This model will allow the researchers at the critical preclinical stages to more accurately determine the antimicrobial efficacy of novel formulations, and gives researchers greater control over drug design and formulation. This model enables rapid reproducible results in a representative wound environment. It reduces the need for purpose-bred animals, and facilitates the faster translation of topical antimicrobials for wound infections to the clinic.
The main challenges are contamination and dexterity. A strict aseptic technique and patience with tissue processing are essential;precise and purposeful flap removal is needed for the success of this method. Begin with the disinfection of forceps by exposing them to dry heat sterilization at 200 degrees Celsius for one hour.
Autoclave all glassware at 121 degrees Celsius for 15 minutes before use. Disinfect the forehead section of the lamp by pouring approximately 100 milliliters of 200 PPM chlorine dioxide solution onto the sample area. Shave the forehead section using electric clippers, followed by a wash with 200 milliliters of 200 PPM chlorine dioxide solution.
Wipe the area using ethanol and a blue roll, and cover the sample area with hair removal cream. After 35 minutes, gently scrape off the hair removal cream using a scraping tool and assess the sample area. Repeat the hair removal process if a significant amount of hair remains.
Rinse the clean sample area with 200 milliliters of chlorine dioxide solution followed by ethanol and a blue roll. Use a sterile eight-millimeter biopsy punch to cut out an eight-millimeter skin sample from the prepared area. Then remove the sample using sterile forceps and number 15 blade scalpel, ensuring that all cutaneous fat is removed.
Place the samples in a sterile 0.5 liter jar filled with sterile PBS. Then transfer them to sterile 50-milliliter tubes filled with 50 milliliters of 200 PPM chlorine dioxide solution. Invert the tube twice and leave it to sterilize for 30 minutes.
Transfer the samples to a 50-milliliter tube filled with 40 milliliters of sterile PBS. After the PBS wash, place each skin sample in a separate well of a 24-well plate. Add 350 microliters of the pre-warmed medium in a well, while maintaining the sample at the air-liquid interface.
Seal the 24-well plates with a gas permeable plate seal before placing the plate for incubation in a humidified tissue incubator at 5%carbon dioxide and 37 degrees Celsius for 24 hours. After incubation, remove the culture medium and rinse the samples in 500 microliters of sterile PBS. Remove the residual antibiotics from the sample by treating them with antibiotic-free media at 5%carbon dioxide and 37 degrees Celsius for 24 hours.
If turbidity or fungal infection develops in the antibiotic free media, discard the sample. Next, prepare a 50-milliliter tube with 10 milliliters of sterile Tryptic Soy Broth. Then transfer several Staphylococcus aureus colonies from a fresh agar plate of S.aureus to the broth, and incubate the tube at 37 degrees Celsius and 150 RPM for 18 hours.
After centrifuging the tubes at 4, 000 G for three minutes, remove the supernatant and resuspend the cell pellet in 10 milliliters of sterile PBS. Repeat the PBS wash twice before adjusting the inoculum to 0.6 optical density at 600 nanometer wavelengths using sterile PBS. Confirm the inoculum load by a manual viable plate count.
To infect the skin sample, prepare a fresh 24-well plate with 400 microliters of pre-warmed antibiotic free media, and add the 24 well inserts using sterile forceps. Remove the media from the skin samples. Wash them with 500 microliters of sterile PBS and remove the wash.
Use sterile forceps to gently hold the sample to the bottom of the well. Use a four-millimeter punch biopsy to pierce through the skin sample to a rough depth of one to two millimeters and make a central wound flap. Then use a number 15 blade scalpel and sterile-toothed Allis tissue forceps to remove the top layer of the wound flap.
Once all the samples have been wounded, transfer them to the 24 well inserts using sterile forceps. Pipette 15 microliters of the bacterial inoculum into the wound bed before incubating for 24 hours at 37 degrees Celsius in a humidified tissue incubator. For longer incubation periods, remove the media, replace it with fresh media every 24 hours, and incubate the plates under the same conditions.
To determine the bacterial load, remove the media from the bottom of the wells. And using sterile forceps, transfer each sample into a separate 50-milliliter tube filled with one milliliter of sterile PBS. Then detach the bacteria from the surface of the wound bed by homogenizing the sample surface with a fine-tipped homogenizer.
Ensure that the wound bed is in direct contact with the tip of the homogenizer. Once all the samples are processed, vortex each sample before transferring 20 microliters of homogenate into the corresponding well of a 96-well plate containing 180 microliters of sterile PBS. Serially dilute each sample homogenate to 1 times 10 to the negative 7, and pipette 10 microliters of the diluted homogenate onto a Tryptic Soy Agar plate in triplicate.
Incubate the Tryptic Soy Agar plate at 37 degrees Celsius, and after 18 hours, count the number of colonies to determine the colony forming units or CFU for each sample. An appropriate skin sterilization regime was identified using different treatments for varying lengths of time. Tissue integrity was monitored by histology, followed by staining with hematoxylin and eosin immediately after the treatment.
A 30-minute treatment with chlorine dioxide was the most effective treatment, with reproducible sterilizing the skin tissue while preserving tissue integrity. A white film in the wound bed was evident on the tissue 48 hours after infection. Although the scratch model harbored a higher average number of CFUs after 24 hours, the flap removal wounding technique produced more consistent results.
After 48 hours, approximately 100 times more bacterial CFUs were recovered from the wounded tissue compared to the inoculum, indicating a successful infection. Gram-stained histology sections of the infected tissue indicated the presence of S.aureus cells in the wound bed, which was absent in the sections of uninfected tissue. Following this procedure, one can perform immunohistochemistry to study tissue response, modified gram stain to visualize the bacterial localization, and viable plate counts after antimicrobial exposure to test antimicrobial efficacy.
For the researchers developing novel antimicrobials, this model will provide greater control of lead optimization, reduce reliance on expensive and tightly regulated animal trials, and enable the rapid translation of antimicrobials to the clinic.
