Full-thickness porcine burns infected with Staphylococcus aureus or Pseudomonas aeruginosa can be effectively treated with topical antibiotics

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1 Full-thickness porcine burns infected with Staphylococcus aureus or Pseudomonas aeruginosa can be effectively treated with topical antibiotics David M. Tsai, MD 1 ; Lauren E. Tracy, MD 1 ; Cameron C. Y. Lee, BS 1 ; Florian Hackl, MD 1 ; Elizabeth Kiwanuka, MD, PhD 1 ; Raquel A. Minasian, BS 1 ; Andrew Onderdonk, PhD 2 ; Johan P. E. Junker, PhD 1 ; Elof Eriksson, MD, PhD 1 ; E. J. Caterson, MD, PhD 1 1. Division of Plastic Surgery, Brigham and Women s Hospital, Harvard Medical School, Boston, Massachusetts, 2. Department of Pathology, Brigham and Women s Hospital, Harvard Medical School, Boston, Massachusetts Reprint requests: E.J. Caterson MD, PhD, Brigham and Women s Hospital, Division of Plastic Surgery, 75 Francis Street, Boston, MA Tel: ; Fax: ; ecaterson@partners.org Funding: No external funding to disclose Manuscript received: January 20, 2015 Accepted in final form: January 17, 2016 DOI: /wrr ABSTRACT Burn and blast injuries are frequently complicated by invasive infections, which lead to poor wound healing, delay in treatment, disability, or death. Traditional approach centers on early debridement, fluid resuscitation, and adjunct intravenous antibiotics. These modalities often prove inadequate in burns, where compromised local vasculature limits the tissue penetration of systemic antibiotics. Here, we demonstrate the treatment of infected burns with topical delivery of ultrahigh concentrations of antibiotics. Standardized burns were inoculated with Staphylococcus aureus or Pseudomonas aeruginosa. After debridement, burns were treated with either gentamicin (2 mg/ml) or minocycline (1 mg/ml) at concentrations greater than 1,000 times the minimum inhibitory concentration. Amount of bacteria was quantified in tissue biopsies and wound fluid following treatment. After six days of gentamicin or minocycline treatment, S. aureus counts decreased from 4.2 to 0.31 and 0.72 log CFU/g in tissue, respectively. Similarly, P. aeruginosa counts decreased from 2.5 to 0.0 and 1.5 log CFU/g in tissue, respectively. Counts of both S. aureus and P. aeruginosa remained at a baseline of 0.0 log CFU/mL in wound fluid for both treatment groups. The findings here demonstrate that super-therapeutic concentrations of antibiotics delivered topically can rapidly reduce bacterial counts in infected full-thickness porcine burns. This treatment approach may aid wound bed preparation and accelerate time to grafting. Burn and blast injuries are devastating consequences of modern military conflicts. 1 5 Successful management hinges on timely, aggressive wound care that focuses on decreasing contamination, removing necrotic tissue, and initiating antimicrobial prophylaxis. Invariably, these wounds are subject to infections, which cause profound morbidity and mortality Thus, the primary goal in treating these injuries should be infection prevention. Unfortunately, current treatment modalities frequently prove inadequate in this regard. Moreover, these injuries often occur in the field, where no frontline measures exist that can reliably curtail invasive infections and prevent subsequent sepsis. 11,12 With the growing threat of terrorist attacks on civilian soil and the increased incidence of civilian casualties in modern conflicts, the need for a safe and effective way to prevent and treat burn infections has become strikingly important to the population as a whole. Unlike systemic antibiotic prophylaxis, topical antimicrobials have become well established as an adjunct to early excision and grafting They are exceptionally advantageous in the context of burns, which are encumbered by devitalized tissue and diminished blood supply. By administering antimicrobials directly to a wound, higher concentrations can be reached without the need for a healthy, intact vascular network. The risk of adverse systemic side effects is also dramatically diminished, making topical antimicrobials much more appealing than their systemic counterparts. Without their use in the immediate postburn period, burn wounds are quickly colonized by staphylococcus, streptococcus, and gram negative species. Of these, Staphyloccocus aureus, Pseudomonas aeruginosa, andklebsiella pneumoniae have the most potential to cause invasive wound infections, and S. aureus and P. aeruginosa are most frequently associated with bacteremia Silver sulfadiazine, mafenide acetate, silver nitrate are some of the most commonly used topical antimicrobials today for burn care. 14,15,21 23 These agents exist as creams or aqueous solutions that are applied with dry or moist dressings, which often require multiple dressing changes. 24 Despite the proven efficacy in decreasing burn infections, using these agents in the correct manner can strain a facility s time and resources. 11,12 Further, these topical antimicrobials need to be applied immediately and are effective only after proper cleansing and debridement. 12 This level of wound care is unrealistic in the battlefield, 356 Wound Rep Reg (2016) VC 2016 by the Wound Healing Society This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.

2 Tsai et al. Topical antibiotics for treatment of infected burns Figure 1. Polyurethane wound enclosure device. Shown here is a schematic of the polyurethane wound enclosure device. A circumferential adhesive rim permits complete enclosure of a wound, and thereby establishes a protected, incubator-like environment. A self-sealing injection port allows easy sampling of wound fluid or delivery of liquid solutions such as antibiotics, growth factors, or analgesia in precise amounts and concentrations. and first responders are often not equipped with these agents. Finally, these topical antimicrobials have no proven ability to treat burn wounds plagued with an existing infection. 21,25 In these cases, a shift to systemic antibiotic therapy is often warranted. 26 Nonetheless, systemic agents prove less than ideal because they generally exhibit poor tissue penetration, especially in burns where local wound vasculature is compromised. Higher doses are thus needed to reach therapeutic concentrations in tissue, which unavoidably increases the risk of adverse side effects associated with systemic antibiotics such as nephropathy and disruption of GI flora. This study is in response to the need for a treatment modality that can safely and effectively decrease contamination of infected burn wounds. Building upon our earlier work on a topical antibiotic delivery system that uses a polyurethane wound enclosure device or wound chamber (Figure 1), we extended the treatment methodology to full-thickness porcine burns infected with S. aureus or P. aeruginosa. We hypothesized that this system would be effective in rapidly reducing the bacterial burden of infected burn wounds. Moreover, by correlating infection clearance over time with changes in levels of inflammatory cytokines, we sought to compare antibiotics and pinpoint an optimal duration of treatment with respect to balancing rapid wound decontamination with a possible delay of local wound healing. MATERIAL AND METHODS Animals All animal procedures performed in this study received prior approval from the Harvard Medical Area Standing Committee on Animals (Protocol # 00693), and conformed to federal statutes and regulations regarding animal use. Nine female Yorkshire pigs (Parson s Farm, Hadley, MA) weighing between 50 and 65 kg were used in the study. The animals were socially housed and acclimatized for 72 hours prior to any procedures. Burn device We used a custom burn device (Figure 2) to create standardized full thickness burn wounds (Figure 3). It consists of an aluminum disk that is insulated with cork on all sides except the base, which comes into direct contact with the skin. A probe is embedded in the device to Figure 2. Custom aluminum burn device. (Left) An aluminum disk, 2 cm in diameter, is insulated with cork on all sides except the base, which comes into direct contact with the skin. An embedded probe allows accurate measure of the temperature drop upon application of device to skin. This allows precise calculation of the amount of heat energy transferred for each wound, ensuring standardized full-thickness burns. (Right) The device is heated up to 230 8C and applied to the skin until the temperature dropped to 180 8C. To ensure equal force of application among all wounds during the burn creation, a custom plunger device calibrated to 10 Newton of force is used to apply the burn device to the skin. This helps eliminate operator variance and allows comparison of wounds from the same pig, from different pigs, and from other investigators. Wound Rep Reg (2016) VC 2016 by the Wound Healing Society 357

3 Topical antibiotics for treatment of infected burns Tsai et al. Figure 3. Standardized full-thickness burns. Histological image of the depth of burn as demonstrated by the homogenization of collagen fibrils. The burns extended through the epidermis, dermis, and parts of the subcutaneous adipose tissue. Immediately after burn, each wound was inoculated with 10 8 CFU/mL of bacteria at a depth of 8 mm. Excisional debridement was performed at approximately the level of the panniculus carnosus. accurately record the drop in temperature upon application of the device to the skin. This allows precise calculation of the amount of heat energy transferred for a defined surface area during the burn (32 calories/cm 2 ). The device is heated up to 230 8C and applied to the skin until the temperature dropped to 180 8C. To ensure equal force of application among all wounds during the burn creation, a custom plunger device calibrated to 10 Newton of force is used to apply the burn device to the skin (Figure 2). This helps eliminate operator variance and allows comparison of wounds from the same pig, from different pigs, and from other investigators. Porcine burn model Anesthesia was induced with intramuscular administration of 4.4 mg/kg zolazepam and tiletamine (Telazol; Fort Dodge Veterinaria, Madrid, Spain) and 2.5 mg/kg xylazine (Xyla-Ject; Phoenix Pharmaceutical, St. Joseph, MO). General anesthesia was maintained with 2% isoflurane (Hospira, Lake Forest, IL) and oxygen via endotracheal intubation. A pulse oximeter was used to monitor oxygen saturation and heart rate. Temperature was maintained at 38 8C with heating blankets. The dorsum of the pig was waxed and shaved. Skin was disinfected with 10% povidone-iodine scrub (Purdue Pharma, Stamford, CT) and 70% isopropanol (Aaron Industries, Clinton, SC). Up to 26 full thickness circular burns, 2 cm in diameter were created on each pig using the burn device described above. The depths of burns were verified histologically (Figure 3). With a 32-gauge needle, each wound was inoculated with 10 8 colony-forming units (CFU) of the chosen pathogen diluted in 1 ml saline, at a depth of 8 mm (Figures 3 and 4A), in the subeschar plane. To prevent cross-contamination and confounding, each pig was only inoculated with one type of microbe, either S. aureus (ATCC 12600) or P. aeruginosa (ATCC 27853). The burns were subsequently covered with sterile gauze and leukotape (BSN Medical, Hamburg, Germany), and allowed to incubate for 3 days (Figure 4B). On day 3 postburn, the eschar and necrotic tissue were debrided from all wounds in a standardized fashion, at the level of the panniculus carnosus and beyond the zone of coagulation at the zone of stasis with a standardized sized cutting tool (Figure 4C). Wounds were divided into those receiving dry treatment and topical treatment. For those receiving the latter, polyurethane enclosure devices were applied (Figure 4D), and randomly assigned to one of two groups using a random number generator ( random.org): one receiving 10 ml of saline, and the other receiving 10 ml of the chosen antibiotic (2 mg/ml gentamicin or 1 mg/ml minocycline, >1,000 times minimum inhibitory concentration). Concentrations were chosen to roughly equate MICs and to allow comparison with previous studies. Each pig received only one antibiotic (gentamicin or minocycline) during the length of the experiment. For accuracy and consistency, devices were emptied daily, sampled, and reinjected with 10 ml of the chosen treatment solution; effectively diluting bona fide wound fluid by a factor of 10. This was later corrected in subsequent analyses. Daily fluid was collected from the polyurethane devices with an 18-gauge needle and flash frozen for later analysis. On days 3, 6, and 9 after burn creation, tissue was sampled at an appropriate depth using a 6 mm punch biopsy, making sure to obtain a small portion of healthy underlying tissue. Specimens were placed in dry, sterile cryogenic vials and flash frozen for later analysis. Biopsied wounds were subsequently excluded from the remainder of the experiment. Wounds were treated as separate entities and as N, with at least 6 wounds for each time point and treatment group. Quantitative bacterial cultures Tissue punch biopsies and fluid samples were collected as described above. Analysis was performed by the Brigham and Women s Hospital Clinical Microbiology Laboratory. Quantitative bacteriology was performed to assess infections levels of S. aureus and P. aeruginosa remaining in tissue and fluid after excisional debridement, prior to subsequent treatment. Counts were then subsequently performed to quantify infection clearance on days 3, 6, Wound Rep Reg (2016) VC 2016 by the Wound Healing Society

4 Tsai et al. Topical antibiotics for treatment of infected burns Figure 4. Inoculation, debridement, and treatment. (A) On day 0, after the burns were created, Jackson s zones of coagulation, stasis, and hyperemia were immediately evident. A 27-gauge needle with a custom needle-stopper was then used to inoculate the burns with 10 8 CFU/mL of bacteria at a depth of 8 mm. (B) Appearance of burns after 3 days of incubation. (C) Wounds after excisional debridement. (D) Application of polyurethane device on debrided wound. Devices were then filled with 10 ml of either saline or antibiotics. Samples were serially diluted, plated on blood agar plates, and examined for S. aureus and P. aeruginosa growth. Peripheral blood analysis At appropriate time points, venous blood was drawn from the ear veins and sent to the Clinical Laboratory at Brigham and Women s Hospital for analysis. Blood counts for white blood cells, red blood cells, hemoglobin, and hematocrit were determined. For gentamicin-treated pigs, serum concentrations of gentamicin were assessed to monitor systemic uptake and risk of toxicity. Inflammatory mediators Fluid samples from the polyurethane devices were diluted, activated, and analyzed with a sandwich enzyme linked immunosorbent assay using kits specific for porcine IL-1b (DNF ), IL-6 (CBA ), TNF-a (EKQ ; R&D Systems, Minneapolis, MN) per manufacturer s protocol. Reactions were stopped with 2M H 2 SO 4 (Sigma Aldrich, St. Louis, MO) and plates read with a SpectraMax M5 Microplate Reader and Soft- Max Pro software (Molecular Devices, Sunnyvale, CA). Statistical analysis Statistical analysis was performed using GraphPad Prism 6.0 (GraphPad, La Jolla, CA). Data is presented as mean 6 SEM. Statistical comparisons were performed using a twoway ANOVA with a Bonferroni posttest. p-values under 0.05 were considered significant. RESULTS Reduction of S. aureus levels in tissue After excisional debridement on day three, an infection level of log CFU/g remained in the wound tissue. Treatment with gentamicin at 2 mg/ml (>1,000 times MIC) further decreased S. aureus levels to log CFU/g (n 5 6) by day 6 (after 3 days of treatment) and log CFU/g (n 5 9) by day 9 (after 6 days of treatment; Figure 5). Treatment with minocycline at 1 mg/ ml (>1,000 times MIC) decreased S. aureus counts to log CFU/g (n 5 8) and log CFU/g (n 5 6) by day 6 and 9 (after 3 and 6 days of treatment), respectively. Levels in saline-treated tissue increased to log CFU/g (n 5 12) by day 6 and subsequently decreased to (n 5 16) by the terminal endpoint, day 9. In dry-treated wounds, levels of S. aureus increased to log CFU/g (n 5 11) by day 6 and decreased to log CFU/g (n 5 14) by the terminal endpoint. Compared with saline treatment and dry treatment, the differences observed with the antibiotic treated groups were statistically significant (p < ). Reduction of S. aureus levels in wound fluid S. aureus levels in wound fluid from both gentamicintreated wounds and minocycline-treated wounds remained at the baseline of log CFU/mL from the start of treatment on day 3 to the terminal endpoint, day 9 Wound Rep Reg (2016) VC 2016 by the Wound Healing Society 359

5 Topical antibiotics for treatment of infected burns Tsai et al. Figure 5. Quantitative bacterial cultures in wound tissue. Tissue biopsies from wounds infected with S. aureus (left) and P. aeruginosa (right) and treated with either gentamicin or minocycline were analyzed for amount of bacteria, in CFU, per gram of tissue. Treatment with saline or dry dressings served as controls. Both topical administration of gentamicin and minocycline significantly reduced bacterial counts at all time points; whereas bacterial counts were increased in the control groups. Error bars represent SEM. (Figure 6). In contrast, wound fluid from saline-treated wounds increased from the baseline of log CFU/mL to log CFU/mL, and log CFU/mL by day 6 and 9, respectively (Figure 6). The difference was statistically significant (p <0.0001). Reduction of P. aeruginosa levels in tissue An infection level of log CFU/g remained in tissue after excisional debridement three days after burn creation. Treatment with gentamicin at 2 mg/ml (>1,000 times MIC) reduced levels to log CFU/g (n 5 7) Figure 6. Quantitative bacterial cultures in wound fluid. Fluid samples from wounds infected with S. aureus (left) and P. aeruginosa (right) and treated with either gentamicin or minocycline were analyzed for amount of bacteria, CFU, per millimeter of fluid. Treatment with saline served as controls. No fluid was collected with dry dressings. Addition of either gentamicin or minocycline rapidly reduced bacterial counts in fluid compared with saline treatment. Error bars represent SEM. 360 Wound Rep Reg (2016) VC 2016 by the Wound Healing Society

6 Tsai et al. Topical antibiotics for treatment of infected burns by day 6 (after 3 days of treatment) and to log CFU/g (n 5 7) by day 9 (after 6 days of treatment). Treatment with minocycline at 1 mg/ml (>1,000 times MIC) decreased initial levels to log CFU/g (n 5 7) and log CFU/g (n 55) by day 6 and 9 (after 3 and 6 days of treatment), respectively (Figure 5). In contrast, saline-treated wounds increased to log CFU/g (n 5 14) and log CFU/g (n 5 14) by day 6 and 9, respectively. In dry-treated wounds, levels increased to log CFU/g (n 5 12) by day 6 and increased slightly to log CFU/g (n 5 12) by the terminal endpoint, day 9. Again, compared with saline treatment and dry treatment, the differences observed with gentamicin treatment and minocycline treatment were statistically significant (p <0.0001). Reduction of P. aeruginosa levels in wound fluid P. aeruginosa levels in wound fluid from gentamicintreated wounds and minocycline-treated wounds remained at the baseline of log CFU/mL from the start of treatment on day 3 to the terminal endpoint, day 9 (Figure 6). In contrast, wound fluid from saline-treated wounds increased from the baseline of log CFU/mL to log CFU/mL and log CFU/mL by day 6 and 9, respectively. The difference was statistically significant (p < ). Systemic uptake Serum concentrations of gentamicin were undetectable using the in-house assay (<0.4 mg/ml). No signs of systemic toxicity were observed in any pigs. Inflammatory response On day 6, in S. aureus infected burns, levels of IL-6 and TNF-a were significantly lower in minocycline-treated and gentamicin-treated wounds, respectively, compared with saline controls (Figure 7). On day 9, levels of IL-1b were significantly higher in gentamicin-treated wounds compared with saline controls. There was no significant difference observed between gentamicin and minocycline groups. In P. aeruginosa infected wounds, levels of Il-6 and TNF-a were significantly lower in both gentamicin-treated and minocycline-treated wounds compared with saline controls on day 6 (Figure 7). Levels of IL-1b in minocyclinetreated wounds were significantly increased by day 9 compared with both saline controls and gentamicin-treated wounds. Conversely, IL-6 levels were significantly decreased in minocycline-treated wounds compared to both saline controls and gentamicin-treated wounds on day 9. Levels of Il-6 in gentamicin-treated wounds were significantly increased compared with saline controls on day 9. Wound bed preparation After six days of treatment, the antibiotic-treated wounds exhibited red, healthy granulation tissue, which appeared ready for skin grafting (Figure 8A,B). In comparison, the saline-treated wounds were covered with what appeared to be a layer of biofilm, and exhibited erythema and induration. Unsurprisingly, these wounds demonstrated high levels of infection when biopsied (Figure 8C). The drytreated wounds had a slightly better appearance than saline-treated wounds; however, bacteria levels in these wounds still exceeded 10 5 CFU/g at the terminal endpoint (Figure 8D). DISCUSSION This study demonstrates the effective treatment of infected full-thickness porcine burns with a topical antibiotic treatment platform. Building upon the principles and advantages of topical antimicrobials, this modality improves on existing formulations. In particular, the polyurethane enclosure device can establish a controlled incubator-like wet environment, which is conducive to optimal wound healing and allows direct delivery of antibiotic solutions (Figure 1). By diluting a typical single IV antibiotic dose in a small volume in contrast to systemic distribution, we can achieve concentrations in orders of magnitude greater than intravenous delivery (>1,000 times MIC). This allows for rapid reduction of bacterial burden while isolating and protecting the wound from both the environment and potential contamination from other wounds. From a practical standpoint, the device has a self-adhesive rim that facilitates application to the wound. The injection port allows timely administration of antibiotics that can be reconstituted with water, or analgesic solutions to provide immediate pain relief. In recent years, several other topical delivery strategies have been described. Collectively, these efforts represent a paradigm shift in the way clinicians are thinking about treating and managing wounds. For instance, irrigationbased systems exist as described by Svedman, 32 Kinetic Concepts Incorporated, 33 and Zamirowski, 34 in which antimicrobials are delivered via pulsatile irrigation. Although promising, these systems have difficulty delivering precise amounts of antibiotics since it is difficult to gauge how much is actually absorbed. Other strategies have included antimicrobial polymers in the form of dressings, sponges, or patches. These polymers are engineered with hydrocolloids, alginates, collagen, and others. 35,36 Although some studies have been encouraging, a shared limitation among these different strategies is that they are inherently fixed systems, and lack the capability of altering the antimicrobial agent or titrating the dose. In comparison, our delivery system enables the clinician to precisely administer an antibiotic in varying concentrations at one s discretion. Further, the injection port allows fluid sampling and removal or exchange of fluid. This is conducive to initial broad-spectrum antibiotic administration and a facile switch to culture-directed narrow-spectrum antibiotics. To evaluate our treatment system, we developed a porcine model to study infected full-thickness burns. Using an aluminum burn device, we delivered equivalent amounts of heat energy to each wound under equal pressure to ensure full-thickness burns were standardized across all animals (Figure 2). Subsequently, we tested the treatment on burns infected with either S. aureus or P. aeruginosa, two of the most common pathogens associated with bacteremia and invasive burn infections To properly assess bacterial clearance, we performed tissue biopsies, which has been the historical standard in diagnosing invasive burn infections and correlating counts with burn Wound Rep Reg (2016) VC 2016 by the Wound Healing Society 361

7 Topical antibiotics for treatment of infected burns Tsai et al. Figure 7. Levels of inflammatory cytokines in wound fluid. Fluid samples from wounds infected with S. aureus (left) and P. aeruginosa (right) and treated with either gentamicin or minocycline were analyzed for concentrations of IL-1b, IL-6, and TNF-a. Levels of inflammatory markers were generally decreased after 3 days of treatment relative to saline control. This was primarily noted in IL-6 and TNF-a levels. After 6 days of treatment, some inflammatory markers increased in antibiotic-treated groups relative to saline control such as IL-1b and IL-6. Error bars represent SEM. 362 Wound Rep Reg (2016) VC 2016 by the Wound Healing Society

8 Tsai et al. Topical antibiotics for treatment of infected burns Figure 8. Wound bed preparation six days posttreatment. Clinical appearance of burns after treatment with gentamicin (A), minocycline (B), saline (C), and dry dressings (D). sepsis. 25,37,38 We also assessed bacterial levels in wound fluid, since high levels of bacteria in fluid can often serve as a continuing inoculant in burn wounds, and in a healthcare environment can act a route of nosocomial transmission or cross-contamination among wounds. 25 Additionally, since gentamicin is commonly associated with systemic toxicity, serum concentrations were monitored and served as a measure for systemic uptake for our delivery system. Our results establish the capability of ultrahigh concentrations of topical antibiotics to dramatically reduce bacterial counts in infected burn wounds. Despite the standard practice of excisional debridement, there remained considerable levels of bacteria in the tissue, up to 10 4 CFU/g in S. aureus infected burns, serving to illustrate that debridement alone is insufficient and adjunct antibiotics are necessary. Subsequently, bacterial counts in tissue from S. aureus infected burns dropped by 10,000 fold after 6 days of treatment with topical gentamicin or minocycline. In contrast, bacterial counts dramatically increased in the control groups, and by the terminal endpoint, there was 100,000-fold difference in bacterial counts between control groups and treatment groups. In P. aeruginosa infected burns, less bacteria remained in tissue after initial debridement, but by the terminal endpoint in dry and saline control groups, bacteria levels increased to 10 5 and 10 7 CFU/g, respectively, compared with negligible levels in gentamicin and minocycline groups. Overall, this data indicates that this treatment method is not only effective for rapidly reducing high bacterial burden in burns but can doubly serve as a prophylactic measure. The ability to achieve two crucial aims of burn management simultaneously is a powerful advantage when compared with current modalities such as topical antimicrobials and systemic antibiotics, which can only accomplish one. One of the key benefits of reducing bacterial burden is wound bed preparation, which is in line with the ultimate goal of skin closure. It is well known that infections can impede wound healing and graft take, and increase scarring. As shown in Figure 8, antibiotic treated wounds exhibited a healthy wound bed that appeared prime for skin grafting. As an additional measure of the efficacy of topical antibiotics, we assessed relative changes in levels of inflammatory markers after 3 and 6 days of treatment. We acknowledge these changes to be a complex interplay of five factors: (1) initial burn, (2) trauma from excisional debridement, (3) type of pathogen, (4) bacterial levels, and (5) type of antibiotic administered. Since all wounds were subjected to the same degree of burn and surgical trauma, it is reasonable to conclude that the bacteria and antibiotics are primarily responsible for any differences in cytokine levels. Although difficult to interpret in isolation, when viewed as a whole we recognize a trend in which the levels of inflammatory markers were decreased after 3 days of treatment relative to saline control (Figure 7). This can be primarily noted in IL-6 and TNF-a levels. Ostensibly, these differences can be attributed to the respective Wound Rep Reg (2016) VC 2016 by the Wound Healing Society 363

9 Topical antibiotics for treatment of infected burns Tsai et al. reduction in bacterial counts. After 6 days of treatment, however, some inflammatory markers increased in antibiotic-treated groups relative to saline control such as IL-1b and IL-6. The challenging question to answer is whether this is a consequence of local inflammation and cytotoxicity caused by prolonged exposure to ultrahigh concentrations of antibiotics or if the increased levels are due to a mass release of products from lysed bacteria that subsequently triggers a surge in inflammatory cytokines. Future work focused on further detailing the inflammatory response will undoubtedly unveil more about these intricate interactions. Within this study, however, we can view these inflammatory markers in conjunction with levels of bacteria on corresponding days. By day 6, most of the bacterial contaminants have been dramatically reduced. Taking this into account, we aim to titrate or cease antibiotics in the future after 3 days of treatment as a way to balance bacterial reduction with any inflammatory response that may potentially impede healing. Nevertheless, we believe the benefits of bacterial reduction outweigh the temporary delay in wound healing, as it can profoundly prepare the wound bed for grafting. With the application of this treatment to large burns, we expect the toxicity to remain minimal relative to systemic antibiotics, since the uptake is limited by the compromised vasculature of burns. Higher concentrations can then be used, as we have exhibited in this study, and greater therapeutic levels reached at a much lower risk of toxicity. Nevertheless, the use of any antibiotics regardless of formulation should be accompanied with vigilant monitoring and should be guided by serum levels. To further mitigate risk, the concentrations of the topical antibiotics are easily titrated. In conclusion, ultrahigh concentrations of topical gentamicin or topical minocycline can rapidly reduce the bacterial burden of full-thickness porcine burns infected with either S. aureus or P. aeruginosa. The polyurethane enclosure device permits precise, controlled delivery while minimizing the risk of systemic toxicity. Treatment with this proposed methodology may aid wound bed preparation and accelerate time to subsequent grafting. ACKNOWLEDGMENTS We would like to thank Andrea Dubois for her exceptional technical assistance and Anders Carlsson and Olle Engstrom for their contributions to the porcine experiments. Conflict of Interest/Disclosure Statement: Dr. Eriksson has filed a number of patents related to the wound chamber platform technology, and is a member of a LLC that deals with wound healing. The remaining authors have no commercial associations or financial disclosures that might pose or create a conflict of interest. REFERENCES 1. Atiyeh BS, Gunn SWA, Hayek SN. 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