Swine Model of Biofilm Infection and Invisible Wounds

Biofilm infection significantly contributes to wound chronicity. This preclinical model infects full thickness Q-TDS swine wounds with clinically isolated pathogens to study wound biofilm formation and biofilm dependent changes in wound outcomes. By including both microbial as well as host factors, we aim to investigate the complex host microbe interactions underlying pathogenic biofilms in chronic wounds.

Although the current literature does not discriminate between the two wound biofilm aggregates differs significantly from biofilm aggregates found by microbial interactions in the absence of host defenses. For example, biofilm grown on plastic surface or on say, dead skin. The iterative interplay between microbial processes and host immune defense systems leads to wound biofilm aggregate formation.

Since this process takes several days, having a long-term model of wound infection is crucial. Both in vitro and ex vivo systems lack the necessary host immune response required to study wound biofilm. While short term in vivo studies yield acute response and that do not allow for biofilm maturation, our standardized preclinical experimental system with reported clinically relevant findings addresses these gaps.

While biofilm-infected wound might close structurally, they may not achieve functional wound closure as the repaired skin often fail to reestablish the barrier function. The breached affected skin with compromised barrier function constitutes an open, invisible wound that is structurally covered but functionally open. Our study highlights the importance of measuring skin barrier function and the site of wound repair to determine functional wound closure.

The barrier function is measured by clinically validated point of care measurement of transdermal water loss. To begin the procedure inside the properly arranged surgery room, place the anesthetized pig in the sternal recumbency position and ensure all the connected lines and tubes are secured. Remove any residual hairs from the shaved area designated for wounding using hair removal cream.

After sterilizing the wound sites, mark the area using a sterile burn template and a surgical skin marker. Set the burner by adjusting the set point on the control unit to 150 degrees Celsius, the low set point to 145 degrees Celsius and the high set point to 155 degrees Celsius. Using a heated stainless steel blocks connected to the burn device, create a full thickness burn wound by placing the blocks on the skin for 60 seconds.

Take a digital photo of the whole back of the pig, including a placard with the pig's identification time point and date. Then separately take images for each wound along with a ruler and a placard displaying the pig ID, wound ID and time point. To assess the wound microvascular perfusion in real time using laser speckle imaging or LSI, image all the wounds in a single recording using a LASCA blood perfusion imager.

To measure trans epidermal water loss or TEWL, place a clean probe cover over the TEWL probe tip, which will be in contact with the wound tissue. Measure each wound five times, first in the center, followed by each corner. Then export all the readings to a spreadsheet.

Perform a harmonic ultrasound or HUSD mapping by scanning the wound with an ultrasound probe from the midline or vertebral column, starting from normal skin and moving towards the lateral side of the pig, over the wounds until the normal skin is again reached. Follow the scanning pattern for each wound in both B mode and tissue elastography mode. Change the annotation after each wound is imaged in both B mode and elasto mode, which is two recordings per wound.

Once all the imaging is complete, cover the burn wounds individually with transparent film dressings or the test dressing. Then place a larger transparent film dressing over the entire wound area. Apply a second layer of rolled gauze loosely around the entire trunk of the pig to absorb any fluid exuding from the wounds.

Cover the gauze loosely with a layer of flexible elastic bandage. Cover the elastic bandage with a final layer of four inch elastic tape. After discontinuing the anesthetic gas upon completion of the bandaging, allow the pig to remain on oxygen for at least five minutes.

To inoculate the burn wounds on day three post burn, disperse the inoculum across the surface of the exposed wounds using a pipette and spread it evenly using a disposable spreader to collect the tissue biopsies for analysis following imaging, infiltrate the area around the wound with 0.5%bupivacaine. Using a disposable scalpel with a size 10 blade, cut a three to four millimeter wide strip from one edge of the wound to the other, keeping small margins of normal skin on both sides. Cut a six millimeter punch biopsy from the wound, either from the wound bed or wound edge, achieve hemostasis after the samples are collected by gently pressing the wound with a sterile gauze, cover the wound with a non-adherent dressing and bandage it as demonstrated previously.

Analysis of the biopsy sample by hematoxylin and neositin staining showed distortion and necrosis of all the histological skin layers. The established biofilm was further validated at day seven post inoculation by colony forming unit. Scan the electron microscopy imaging and immunofluorescence staining.

The burn wounds showed a thick biogenic membrane on the wound surface by day seven post inoculation, confirming the infection and establishment of biofilm. The wound area started to shrink by day 14, but incomplete healing to about 25%of the original wound size was observed by day 56, indicating wound chronicity. Wound chronicity and impaired wound healing were further confirmed by the TEWL, showing high transepidermal water loss at all measured time points.

This was also confirmed by the suppression of the tight junctional proteins, ZO-1 and 2, and the impaired restoration of skin barrier function.