ABSTRACT
The photodynamic antimicrobial process is a multi-target method to inactivate pathogenic microorganisms by exciting a photoantimicrobial agent with visible light of appropriate wavelength in the presence of molecular oxygen (3O2). There are two major pathways by which reactive oxygen species (ROS) are produced. In type-1 reactions, radicals such as superoxide (O2•−) and hydroxyl radicals (•OH) are generated by electron transfer. In type-2 reactions, highly reactive singlet oxygen (1O2) is produced by direct energy transfer. This study investigated the efficiency of the photodynamic antimicrobial process in Escherichia coli wild type (EC WT) and the mutant Escherichia coli PN134 (EC PN134) which is not able to produce SOD A and SOD B, by means of two different photoantimicrobials from different chemical classes with different 1O2 quantum yields: methylene blue (MB) and 5,10,15,20-tetrakis(1-methyl-4-pyridinio)porphyrin tetra(p-toluenesulfonate) (TMPyP). Mutants, which lack antioxidant enzymes, were particularly susceptible towards type-1 reactions. When using light-activated MB, quenching agents such as superoxide dismutase (SOD) and catalase (CAT) were sufficient for protecting both the wild type and the mutant, whereas they were not able to prevent bacterial killing sufficiently using light-activated TMPyP.
Summary
The susceptibility of EC PN134 and EC WT differed towards photodynamic inactivation via the type-1 mechanism of action. Thus, already existing defense mechanisms against ROS in bacteria might influence the susceptibility against type-1 photodynamic mechanism of action, while this was not the case using type-2 photoantimicrobials.
Contamination of food takes place in all stages of production process, packaging, storage, transportation, sales and cooking. Therefore large amounts of clean water are used throughout the food production process such as cleaning, sanitizing, peeling, cooling or cooking. Besides physical or chemical contamination a major part represents the biological contamination by different microbial species. In recent years Enterohaemorrhagic Escherichia coli (EHEC) caused a serious outbreak of food borne illness worldwide, where patients suffered from severe diarrhoea and in some cases from haemolytic-uremic syndrome. The source of infection for humans was contaminated food (e.g. bean sprout) with EHEC. Furthermore pigs, colonised with methicillin resistant Staphylococcus aureus (MRSA), has become an emerging risk to be a source of infection for humans. Therefore one important task will be preventing contamination of food with pathogens and spreading from people, pets and pests. One new technology for successful eradication of bacteria is the photodynamic process. This talk summarizes the potential of photodynamic decontamination of foodstuff and decolonization of porcine skin as a novel approach for improving hygiene standards as well as to safe clean water in the future.
Summary
In general photodynamic decontamination of living surfaces like plant food or porcine skin is possible. However successful eradication of pathogens (killing efficacy of > 99.9%) depends on the constitution and geometry of the living object.
Singlet oxygen (O 2 1 ) is an important reactive intermediate in photodynamic reactions, particularly in antimicrobial PDT (aPDT). The detection of O 2 1 luminescence is frequently used to elucidate the role of O 2 1 in various environments, particularly in microorganisms and human cells. When incubating the fungus, Candida albicans, with porphyrins XF73 (5,15-bis-[4-(3-Trimethylammonio-propyloxy)-phenyl]-porphyrin) or TMPyP (5,10,15,20-Tetrakis(1-methyl-4-pyridinio)-porphyrin tetra(p-toluenesulfonate)), the O 2 1 luminescence signals were excellent for TMPyP. In case of XF73, the signals showed strange rise and decay times. Thus, O 2 1 generation of XF73 was investigated and compared with TMPyP. Absorption spectroscopy of XF73 showed a change in absorption cross section when there was a change in the concentration from 1×10 −6 M to 1×10 −3 M indicating an aggregation process. The addition of phosphate buffered saline (PBS) substantially changed O 2 1 luminescence in XF73 solution. Detailed experiments provided evidence that the PBS constituents NaCl and KCl caused the change of O 2 1 luminescence. The results also indicate that Cl − ions may cause aggregation of XF73 molecules, which in turn enhances self-quenching of O 2 1 via photosensitizer molecules. These results show that some ions, e.g., those present in cells in vitro or added by PBS, can considerably affect the detection and the interpretation of time-resolved luminescence signals of O 2 1 , particularly in in vitro and in vivo. These effects should be considered for any other photosensitizer used in photodynamic processes.
Ultraviolet A (UVA) radiation has been known to generate reactive oxygen species, such as singlet oxygen, in skin, leading to the oxidation of lipids and proteins. This oxidation influences cellular metabolism and can trigger cellular signaling cascades, since cellular membranes and the stratum corneum contain a substantial amount of fatty acids and lipids. Using highly sensitive IR-photomultiplier technology, we investigated the generation of singlet oxygen by fatty acids and lipids. In combination with their oxidized products, the fatty acids or lipids produced singlet oxygen under UVA radiation at 355 nm that is directly shown by luminescence detection. Linolenic or arachidonic acid showed the strongest luminescence signals, followed by linoleic acid and docohexaenoic acid. The amount of singlet oxygen induced by lipids such as phosphatidylcholine was significantly higher compared to the corresponding fatty acids within phospholipids. This result indicates a synergistic process of oxygen radicals and singlet oxygen during irradiation. UVA radiation initiates singlet oxygen generation, which subsequently oxidizes other fatty acids that in turn produce additional singlet oxygen. This leads to an enhancement of UVA-induced damage of fatty acids and lipids, which must enhance the oxidative damages in cells.
Singlet oxygen plays a major role in photodynamic inactivation of tumor cells or bacteria. Its efficacy depends critically on the oxygen concentration [O2], which can decrease in case oxygen is consumed caused by oxidative reactions. When detecting singlet oxygen directly by its luminescence at 1270 nm, the course of the luminescence signal is critically affected by [O2]. Thus, it should be feasible to monitor oxygen consumption during photo-oxidative processes. Singlet oxygen was generated by exciting a photosensitizer (TMPyP) in aqueous solution (H2O or D2O) of albumin. Chromatography shows that most of the TMPyP molecules are unbound, and therefore singlet oxygen molecules can diffuse in the solution. A sensor device for oxygen concentration revealed a rapid decrease of [O2] (oxygen depletion) in the solution during irradiation. The extent of oxygen depletion in aqueous albumin solution depends on the radiant exposure and the solvent. When detecting the luminescence signal of singlet oxygen, the shape of the luminescence signal significantly changed with irradiation time. Thus, local oxygen consumption could be monitored during photodynamic action by evaluating the course of singlet oxygen luminescence.
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