Begin by preparing a Pseudomonas aeruginosa infection stock of known colony forming units per milliliter prior to infection. To pellet the infection stock, centrifuge at 10, 000 x g for five minutes at room temperature. Resuspend the pellet in PBS.
Next, prepare three one milliliter microfuge tubes containing either sterile distilled water, ethanol, or sterile PBS. Inspect a sterile 100 microliter Hamilton syringe, ensuring that the needle tip is sharp and has not developed a hook which could cause significant trauma to the infection site. Now wash the syringe sequentially with distilled water, ethanol, and PBS prior to infections, drawing up and discarding the maximum capacity.
Then vortex the resuspended infection inoculum and draw up a maximum of 100 microliters into the Hamilton syringe sufficient to inject 10 microliters per larva. Afterward, inject each Galleria mellonella larvae with 10 microliters of inoculum in the rear right proleg. Incubate the infected larvae between 20 degrees Celsius and 42 degrees Celsius, which is an appropriate range for Galleria mellonella survival.
Prepare a broad range of doses exceeding the expected therapeutic range of the antimicrobial agent guided by in vitro toxicity data. Inject healthy, sterile Galleria mellonella in appropriate group sizes with the different antimicrobial and PBS vehicle concentrations. To optimize the treatment of bacterial infection in Galleria mellonella, prepare a suitable range of antimicrobial agent doses based on preliminary toxicity testing.
Infect groups of larvae with bacteria as previously described at two hours post infection. Treat the larvae with the prepared antimicrobial agent or vehicle control solution. Monitor Galleria mellonella survival at 30 minute intervals or more frequently from the time at which mortality is usually first observed.
Remove any dead larvae and record their time of death. To compare how the timing of treatment impacts mortality, treat the larvae at 2, 4, 6, 9 or 12 hours post-infection and monitor their survival at a 30 minute interval. The effective dose of tobramycin varied by strain.
Five milligrams per kilogram was optimal for PAO1 and PA14.2.5 milligrams per kilogram for PAK and 10 milligrams per kilogram for IST27 mucoid. No dose was effective for LESB58. Treatment with five milligrams per kilogram of tobramycin at two, four, and six hours post-infection resulted in over 50%survival of Plasmodium aeruginosa infected Galleria mellonella larvae.
