Data supporting the concept that microbial biofilms are a major cause of non-healing ulcers remain limited. A porcine model was established where delayed healing resulted from methicillin-resistant Staphylococcus aureus (MRSA) infection in full-thickness wounds. At the end of one study a wound remaining open was sampled and a MRSA strain was isolated. This pig-passaged strain was used as the inoculating strain in several subsequent studies. The resulting MRSA wound infections exhibited a greater, more stable tissue bioburden than seen in studies using the parent strain. Furthermore, wounds infected with the passaged strain experienced a greater delay in healing. To understand whether these changes corresponded to an increased biofilm character of the wound infection, wound biopsy samples from studies using either the parent or passaged MRSA strains were examined microscopically. Evidence of biofilm was observed for both strains, as most samples at a minimum had multiple isolated, dense microcolonies of bacteria. However, the passaged MRSA resulted in bacterial colonies of greater frequency and size that occurred more often in concatenated fashion to generate extended sections of biofilm. These results provide a model case in which increasing biofilm character of a wound infection corresponded with a greater delay in wound healing.

Examination of clinical samples indicates bacterial biofilms are present in the majority of chronic wounds, and substantial evidence suggests biofilms contribute significantly to delayed healing. Bacteria in biofilms are highly tolerant of antimicrobials, and little data exist to guide the choice of anti-biofilm wound therapy. Cadexomer iodine (CI) was recently reported to have superior efficacy compared to diverse wound dressings against Pseudomonas aeruginosa biofilms in an ex vivo model. In the current study, the strong performance of CI vs. P. aeruginosa biofilm was confirmed using colony and colony drip-flow in vitro wound biofilm models. Similar in vitro efficacy of CI was also demonstrated against mature Staphylococcus aureus biofilms using the same models. Additionally, the rapid kill of mature S. aureus and P. aeruginosa colony biofilms was visualized by confocal microscopy using Live/Dead fluorescent stains. Superior in vitro efficacy of CI vs. staphylococcal biofilms was further demonstrated against methicillin-resistant S. aureus (MRSA) using multiple biofilm models with log reduction, Live/Dead, and metabolic endpoints. Comparator antimicrobial dressings, including silver-based dressings used throughout and other active agents used in individual models, elucidated only limited effects against the mature biofilms. Given the promising in vitro activity, CI was tested in an established mouse model of MRSA wound biofilm. CI had significantly greater impact on MRSA biofilm in mouse wounds than silver dressings or mupirocin based on Gram-stained histology sections and quantitative microbiology from biopsy samples (>4 log reduction in CFU/g vs. 0.7-1.6, p < 0.0001). The superior efficacy for CI in these in vitro and in vivo models suggests CI topical products may represent a better choice to address established bacterial biofilm in chronic wounds.

A wound biofilm model was created by adapting a superficial infection model. Partial-thickness murine wounds were inoculated with methicillin-resistant Staphylococcus aureus (MRSA). Dense biofilm communities developed at the wound surface after 24 h as demonstrated by microscopy and quantitative microbiology. Common topical antimicrobial agents had reduced efficacy when treatment was initiated 24 h after inoculation compared to 4 h after inoculation. This model provides a rapid in vivo test for new agents to treat wound biofilm infections.

ABSTRACT Necrotic tissue generated by a thermal injury is typically removed via surgical debridement. However, this procedure is commonly associated with blood loss and removal of viable healthy tissue. For some patients and contexts such as extended care on the battlefield, it would be preferable to remove devitalized tissue with a non-surgical debridement agent. In this paper, a proprietary debridement gel (SN514) was evaluated for the ability to debride both deep-partial thickness and full-thickness burn wounds using an established porcine thermal injury model. Burn wounds were treated daily for four days and visualized with both digital imaging and laser speckle imaging. Strip biopsies were taken at the end of the procedure. Histological analyses confirmed a greater debridement of the porcine burn wounds by SN514 than the vehicle-treated controls. Laser speckle imaging detected significant increases in the perfusion status after 4 days of SN514 treatment on deep-partial thickness wounds. Importantly, histological analyses and clinical observations suggest that SN514 gel treatment did not damage uninjured tissue as no edema, erythema, or inflammation was observed on intact skin surrounding the treated wounds. A blinded evaluation of the digital images by a burn surgeon indicated SN514 debrided more necrotic tissue than the control groups after 1, 2, and 3 days of treatment. Additionally, SN514 gel was evaluated using an in vitro burn model that used human discarded skin. Treatment of human burned tissue with SN514 gel resulted in greater than 80% weight reduction compared to untreated samples. Together, these data demonstrate that SN514 gel is capable of debriding necrotic tissue, and suggest that SN514 gel could be a useful option for austere conditions such as military multi-domain operations (MDO) and prolonged field care (PFC) scenarios.

Introduction The future multi-domain battlefield will require prolonged care in theater with evacuation delays of up to seven days. This creates a great risk of infection by not removing any necrotic tissue from burns in a timely manner. Products that can provide non-surgical debridement (NSD) prior to definitive care are essential. The purpose of this study was to evaluate a novel protease (SN514) as a potential NSD agent on deep partial (DPT) and full thickness (FT) burn wounds in a pig and on discarded human skin. Methods On day -4, twenty DPT and FT 3 cm diameter burn wounds were created on the dorsum of 8 anesthetized Yorkshire pigs using appropriate pain control methods. Burns were treated daily from day 0 to day 4 with one of five treatment groups: 1) no treatment, 2) collagenase, 3) 0.5% SN514, 4) 1.0% SN514, or 5) hydrogel vehicle. The wounds were assessed every day, rinsed with 4% chlorohexidine and sterile water, digital and laser speckle images (LSI) were acquired, the treatments reapplied, and then rebandaged. Biopsy strips were harvested across the middle of wounds on day 3 or 4. Samples were paraffin embedded, sectioned, stained with H&E and Masson’s trichrome, and imaged. For the human skin cohort, an abdominoplasty sample was burned to create FT burns. Burned and unburned samples were treated daily with SN514 for up to 4 days. Results During the daily assessment, SN514 treated wounds appeared more debrided than controls. No bleeding, infection, inflammation, or redness were observed. LSI detected significant increases in the perfusion status of SN514 treated wounds for the DPT burns on day 4. Histological analysis indicated a greater debridement of the porcine burn wounds for SN514. Treatment of human burned tissue with SN514 resulted in an 80% weight reduction compared to untreated samples. Conclusions SN514 treatment removed significant amounts of necrotic tissue from DPT and FT burns. SN514 achieved this in the absence of blood loss, without harming adjacent normal skin, and may have potentiated the wounds to allow healing to begin. The observed increase in perfusion after treatments suggests that the wound bed was optimal for spontaneous healing or a temporary covering. Application of SN514 on the burned human tissue successfully debrided both the dermis and hypodermis. Applicability of Research to Practice The ultimate aim is to identify NSD products for prolonged care scenarios that are easy to use, safe, and may improve healing of burns without the need for surgical debridement.