Pre-clinical Evaluation of a New Antimicrobial Enzyme for the Control of Wound Bioburden
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Abstract: A new, optimized, antimicrobial enzyme system was developed for the control of wound bioburden. This Glucose oxidase-Lactoperoxidase-Guaiacol (GLG) system was analyzed for antimicrobial activity and cytotoxicity. The susceptibility of a wide range of antibiotic-resistant bacterial strains to the GLG-enzyme system was analyzed using minimum inhibitory concentration (MIC90), minimum bactericidal concentration (MBC) determination, and growth kinetics analysis. Additionally, challenge tests and cytotoxicity tests were performed with a new hydroactive alginate gel dressing with antimicrobial activity obtained by the presence of the GLG-enzyme system (Flaminal® Forte, Flen Pharma, Kontich, Belgium). All bacterial strains were susceptible to the GLG-enzyme system at low concentrations. The exact concentration required for growth arrest and cell death was dependent on the experimental design. Further, a 20% (w/v) GLG dilution showed no cytotoxicity toward fibroblasts and keratinocytes. Conversely, other antimicrobial wound-care products applied with the same dilution showed a high degree of cytotoxicity. With increasing concerns about bacterial resistance to antibiotics, this study shows that low concentrations of the GLG-enzyme system are successful in killing antibiotic-resistant bacterial strains. Furthermore, results show that GLG-enzyme system combines strong antimicrobial activity with non-cytotoxicity and promotes optimal wound healing.
Address correspondence to:
Kris De Smet, PhD
Flen Pharma NV
Blauwesteenstraat 87, B-2550
Kontich, Belgium
Phone: +32-3-825-7063
E-mail: kris.desmet@flenpharma.com
Microbial drug resistance is a growing problem. The rise in multiresistant strains of bacteria observed over recent decades emphasizes the importance of the development of new, innovative, antimicrobial drugs. There is a great deal of interest in naturally occurring antimicrobial products, and intensive research aimed at the development of drugs based on naturally occurring antimicrobial peptides (AMPs) of various origins is being performed.1–3 Despite such efforts over the past 2 decades, clinical success has been limited.4 Other naturally occurring antimicrobial products with clinical potential are enzymes, which produce antimicrobial products. Hydrogen peroxide (H2O2) is such a potent antimicrobial product of enzyme activity that killing microorganisms through the oxidation of biologically important molecules readily diffuses across cell membranes.
H2O2-mediated cellular damage is caused by the oxidation of membranes and enzymes, DNA damage and mutation, and the inhibition of membrane transport.5 It is broadly used as a disinfective agent across a wide range of industries.6 Furthermore, H2O2 is used in vivo in a wide range of biological systems in the defense against pathogenic microorganisms.7–11 The disadvantage of the use of pure H2O2 in clinical practice is its toxicity and the fact that it can cause embolism.12–17 Therefore, a subtle approach in the use of H2O2 is suggested. This can be accomplished through the use of enzyme systems that produce H2O2 as an intermediate with the generation of other, less dangerous and less toxic, antimicrobial molecules.
The oxidase-peroxidase system, which is part of the innate salivary defence system, is such a naturally occurring antimicrobial enzyme system and is constituted of glucose oxidase, lactoperoxidase, and iodide (I–) or thiocyanate (SCN–).18 Glucose oxidase catalyzes the oxidation of β-D-glucose to glucono-β-lactone, concomitantly producing hydrogen peroxide.
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