A Delayed Inoculation Model of Chronic Pseudomonas aeruginosa Wound Infection

This protocol is significant because it describes a model for chronic Pseudomonas aeruginosa wound infection without the need for foreign material implantation or immune suppression. Delayed inoculation with Pseudomonas aeruginosa 24 hours after full thickness excisional wounding prostalls the rapid clearance and dissemination of the bacteria, establishing an infection that lasts seven to 10 days. This unique model will be a useful tool for the investigations of bacterial pathogenesis, host pathogen interactions, and the development of new therapies for chronic Pseudomonas aeruginosa wound infections.

Assisting me in a demonstration of the procedure will be Dan Liu, a medical student from the Bollyky Laboratory. Before beginning the procedure, weigh an anesthetized eight to 12 week old mouse to obtain a baseline weight and place the mouse in the prone position. After confirming a lack of response to the pedal reflex, subcutaneously inject the mouse with 250 microliters of pre-warmed sterile 0.9%sodium chloride on each flank.

Use an electric shaver to remove the hair from the dorsal area of the mouse and apply a thin layer of depilatory cream to the exposed skin. After 20 to 60 seconds, remove the hair and excess lotion with gauze moistened in warm water. For full thickness excisional wound surgery, first use a 25 gauge needle to subcutaneously inject 0.6 to one milligrams per kilogram of sustained release buprenorphine at the mid-dorsal area of the mouse and wipe the surgical site in a circular manner with three alternative sterile Betadine and alcohol swabs.

Place a plastic cling-wrap drap over the surgical site and stretch the skin taut caudally. When the skin has dried, use a sterile six millimeter diameter skin biopsy punch to make an initial incision through the left dorsal epidermis, followed by a second incision on the right dorsal epidermis. Use forceps to tent the skin from the center of the left outlined wound area and use scissors to excise the epidermal and dermal layers.

After repeating the incision on the right outlined wound area to create symmetrical excisional wounds, wash both wounds with 50 microliters of sterile saline. When the surgical site and surrounding skin have dried, cover the wounds and dorsum with a transparent film dressing and place the mouse in a clean cage on a heating pad with monitoring until full recumbency. 24 hours after the surgery, weigh the re-anesthetized mouse again and inject the animal subcutaneously with 250 microliters of pre-warmed sterile 0.9%sodium chloride in both flanks.

Next, load 100 microliters of the luminescent P.aeruginosa strain suspension into a 500 microliter tuberculin safety cap syringe equipped with a 27 gauge needle and inject 40 microliters of bacteria suspension through the transparent film dressing into each wound. Ensure that the bacterial suspension is well mixed and that the transparent dressing is intact. Make sure that you puncture the transparent dressing only once with the needle bezel side up.

Then return the mouse to its cage on a heating page with monitoring and provide high calorie nutritional supplement paste sandwiched between food pellets on the floor of the cage. Plate the remaining inoculum on an LB agar plate and count the colonies to confirm the number of bacteria that have been administered. For in vivo imaging of the infected wounds, follow bio safety level two containment protocols for transport of the mice to and from the imaging instrument and place the anesthetized mouse in the prone position within the imaging chamber.

In the imaging software, set the exposure time, binning, f-stop, and field of view as appropriate for the experiment. Next, create a region of interest at the wound site and measure the average detected flux in photons per second. To measure the background, create a region of interest over a random area on the imaging platform and subtract the background number of photons per second.

At the end of the experiment, in a bio safety cabinet, use sterile scissors and forceps to excise the wound beds and place each bed in one milliliter of sterile PBS in a 1.5 milliliter polypropylene tube. Mince the wound tissue with scissors and incubate the tissue pieces on a shaker at four degrees Celsius in 300 rotations per minute for two hours. At the end of the incubation, vortex each tube for 10 seconds before serially diluted the bacterial effluent in PBS and plating the diluted bacterial effluent on LB agar to quantify the bacterial burden.

This representative image obtained using an imaging optical system demonstrates that this model results in detectable luminescence. In this experiment, the infection peaked at day three post inoculation and persisted seven days post inoculation based on both the bioluminescence and colony counts. Culture of the bacteria isolated from the wound further demonstrate that the quantified colony forming units per wound correlate with the detected luminescence.

Though there is an initial rapid weight loss immediately after the injection, saline injections and supplemental nutrition can restore the mice to their initial weights. The calculated inoculation dose of which 50%of the wounds will become substantially infected has been determined to be about 7.7 times 10 to the second colony forming units per milliliter. Doses higher than one times 10 to the fourth colony forming units per milliliter result in a 100%infection rate.

Various topical and systemic treatments can be applied after the wounding and inoculation procedures to investigate new potential therapies for Pseudomonas aeruginosa. All the work with Pseudomonas aeruginosa and infected mice should be conducted with BSL-2 safety precautions in accordance with the researcher's institutional bio safety and animal use committee guidelines.