Modelo ex vivo de ferida em pele porcina para avaliação de compostos com atividade antibiofilme bacteriano

Abstract

Wounds result from the breakdown of skin integrity through pathological processes or exogenous damage. One of the main factors for the chronicity of wounds is infection, especially when this is associated with the presence of biofilms. To evaluate new therapies that are effective against bacterial biofilm, it is necessary to have a study model of antibiofilm compounds that mimic the treatment of wounds in vivo, and an option for this is the ex vivo model. Thus, this study aimed to propose a new sensitivity methodology in an ex vivo wound model for the evaluation of compounds with an antibiofilm effect. For this, a strain of S. aureus (ATCC 29213) and a strain of P. aeruginosa (ATCC 27853) had their sensitivity profile tested in vitro in planktonic and biofilm form for ciprofloxacin and polyhexanide biguanide (PHMB), compounds used in the wound care. Subsequently, the porcine skin was cut and disinfected with 70% alcohol and 0.615% sodium hypochlorite, and the wound was made using a number 8 punch and a scalpel. Biofilms were formed in the ex vivo model using a 1.8 x 109 inoculum in BHI medium + 1% glucose and grown for 72 hours at 37 ºC in a bacteriological incubator. Afterwards, treatment was performed with gauze impregnated with ciprofloxacin at CEMB and 2x CEMB concentrations, 2 µg/mL and 4 µg/mL and 8 µg/mL and 16 µg/mL for Staphylococcus aureus and Pseudomonas aeruginosa, respectively. PHMB was used neat (100 µg/ml). The skins crafts were again placed in a bacteriological incubator for 24 hours at 37 ºC. Debridement of the wound was performed and a swab was introduced into the wound. The effect of the compounds was evaluated by counting Colony Forming Units (CFU) and, later, the architecture and viability of the biofilms were observed through confocal microscopy. The reduction in CFU count was 96% and 100% for ciprofloxacin, Staphylococcus aureus and Pseudomonas aeruginosa, respectively. PHMB showed a CFU reduction greater than 70% for both microorganisms. The structure of the Staphylococcus aureus biofilm showed greater dispersion (P=0.0355) compared to the control and lower biomass; the biofilm of Pseudomonas aeruginosa had reduced biomass for both compounds, ciprofloxacin (P=0.0336) and PHMB (P=0.0057). The use of this model brings the possibility of testing new alternatives for the treatment of biofilms in wounds, as it mimics the growth of biofilms in vivo and the treatment in clinical practice.