Swine Model of Biofilm Infection and Invisible Wounds

All the animal studies were performed in accordance with protocols approved by the Institutional Animal Care and Use Committee (IACUC) #21147. The study was conducted at the Laboratory Animal Resource Center (LARC), Indiana University. We used a female domestic white pig (70-80 lb) in this protocol.

1. Animal acclimatization

1. Upon the arrival of the pigs to the facility, house the animals individually within the same room for at least 3 days for acclimatization and social interaction.
2. Feed the pigs a well-balanced diet. Decide the amount fed based on weight, and follow the recommendations from the manufacturer.
3. Ensure that the animal is fasted for 6-12 h prior to the procedure to prevent nausea, vomiting, and the aspiration of stomach fluids while under anesthesia.

2. Surgery room setup

1. Prepare the anesthesia machine, and make sure it is ready with the rebreathing circuit.
2. Arrange the room for surgery, as described below (Figure 1A).
   1. Cover the procedure table with a sterile drape, and place a circulating water blanket underneath to aid in thermoregulation.
   2. Set up a table with induction supplies and surgery preparation materials. Set up a table with the burner devices and control boxes. Set up the imaging equipment, and make sure that it is turned on.

3. Sedation of the pig

1. Sedate the pig with an intramuscular injection of TKX (Telazol 4.4 mg/kg; ketamine 2.2 mg/kg; xylazine 2.2 mg/kg) at a dose of 1 mL/50 lb. Maintain the pig in the procedure room on 1%-3% isoflurane delivered via a mask.
2. Administer the (pre-operative) analgesics to the pigs according to the IACUC protocol; some examples are as follows: buprenorphine 0.3 mg/mL, 0.01-0.05 mg/kg IM; carprofen 50 mg/mL, 4 mg/kg IM or SQ; fentanyl transdermal 100 mcg/h placed on the pinna of the ear; gabapentin 300 mg capsules, 3-10 mg/kg PO.
   NOTE: For all burn and biopsy procedures, 1 dose of gabapentin will be given the day prior to surgery and 1 dose of carprofen will be given on the day of the procedure. For the Main burning procedure, a fentanyl patch will be placed, and 1 full dose of buprenorphine will be given during surgical prep.

4. Induction of anesthesia

1. Sterilize the ear with alternating 2% chlorhexidine scrub and alcohol at least three times. Insert A 22-18 G 1 in intravenous catheter into the marginal ear vein, and confirm the blood flow. Flush the catheter with saline, and fix the catheter with surgical tape (Figure 1B).
2. Intubate the pig with an appropriately sized endotracheal tube (7-9 mm) once muscle relaxation has been achieved by the inhalation of anesthesia via the mask. Check the muscle relaxation by a loss of jaw tone and a palpebral reflex being observed.
   1. Open the tube, and test the cuff leak using a syringe of air. Insert the tube with the aid of a laryngoscope³⁰.
   2. Inflate the cuff, and secure the tube once proper placement is confirmed. Connect the pig to the rebreathing circuit.
      NOTE: The tube is tied into place over the snout, and roll gauze is used to secure it. Auscultation of the chest is performed with a stethoscope to confirm the proper placement of the tube.
      NOTE: During anesthesia, air is supplied every 5-10 min by closing the pop-off valve and depressing the rebreathing bag until the pressure manometer reaches 20 mm/Hg to prevent positional atelectasis.
3. Monitor the animal and the depth of anesthesia.
   1. Connect the pig to a multi-parameter monitor. The monitor will continuously read the oxygen saturation (SpO₂), pulse rate, end tidal carbon dioxide (EtCO₂), respiratory rate, and temperature. Record the vitals every 10 min throughout the procedure.
   2. Assess the depth of anesthesia by testing the pain reflexes with a hind leg toe pinch prior to beginning the wounding.
      ​NOTE: When necessary, adjust the anesthetic vaporizer to administer additional anesthesia, or wait for a few minutes. Check the pain reflexes and palpebral reflexes regularly throughout the surgery.

5. Animal preparation for burn wounding

1. Disconnect the pig from the anesthesia machine, and move it to the procedure table. Place the pig in the sternal recumbency position, and make sure to secure all the connected lines and tubes (Figure 1C).
2. Reconnect the pig to the anesthesia machine, and maintain the O₂ at 0.8-1.5 L/min and the isoflurane at 1%-3% until the end of the procedure.
3. Administer IV fluids (LRS) to the pig at a drip rate of 8-10 mL/kg/h. Monitor the anesthesia as in step 4.3.

6. Antiseptic preparation and marking of the skin burn site

1. Prepare the wound area by shaving and applying the hair removal cream, as described below (Figure 2).
   1. Shave the pig dorsum in an area of approximately 25 cm width from the vertebral column all the way to the axilla on both sides using electrical clippers.
   2. Apply the hair removal cream to the clipped area, and allow to sit for 3-7 min. Remove the cream along with the hair using clean absorbent towels.
2. Preparation of the burn site
   1. Scrub the area to be wounded with alternating 2% chlorhexidine scrub and 70% isopropyl alcohol at least three times for approximately 5 min. Ensure the scrub is applied in a bullseye pattern (starting at the center and moving outward in a spiral) by personnel wearing sterile gloves.
   2. Mark the wound sites using a sterile burn template and a surgical skin marker (Figure 2B). Mark six to eight wounds (2 in x 2 in) symmetrically on the dorsum.
   3. Cover the areas around the marked sites with a sterile drape to reduce contamination (Figure 2C).

7. Burn wounding procedure

1. Use a burn device, such as an in-house-fabricated custom burner consisting of a 2 in x 2 in stainless steel block (weight: 352 g) connected to a metal stylus, an electronic microstat, and an electronic scale (total weight: 1,714 g; Figure 3).
   1. Set the burner to the desired temperature. Adjust the target temperature for full-thickness wounds at 150 °C (Figure 3A). To do this, adjust the set point (SP) on the control unit to 150 °C. Set the low setpoint to 145 °C and the high setpoint to 155 °C (Figure 1D).
2. Create a full-thickness burn wound of 2 in x 2 in by using heated stainless-steel blocks connected to the burn device and placing them on the skin for 60 s (Figure 3B, C). During the application of the burn, use the electronic scale to ensure uniform pressure is being applied by the burner.

8. Burn wound assessment and imaging

1. Digital photography
   1. Image the wounds using a DSLR camera and an electro-focus short back focus (EFS) 17-55 mm ultrasonic wide-angle lens and a flashlight.
   2. Take a digital photo of the whole pig back, including a placard with the pig identification, timepoint, and date. Then, take images for each wound separately showing a placard with the pig ID, wound ID and timepoint, and a ruler.
   3. Calculate the wound area as the percentage of the original wound size at each timepoint of collection until day 56.
      NOTE: In this work, the wound area was calculated at each timepoint (d0, d7, d14, d28, and d56) as a percentage of the original wound area on d0.
2. Laser speckle imaging (LSI)
   1. For laser speckle imaging, use a blood perfusion imager based on the laser speckle contrast analysis (LASCA) technology to assess the wound microvascular perfusion in real-time.
   2. Take the images of all the wounds in a single recording. Adjust the measured value of the working distance from the laser camera to the wound so that it is consistent for the imaging of each wound (Figure 4A).
   3. Record the perfusion by a series of images taken over a span of 10-15 s. After a wound is imaged, the recording automatically pauses, and the recording is resumed once the camera is adjusted for the subsequent wound. Each time the recording pauses, a marker is added to identify the wound.
3. Transepidermal water loss (TEWL)
   1. Measure the TEWL for each wound using a standard unit, a TEWL probe, and software (Figure 4B). For each wound, place a clean probe cover over the probe tip, which will be in contact with the wound tissue.
   2. Place the probe gently and evenly onto the skin, and start the reading by pressing the Start button on the unit.
   3. Measure each wound five times, first in the center and then on each corner. Then, export all the readings to a spreadsheet (Figure 4B).
4. Harmonic ultrasound (HUSD)
   1. Perform HUSD mapping by scanning the wound with an ultrasound (US) probe from the midline (vertebral column) starting from normal skin toward the lateral side of the pig where there is normal skin again. Follow this scanning pattern for each wound in both B-mode and tissue elastography mode using the ultrasound machine (Figure 4C).
      1. For B-mode scanning, apply sterile ultrasound gel to the wound area, and apply some on the ML-615 high-resolution probe. Annotate each recording with the wound identification label. Start the recording, and move the probe slowly from the midline down the wound until the normal skin on the other side is reached.
         NOTE: After finishing the scanning, the recording is saved and exported from the machine for analysis.
      2. For elastography, switch the ultrasound machine to elasto mode by pressing the Elasto button. Scan the wound again in the same manner as in the B-mode scanning, ensuring that uniform pressure of the probe is maintained to allow for the elastography color indicator (green bars) to remain visible throughout the recording.
         NOTE: Appropriate pressure can be determined by the scale bar on the recording, which appears green when the correct contact is being made (Figure 4D).
      3. Change the annotation after each wound is imaged in both B-mode and elasto mode (two recordings per wound). Change the comment in the software to include the information for the next wound, and repeat the process for the subsequent wounds.

9. Bandaging and dressing

1. Cover the burn wounds individually with transparent film dressings or the test dressing (Figure 5A, B). Place a larger transparent film dressing over the entire wound area (Figure 5C).
2. Apply a second layer of roll gauze loosely around the entire trunk of the pig to absorb any fluid exudate that comes from the wounds. Roll the pig back and forth from its side to slightly on its back to wrap the bandaging material around the pig.
3. Cover the gauze loosely with a layer of flexible elastic bandage (Figure 5D). Ensure that the bandage is not too tight, as applying it too tightly can restrict the breathing and put pressure on the abdomen, which can result in rectal prolapse or different complications.
   NOTE: The elastic bandage is stretchy and can easily be overtightened during application. Pulling it off the roll and allowing it to lay over the edge of the previous wrap can help to prevent overtightening.
4. Cover the elastic bandage with a final layer of 4 in elastic tape (Figure 4E). Again, ensure the application is not too tight, but make sure that the dressing is secured at the top and bottom edge to prevent it from slipping down as the pig moves around post-procedure.

10. Animal recovery and postoperative care

1. Recovery
   1. Discontinue the anesthetic gas upon completion of the wounding, imaging procedure, and bandaging. Allow the pig to remain on oxygen for at least 5 min.
   2. Move the pig, after returning to the primary enclosure, from the transport/lift table to a foam recovery mat in the cage. Raise the automatic waterer, and remove the j-feeder to prevent injury to the pig during recovery.
   3. Cover the pig with blankets (including a warm air blanket) if hypothermia is present. Monitor and record the vitals including the temperature, pulse, respiration rate, and SpO₂ every 10-15 min.
   4. Continuously monitor the pig until it is able to maintain sternal recumbency independently. Once the pig is fully recovered, lower the nipple waterer, and then the pig can also be fed.
2. Pain assessment
   1. Perform a post-operative pain assessment using a modified Glasgow pain scoring form. Ensure the pain assessments are completed by either lab or LARC staff at least every 12 h for the first 3-4 days postoperatively. The frequency of pain scoring is determined by the attending veterinarian. If the animal scores above 5, administer rescue analgesia (buprenorphine or hydromorphone).
   2. Provide analgesia by administering a dose of buprenorphine 0.01-0.05 mg/kg IM pre-procedure, with a second dose given 8-12 h later.
   3. Place a fentanyl patch (100 mcg/h) on the pinna of the ear prior to the burn wounding.
   4. Inject carprofen 4 mg/kg IM or SQ pre-procedure, and then once daily IM, SQ, or PO for 2 days or as directed by the LARC veterinarian.
   5. Give gabapentin 3-10 mg/kg orally, with a dose being given the day prior to the procedure, the morning of the procedure, the evening following the procedure, and then every 12 h for 3-5 days.
3. Diet
   1. Ensure the pigs are recovered, and then allow free access to water and food according to their weight-based ration twice daily.
   2. Provide food enrichment (fresh fruit and vegetables, frozen fruit, marshmallows, yogurt, pudding, etc.), and use these to entice eating if a decreased appetite is observed.
4. Dressing change
   1. Change the bandages at least once weekly or more often if the bandages become soiled or to accommodate treatment strategies.
   2. Change the bandages after imaging while still under anesthesia, or sedate the pig with only TKX for a dressing change.
   3. To replace the bandage, start by carefully removing the soiled bandage using Lister bandage scissors or trauma shears, being mindful to not allow the outside of the dressing to come into contact with the wounds.
   4. Clean the area around the wounds if needed using 0.9% NaCl on clean gauze, and dry the area gently. Follow the procedure steps for bandaging outlined in section 9.
      ​NOTE: If experimental dressings are being applied, these can be applied prior to covering the wounds with the transparent film dressing.
5. Imaging frequency
   1. Obtain imaging (digital photos, LSI, TEWL, and HUSD) at various timepoints throughout the study. Collect imaging data on day −3 (burn wounding), day 0 (inoculation), and day 7, day 14, day 28, day 35, and day 56 post-inoculation.

11. Biofilm preparation and inoculation

1. Inoculum preparation
   1. Prepare a starter plate from a glycerol freezer stock of Pseudomonas aeruginosa (PA01) for a pure culture of the bacterium. Grow a P. aeruginosa culture in low-salt Luria−Bertani agar (LBA), and incubate at 37 °C overnight.
   2. Inoculate 5 mL of low-salt Luria−Bertani broth (LBB) with a single P. aeruginosa colony on the next day, and incubate overnight at 37 °C with shaking at 200 rpm.
   3. To obtain log phase cells, inoculate 200 µL of the overnight culture into 5 mL of LBB, and incubate in the shaker at 200 rpm at 37 °C for 2.5 h.
   4. Measure the optical density at 600 nm (OD600) using a spectrophotometer. Prepare serial dilutions up to 1 x 10⁻⁹ using 100 µL from the culture in 900 µL of sterile LBB.
      NOTE: We started with undiluted samples and ended with 1 x 10⁷ CFU/mL.We obtained countable colonies in the 1 x 10⁷ dilution, so we considered this dilution as the ending dilution.
   5. Spread 100 µL of each dilution on LBA, and incubate overnight at 37 °C. As per standard microbiological protocols, use dilutions showing countable colonies (30-300) for the colony count, and obtain the colony forming units (CFU).
2. Inoculation of the wound
   1. Inoculate 200 µL from the overnight culture into 5mL of LB broth, and incubate in the shaker at 37 °C for 2.5 h.
   2. Measure the optical density of the day culture at 600 nm (OD600). For PA01 inoculation, use 3 x 10⁸ CFU/mL (250 µL of 1 x 10⁸ CFU/mL PA01 is inoculated per wound). Transport the inoculum to the animal facility in a biohazard container.
   3. Disperse the inoculum across the surface of the exposed wounds on day 3 post-burn using a pipette, and spread evenly using a disposable spreader (Figure 6). Keep the wounds open for approximately 15 min before bandaging.
      NOTE: All surgical procedures, inoculation, tissue biopsies, imaging, and bandaging are done under general anesthesia as in sections 3 and 4.
3. Confirming the establishment of infection
   NOTE: To confirm that the wounds have become successfully infected following the inoculation, several approaches are utilized, and wound samples are compared to samples collected from normal skin; below are some examples.
   1. For the pathology-based analysis of samples collected at different timepoints, use the count of colony forming units to estimate an infection (CFU; Figure 7E, F).
      1. Collect 6 mm of wound tissues by punch biopsy. Label and weigh empty 5 mL round-bottom tubes. Transfer the samples to the tubes, and weigh the tubes with the samples.
      2. Dice the tissue with a scalpel on a sterile surface. Perform all the steps in a BSL2 hood.
         NOTE: To make sure that the tissues are easily homogenized, the size should be very small (but not less than 0.5 mm)
      3. Put the sample in the tube, and add 1mL of PBS. Mix and grind the tissue using a hard tissue grinding probe.
      4. Serially dilute (undiluted to 1 x 10⁻⁵) the homogenate, and plate 50 µL of each dilution in selective (Pseudomonas Isolation Agar, PIA) and nonselective (LBA) media.
      5. Incubate all the dilutions under aerobic conditions at 37 °C for 18-24 h. Image the plates with proper lighting conditions.
      6. Select plates with 30-300 colonies, if none of the plates have reached that concentration, use the undiluted plate. Use ImageJ to count the colony numbers, and calculate the CFU per plate by multiplying the average value by the final dilution factor.
   2. Acquire the images from samples collected from day 7 post-inoculation and other timepoints using scanning electron microscopy (SEM) to confirm the presence of the bacterial biofilms (Figure 7G).
      NOTE: Day 7 post-inoculation was selected because it is the day of establishment of biofilm infection and the beginning of burn eschar softening, which allows the penetration of the US waves and, thus, the visualization of the deeper tissues. In Figure 4, check the day 3 US burn wound image, which shows the thick leathery eschar that prevents the US waves from passing through to the deeper tissues.
   3. Stain the sections of the wound biopsies with specific antibodies against P. aeruginosa to confirm the presence of the specific bacteria, as shown in a previous publication¹³ (Figure 7H).
   4. Perform next-generation sequencing (NGS), as published in Sinha et al.³¹. Quantitate the bacteria 16srRNA from the infected wounds and the normal uninfected skin samples collected at different timepoints starting at day 7 post-inoculation until the end of the study.

12. Biopsy collection

1. Collect the tissue biopsies for analysis following imaging on day 7, day 14, day 28, and day 56 post-inoculation. Collect biopsies from each wound only one time to minimize the interference with the healing process.
   NOTE: All surgical procedures, inoculation, tissue biopsies, imaging, and bandaging are done under general anesthesia as in sections 3 and 4.
   1. Infiltrate the area around the wound with 0.5% bupivacaine. Cut a 3-4 mm wide strip from one edge of the wound to the other, keeping small margins of normal skin in both sides, using a disposable scalpel with a size 10 blade. Place the strip into a labeled conical tube filled with 4% buffered formalin for fixation.
      NOTE: For early timepoint imaging and biopsy procedures, a full dose of buprenorphine will be given during surgical prep. For late timepoint biopsy procedures, a half dose of buprenorphine will be given during surgical prep. After all burn and biopsy procedures, gabapentin will be given BID for up to 7 days as advised by the attending veterinarian. Carprofen will be given for days post-op or as advised by the attending veterinarian.
   2. Cut a 6 mm punch biopsy from the wound (either from the wound bed or wound edge). Collect from the wound edge, including part of the normal skin and the wound bed, for different types of analysis.
   3. Remove the sample using sterilized forceps and dissecting scissors. Place the biopsy sample into the appropriate tube or cassette for processing and analysis.
   4. For CFU, SEM, RNA, and FPPE, preserve the samples in tubes with an appropriate buffer. For example, samples can be placed into OCT in cassettes for laser capture microscopy (LCM) and immunohistochemistry (IHC).
   5. Achieve hemostasis after the samples are collected by gently pressing the wound with a sterile gauze. Cover the wound with non-adherent dressing, and bandage as in section 9.

13. Euthanasia and tissue collection

1. Sedate the pig on the day of euthanasia with TKX, and anesthetize with isoflurane. Place an intravenous catheter in the marginal ear vein following the steps outlined in section 3. Intubate the pig following the steps in section 4.
2. Remove the bandage once the pig is anesthetized, and clean the area around the wounds.
3. Complete digital photography, LSI, TEWL, and HUSD imaging. Collect the samples from the wounds and normal skin following the steps outlined in section 12.
4. Once all the required samples are collected, humanely euthanize the pig while still under anesthesia via an intravenous injection of commercially available euthanasia solution (sodium pentobarbital). Use a stethoscope to auscultate to confirm the cessation of the heartbeat and spontaneous respiration.
5. Perform a secondary method of euthanasia, as required by SOM IACUC, by using a scalpel to induce pneumothorax. Transfer the pig carcass into a barrel, and transport to the freezer to be picked up for incineration.