Our lab seeks to understand how bacteria sense and respond to the environment in human airways. We characterize airway environments and use the information gained in the design of novel models for studying bacterial physiology during airway infection, and in the identification of novel antimicrobial agents. CFTR modulator therapies have greatly improved lung function and quality of life for individuals with cystic fibrosis.
This improvement may cause changes in bacterial expression and factors involved in both virulence and antimicrobial resistance, which may pose challenges in adapting current preclinical infection models. The development of robust cell culture models has been accelerated through the use of air liquid interface, epithelial cell cultures, which were available in both cystic fibrosis and non-CF variants. At the same time, the use of high throughput approaches like transcriptomics to understand bacterial physiology in infection relevant conditions has helped us understand whether models recapitulate infection scenarios.
Developing new antimicrobial agents for treating chronic respiratory infections is challenging. Most preclinical drug screening processes do not fully consider the unique challenges in treating infections in the complex and difficult to access environment of the chronically infected lung. We are working with a network of collaborators to try and standardize a preclinical framework for the development of novel antimicrobial agents for the treatment of chronic respiratory infection.
We aim to use the models generated to accelerate drug discovery and to better understand microbial pathogenesis in an airway environment. Begin by weighing the galleria mellonella larvae. To sterilize the surface of the galleria mellonella larvae in batches of 10 to 20 larvae, place each batch individually in the dish and spray the larvae twice with 70%ethanol.
Using tweezers, roll the larvae to ensure full coverage with ethanol. Then, remove the larvae from the ethanol, place them in a sterile petri dish and leave the dish uncovered to allow them to dry. After two hours, about 90%of the larvae regain activity, separate them into groups based on power calculations detailed in this table.
Begin by preparing the pseudomonas aerogenosa infection stock of known colony forming units per milliliter prior to infection. To pellet the infection stock, centrifuge at 10, 000 G for five minutes at room temperature. Resuspend the pellet in PBS.
Next, prepare three one milliliter microfuged 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 larvae. Afterward, inject each galleria mellonella larvae with 10 microliters of inoculum in the rear right pro leg. 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 invitro 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 two, four, six, nine 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-M.
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.
