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- MATERIALS AND METHODS
The purpose of the present study was to evaluate the mechanism of microbial resistance to oxidative stress induced by photolysis of hydrogen peroxide (H2O2) in relation to microbial catalase activity. In microbicidal tests, Staphylococcus aureus and Candida albicans were killed and this was accompanied by production of hydroxyl radicals. C. albicans was more resistant to hydroxyl radicals generated by photolysis of H2O2 than was S. aureus. A catalase activity assay demonstrated that C. albicans had stronger catalase activity; accordingly, catalase activity could be one of the reasons for the resistance of the fungus to photolysis of H2O2. Indeed, it was demonstrated that C. albicans with strong catalase activity was more resistant to photolysis of H2O2 than that with weak catalase activity. Kinetic analysis using a modified Lineweaver-Burk plot also demonstrated that the microorganisms reacted directly with hydroxyl radicals and that this was accompanied by decomposition of H2O2. The results of the present study suggest that the microbicidal effects of hydroxyl radicals generated by photolysis of H2O2 can be alleviated by decomposition of H2O2 by catalase in microorganisms.
A novel disinfection method whereby hydroxyl radicals, a ROS, are artificially generated by photolysis of H2O2 has been developed recently. In a previous study, we demonstrated that hydroxyl radicals generated by laser irradiation of H2O2 could kill pathogens of oral infectious diseases (1). Although laser irradiation of bacterial suspensions in 1 M H2O2 resulted in a >99.99% reduction in the numbers of bacteria within 3 min, the sensitivity of the bacteria to this disinfection system varied somewhat according to the species. This finding suggests that bacterial species have varying degrees of resistance to this disinfection system.
It is well known that some species of microorganisms are equipped with enzymatic defense mechanisms that suppress oxidative stress caused by ROS. As for defense mechanisms against H2O2, catalase, one such enzyme, is mainly responsible for these defense mechanisms, catalyzing a reaction in which H2O2 decomposes into water and oxygen (2–4). Hence, the catalase activity of microorganisms might affect the microbicidal effect of photolysis of H2O2. We have recently established an analytical method for evaluating the catalase activity of microorganisms at a cellular level (5). The assay enables us to measure the catalase activity of cell suspensions rather than cell lysates. Analysis of microbial resistance to photolysis of H2O2 associated with catalase activity would probably provide important information for characterizing this disinfection system.
To evaluate whether microorganisms have resistance to this disinfection system in relation to catalase activity, we applied kinetic analysis to the reactions between microorganisms and hydroxyl radicals generated by photolysis of H2O2. The Lineweaver-Burk plot has often been used for analysis of the kinetics of enzyme reactions with an inhibitor (6). In addition to enzyme reactions, Niwano et al. demonstrated that this analytical method can be used for kinetic analysis of the reactions between ROS and antioxidants using an ESR spin trapping technique (7). According to this theory, it is possible to determine whether the antioxidant directly scavenges ROS or suppresses its generation (7, 8). Thus, the same analytic method, using microorganisms instead of antioxidants, could clarify whether the microorganisms react directly with hydroxyl radicals or interrupt photolysis of H2O2.
The purpose of the present study was, therefore, to evaluate the mechanism of microbial resistance to oxidative stress induced by photolysis of H2O2 in relation to microbial catalase activity.
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- MATERIALS AND METHODS
Both S. aureus and C. albicans are frequently detected in the oral cavity and sometimes cause serious infectious diseases such as pneumonia, toxic shock syndrome and septicemia, especially in elderly people (15–17). Furthermore, since both microbial species contain catalase to protect themselves from oxidative stress, we selected them as the subjects for a study on the assumption that they might be resistant to a disinfection system utilizing photolysis of H2O2. Indeed, 250 mM H2O2 alone hardly killed any microorganisms. We believe that the concentration of 250 mM was too low to exert fungicidal and bactericidal effects because 3% H2O2, corresponding to approximately 890 mM, is usually used as a disinfectant. Meanwhile, when H2O2 was photolyzed by LED irradiation, sufficient bactericidal and fungicidal effects resulted, demonstrating that these effects are attributable to hydroxyl radicals generated by photolysis of H2O2. The bactericidal and the fungicidal effects were enhanced by longer LED irradiation times, suggesting that the microbicidal effect depends on the yield of hydroxyl radicals, as shown in our previous study (1). Even though the concentration of H2O2 used in the present study was lower than that used in our previous study (1), photolysis of H2O2 generated enough hydroxyl radicals to kill S. aureus and C. albicans with four or more logarithmic reduction within 5 and 10 min, respectively (Fig. 1a, b).
Since S. aureus and C. albicans contain catalase, they could be resistant to the disinfection system by decomposing H2O2. We measured the catalase activity of the microorganisms to evaluate how much their catalase contributed to their defense mechanisms. In a previous study, we proposed a new analytical method for determining catalase activity at a cellular level (5). The glutathione peroxidase system is one possible candidate for decomposing H2O2. However, as demonstrated in our previous study, the system not at all or only slightly affected the assay for catalase activity because it did not reduce the concentration of H2O2 under the condition applied in this catalase assay (5). Thus, we suggest that this assay specifically determines catalase activity. Analysis of catalase activity by the method described demonstrated that C. albicans has stronger catalase activity than S. aurues. The catalase activities of C. albicans and S. aureus in this study were comparable with those of a different stock strain of C. albicans and other aerobes used in our previous study (5).
To evaluate the contribution of catalase activity to the microbial defense mechanisms against photolysis of H2O2, C. albicans with different catalase activities, grown in the same medium except for the carbon source, were prepared. Analysis of catalase activity showed that the fungus grown in the medium containing n-alkane mixture had significantly stronger catalase activity than that grown in the medium containing glucose, as reported previously (9). Furthermore, the fungus with stronger catalase activity showed greater resistance to photolysis of H2O2. This suggests that catalase of C. albicans plays an important role in defense against photolysis of H2O2.
Analysis of the inhibition curve showed that DMPO-OH formation decreased with increasing concentrations of microorganisms. The IC50 of C. albicans (1.8 × 107 CFU/mL) was smaller than that of S. aureus (5.9 × 107 CFU/mL). This finding indicates that each C. albicans cell suppresses the formation of DMPO-OH more strongly than does each S. aureus cell. Because C. albicans cells are larger than S. aureus cells, each C. albicans cell is obviously exposed to larger amounts of hydroxyl radicals than each S. aureus cell. Nevertheless, C. albicans was more resistant to hydroxyl radicals than S. aureus. One possible explanation for the resistance of C. albicans is that it decomposes H2O2 more effectively than does S. aureus. Indeed, C. albicans showed stronger catalase activity per cell than did S. aureus, as discussed above. In addition, the differences between eukaryotic fungal cells and prokaryotic bacterial cells in cellular structure as well as gene expression associated with resistance to oxidative stress might allow C. albicans to tolerate larger amounts of hydroxyl radicals.
In the ESR spin trapping analyses, detection of DMPO-OH depends on two reactions, namely generation of hydroxyl radicals and formation of DMPO-OH, as shown in equations 1 and 2. Under the condition used in the present study, the formation of DMPO-OH was inhibited by hydroxyl radical scavengers, dimethyl sulfoxide and mannitol, but not by the superoxide radical scavenger, superoxide dismutase (data not shown). This finding suggests that DMPO-OH is formed by the reaction between DMPO and the hydroxyl radical as defined in equations 1 and 2. When DMPO-OH formation from the reaction between DMPO and hydroxyl radicals was inhibited, analysis using the modified Lineweaver-Burk plot enabled us to evaluate how that inhibition occurs. The theory is that a linear pattern with an intersection on the y axis means competitive inhibition, a parallel linear pattern means noncompetitive inhibition, and a linear pattern with an intersection on the negative side of x axis means mixed inhibition (7, 6, 18). Analysis using different concentrations of the microorganisms and catalase clearly demonstrated that there were different kinetic patterns between the microorganisms and catalase against DMPO-OH formation. The linear patterns of the double reciprocal plots for microorganisms showed different slopes and intersections shifted to the negative side of the x axis, whereas the linear patterns for catalase showed parallel lines without intersection. These findings suggest that the microorganisms function as mixed inhibitors whereas catalase functions as a noncompetitive inhibitor. This means that the microorganisms react directly to the hydroxyl radicals and simultaneously decompose H2O2 with their catalase, the latter reaction probably conferring some resistance to the disinfection system. Hydroxyl radicals are also generated by a Fenton-like reaction, in which hydrogen peroxide formed by dismutation of superoxide anion is decomposed in the presence of transition metals. Catalase probably contributes to reduced formation of such hydroxyl radicals in microorganisms.
Indeed, it has been demonstrated that the bactericidal effect of several hydroxyl radical generation systems is potentiated by an increased yield of hydroxyl radicals (19–21), and suppressed by the presence of hydroxyl radical scavengers (22, 23). Similarly, the kinetic analysis suggests that microbial catalase suppresses generation of hydroxyl radicals in the disinfection system. Therefore, it is possible that inactivation of catalase potentiates the bactericidal and the fungicidal effects of the disinfection system.
Hydroxyl radicals generated by photolysis of H2O2 using LED could potentially be adopted as a novel disinfection system. Besides having strong bactericidal and fungicidal effects, disinfection by ROS probably would not lead to development of bacterial and fungal resistance to these agents because ROS interact directly with several cell structures and different metabolic pathways (24, 25). In particular, hydroxyl radicals and singlet oxygen are thought to be free from induction of resistance because no defense mechanisms against these two ROS have been reported in living cells. From a clinical point of view, this is a major advantage of this disinfection system.