Hiroyo Ikai

Bactericidal Action of Photogenerated Singlet Oxygen from Photosensitizers Used in Plaque Disclosing Agents

Background Photodynamic therapy (PDT) has been suggested as an efficient clinical approach for the treatment of dental plaque in the field of dental care. In PDT, once the photosensitizer is irradiated with light of a specific wavelength, it transfers the excitation energy to molecular oxygen, which gives rise to singlet oxygen. Methodology/Principal Findings Since plaque disclosing agents usually contain photosensitizers such as rose bengal, erythrosine, and phloxine, they could be used for PTD upon photoactivation. The aim of the present study is to compare the ability of these three photosensitizers to produce singlet oxygen in relation to their bactericidal activity. The generation rates of singlet oxygen determined by applying an electron spin resonance technique were in the order phloxine > erythrosine ≒ rose bengal. On the other hand, rose bengal showed the highest bactericidal activity against Streptococcus mutans, a major causative pathogen of caries, followed by erythrosine and phloxine, both of which showed activity similar to each other. One of the reasons for the discrepancy between the singlet oxygen generating ability and bactericidal activity was the incorporation efficiency of the photosensitizers into the bacterial cells. The incorporation rate of rose bengal was the highest among the three photosensitizers examined in the present study, likely leading to the highest bactericidal activity. Meanwhile, the addition of L-histidine, a singlet oxygen quencher, cancelled the bactericidal activity of any of the three photoactivated photosensitizers, proving that singlet oxygen was responsible for the bactericidal action. Conclusions It is strongly suggested that rose bengal is a suitable photosensitizer for the plaque disclosing agents as compared to the other two photosensitizers, phloxine and erythrosine, when used for PDT.

Bactericidal action of photoirradiated gallic acid via reactive oxygen species formation.

It is known that gallic acid shows antimicrobial activity. In the present study, photoirradiation induced reactive oxygen species formation was investigated for augmentation of the antimicrobial activity of gallic acid. Staphylococcus aureus suspended in 4 mmol/L gallic acid was exposed to blue light of a LED at 400 nm. This treatment killed the bacteria, and a >5-log reduction of the viable counts was observed within 15 min. By contrast, neither the LED treatment alone nor the treatment with gallic acid alone showed substantial bactericidal effect. When hydroxyl radical scavengers were added to the suspension, the bactericidal effect of photoirradiated gallic acid was attenuated. Furthermore, electron spin resonance analysis demonstrated that hydroxyl radicals were generated by the photoirradiation of gallic acid. Thus, the present study suggests that the photo-oxidation can enhance the antimicrobial activity of gallic acid via hydroxyl radical formation.

Bactericidal Activity and Mechanism of Photoirradiated Polyphenols against Gram-Positive and -Negative Bacteria.

The bactericidal effect of various types of photoirradiated polyphenols against Gram-positive and -negative bacteria was evaluated in relation to the mode of action. Gram-positive bacteria (Enterococcus faecalis, Staphylococcus aureus, and Streptococcus mutans) and Gram-negative bacteria (Aggregatibacter actinomycetemcomitans, Escherichia coli, and Pseudomonas aeruginosa) suspended in a 1 mg/mL polyphenol aqueous solution (caffeic acid, gallic acid, chlorogenic acid, epigallocatechin, epigallocatechin gallate, and proanthocyanidin) were exposed to LED light (wavelength, 400 nm; irradiance, 260 mW/cm(2)) for 5 or 10 min. Caffeic acid and chlorogenic acid exerted the highest bactericidal activity followed by gallic acid and proanthocyanidin against both Gram-positive and -negative bacteria. It was also demonstrated that the disinfection treatment induced oxidative damage of bacterial DNA, which suggests that polyphenols are incorporated into bacterial cells. The present study suggests that blue light irradiation of polyphenols could be a novel disinfection treatment.

Synergistic Effect of Thermal Energy on Bactericidal Action of Photolysis of H2O2 in Relation to Acceleration of Hydroxyl Radical Generation

ABSTRACT The purpose of the present study is to evaluate the effect of thermal energy on the yield of and the bactericidal action of hydroxyl radical generated by photolysis of H2O2. Different concentrations of H2O2 (250, 500, 750, and 1,000 mM) were irradiated with light-emitting diodes (LEDs) at a wavelength of 400 ± 20 nm at 25°C to generate hydroxyl radical. The 500 mM H2O2 was irradiated with the LEDs at different temperatures (25, 35, 45, and 55°C). Electron spin resonance spin trapping analysis showed that the yield of hydroxyl radicals increased with the temperature, as well as the concentration of H2O2. Streptococcus mutans and Enterococcus faecalis were used in the bactericidal assay. The LED-light irradiation of the bacterial suspensions in 500 mM H2O2 at 25°C could hardly kill the bacteria within 3 min, while the bactericidal effect was markedly enhanced with the temperature rise. For instance, a temperature increase to 55°C resulted in >99.999% reduction of viable counts of both bacterial species only within 1 min. The photolysis of 500 mM H2O2 at 55°C could reduce the viable counts of bacteria more efficiently than did the photolysis of 1,000 mM H2O2 at 25°C, although the yields of hydroxyl radical were almost the same under the both conditions. These findings suggest that the thermal energy accelerates the generation of hydroxyl radical by photolysis of H2O2, which in turn results in a synergistic bactericidal effect of hydroxyl radical and thermal energy.

In vitro evaluation of the risk of inducing bacterial resistance to disinfection treatment with photolysis of hydrogen peroxide.

The purpose of the present study was to evaluate the risk of inducing bacterial resistance to disinfection treatment with photolysis of H2O2 and comparing this with existing antibacterial agents. We tested seven antibacterial agents, including amoxicillin, cefepime hydrochloride, erythromycin, ofloxacin, clindamycin hydrochloride, ciprofloxacin hydrochloride, and minocycline hydrochloride, as positive controls for validation of the assay protocol. For all of the agents tested, at least one of the four bacterial species (Staphylococcus aureus, Enterococcus faecalis, Escherichia coli, and Streptococcus salivarius) was resistant to these agents by repeated exposure to subinhibitory concentrations of the agents up to 10 times. In contrast, antibacterial activity against any of the bacterial species tested (S. aureus, E. faecalis, E. coli, S. salivarius, Pseudomonas aeruginosa, Streptococcus mutans, and Aggregatibacter actinomycetemcomitans) was not affected by repeated exposure to the disinfection treatment up to 40 times. This finding suggested that the risk of inducing bacterial resistance by disinfection treatment was low. The active ingredient of this disinfection treatment is hydroxyl radicals generated by photolysis of H2O2. Therefore, hydroxyl radicals interact with several cell structures and different metabolic pathways in microbial cells, probably resulting in a lack of development of bacterial resistance. In conclusion, disinfection treatment with photolysis of H2O2 appears to be a potential alternative for existing antimicrobial agents in terms of a low risk of inducing bacterial resistance.

Photo-Irradiation of Proanthocyanidin as a New Disinfection Technique via Reactive Oxygen Species Formation

In the present study, the bactericidal effect of photo-irradiated proanthocyanidin was evaluated in relation to reactive oxygen species formation. Staphylococcus aureus suspended in proanthocyanidin aqueous solution was irradiated with light from a laser at 405 nm. The bactericidal effect of photo-irradiated proanthocyanidin depended on the concentration of proanthocyanidin, the laser irradiation time, and the laser output power. When proanthocyanidin was used at the concentration of 1 mg/mL, the laser irradiation of the bacterial suspension could kill the bacteria with a >5-log reduction of viable cell counts. By contrast, bactericidal effect was not observed when proanthocyanidin was not irradiated. In electron spin resonance analysis, reactive oxygen species, such as hydroxyl radicals, superoxide anion radicals, and hydrogen peroxide, were detected in the photo-irradiated proanthocyanidin aqueous solution. The yields of the reactive oxygen species also depended on the concentration of proanthocyanidin, the laser irradiation time, and the laser output power as is the case with the bactericidal assay. Thus, it is indicated that the bactericidal effect of photo-irradiated proanthocyanidin is exerted via the reactive oxygen species formation. The bactericidal effect as well as the yield of the oxygen radicals increased with the concentration of proanthocyanidin up to 4 mg/mL, and then decreased with the concentration. These findings suggest that the antioxidative activity of proanthocyanidin might prevail against the radical generation potency of photo-irradiated proanthocyanidin resulting in the decreased bactericidal effect when the concentration is over 4 mg/mL. The present study suggests that photo-irradiated proanthocyanidin whenever used in an optimal concentration range can be a new disinfection technique.

Literature review of the role of hydroxyl radicals in chemically-induced mutagenicity and carcinogenicity for the risk assessment of a disinfection system utilizing photolysis of hydrogen peroxide

We have developed a new disinfection system for oral hygiene, proving that hydroxyl radicals generated by the photolysis of 1 M hydrogen peroxide could effectively kill oral pathogenic microorganisms. Prior to any clinical testing, the safety of the system especially in terms of the risk of carcinogenicity is examined by reviewing the literature. Previous studies have investigated indirectly the kinds of reactive oxygen species involved in some sort of chemically-induced mutagenicity in vitro by using reactive oxygen species scavengers, suggesting the possible involvement of hydroxyl radicals. Similarly, possible involvement of hydroxyl radicals in some sort of chemically-induced carcinogenicity has been proposed. Notably, it is suggested that the hydroxyl radical can play a role in heavy metal-induced carcinogenicity that requires chronic exposure to the carcinogen. In these cases, hydroxyl radicals produced by Fenton-like reactions may be involved in the carcinogenicity. Meanwhile, potential advantages have been reported on the use of the hydroxyl radical, being included in host immune defense by polymorphonuclear leukocytes, and medical applications such as for cancer treatment and antibiotics. From these, we conclude that there would seem to be little to no risk in using the hydroxyl radical as a disinfectant for short-term treatment of the oral cavity.

Synergistic interaction between wavelength of light and concentration of H2O2 in bactericidal activity of photolysis of H2O2

The present study aimed to evaluate the interaction between wavelength of light in the range of ultra violet A-visible and concentration of H2O2 in the reaction of photolysis of H2O2 from the point of view of hydroxyl radical (·OH) generation and the bactericidal activity. Light emitting diodes (LEDs) emitting the light at wavelengths of 365, 385, 400 and 465 nm were used at an irradiance of 1000 mW/cm(2). H2O2 was used at the final concentrations of 0, 250, 500, and 1000 mM. Quantitative analysis of ·OH generated by the LED irradiation of H2O2 were performed using an electron spin resonance-spin trapping technique. In a bactericidal assay, a bacterial suspension of Staphylococcus aureus prepared in sterile physiological saline was irradiated with the LEDs. The bactericidal activity of each test condition was evaluated by viable counts. When H2O2 was irradiated with the LEDs, ·OH was generated and bacteria were killed dependently on the concentration of H2O2 and the wavelength of LED. The two-way analysis of variance revealed that the wavelength, the H2O2 concentration and their interaction significantly affected the yield of ·OH and the bactericidal activity of the photolysis of H2O2. Therefore, it is suggested that bactericidal activity of photolysis of H2O2 could be enhanced by controlling the wavelength and the concentration of H2O2, which may contributes to shortening the treatment time and/or to reducing the concentration of H2O2.

Postantibiotic effect of disinfection treatment by photolysis of hydrogen peroxide

Abstract The purpose of the present study was to evaluate the postantibiotic effect (PAE) of the disinfection treatment by photolysis of H2O2. Postantibiotic effect was induced in Staphylococcus aureus and Streptococcus salivarius by exposing the bacteria to H2O2 at concentrations of 250–1000 mmol/l, laser irradiation at a wavelength of 405 nm, and the combination of both (photolysis of H2O2) for 10–30 seconds. The photolysis of H2O2 induced significantly longer PAE than other treatments. The PAE was augmented dependently on not only the concentration of H2O2 but the laser irradiation time. Electron spin resonance analysis showed that the hydroxyl radical was also generated dependently on both the concentration of H2O2 and the laser irradiation time, suggesting that the hydroxyl radicals contribute to the PAE. These results suggest that the disinfection treatment by photolysis of H2O2 induces PAE in S. aureus and S. salivarius even though they were treated for only 10–30 seconds.

Booster Effect of Thermal Energy on Bactericidal Action of Hydroxyl Radical Generated by Photolysis of H2O2

The effect of thermal energy on the yield of and the bactericidal action of hydroxyl radical generated by photolysis of H2O2 is discussed in this review article.