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 cycle....

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 ....

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