In vitro toxicity and activity of Dakin's solution, mafenide acetate, and amphotericin B on filamentous fungi and human cells.

OBJECTIVES: Posttraumatic invasive fungal infections threaten critically injured combat-related injuries and require a combination of extensive surgery and systemic antifungal therapy, along with topical antimicrobials used adjunctively to control the infection. We evaluated the in vitro activity of topical agents in varying combinations and concentrations against molds from patients that were responsible for wound invasive fungal infections and the topical agents' toxicity to human cells. METHODS: Mafenide acetate solutions (2.5%, 5%, and 7.5%), amphotericin B solutions (2 µg/mL, 2 mg/mL, and 20 mg/mL), SMAT (5% mafenide acetate in combination with 2 µg/mL, 2 mg/mL, and 20 mg/mL amphotericin B), and Dakin's solutions (buffered sodium hypochlorite) (0.5%, 0.25%, and 0.125% and 10-fold serial dilutions of 0.25%-0.00000025%) were evaluated for antifungal activity against 4 molds using a time-kill assay using standard conidial suspensions of 5 × 10(4) colony-forming units per milliliter. To assess cellular toxicity, confluent monolayers of human keratinocytes, dermal fibroblasts, and osteoblasts were exposed to these topical agents. Based upon efficacy and toxicity ratios, an additional 10 molds were screened with selected concentrations of the topical agents for antifungal activity and toxicity. RESULTS: All the topical agents seemed to have a dose-dependent killing with only mafenide acetate showing time killing associated with prolonged contact. There was overall evidence of dose-dependent cytotoxicity of the various topical agents against the various cell lines tested, but there did not seem to be increased cell death with continued exposure to the agents over time. Dakin's solution exhibited dose-dependent toxicity and efficacy with 0.00025% appearing to optimize those parameters. CONCLUSIONS: Mafenide acetate and amphotericin B did not seem to persistently meet the toxicity and efficacy balance as consistently as Dakin's solution.

Cytotoxicity to human leukocytes by topical antimicrobial agents used for burn care.

We tested two topical antimicrobial agents (TAAs), silver sulfadiazine and mafenide acetate, to determine their cytotoxic effects when human lymphocytes and neutrophils were incubated with the agents in vitro for 30 minutes. Dilute concentrations of both TAAs markedly inhibited neutrophil respiratory burst activity and mitogen-stimulated lymphocyte proliferation (p < 0.05). The components of silver sulfadiazine (silver and sulfadiazine) were separately tested, and each component inhibited both neutrophil and lymphocyte functions. Mafenide acetate markedly decreased intracellular Ca+2 flux in lymphocytes. The effects of the TAAs were partially reversed when cells were washed and resuspended in medium after they were exposed in vitro to the TAAs. Commonly used TAAs may contribute to local immune dysfunction in the patient with burns. Because evidence suggests that T lymphocytes may participate in wound healing, prolonged treatment with TAAs may also effect certain aspects of wound healing.

The cytotoxic effects of commonly used topical antimicrobial agents on human fibroblasts and keratinocytes.

This study evaluated the cytotoxicity of commonly used topical agents to human dermal fibroblasts and epidermal keratinocytes, which play a prominent role in wound healing. The effects of these topical agents were assessed using two separate assays for the fibroblasts--tritiated thymidine incorporation and the uptake of a vital dye (neutral red). Keratinocytes were evaluated with the neutral red assay. Serial dilutions of each of 10 commonly used topical agents produced decreases in both the uptake of neutral red and the incorporation of thymidine at clinically relevant doses. Only Neosporin G.U. irrigant showed no significant difference compared with controls in the assays for both the fibroblasts and the keratinocytes. Careful attention must be paid to which agent is used in the clinical setting, since many of these can have profound effects on cells that influence wound healing.

Cytotoxicity to cultured human keratinocytes of topical antimicrobial agents.

Cultured skin grafts administered clinically for closure of burn wounds may be contacted by topically applied antimicrobial agents. A study was performed to assess whether commonly used topical antimicrobial agents are toxic to cultured human keratinocytes (HK) in vitro. Serum-free MCDB 153 culture medium containing Neosporin G.U. irrigant (Neomycin, 40 micrograms/ml-polymyxin B sulfate, 200 units/ml) and a standard tissue culture antimicrobial agent of penicillin (10,000 units/ml)-streptomycin (10,000 micrograms/ml)-amphotericin B (25 micrograms/ml) had no effect on the keratinocyte growth rates when compared to standard MCDB 153 medium without antibiotics. Medium containing Sulfamylon (mafenide acetate, 0.85%), Polysporin (polymyxin B sulfate, 1 x 10(4) units/ml-bacitracin, 500 units/ml), gentamicin sulfate (0.1%), modified Dakins solution (25%), and acetic acid (0.25%) all showed statistically significant (P less than 0.01) decreases in keratinocyte growth rates. This data suggests that commonly applied antimicrobials may not be appropriate for cultured grafts in the concentrations that are used clinically.

In vitro toxicity of topical antimicrobial agents to human fibroblasts.

Topical antimicrobial agents are essential to optimal burn care. However, exposure of WI-38 human diploid fibroblasts (ATCC CCL 75) and fresh donor human dermal fibroblasts to silver sulfadiazine and mafenide acetate results in a significant reduction in cell proliferation, as determined by hemocytometer cell counts and total matrix protein assays, within 48 hr of exposure. Changes in cellular morphology and progressive deterioration of cytoplasmic organelles and the nucleus are seen with phase-contrast microscopy and transmission electron microscopy. These findings may explain the clinical observation of delayed wound healing after the use of topical antimicrobial agents.