Singlet oxygen generated by a new nonthermal atmospheric pressure air plasma device exhibits
bactericidal effect against oral pathogens
Yoriyuki Hirano
Nihon University Graduate School of Dentistry, Major in Endodontics
(Directors: Prof. Bunnai Ogiso, Assoc. Prof. Makoto Hayashi, and Assoc. Prof. Muneaki Tamura)
Contents
Abstract Page 1 Introduction Page 3 Materials and Methods Page 6 Results Page 12 Discussion Page 15 Conclusion Page 20 Acknowledgements Page 21 References Page 22 Figures Page 26
This thesis is composed by an article ahead of print and an additional data listed below.
1. Hirano Y, HayashiM, TamuraM, YoshinoF, YoshidaA, MasubuchiM, ImaiK, and OgisoB. The bactericidal effect of singlet oxygen generated by a new
nonthermal atmospheric pressure air plasma device on oral pathogens. Journal of Oral Science, in press
2. The results of bactericidal activity and enhancement of oxidative stress level for Porphyromonas endodontalis shown in Figs. 3D, 4D, 5D, and 6D.
1 Abstract
Certain bacteria are associated with oral diseases. Plasma generated at atmospheric pressure and room temperature is termed nonthermal atmospheric pressure plasma (NTAP), and incorporates several molecules, including reactive oxygen species (ROS), that can inactivate various bacteria including oral pathogens. Thus, several NTAP devices have been developed to treat oral diseases. Noble gases, which are often used as the working gases, enhance bactericidal efficacy. However, it requires additional gas supply equipment. Therefore, a new NTAP device that uses ambient air as the working gas was developed. The device generates nonthermal atmospheric pressure air plasma.
Here, the singlet oxygen (1O2) levels generated, their bactericidal effects on the oral pathogens (Streptococcus mutans, Porphyromonas gingivalis, Enterococcus faecalis, and Porphyromonas endodontalis), and their bacterial oxidative stresses were measured.
1O2 generation in phosphate-buffered saline was qualitatively assessed via electron spin resonance (ESR) spectroscopy, and bactericidal efficacy was evaluated by measuring colony-forming units/mL. Bacterial oxidative stress was analyzed by hydrogen peroxide (H2O2) and superoxide dismutase (SOD) activity measurements. ESR indicated that the level of 1O2 increased significantly in time- and inversely distance-dependent manners, but the bactericidal effects varied by treatment time only (not by distance) with
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increasing H2O2 and decreasing SOD levels, suggesting that the new device has a potential for being used in treating oral disease.
3 Introduction
Dental caries and periodontal disease are the most common bacterial infections encountered in dentistry. Caries features demineralization of hard tissue by acids produced during bacterial glycolysis (1). Streptococcus mutans (S. mutans) is the primary causative agent for dental caries (2). Periodontal disease is characterized by progressive destruction of the periodontal tissue induced by bacterial infections (3,4).
Porphyromonas gingivalis (P. gingivalis) is the prime pathogen for periodontal disease
(5,6). Also, periapical periodontitis is a frequent cause of oral inflammation after root canal infection (7). Many studies have indicated that persistent endodontic infections and periapical lesions with acute symptoms are frequently caused by Enterococcus faecalis (E. faecalis) (8-10) and Porphyromonas endodontalis (P. endodontalis) (11,12),
respectively.
Plasmas are completely or partially ionized gases, which contain anionic, cationic, and neutral molecules (13). Plasma generated at atmospheric pressure and room temperature is termed nonthermal atmospheric pressure plasma (NTAP), the temperature of which is less than 40°C just before ejected from generators (14). NTAP produces reactive oxygen species (ROS), reactive nitrogen species (RNS), and ultraviolet (UV) radiation, which can affect both prokaryotic and eukaryotic cells (15).
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Therefore, NTAPs are used for sterilization, hemostasis induction, and treatment with skin disease and cancer (16). NTAPs have also been employed to treat oral diseases (17).
Yamazaki et al. (18) demonstrated that treatment with NTAP using low frequency atmospheric pressure plasma jets kills S. mutans and E. faecalis. This indicates that NTAP may be useful to treat dental caries and periapical periodontitis. In addition, NTAP treatment reportedly can inactivate P. gingivalis (19). This evidence presents that NTAP might be used for nonsurgical periodontal therapy.
Several NTAP devices have been developed to inactivate pathogens (16-19). The operational conditions vary, particularly in terms of the working gas, the nature of which affects the range and type of reactive species generated, in turn modulating the antibacterial effects (20). Noble gases such as argon or helium, or mixtures thereof with oxygen, are commonly used and are associated with efficient generation of reactive species (16-19). However, additional equipment (such as a gas bottle) is required.
Although ambient air can serve as the working gas, only a few such devices have been developed, because it is difficult to control the excitation conditions (21). Therefore, a new NTAP jet device using ambient air as the working gas was developed. The device generates nonthermal atmospheric pressure air plasma (NTAAP), not noble gas plasma.
Here, Singlet oxygen (1O2), which is a critical ROS, generation by the device and the
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bactericidal effects thereof on the oral pathogens: S. mutans, P. gingivalis, E. faecalis, and P. endodontalis were qualitatively assessed. Additionally, the bacterial oxidative stress levels, such as hydrogen peroxide concentration (H2O2) and superoxide dismutase (SOD)-level, after treatment with the NTAAP device were examined.
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Materials and Methods Reagents
2,2,6,6-tetramethyl-4-hydroxy-piperidinol (4-OH-TEMP) was obtained from Sigma-Aldrich (St. Louis, MO, USA). L-histidine was purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan) and phosphate-buffered saline (pH 7.2) (PBS) from Invitrogen (Carlsbad, CA, USA). All reagents were of analytical grade.
NTAAP device
Figure 1 shows a schematic of the laboratory-developed NTAAP device. The device consists of a handpiece for the reactor and a controller including a power supply and air compressor. The handpiece (diameter: 12 mm, length: 120 mm) contains two electrodes; a central thin inner tungsten wire and an outer stainless steel cylinder. Both electrodes are connected to the power supply, but only the outer electrode is grounded.
A solid-state pulsed-power generator served as the power supply (output voltage: 7.5 ± 0.5 kV, pulse repetition frequency: 200 Hz); very short high-voltage pulses efficiently generate NTAAP (22) that is ignited between the two electrodes, and the excited plasma is expelled by the force imparted by compressed air. The working gas (air) flow was held at 1.0 L/min.
7 Experimental solution and NTAAP-treatment
PBS in 24-well plates served as the experimental solution. The distance between the surface of the experimental solution and the head of the handpiece was fixed at 1, 3, and 5 mm. The experimental solution was then treated with the NTAAP device for 1, 3, 5, and 7 min, during which the head of handpiece was maintained in the center of each well.
1O2 measurements
The 1O2 generation was quantitatively analyzed via electron spin resonance (ESR) spin-trapping. In the presence of 1O2, 4-OH-TEMP is oxidized and generate 2,2,6,6-tetramethyl-4-hydroxy-piperidinyloxy (4-OH-TEMPO) radical, which can be detected using ESR (23). Briefly, 4-OH-TEMP (100 mM) containing PBS (500 µL/well) were treated with NTAAP. Generated 4-OH-TEMPO radical were measured using JES-RE1X, an ESR spectrometer (JEOL, Tokyo, Japan). ESR data were analyzed using a WIN-RAD ESR data analyzer (Shidai System, Saitama, Japan); the experimental settings were: microwave power, 8.00 mW; magnetic field, 335.8 ± 5.0 mT; field modulation width, 0.1 mT; sweep time, 1 min; and time constant, 0.03 s. To
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quantify the spin adducts detected, the ESR spectrum for manganese oxide (MnO) was used, which appears at either side of ESR spectra. After signal intensity (peak height) of 4-OH-TEMPO was measured, 1O2 generation was expressed as the relative signal intensity that normalized against the standard’s signal intensity of the MnO marker. A solution not subjected to NTAAP-treatment served as a control. Furthermore, the inhibitory effect of 1O2 was evaluated using the scavenger L-histidine (5 mM) (24).
Bactericidal activity
The bactericidal effects of 1O2 on four oral pathogens were evaluated as described previously (25), with minor modifications. Briefly, stock cultures of S. mutans ATCC 25175, P. gingivalis ATCC 33277, E. faecalis JCM 5803, and P. endodontalis JCM8526 were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and the Japan Collection of Microorganisms (RIKEN BioResource Center, Tsukuba, Japan). S. mutans and E. faecalis were aerobically cultured in brain-heart infusion (BHI) broth (Becton Dickinson Labware, Franklin Lakes, NJ, USA) at 37°C, harvested by centrifugation, washed once in PBS, and resuspended in broth. Bacterial cell densities were adjusted to 1.0 × 107 colony-forming units (CFU)/mL. Then, 500-μL amounts of suspensions were placed into wells of a 24-well plate and treated with
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NTAAP as described above. Cell suspensions not subjected to NTAAP-treatment served as controls. One-hundred-fold serial dilutions of all suspensions were prepared using PBS, and 50-μL aliquot of diluted cell suspension were spread on BHI agar. The plates were cultured at 37°C for 24 h (S. mutans) or 18 h (E. faecalis) as described above, and the CFU/mL determined.
P. gingivalis or P. endodontalis was grown in BHI broth supplemented with 5 ppm
hemin and 0.5 ppm menadione. Five-hundred-microliter amounts of washed P.
gingivalis or P. endodontalis (1.0 × 107 CFU/mL) were placed into wells of a 24-well plate and treated with NTAAP as described above. One-hundred-fold serial dilutions were then prepared in PBS, and 50-L amounts spread on BHI agar supplemented with hemin and menadione. The plates were cultured under anaerobic conditions (85% N2, 10% H2, and 5% CO2; v/v) at 37°C for 5 days, and CFU/mL counts determined as described above.
Furthermore, the bactericidal effects of treatment on all four pathogens were evaluated after the addition of 5 mM L-histidine (24).
10 Oxidative stress level measurements
Red Hydrogen Peroxide Assay Kit (Enzo Life Sciences, Plymouth Meeting, PA, USA) and Superoxide Dismutase Assay Kit (Cayman Chemical Company, Ann Arbor, MI, USA) were used to measure bacterial H2O2 and SOD levels, respectively, according to the manufacturer’s instructions. In brief, the bacterial cell suspension described above was treated with NTAAP device for 5 min in S. mutans, 1 min in P. gingivalis, 7 min in E. faecalis, and P. endodontalis. The treatment distance was fixed at 1mm. After
NTAAP-treatment, they were lysed with the ZircoPrep Mini Kit (Nippon Genetics Co., Ltd, Tokyo, Japan). For H2O2 quantification, the H2O2 detection solution was mixed with bacterial lysate and incubated at room temperature for 30 min. Sample densities were measured using a spectrometer (TriStar LB 941, Berthold Technologies, Bad Wildbad, Germany) at 570 nm. For SOD quantification, the SOD detection solution was mixed with bacterial lysate and incubated in a plate shaker for 20 min at room temperature. Absorbance was then measured at 405 nm using a spectrometer. The protein content in bacterial lysate was also quantitatively determined using the BCA Protein Assay Kit (Thermo Scientific, Rockford, IL, USA) and both the H2O2 and the SOD levels were normalized to the protein content.
11 Statistical analysis
All experiments were performed three times in duplicate (n = 6). To assess the significance of among-group differences in terms of 4-OH-TEMPO signal intensity and CFU, one-way analysis of variance (ANOVA) and Tukey’s test or unpaired t-tests were used. Additionally, unpaired t-tests were performed to investigate the differences in the H2O2 and SOD levels. A P-value less than 0.05 was considered statistically significant in all analyses using SPSS software ver. 17 (SPSS Inc., Chicago, IL, USA).
12 Results
1O2 measurements
The 1O2 generatedby the NTAAP device under the aforementioned conditions was evaluated using an ESR spin-trapping technique. When 4-OH-TEMPO radical is generated from 4-OH-TEMP by 1O2, it has one nitrogen atom (n = 1) with nuclear spin (I = 1). Because n nuclei of spin I can give rise to 2nI + 1 resonances, ESR spectrum of
1O2 indicates three (2 × 1 × 1 + 1 = 3) intense lines (26). Therefore, 1O2 generation by the NTAAP device was confirmed (Fig. 2A). When experimental solutions were treated, all signal intensities increased significantly in a time-dependent manner (Fig. 2B). The amount of 1O2 generation is inversely dependent of the distance between the tip of handpiece of NTAAP generator and experimental fluid with significant difference except for treatment time of 1 min (Fig. 2B). However, the controls exhibited minimal signals, regardless of treatment time (Fig. 2B). Signal intensity of 1O2 in the NTAAP-treatment was strongly inhibited by L-histidine (P < 0.05) (Fig. 2C).
Bactericidal activity
The bactericidal effects of the NTAAP-treatment on oral pathogens were examined under the aforementioned conditions. For S. mutans, NTAAP-treatment cause more than
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4-log significant reduction in the numbers of the bacterium within 3 min (P < 0.05) and almost completely killed after 5 min of the treatment; killing rate was not significantly affected by distance between tip of handpiece and cell suspension (P > 0.05) (Fig. 3A).
However, little bactericidal activity was observed in the control group (Fig. 3A). P.
gingivalis and P. endodontalis were completely killed within 1 min of the treatment;
killing rate was not significantly affected by treatment distance (P > 0.05) (Figs. 3B and D). Control group showed no bactericidal effect (Figs. 3B and D). Viable counting of E.
faecalis was significantly decreased with NTAAP-treatment in a time-dependent
manner; complete killing was observed within 7 min of the treatment (Fig. 3C). Their killing rates were not significantly affected by treatment distance (P > 0.05) (Fig. 3C).
Additionally, all bactericidal effects were inhibited by the addition of L-histidine (Figs.
4A-D).
Oxidative stress level measurements
The 1O2 is a ROS that is known to activate H2O2 production in cells, which in-turn affects SOD activity (27,28). As 1O2 generation by NTAAP-treatment and its bactericidal activity were confirmed, H2O2 and SOD activity levels were measured to assess cellular oxidative stress. The H2O2 levels in NTAAP-treatment were significantly
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higher than those of control condition for all pathogens (P < 0.05) (Figs. 5A-D). In contrast, significantly lower SOD levels in NTAAP-treatment were observed when compared to those of control condition for all pathogens (P < 0.05) (Figs. 6A-D).
15 Discussion
A key role of bactericidal effect in NTAP is attributed to ROS (29), although how plasma affects microbes and which agent drive bactericidal efficacy is not yet fully understood. However, the ability to detect ROS generation directly in vitro is limited due to analysis difficulties, mainly because of the very short half-life involved. The ESR spin-trapping technique is useful tool for direct measuring the ROS generated in experimental models (30), which was used in this study.
Initially, identification of 1O2 generated by the NTAAP device was investigated.
The amount of 1O2 increased significantly in a time-dependent manner (Fig. 2B). This finding indicated a time-dependent accumulation of 4-OH-TEMPO in the solution, suggesting that the NTAAP device could continuously generate 1O2 within the treatment time. Also, a significant increase of 1O2 was observed in an inversely distance-dependent manner (Fig. 2B). Various ROS and RNS exist in aqueous solution treated with NTAP (31); therefore, the detection of 1O2 in the experimental solution in this study indicates that 1O2 might be diffused into the solution after its generation with the NTAAP device. Received air pressure on the surface of solution differs in each treatment distance. This difference may be attributed to inversely distance-dependent increase of 1O2. The 1O2 is a highly oxidizing molecule that introduces further oxidative
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reactions in areas, such as the bacterial cell wall, lipid membranes, or nucleic acids (32).
Thus, 1O2 generation with the NTAAP device raised the possibility of bactericidal effect of NTAAP-treatment.
Next, the bactericidal effects of NTAAP-treatmentagainst typical oral pathogens were explored. The effects were evident within 5 min for S. mutans (Fig. 3A), 1 min for P. gingivalis (Fig. 3B), 7 min for E. faecalis (Fig. 3C), and 1 min for P. endodontalis
(Fig. 3D). In addition, the inhibitory effect was confirmed using the scavenger L-histidine (Figs. 4A-D), suggesting that 1O2 generated by the NTAAP device inactivated oral pathogens. These results were supported by previous findings that NTAP devices inactivated oral pathogens such as S. mutans, P. gingivalis, and E.
faecalis (18,19). However, few reports have been published on the bactericidal effects on oral pathogens of 1O2 generated by NTAAP devices.
In terms of treatment time, the device killed P. gingivalis and P. endodontalis more rapidly than S. mutans or E. faecalis. Depending on the diverse structures of the cell wall of the bacteria, different bactericidal responses were reported in NTAP treatment;
Gram-positive bacteria such as S. mutans and E. faecalis are more resistant to NTAP treatment than Gram-negative bacteria such as P. gingivalis and P. endodontalis (33,34).
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Notably, the same bactericidal levels were observed in each pathogen, even when the treatment distance was changed. A monotonic decrease in bactericidal level with increasing distance would be expected. Li et al. (35) found that the maximal bactericidal efficacy of NTAP treatment was apparent when the distance from the electrode to the sample was approximately 2-4 mm. The conditions of the treatment distance in this study were 1-5 mm, similar to the distance described above. Therefore, a distance of 1-5 mm may optimize the effects of the NTAAP device.
In this study, only 1O2 generation by the NTAAP device and its bactericidal effect were examined. However, other NTAP devices generate several types of ROS and RNS, such as hydroxy radical, H2O2, ozone, and nitric oxide; these devices also indicates bactericidal activity (15). For that reason, it is possible that NTAAP device used in this study generates not only 1O2 but also other ROS or RNS, which may contribute to bactericidal activity. Further study about other ROS and RNS generations are the focus of future work.
ROS are normally in balance with antioxidant molecules, and oxidative stress arises when this balance is disturbed, due to the depletion of antioxidants or the excess accumulation of ROS (36,37). As the bactericidal effect of 1O2 generated by the NTAAP device was confirmed, it is possible that the NTAAP-treatment enhances the
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intracellular oxidative stress level. Therefore, the amount of H2O2 and SOD activity were assessed. The H2O2, which is one of ROS, is a reactive oxygen metabolic by-product that serves as a key regulator for several oxidative stress-related states, whereas SOD, which is one of antioxidant, is related to a crucial part of the cellular antioxidant defense mechanism (38). In present results, significantly higher H2O2 and lower SOD levels were observed with the NTAAP-treatment. Ohya et al. (37) and Cueno et al. (39) have reported that lower SOD levels compared to H2O2 amounts in P.
gingivalis are classified toxic oxidative stress. On the basis of this classification, present results indicated that the NTAAP-treatment might enhance bacterial oxidative stress. Although SOD is metalloenzyme that catalyze the dismutation of the superoxide anion to H2O2 and molecular oxygen, H2O2 is generated from not only reaction product of SOD but also cellular metabolism (40,41). Thus, high oxidative stress may be indicated as higher H2O2 and lower SOD levels rather than both higher levels.
SOD activities of P. gingivalis and P. endodontalis were lower than those of S.
mutans and E. faecalis (Figs. 6A-D), suggesting the characteristics of lower ROS-erasing activity in P. gingivalis and P. endodontalis. These results may be
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another reason why bactericidal treatment times for P. gingivalis and P. endodontalis were shorter than those for S. mutans and E. faecalis (Figs. 3A-D).
Ambient air (thus principally oxygen and nitrogen) was the working gas used in this study. As both oxygen and nitrogen are diatomic, and as oxygen is highly electronegative, air excitation is more difficult than noble gas excitation (21). Thus, a power supply featuring a high repetition rate (a pulsed-power generator featuring semiconductor switches) was used for the NTAAP device in this study (22). Thus, a working gas supply is not required, and the device is sufficiently compact for use in dental clinics.
20 Conclusion
Results in this study demonstrate that a new NTAAP device generated 1O2 in the liquid phase and exerted bactericidal effects on the oral pathogens: S. mutans, P. gingivalis, E.
faecalis, and P. endodontalis. The 1O2 played the primary role in the inactivation of oral pathogens with the oxidative stress. This device has a potential for being used in treating oral diseases.
21
Acknowledgements
I wish to thank Prof. B. Ogiso and Associate Prof. M. Hayashi of Department of Endodontics and Associate Prof. M. Tamura of Department of Microbiology for their valuable guidance.
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Figures
Figure 1. Schematic diagram of the new nonthermal atmospheric pressure air plasma (NTAAP) device. Ambient air was used as working gas. An electric discharge occured between the tips of electrode 1 and electrode 2. Excited plasma was generated when the air flowed out through this area.
Figure 2. Singlet oxygen (1O2) generation by the NTAAP device. (A) Typical in vitro electron spin resonance (ESR) spectra. The experimental solution was treated by the NTAAP device at different treatment times and measured with ESR spectrometer. Manganese oxide (MnO) was used for standard’s signal intensity. (B) Relative signal intensities of 4-OH-TEMPO. The experimental solution was treated by the NTAAP device at different treatment times and distances. 1O2 generations were expressed as the relative signal intensities that normalized against the standard’s signal intensity of the MnO marker. All results are means ± standard deviations (SDs) (n = 6). Within the same distance, different letters represent significantly different (P < 0.05). Within the same treatment time, n.s. represents no significant different (P > 0.05). (C) NTAAP-induced 1O2 generation in the presence or absence of L-histidine. The experimental solution was treated with NTAAP device for 7 min at 1 mm-treatment distance in the presence or absence of L-histidine. 1O2 generations were expressed as the relative signal intensities that normalized against the standard’s signal intensity of the MnO marker.
Figure 3. Bactericidal activity of NTAAP-treatment. Bacterial cell suspensions were treated with the NTAAP device for 1, 3, 5, and 7 min at 1, 3, and 5-treatment distance. The suspensions were incubated on the BHI agar, and then the CFU/mL were determined. (A) Effect of NTAAP-treatment on the viability of S. mutans. (B) Effect of NTAAP-treatment on the viability of P. gingivalis. (C) Effect of NTAAP-treatment on the viability of E. faecalis. (D) Effect of NTAAP treatment on the viability of P. endodontalis.
Figure 4. Effect of NTAAP-treatment on the viabilities of oral pathogen in the presence of L-histidine, a 1O2 scavenger. (A) Viability of S.
mutans 5 min after NTAAP-treatment in the presence of 5 mM L-histidine. (B) Viability of P. gingivalis 1 min after NTAAP-treatment in the presence of 5 mM L-histidine. (C) Viability of E. faecalis 7 min after NTAAP-treatment in the presence of 5 mM L-histidine. (D) Viability of P. endodontalis 1 min after NTAAP-treatment in the presence of 5 mM L-histidine.
Figure 5. Hydrogen peroxide (H2O2) levels of oral pathogens after NTAAP-treatment. Treatment distance was fixed at 1 mm and treatment time was 5 min for S. mutans, 1 min for P. gingivalis, 7 min for E. faecalis, and 1 min for P. endodontalis. (A) H2O2 levels of S. mutans.
(B) H2O2 levels of P. gingivalis. (C) H2O2 levels of E. faecalis. (D) H2O2 levels of P. endodontalis.
Figure 6. Superoxide dismutase (SOD) activities of oral pathogens after NTAAP-treatment. Treatment distance was fixed at 1 mm and treatment time was 5 min for S. mutans, 1 min for P. gingivalis, 7 min for E. faecalis, and 1 min for P. endodontalis. (A) SOD levels of S.
mutans. (B) SOD levels of P. gingivalis. (C) SOD levels of E. faecalis. (D) SOD levels of P. endodontalis.
