Come to the Light Side: In Vivo Monitoring of Pseudomonas aeruginosa Biofilm Infections in Chronic Wounds in a Diabetic Hairless Murine Model

Here we describe a novel diabetic murine model utilizing hairless mice for real-time, non-invasive, monitoring of biofilm wound infections of bioluminescent Pseudomonas aeruginosa. This method can be adapted to evaluate infection of other bacterial species and genetically modified microorganisms, including multi-species biofilms, and test the efficacy of antibiofilm strategies.

Transcript

The overall goal of this novel model is to allow real time in vivo monitoring of Biofilm wound infection and healing in diabetic mice. This method can help answer key questions in the wound healing field such as what are they underlying interactions between Biofilms and the hosts suffering from diabetes and their impact in would closure. The main advantage of this technique is that in vivo Biofilm progression and their impact in wound healing can be assessed without having to serially euthanize animals over time or at specific time points.

The implications of this technique extend to our treatment, therapy or diagnosis of chronic wounds because the model can be used to test a wide variety of therapies including systemic drugs and topical treatments such as antimicrobial dressings. Demonstrating the procedure will be Svetlana Navitskaya, a technician in a Busik Lab, Cassie Larrivee, a Master's student at Michigan State University and Sandra O'Reilly, Research Assistant Professor at MSU. To induce diabetes, administer five consecutive daily intraperitoneal injections of streptozotocin in citric acid to eight week old male SKH-1 mice.

14 days after the last injection, use a glucometer to confirm hyperglycemia by blood glucose monitoring. Outline 10 millimeter circles on a 0.5 millimeter thick silicone sheet, centering one five millimeter biopsy punch in the middle of each circle and press the punch firmly to create donut-like discs that will be used as splints. Use scissors to cut a transparent occlusive dressing into one by once centimeter pieces.

UV sterilize polycarbonate membrane filters with a 0.2 micron pore size in a biological safety hood for 15 minutes per side. Two days before the would procedure, inoculate a culture of bioluminescent P.aeruginosa-Xen41 in tryptic soy broth overnight at 37 degrees Celsius and 200 rpm. The next morning, pellet the bacterial cells and perform three washes in one milliliter of DPBS per wash.

After the third wash, dilute the bacterial suspension in fresh DPBS to an absorbance of 05 at 600 nanometers. Place the UV sterilized filters into a tryptic soy agar plate and add 10 microliters of the diluted bacteria onto each filter. When the membranes are dry place the plate at 37 degrees Celsius, transferring the filters to fresh tryptic soy agar plates every 24 hours.

The next day confirm a lack of response to toe pinch and wipe the skin on the back of a diabetic SKH-1 animal with 10 per cent povidone iodine and an isopropanol pad. Using a sterile four millimeter biopsy punch, use a permanent marker to outline a circular pattern for the wound on one side of the animal's midline at the shoulder level. Next, use serrated forceps to lift the skin in the middle of the outline and use iris scissors to create a full thickness wound extending through the subcutaneous tissue and including the panniculus carnosus.

Excise the circular piece of tissue and apply a waterproof medical skin-adhesive glue to the animal. Using mild pressure, place a silicone splint onto the adhesive and hold the splint for about 30 seconds until the glue dries. Then cover the wound with one of the transparent occlusive dressings, administering analgesia once daily for two days via subcutaneous injection for post-operative pain relief.

Place the animal into its own recovery cage. 48 hours after the surgery, use a sterile spatula to transfer several Biofilms from their filters into a syringe. Then use the syringe to dispense the Biofilms into individual micro centrifuge tubes.

Dilute the Biofilms at a one to two ratio in DPBS with brief mixing and further break down the Biofilm inoculum with two one minute vortexes intercalated by a two minute sonication at 40 kilohertz in an untrasonic cleaner. Serially dilute the broken down inoculum up to a one times 10 to the negative eight concentration for plating and plate the inoculum onto tryptic soy agar plates. After 24 hours at 37 degrees Celsius, count the bacteria to calculate the Biofilm colony-forming units.

To evaluate the bioluminescence to bacterial count ratio, make one half to one twenty fourth serial Biofilm dilutions in DPBS, vortexing the dilutions until visually homogeneous bacterial suspensions are produced. Tranfer 200 microliters of the diluted solutions into individual wells of a black 96 well plate and image each well using an in vivo imaging system. Then plate the dilutions onto new TSA plates for a 24 incubation at 37 degrees Celsius, counting the colony forming units the next day to create a standard curve of the bioluminescence for specterial counts.

Next remove the dressing cover and silicone splints from the wounds and use a microscope with an attached camera to obtain micro graphs of the wounds. Using a 200 microliter pipette tip with a cut end, pipette 10 microliters of the Biofilm inoculum onto each wound and cover the wounds with fresh dressings. Then use an in vivo imaging system to image the wounds daily in an isolation chamber equipped with a HEPA filter to assess the bioluminescence in the wounds until the luminescence values fall below the background level.

Obtain daily images and measurements of the wound closure progress until the wounds are healed. Some advantages of using SKH-1 mice for this diabetic wound healing model are that the mice do not demonstrate a significant weight loss after diabetes induction, fewer deaths occur in the diabetic SKH-1 animal cohort and the SKH-1 animals do not demonstrate short term skin damage or hair regrowth interference compared to C 57 black six animals. Daily in vivo imaging of the Biofilm containing wounds allows monitoring of the wound-infection development and the evolution of the Biofilms.

A standard curve can then be generated to compare the bioluminescence recorded as the total flux with the bacterial density within the wounds. Removal of the splint and dressing on day eight facilitates visualization of the wound healing and the daily measurement of the wound closure progress. Once mastered, this technique can be done in a short period of time if it is performed properly, reducing the time the animals are under anesthesia.

Following this procedure, other experimental conditions like the use of different microorganisms or their combinations as well as the application of treatments can be performed to answer additional questions such as how multi species Biofilms adversely impact wound healing or how well wounds respond to novel drugs. After its development this technique paved the way to allow researchers in the field to explore the impact of Biofilms in wound healing by monitoring bacterial growth through direct and continuous luminescence measurements while reducing the number of animals necessary per experiment. After watching this video you should have a good understanding of how to perform the wound surgery and conduct the Biofilm preparation, inoculation and infection protocol.

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Journal (Immunology and Infection)

Journal (Immunology and Infection)

Come to the Light Side: In Vivo Monitoring of Pseudomonas aeruginosa Biofilm Infections in Chronic Wounds in a Diabetic Hairless Murine Model

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