# Modeling Persistent Pseudomonas aeruginosa Infection in Wounded Zebrafish Larvae

Nilly Flore, Anne-beatrice Blanc-potard

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Nilly Flore, Anne-beatrice Blanc-potard. Modeling Persistent Pseudomonas aeruginosa Infection in Wounded Zebrafish Larvae. Journal of visualized experiments: JoVE, 2025, 220, Online ahead of print. 10.3791/68464. inserm-05384104v2

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# Modeling Persistent *Pseudomonas aeruginosa* Infection in Wounded Zebrafish Larvae

Flore Nilly<fnref n="1" />, Anne-Béatrice Blanc-Potard<fnref n="1" />

## Corresponding Author

Flore Nilly

flore.nilly@umontpellier.fr

## Citation

Nilly, F., Blanc-Potard, A.B. Modeling Persistent *Pseudomonas aeruginosa* Infection in Wounded Zebrafish Larvae. *J. Vis. Exp.* (220), e68464, doi:10.3791/68464 (2025).

## Date Published

June 13, 2025

## DOI

10.3791/68464

## URL

jove.com/video/68464

## 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<fnref n="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

<footnote n="1">¹Laboratory of Pathogens and Host Immunity (LPHI), CNRS, Inserm, Université de Montpellier</footnote>
(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 model¹¹,¹², 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><img src="image_2.png" /><figcaption>**Figure 1:** Experimental timeline to assess bacterial virulence and persistence in zebrafish embryo. Abbreviations:</figcaption></figure>

CFU = Colony forming units, hpi = hours post-infection. The drawing was created with BioRender.com. The figure has been modified from<sup>13</sup>. 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><img src="image_3.png" /><figcaption>**Figure 2: Experimental procedure used to assess the efficacy of antibiotic treatments on infected embryos.**</figcaption></figure>

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 from<sup>13</sup>. Please click here to view a larger version of this figure.

Representative 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 airways¹⁴,¹⁵. All four CF isolates were
highly attenuated when compared to the reference strain
PAO1 (Figure 3A and Figure 3B).

To discriminate between bacterial elimination and
persistence, we quantified the bacterial load per embryo
over 3 days (up to 72 hpi). To easily count *P. aeruginosa*
bacteria, we used green-fluorescent strains harboring
a chromosomally integrated *GFP* gene with constitutive
expression to avoid the risk of GFP signal loss during
infection. In surviving embryos infected with the reference
PAO1 strain, a continuous decrease of bacterial load was
observed over time, with few persisting bacteria. With isolates
A6520 and C6490, most embryos eliminated bacteria after 3
days (**Figure 3C**). In contrast, with strains B6513 and RP73,

the bacterial load stayed important and relatively constant
in numbers between 18 hpi and 65 hpi after an initial drop
between 1.5 hpi and 18 hpi (Figure 3C). Thus, isolates B6513
and RP73 developed a persistent infection in this model,
whereby bacteria can survive after 18 hpi in a living host.

Persistent *P. aeruginosa* bacteria exhibit adaptive
resistance to antibiotic treatment

We investigated antibiotic efficacy on embryos infected by the isolate B6513 by applying a 30 min tobramycin treatment (at 40x MIC, 40 µg/mL) at 1.5, 24, and 48 hpi (Figure 2). A sharp reduction in bacterial load was observed when tobramycin was applied just after the initiation of infection (1.5 hpi; **Figure 4A**). However, tobramycin had no significant effect when used at 24 hpi or 48 hpi (Figure 4A), i.e., when a persistent infection has been established in the embryos.

Antibiotics differ in their ability to penetrate eukaryotic cells, and their activity varies against intracellular *P. aeruginosa* in cultured phagocytes<sup>16</sup>. To assess if the intracellular niche may drive treatment failure in this model, we tested the *in vivo* activity of a fluoroquinolone, ofloxacin, that is known to be host cell-permeable, in contrast to tobramycin<sup>17</sup>.
Persistent bacteria at 24 hpi and 48 hpi exhibited some
tolerance towards ofloxacin (used at 40x MIC, 20 µg/mL)
when compared to bacteria treated very early after the start

of infection (Figure 4B). However, in contrast to tobramycin,
ofloxacin retained efficacy at the persistent stages (24 hpi and
48 hpi).

<figure><img src="image_4.png" /></figure>

**Figure 3: Cystic fibrosis (CF) isolates can establish a persistent infection in zebrafish embryos. (A) and (B) Embryo survival following infection by immersion with indicated *P. aeruginosa* strains at bacterial concentrations ranging from 7.2 x 10⁷ to 1.2 x 10⁸ CFU/mL. Survival was monitored for > 40 h following the infection (n=3, 60 larvae in total). Logrank test: \* p < 0.05, \*\* p < 0.01 and \*\*\*\* p < 0.0001. (C) Evolution of the bacterial load per embryo over time (until 72 hpi). Following infection by immersion with fluorescent (*GFP*⁺) *P. aeruginosa* isolates, embryos were crushed at the indicated time points and plated for CFU counting (n=3, 15 larvae). The bars represent the minimum and maximum values. GraphPad Prism 8.3.0 was used to perform statistical tests and create graphs. The figure has been modified from¹³. Please click here to view a larger version of this figure.
<figure><img src="image_5.png" /></figure>

<figure><img src="image_6.png" /></figure>

**Figure 4:** Antibiotics have a reduced efficacy on persistent *P. aeruginosa* in infected zebrafish. Abbreviations: ns = not significant, hpi = hours post infection, ATB = antibiotic (A) Efficacy of tobramycin on strain B6513 with respect to the time post-infection (n=3 to 4, 15 to 21 larvae). Embryos colonized for 1.5, 24, or 48 hpi were subjected to the indicated antibiotic challenge or incubated in water for the control condition. Following this 30 min treatment, bacterial load per embryo was determined in both groups. Mann-Whitney test: ** p < 0.01, *** p < 0.001, and **** p < 0.0001. The bars represent the minimum and maximum values. (B) Efficacy of ofloxacin, an antibiotic known to enter eukaryotic cells with high efficiency¹⁷, on strain B6513 (same analysis as in (A)). GraphPad Prism 8.3.0 was used to perform statistical tests and create graphs.
The figure has been modified from¹³. Please click here to view a larger version of this figure.
<table><caption>Table 1: Preparation of solutions.</caption><thead><tr><td>Solutions or Materials</td><td>Preparation</td></tr></thead><tbody><tr><td>Anaesthetic stock solution 20X</td><td>4 mg/mL of Tricaine (Ethyl-3-aminobenzoate methanesulfonate) in Tris 22,5 µM pH 8,8. Store at -20 °C.</td></tr><tr><td>Anaesthetic working solution 1X</td><td>dilute in fish water without methylene blue the tricaine stock solution 20X preparation to reach the 1X concentration (0,02 %). Store at 4°C.</td></tr><tr><td>Fish water with methylene blue</td><td>60 µg/mL sea salts (Instant Ocean), 0,4 mM NaOH, 0,1 % methylene blue in distilled H2O. Store at RT.</td></tr><tr><td>Fish water without methylene blue</td><td>60 µg/mL sea salts (Instant Ocean), 0,4 mM NaOH in distilled H2O. Store at RT.</td></tr><tr><td>LB broth</td><td>10 g/L tryptone, 5 g/L yeast extract, 10 g/L NaCl in distilled H2O and autoclave 20 min at 120 °C and 103 kPa.</td></tr><tr><td>LB agar</td><td>10 g/L tryptone, 5 g/L yeast extract, 10 g/L NaCl, 10 g/L agar in distilled H2O and autoclave 20 min at 120 °C and 103 kPa.</td></tr><tr><td>Triton 2 %-PBS</td><td>dilute in PBS 1X the triton-X100 stock 100 %. Store at 4 °C.</td></tr></tbody></table>

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 optimized compounds/antibiotics. This model of persistent *P. aeruginosa* infection in zebrafish is based on an original methodology: (i) analysis of *P. aeruginosa* isolates from CF patients, including an isolate reported to establish chronic pneumonia in mice¹⁴, (ii) an analysis over

a long period (3 days) after infection that includes counting of bacterial load, and (iii) a wound mode of infection.

We describe a reproducible and relatively simple method to model *P. aeruginosa* persistent infection in zebrafish embryos through wound healing and bacterial immersion in water. In comparison to mammalian models, all the described procedures in zebrafish follow the 3Rs principles and EU animal care regulations. Moreover, we have validated the translation of our non-mammalian model in mice since the virulence and bacterial load profiles of clinical isolates in zebrafish were reproduced in a mouse model of chronic wound infection¹³.
Persistent bacteria in the zebrafish model were tolerant to antibiotics, as reported during chronic infection in mammals and humans¹⁸. Intracellular stages can contribute to evade antibiotics, even for pathogens considered as extracellular as *P. aeruginosa*⁵,¹⁹. The finding that a cell-permeant antibiotic, ofloxacin, demonstrated higher efficacy than a poorly permeant antibiotic (tobramycin) supports the hypothesis that intracellular persisting bacteria are poorly accessible to antibiotics. Zebrafish thus provides a novel pre-clinical model to screen and evaluate *in vivo* the efficacy of treatments against persistent *P. aeruginosa* before being transposed and refined in mammalian models. Additional experiments, including controls, as well as live imaging, possible thanks to optical transparency of the embryo, are shown in a companion article that uses this methodology¹³.

## Disclosures

The authors declare no competing interests.

## Acknowledgments

We thank Stéphane Pont (LPHI, Montpellier), who largely contributed to setting up the methodology and generated results shown in **Figure 3**, as described in the companion article¹³. We thank P. Plésiat (French National Reference Center for antimicrobial resistance, Besançon, France) for providing the clinical strains A6520, B6513, and C6490, and A. Bragonzi (Milano, Italy) for providing the strain RP73. We thank C. Gonzalez and V. Goulian for the Aquatic model facility ZEFIX from LPHI. This work was supported by Vaincre La Mucoviscidose (RF20200502703, RF20210502864, RF20220503060) and Association Gregory Lemarchal. The aquatic facility is supported by the European Community's H2020 Program [Marie-Curie Innovative Training Network Inflanet: Grant Agreement n° 955576].

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