Characterization of a novel antimicrobial agent inspired by peroxidase-catalyzed systems
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The phenomenon of antibiotic resistance is expanding and the threat regarding our future ability to combat infection is increasing. Thus, key challenges for society and for researchers are to address microbial drug resistance and to develop non-antibiotic based therapies and disinfectants that can avoid induction of resistance. A novel antibacterial complex was developed, drawing the inspiration from naturally occurring peroxidase-catalyzed systems that play a role in immune defense against invading microbes. In the peroxidase system, a particular peroxidase enzyme catalyzes the oxidation of a halide/pseudohalide, at the expense of hydrogen peroxide (H2O2), to generate reactive products with broad antimicrobial properties. However, producing enzymes in quantities widely usable for antibacterial treatments would be both expensive and impractical. In the new peroxidase-like antibacterial complex, it is H2O2 that oxidizes the two halide/pseudohalide substrates (iodide and thiocyanate) in the absence of a peroxidase. This enzyme-free iodo-thiocyanate complex (ITC) is a mixture of highly reactive oxygen and iodine species that can damage bacterial cells, resulting in their death. The objective of this thesis was, firstly, to evaluate the antibacterial properties of ITC. The antibacterial potential of the novel ITC was tested on two Gram-negative and two Gram-positive bacterial strains, including the multidrug-resistant Staphylococcus aureus, in both planktonic and biofilm forms. The results of this study showed that the iodo-thiocyanate complex caused rapid bacterial death in all tested strains, both in biofilms and planktonic cells. Furthermore, the attempts to introduce resistance in these bacteria towards the “killer cocktail”, employing a sequential passage of bacteria in the presence of a sub-lethal concentration of ITC, proved to be not successful. Though the knowledge on the mode of action of the antimicrobial complex is still incomplete, there are indications that its antimicrobial activity is most likely the combinational effect of powerful species capable of oxidizing the essential biomolecules of bacteria, and, perhaps, is a result of simultaneous events. It is essential to take into account the emergence of bacterial resistance when designing a novel antimicrobial. Thus, the next step in this thesis was to elaborate the studies on the emergence of resistance. A continuous culturing was used to generate antimicrobial resistant mutations, coupled with whole-genome sequencing (WGS) to identify those mutations underpinning resistance. An attempt was made to generate de novo resistance in antimicrobial-sensitive Escherichia coli ATCC 25922 during 20-days of continuous culturing when exposed to gradually increasing concentrations of the newly described ITC and a common antibiotic, levofloxacin (LVX). In contrast to antibiotic LVX, the long-term exposure of E. coli to ITC did not induce resistance to ITC, nor cross-resistance to LVX, and no mutational pattern was evidenced during WGS-based comparisons between exposed and unexposed bacterial populations. To derive a biocompatible novel antibacterial agent, both the bacterial and mammalian toxicities must be taken into consideration. For biocompatibility testing, in vitro cytotoxicity, in parallel with antimicrobial activity, hemolytic activity and genotoxicity evaluations were carried out. The cytotoxicity of ITC towards human epithelial HeLa cells was evaluated by comparison with some of the oldest, and most widely used, antiseptics hydrogen peroxide, povidone-iodine (PVP-I) and Lugol’s iodine. The cytotoxic concentrations of ITC were equivalent to those resulting in potent bactericidal activity. By contrast, the cellular toxicities of H2O2, PVP-I and Lugol were apparent at sub-bactericidal levels. The activity of ITC was not quenched by organic matter, whereas the activities of the other antiseptics were suppressed. Hemolytic activity was also assessed as another measure of cytotoxicity. ITC, PVP-I and Lugol had dose-dependent effects on the viability of horse erythrocytes, while H2O2 showed no hemolytic impact. HeLa DNA damage caused by ITC was evaluated by in vitro comet assay as a measure of genotoxicity. ITC did not generate DNA breakage, while H2O2 resulted in extensive single-strand DNA breaks. Overall, this research indicates that the novel iodo-thiocyanate complex exhibits a broad-spectrum bactericidal activity against pathogenic bacteria in planktonic and biofilm forms, without triggering the emergence of resistance. The use of this composition may provide an effective and efficient method for killing potential pathogens, as well as for disinfecting and removing biofilm contamination.
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