transfer five embryos from the wash step per treatment into the 24-well plate, adding as little liquid as possible.
4. Incubate with the antibiotics for 30 min at room temperature. Return to step 7.3. to quantify the bacterial load after treatment.

Figure 2: Experimental procedure used to assess the efficacy of antibiotic treatments on infected embryos. Abbreviations: ATB 30' = antibiotic treatment for 30 min, CFU = Colony forming units, hpi = hours post infection. The drawing was created with BioRender.com. The figure has been modified from¹³. Please click here to view a larger version of this figure.

Results

Zebrafish embryo is an appropriate model to monitor a persistent infection with P. aeruginosa CF isolates
We used the immersion of injured embryos mode to evaluate in vivo the virulence of three CF isolates (A6520, B6513, and C6490)⁹. We also assessed the behavior of a well-characterized late CF isolate, RP73, known for long-term colonization in mouse airways^14,15. Al...

Discussion

Modeling bacterial chronic infection in vivo is essential to understand pathogenesis and evaluate treatment efficacy. Here, we established a persistent wound infection model in zebrafish suitable for testing drugs against P. aeruginosa. This fills the gap of current methodology with a lack of in vivo models amenable to drug screening against chronic bacterial infections. Notably, this model mimics a major feature of chronicity, i.e., adaptive antibiotic resistance, and allows the search for opt...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Ethyl-3-aminobenzoate methanesulfonate	Sigma	E50521
Piston pellet	Eppendorf, Dutscher	33522
sea salts	Instant Ocean, Aquarium systems	218035
Triton	Euromedex	2000-B