Antimicrobial and Antibiofilm Efficacy of a Next Generation Multifunctional metal EDTA Complex in an in vitro artificial wound slough biofilm model.

Abstract: Introduction: The presence of slough, which serves as a reservoir for microbes and biofilms, damaged/devitalized cells and inflammatory chemokines, is a hallmark of chronic wounds. Slough attracts microbes to the wound bed and form biofilms, which are particularly hard to treat with conventional topical antimicrobials. In a previous study, multifunctional metal complexes (MMC) showed strong antimicrobial and antibiofilm efficacy in minimum inhibitory (MIC) and minimum bactericidal (MBC) tests and in vitro biofilm models, such as the CDC bioreactor and drip flow models. We demonstrated an artificial wound slough (AWS) model that can be mimetic of wound eschar and slough, and which has been successfully applied to analyse the efficacy of debridement. In this study, the AWS model has been further developed to include the formation of biofilm under the slough layer to produce a new cost-effective artificial wound eschar – biofilm model. The AWS-biofilm model was applied to evaluate antibacterial and antibiofilm efficacy of patent protected EDTA and silver based containing multifunctional complexes. Method: Ag-Zn EDTA complex was prepared by complexing AgNO3, Zn(NO3)2 and EDTA solutions and then diluting these solutions to 3.0% (w/v) for antibiofilm evaluation. PBS and 1.1% AgNO3 were used as negative and positive control groups. An in vitro AWE-biofilm model which involved a pre-formed Pseudomonas aeruginosa biofilm under a layer of artificial wound eschar (AWE) was set up. The AWE was composed of the main protein components in wound eschar/slough: collagen, elastin and fibrin. The test antibacterials (Ag-Zn EDTA complex and AgNO3 solutions) were added onto the AWE-biofilm substrate to validate the AWE-biofilm model. After 2.5 and 24 hours treatment, the bacterial cell density in the medium was determined by taking a sample and serial diluting it in PBS, before plating each dilution onto Tryptone Soya Agar in duplicate. The penetration of antibacterials (e.g. silver ions) through the AWE membrane was monitored electrochemically using a printed carbon sensor. The disruption of P. aeruginosa biofilm by the treatments was imaged by LSM 780 Zeiss confocal microscope. Result/Discussion: The results showed that silver ions penetrated through the AWE membrane to reach the biofilm underneath. The penetration rate of silver ion was inversely proportional to the thicker of AWE thickness. The antibiofilm test results indicated that Ag-Zn MMC had statistically significantly higher antibiofilm efficacy than AgNO3 solution at the same silver concentration. Conclusion: The results demonstrated the potential for the patent protected EDTA mixed metal complexes, such as Ag-Zn EDTA to be used for treatment of biofilms, such as those that formed in the slough of chronic and acute wounds.

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