Library

Notes: Biofilms are a combination of microorganisms and extrapolysaccharide matrices. Our laboratory has previously demonstrated that biofilms are present in both acute and chronic wounds. Once biofilms are established, phagocytosis and diffusion of antibiotics are impaired thus contributing to antimicrobial resistance due to increased bacterial virulence. The purpose of this study was to determine the efficacy of two topical antimicrobial agents on partial thickness wounds containing Staphylococcus aureus biofilms. All wounds were inoculated with 106 CFU/ml and covered for 48 hrs under a polyurthene film to promote biofilm formation. Wounds were divided into three treatment groups; triple antibiotic ointment (Polymyxin B sulfate, bacitracin zinc, neomycin), mupirocin cream and untreated control. Wounds were treated twice daily. Wounds were cultured for bacterial quantitation at 24, 48, 72, 96, and 120 hrs. Significant reduction in CFU/ml was observed only after several days of treatments. This finding supports the antimicrobial resistance that occurs when bacteria live within biofilms. Our previous studies demonstrated that both of these agents were able to completely eliminate planktonic S. aureus(106) at the early time points. This study demonstrates that when bacterial biofilms are established in wounds there is a longer response time for topical antimicrobial activity suggesting bacterial resistance.

Notes: Many silver-dressings have been recently developed to prevent infection of burn and chronic wounds. Antimicrobial therapies are usually studied in in-vitro assays, which evaluate the antimicrobial efficacy against free-floating bacteria. In a wound environment bacteria attach to tissues and establish bacterial biofilms. Biofilms are communities of colonies of bacteria and other microorganisms encased in a self produced exopolymeric substance. Biofilms help protect the bacteria from the environment and limit the effectiveness of antimicrobials. In this study we evaluated the efficacy of two silver dressings to eradicate biofilm-associated and planktonic Pseudomonas aeruginosa cells using our biofilm burn wound animal model.Three pigs were used in this study. Second-degree burn wounds were made on one-half of the animal’s back and inoculated with a burn wound isolate of P. aeruginosa. These wounds were then covered for 72 hours with a polyurethane dressing to allow biofilm formation. After 72 hours, additional burn wounds were made on the unwounded half of the animal. The new burns were then inoculated with the same P. aeruginosa strain. At this 72 hour point we had established two bacterial groups, one side of the animal had burns with planktonic bacteria and the other half with biofilm bacteria.Both sides of the animal were treated 20 minutes after inoculation of the planktonic group with the following dressings: 1) Nanocrystalline Silver, 2) Hydrocolloid Silver, or 3) untreated. Wounds were cultured from all treatment groups at 24, 48 and 72 hours post treatment. Sites were cultured quantitatively using a novel flush-scrub technique to obtain both planktonic and biofilm bacterial counts. The baseline biofilm bacterial count was 7.56 LogCFU/ml (prior to treatment).The hydrocolloid dressing significantly reduced planktonic bacteria counts as compared to untreated and nanocrystalline silver dressing at 24, 48 and 72 hours. However, biofilm bacteria counts for both dressings were similar to untreated control at all sample points. Our study demonstrates that both silver dressings showed limited effect against P. aeruginosa biofilm-associated cells when compared to untreated wounds.Based on our results we question the effectiveness of silver dressings for infected wounds that are colonized with biofilm associated-cells. We conclude that anti-biofilm susceptibility models may improve on current antimicrobial sensitivity assays.

Notes: A porcine model of second-degree burn wound was used to evaluate the effect of a newly developed topical oxygen emulsion (TOE) on collagen deposition during wound repair. A total of 6 pigs were used for the study. The burn wounds were treated with air exposure (no treatment), vehicle control or TOE, which contains super-saturated oxygen and releases oxygen in sustained high level when applied topically. Skin wound samples were collected at days 0, 1, 4, 7, 10, 14 and 21 after wounding. Semi-quantitative Reverse Transcription and Polymerase Chain Reactions (RT-PCR) were used to examine the mRNA expressions for type I and type III collagens and matrix metalloprotease-1 (MMP-1). RT-PCR products were run on ethidium bromide gel and analyzed under UV light with Bio-Rad Gel Document 2000 system. The expression intensity was recorded as the mean gray value. One-way analysis of variance was used for statistical analysis. The results showed: 1). Higher mRNA expressions of collagen III and MMP-1 were observed in TOE treatment group compared with air exposure and vehicle control groups. 2). MMP-1 expression increased shortly after wounding with peak at days 4 and 7. 3). Collagen III expression increased earlier with peak at day 10 and day 14. 4). Collagen I expression level increased later than that of collagen III. Significant increase was seen after day 10 with the highest at day 21. However, there was no significant difference between vehicle control and TOE treatment groups. The data suggests that sustained high level of oxygen release by TOE may promote wound repair through the mechanism of increased expressions of type I and type III collagens. The TOE also increased the expression of MMP-1, which might accelerate clearing damaged collagens in the earlier phase of wound healing and promote collagen remodeling in the later phase.This study was supported by grants from DOD/DARPA and the Dermatology Foundation of South Florida.

Notes: To better understand the mechanisms of dermal restoration in burn wound we examined the mRNA expressions, using a porcine secondary-degree burn wound model, of major dermal matrices including type I, III collagens and elastin, basement membrane components of collagen IV and laminins, regulatory Matrix Metalloproteinase MMP 1 and MMP 9, as well as growth factors of bFGF, CTGF, TGFβ, and VEGF. Fresh skin burn wound samples from six pigs were collected at days 0, 1, 4, 7, 10, 14, and 21. Semiquantitative Reverse Transcription and Polymerase Chain Reactions (RT-PCR) were used with gene-specific primers. RT-PCR products were run on ethidium bromide gel and analyzed under UV light with Bio-Rad Gel Document 2000 system. One-way analysis of variance was used for statistical analysis. The results showed:'1) There were increases of all dermal extracellular matrices and growth factors tested; 2) A significant increase of type IV collagen occurred the earliest, while expressions of type I collagen and elastin increased at the latest; 3) A dramatic induction of MMP 1 and MMP 9 was observed shortly after wounding, which lasted through entire experiment; 4) The increased expression of VEGF, which is critical to angiogenesis, occurred early with peak at days 1 and 4, respectively. The expression pattern of bFGF, which has strong promoting effects on both endothelial cells and fibroblasts, is similar to that of VEGF; 5) For growth factors important for matrix deposition, both TGF beta 1 and 3 expressions increased gradually with sustained high level during entire experiment. The expression of CTGF, which is important in matrix production and remodeling, increased in later stage. This study showed the correlation between growth factor expression and dermal matrix deposition, which may have implications in developing strategies to improve burn wound healing.This study was supported by grants from DOD/DARPA and the Dermatology Foundation of South Florida.