Assessing Biofilm Dispersal in Murine Wounds

Microbiologists and infectious disease physicians are starting to realize how important biofilm-associated infections are in chronic disease. The NIH has even said that over 80%of chronic infections are due to bacterial biofilms. So this has urged on the emphasis on addressing biofilm, and developing therapeutics that address the biofilm problem.

And a lotta those therapeutics are focused on dispersing bacterial cells that are in biofilms out of the biofilm so that they can be more readily killed. But there really aren't a lot of protocols in the literature currently that address how to determine the efficacy of such a dispersal agent. So we feel like this is a good protocol to use.

It is one type of bacterial infection that's biofilm-associated. And we also think it can be optimized pretty easily to study many different types of bacterial species, and how they form biofilms and disperse, as well as different dispersal agents. And we think it's important to deliver this in a video format, because we have seen that there are nuances to especially the delivery of the agent that we have found work optimally in mice.

To begin, assess the plane of anesthesia by toe pinch, and by monitoring the respiratory rate and effort. Shave the dorsal surface and apply a depilatory cream to the shaved region for 10 minutes, or as per product's instructions. Gently remove the depilatory cream.

And ensure the skin is dry. Using an alcohol-proof marker, draw a circle with a diameter of 1.5 centimeters towards the posterior region of the back. For pain management, administer 0.05 milliliters of lidocaine subcutaneously into the area to be excised.

And 0.02 milliliters of buprenorphine subcutaneously in the scruff of the neck. Wait 10 minutes to ensure pain management is reached. Before excision, disinfect the dorsal surface using an alcohol swab.

Maintain a sterile surgical field throughout the procedure, and use autoclaved instruments and sterile gloves to limit contamination. Administer a full thickness excisional skin wound to the level of the panniculus muscle, with surgical scissors. After excision, immediately cover the wound with a semipermeable polyurethane dressing.

Carefully separate the backing from the dressing using two fine-tipped forceps, if applicable. Inject approximately 10 to the fourth CFU of bacteria, and 100 microliters PBS under the dressing and onto the wound bed to establish an infection. Note, wounds can also be infected with multiple species of bacteria and/or fungi simultaneously, or by placing preformed biofilms or even debridement tissue extracted from patients onto the wound bed.

After infection, place mice in cages on top of paper towels to prevent the mice from asphyxiating on the bedding material. Place the cages on heating pads and monitor the mice until they have regained their righting reflex. Remove the paper towel at this point.

Allow wound infections to establish for 48 hours. Inspect the dressing daily for tears in areas of nonadherence and replace it if needed. Place mice under isoflurane anesthesia at 3%and one liter per minute of oxygen, while administering treatments.

Inject 200 microliters of enzyme solution or PBS control onto the wound by lifting the skin slightly anterior of the wound with forceps, carefully piercing the lifted skin with the syringe, and slowly injecting the solution into the area between the wound bed and the dressing. To ensure that the entire wound bed is covered by solution, gently raise the dressing with forceps, pulling it away from the wound bed, while slowly injecting the solution. After treatment, place the mice back in cages for 30 minutes.

After 30 minutes of exposure to the treatment, reanesthetize the mice with isoflurane and aspirate the remaining solution with a syringe, puncturing in the same area as before. Note, this aspirated solution contains dispersed bacterial cells, which may be saved for various downstream analyses. Administer the second and third irrigation treatments as was done for the first using a new syringe each time.

If a luminescent strain of bacteria is utilized to initiate infection, an in-vivo imaging system can be used to visualize dispersal from the wound bed. For analysis, open the images in the IVIS software and select the area to be analyzed with the tool palette. To account for background, place a circle on the background of an image, and then placed the same sized circle on top of the wound bed for each mouse.

From the tool palette, select Measure ROIs and upload the table of measurements to a spreadsheet. Subtract the background circle reading from each of the wound bed measurements to calculate luminescence for each sample. After humane euthanasia, harvest the wound bed tissue by first gently removing the dressing with sterile forceps, and cutting around the perimeter of the wound bed with sterile surgical scissors.

Use forceps to gently lift one end of the wound bed, then use scissors to cut the tissue sample away from the muscle layer. Place the tissue sample in a pre-weighed two-milliliter homogenization tube containing one milliliter of PBS. Weigh the tube again after inserting the sample.

To harvest the spleen, place the mouse in a supine anatomical position and secure by pinning the extremities to a dissection surface. Soak the area to be dissected with 70%ethanol to help prevent contamination. Make a small incision perpendicular to the midline with surgical scissors in the dermis of the lower abdomen.

Make sure to pull the skin up and away from the internal organs. Continue the incision along the midline until the sternum is reached. Once the peritoneal cavity is exposed and the internal organs are visible, separate the spleen from the connective tissue and place in a separate pre-weighed homogenization tube.

Weigh the tube again after inserting the spleen. Homogenize the samples at five meters per second, for 60 seconds, two times. To enumerate CFU, serially dilute and spot plate the homogenized samples.

Serially dilute by pipetting 100 microliters of the homogenized sample into 900 microliters of PBS and a 1.5 milliliter centrifuge tube. Vortex before continuing the dilution by pipetting 100 microliters of the diluted sample into a separate tube containing 900 microliters of PBS. Repeat these steps until six dilutions are achieved.

Pipette 10 microliters of each dilution onto the indicated area on an agar plate as shown. If more precise CFU quantitation is required, spread 100 microliters of each dilution of the sample across separate agar plates. Wound and infect mice with approximately 10 to the fourth CFU of bacteria, as described in the in vivo protocol.

Allow the infection to establish for 48 hours, then extract the wound bed in a similar fashion to the in-vivo protocol. Begin by carefully removing the dressing. Cutting around the perimeter of the wound.

And separating from the muscle layer. The wound bed sample can be divided into multiple sections to increase the number of samples. Then place the divided samples into separate pre-weighed empty homogenization tubes.

Weigh the tube again and calculate the weight of the sample by subtracting the weight of the tube without the sample from the weight of the tube with the sample. Add one milliliter of PBS to the homogenization tube containing the sample, and gently invert the tube a few times to rinse. Remove the PBS wash and discard.

Add one milliliter of GH treatment, or PBS as a negative control, and incubate for two hours at 37 degrees Celsius, with shaking at 80 RPM. Remove the bile from dispersal solution and place in a separate 1.5 milliliter centrifuge tube. This contains the dispersed cells.

Wash the remaining tissue with one milliliter of PBS, as done previously, and discard the PBS wash. Add one milliliter of PBS to the remaining tissue and homogenize at five meters per second, for 60 seconds. Serially dilute both the solution of dispersed cells and the homogenized tissue sample by pipetting 100 microliters of the sample into 900 microliters of PBS in a 1.5 milliliter centrifuge tube.

Vortex the diluted sample and continue the dilution five more times for a total of six dilutions. Spot plate the sample by pipetting 10 microliters of each dilution onto the indicated area on a selective agar plate. Count the CFU and calculate percent dispersal by dividing the dispersed CFU by the total CFU, and multiplying by 100.

In this experiment, eight to 10 week old female Swiss Webster mice were infected with 10 to the fourth CFU of PA01 carrying the luminescence plasmid PQF50-lux. The infection was allowed to establish for 48 hours prior to administrating three 30-minute treatments of either PBS as a vehicle control, or 10%GH as an experimental treatment to digest the biofilm EPS. Shown in section A, mice were imaged pre-treatment, directly after treatment, and at 10 and 20 hours post-treatment.

Using IVIS, an established infection can be visualized within the wound bed generating a bright bioluminescent signal. The dispersal of bacteria out of the wound bed can be visualized immediately after the GH treatment, but not after the PBS treatment. Bacterial dissemination into the organs can also be seen by placing the mouse on its side for imaging, as shown in the lower panels.

In section B, images from both the PBS and GH-treated mice demonstrated an increase in luminescent signal at 20 hours post-treatment, compared to pre-treatment. However, it is still important to quantitate CFU due to the detection threshold of a luminescence in IVIS. At 20 hours post-treatment, the mice were euthanized and the wound beds and spleens were collected to enumerate CFU per gram of tissue.

Section C shows the bacterial load in the wound bed, while section D shows the bacterial load in the spleens. These data suggest disseminated spread of the dispersed bacteria, since bacteria was only detected in the spleens of the mice treated with the EPS-targeting GH solution. Although these protocols are very useful, there are some limitations.

First, it should be noted that there is a detection limitation with the in-vivo imaging system, or IVIS. A decrease in luminescent signal can be seen when the bacteria enter stationary phase. Therefore it is important to collect tissue of interest and measure the CFU present, as there can be a disconnect between the luminescence and CFU enumerated.

However, IVIS is useful for visualizing the efficacy of the treatment throughout a study, without having to actually euthanize the animal. Another limitation to take into consideration is the requirement of close monitoring of the bandages. The bandages are required to reduce contractile healing as well as keep the wound sterile from other bacteria.

Bandages will need to be replaced often, which can lead to accidental debridement of the wound and opportunity for contamination. These protocols describe novel methods to study bile from dispersal, and the evaluation of therapeutic bile from dispersal agents. These models allow for the development of complex polymicrobial biofilm-associated infections that mimic clinically relevant human infections.

Our hope is that these protocols will be utilized and refined to further the development of anti-biofilm dispersal agents for therapeutic use.