Antimicrobial strategies centered around reactive oxygen species--bactericidal antibiotics, photodynamic therapy, and beyond

Abstract

Reactive oxygen species (ROS) can attack a diverse range of targets to exert antimicrobial activity, which accounts for their versatility in mediating host defense against a broad range of pathogens. Most ROS are formed by the partial reduction in molecular oxygen. Four major ROS are recognized comprising superoxide (O2•-), hydrogen peroxide (H2O2), hydroxyl radical (•OH), and singlet oxygen ((1)O2), but they display very different kinetics and levels of activity. The effects of O2•- and H2O2 are less acute than those of •OH and (1)O2, because the former are much less reactive and can be detoxified by endogenous antioxidants (both enzymatic and nonenzymatic) that are induced by oxidative stress. In contrast, no enzyme can detoxify •OH or (1)O2, making them extremely toxic and acutely lethal. The present review will highlight the various methods of ROS formation and their mechanism of action. Antioxidant defenses against ROS in microbial cells and the use of ROS by antimicrobial host defense systems are covered. Antimicrobial approaches primarily utilizing ROS comprise both bactericidal antibiotics and nonpharmacological methods such as photodynamic therapy, titanium dioxide photocatalysis, cold plasma, and medicinal honey. A brief final section covers reactive nitrogen species and related therapeutics, such as acidified nitrite and nitric oxide-releasing nanoparticles.

Keywords: bacteria; hydrogen peroxide; hydroxyl radicals; photosensitizers; singlet oxygen; titanium dioxide.

Figure 1. Production of ROS via leakage of O₂•– from mitochondrial respiratory chain

Further ROS (H₂O₂ and •OH) are formed and defense systems such as catalase and SOD can be induced to mitigate the resulting damage and prevent excessive oxidative stress.

Figure 2. Oxidative degradation of lipids

Lipid peroxidation, starts with an ROS “stealing” an electron from the lipid cell membrane and the process continuous via free-radical chain reaction mechanism. The peroxidation reaction is especially affective in polyunsaturated fatty acids, since they contain multiple double bonds and in between methylene groups (-CH2-). This position of the methylene groups renders the hydrogen atoms especially reactive and susceptible to ROS attack. The degradation process constituting three major steps goes through initiation, propagation, and termination.

Figure 3. Generation and interconversion of physiologically relevant ROS

It is known that ROS sustain homeostasis but may also trigger cell death by apoptosis and/or necrosis (Pourova, et al., 2010). Basal levels of ROS production in cells is beneficial for several physiological functions, however, excessive ROS production above basal levels can impair and oxidatively damage DNA, lipids and proteins, and consequently result in dysfunction of these molecules within cells and finally cell death (Winterbourn, 2008, Mohsenzadegan & Mirshafiey, 2012). Semiquinone-like radicals (SQ^•−) are generated by autoxidation of a range of compounds including adrenaline and DOPA, or by enzymatic reduction of quinones such as ubiquinone or menadione. Flavonoids and other polyphenols can generate both semiquinone and phenoxyl radicals. Phenoxyl radicals (PhO^•) are produced from tyrosine and other phenolic metabolites and xenobiotics. Aromatic amines and indoles are oxidized to radicals with similar properties. Glutathionyl radical (GS^•) are generated from other thiols such as dihydrolipoic acid or cysteine residues. Only myeloperoxidases are capable of generating hypochlorous acid (HOCl).

Figure 4. Antimicrobial host defense relies on ROS generation by macrophages and neutrophils

The microbial cells is engulfed into a phagolysome and this triggers NADPH oxidase generates O₂•–, while myeloperoxidase generates HOCl, and iNOS generates NO•. These ROS and RNS combine to kill the microbes.

Figure 5. Jablonski diagram illustrating the mechanisms of PDT including Type I and Type II photoreactions

The PS absorbs photons from light and causes excitation to the singlet excited state (¹ PS*). The singlet excited PS* can decay back to the ground state with release of energy in the form of fluorescence. It is possible for the singlet to be converted into the long-lived triplet excited state ( ³ PS*) which is able to transfer energy to another triplet (ground state oxygen) or alternatively carry out electron transfer to oxygen producing a range of ROS via superoxide.

Figure 6. Structures of the cell walls of different classes of microbial cells

Gram-positive bacteria have a relatively porous outer cell wall composed of peptidoglycan, teichuronic acids and lipoteichoic acids. Gram-negative bacteria have a thin layer of peptidoglycan and then a second lipid bilayer incorporating lipopolysaccharide and providing a permability barrier. Yeast have a relatively impermeable cell wall composed of beta-gucan and chitin (Sharma, et al., 2011).

Figure 7. Plasma pencil

The five-centimeter-long plasma plume is generated when a stream of helium gas containing a trace of oxygen passes between two high-voltage copper electrodes. Helium is very difficult to ionize, but the plume's oxygen molecules break into two highly reactive oxygen atoms, which then attack the bacteria. The key to keeping the plasma pencil cool is its kilovolt electric field, which switches on and off thousands of times a second.