Modeling Persistent Pseudomonas aeruginosa Infection in Wounded Zebrafish Larvae

Chronic infection caused by the bacterium Pseudomonas aeruginosa are tolerant to antibiotics and very difficult to treat. Our objective is to develop an in vivo model that will accelerate the discovery of efficient therapies. Antibacterial drugs are mostly screened in vitro, and testing drugs in chronically infected mice is a complex challenge. To fill the gap between these two approaches, we propose an alternative pre-clinical model using wounded zebrafish larvae. Our model of persistent infection in zebrafish based on the use of clinical strain of Pseudomonas aeruginosa reproduces antibiotic tolerance. Whereas micro injection is commonly used to infect zebrafish larvae, our wounding method reflect a natural infection mode and is well-suited for screening of therapeutic compounds.

[Narrator] To begin, place 25-gauge needles on top of two chopsticks to facilitate handling. Using a stereo microscope, identify and remove embryos that show abnormal development or are nonviable. Move the dish in a circular motion to gather the remaining embryos at the center. Now, use two vertically oriented needles to isolate each embryo, positioning the left needle at the tail to keep the body straight. With the right needle, make a single cut at the edge of the notochord to remove the fin. Complete each cut swiftly, ensuring all embryos are immersed in bacterial solution within 10 minutes. For the infection procedure, vortex the Pseudomonas aeruginosa solution under a type two biological safety cabinet, and add it at approximately one times 10 to the power of seven colony-forming units per milliliter to a six-well plate. Use a disposable glass Pasteur pipette to collect the wounded embryos, and transfer them into the bacterial solution. Incubate the six-well plate at 28 degrees Celsius for 1.5 hours. After incubation, retrieve the infected embryos and place them under the microbiological safety for washing. Now, transfer the embryos with a glass pipette into 10 milliliters of fish water without methylene blue, minimizing the transferred volume and incubate for 30 minutes at room temperature. Then, transfer the embryos again with a glass pipette into four milliliters of fish water without methylene blue, and incubate briefly. Next, with a pipette, transfer the infected embryos individually into a 24-well plate, adding one milliliter of fish water without methylene blue to each well. Place the 24-well plate into an incubator set at 28 degrees Celsius. Prepare 1.5-milliliter microcentrifuge tubes with 95 microliters of 1X PBS for each infected larva. Transfer the larvae into a six-well plate containing four milliliters of fish water without methylene blue to wash and remove planktonic bacteria. Place each washed embryo into a microcentrifuge tube with PBS, transferring as little liquid as possible. Now, use a pestle to crush each embryo against the side of the microcentrifuge tube, leaving the pestle inside the tube afterward. Then, lift the pestle and add 100 microliters of 2% PBS Triton to rinse residual bacteria from the pestle, achieving a final concentration of 1%. Vortex the tube and incubate for 10 minutes. Next, dispense three 10-microliter drops of undiluted lysate from each embryo onto LB agar plates. Use a multichannel pipette to serially dilute each lysate in a 96-well plate up to a 10 to the power of minus three dilution. Finally, dispense three 10-microliter drops of the dilutions next to the undiluted spots. And incubate overnight at 37 degrees Celsius. All four cystic fibrosis Pseudomonas aeruginosa isolates were significantly less virulent in the injured embryo model compared to the PAO1 reference strain. In embryos infected with two isolates, the bacterial load was markedly reduced over three days, indicating bacterial elimination. In contrast, the two other isolates, B6513 and RP73, maintained a relatively stable bacterial load from 18 to 65 hours post-infection following an initial drop, suggesting persistence. A short 30-minute treatment with tobramycin at 1.5 hours post-infection drastically reduced bacterial load in embryos infected with isolates B6513. Tobramycin had no significant effect on bacterial load when administered at 24 or 48 hours post-infection, demonstrating resistance during persistent infection stages.