Our previous study revealed that the photo-irradiation of rose bengal, erythrosine, and phloxine, xanthene photosensitizers, used as dental plaque disclosing ...
Biocontrol Science, 2016, Vol. 21, No. 3, 187−191
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Bactericidal Action of Photodynamic Antimicrobial Chemotherapy（PACT）with Photosensitizers Used as Plaque-Disclosing Agents against Experimental Biofilm KIRIKA ISHIYAMA, KEISUKE NAKAMURA, TARO KANNO, AND YOSHIMI NIWANO＊ Tohoku University Graduate School of Dentistry, 4-1 Seiryo, Aoba-ku, Sendai 980-8575, Japan Received 22 August, 2015/Accepted 23 April, 2016
Our previous study revealed that the photo-irradiation of rose bengal, erythrosine, and phloxine, xanthene photosensitizers, used as dental plaque disclosing agents, could exert bactericidal action on planktonic Streptococcus mutans via the singlet oxygen. In the present study, the photo-irradiation induced bactericidal activity of the three xanthene compounds against the experimental biofilm of S. mutans was investigated in combination with acid electrolyzed water（AcEW）and alkaline electrolyzed water（AlEW） . As a result, only the photo-irradiated rose bengal in AlEW showed prominent bactericidal activity with a >3-log reduction of the viable bacterial count. Since our previous study showed that the affinity of rose bengal to bacterial cells was superior to that of erythrosine and phloxine, it was speculated that AlEW damaged the extracellular matrix of the experimental biofilm, which would let the rose bengal easily be bound to the bacterial cells. From these results, it is strongly suggested that rose bengal is a suitable photosensitizer for use as a plaque disclosing agent in photodynamic antimicrobial chemotherapy to treat dental plaque. Key words：Photodynamic antimicrobial chemotherapy（PACT）/ Plaque disclosing agent / Photosensitizer / Experimental biofilm. Photodynamic antimicrobial chemotherapy（PACT） or antimicrobial photodynamic therapy（aPDT）has recently been suggested as a novel therapeutic approach for eradicating pathogenic bacteria in oral infectious diseases including dental plaque（Soukos & Goodson, 2011; Rolim et al., 2012; Mielczarek-Badora & Szulc, 2013） , which is a structurally and functionally organized multi-species biofilm colonizing tooth surfaces and epithelial cells covering the gingival sulcus and periodontal pocket（Marsh et al. 2011）. The use of PACT requires mainly three components: light, oxygen, and a non-toxic photosensitizer. Once the photosensitizer binds to the targeted bacteria, it can be activated by light of the appropriate wavelength, resulting in the killing of the bacteria in the presence of oxygen because it absorbs the light photons and transfers the excitation energy to molecular oxygen which is in turn metamorphosed to its diamagnetic form, singlet oxygen （1O2）, a major active ingredient in PACT（Clo et al., ＊
Corresponding author. Tel: +81-22-717-8298, Fax: +81-22717-8299, E-mail : niwano （a）m.tohoku.ac.jp
2007）. Acid electrolyzed water（AcEW）and alkaline electrolyzed water（AlEW）are produced by the electrolysis of dilute sodium chloride（NaCl）solution on the anode side and the cathode side of an instrument, respectively, where the anode and cathode are separated by an ionpermeable diaphragm. AcEW is commonly used in the agricultural field as a disinfectant in farm and food hygiene（Guentzel et al., 2008; Lu et al., 2010; Rahman et al., 2011）. It is also widely used for the disinfection of medical instruments such as dialyzers（Tanaka et al., 2000）, endoscopes（Lee et al., 2004）, and dentures （Nagamatsu et al., 2001）because of its potent antimicrobial potential（Nisola et al., 2011; Feliciano et al., 2012）. There have been attempts to apply AlEW as a supplementary agent for disinfection because of its high potential for decontamination（Kim et al., 2005; Park et al., 2009; Rahman et al. 2011）. In dentistry, several studies have applied AcEW to oral hygiene（Tanaka et al., 1999; Shiba et al., 2000; Qing et al., 2006）, and AcEW was proven effective for the treatment of oral candidiasis（Tanaka et al., 1999）. Regarding AlEW, it
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FIG. 1．Chemical structures of rose bengal, erythrosine, and phloxine.
was reported that AlEW was effective for removal of caries biofilms by dissolving them（Matin et al., 2011）. We have previously shown that rose bengal, erythrosine, and phloxine（Fig.1） , all of which are photosensitizers used as plaque disclosing agents, can be used for PACT（Ishiyama et al., 2012）. These three photosensitizers showed potent bactericidal activity against planktonic Streptococcus mutans, a major causative pathogen of caries. In dentistry, of the initial preparations for periodontal diseases, scaling is an effective technique used for the removal of dental calculus which is a form of hardened dental plaque. However, the technique is not properly applicable to the disinfection or removal of dental plaque. The target of the PACT we proposed is dental plaque since we used dental plaque disclosing agents as photosensitizers to treat dental plaque specifically. The aim of the present study was to examine the bactericidal effect of PACT with these three photosensitizers on experimental biofilm in combination with AcEW and AlEW as putative enhancers of the bactericidal action of PACT by causing damage to the biofilm.
1O2 was generated by irradiation using laser light, as described in our previous study（Ishiyama et al., 2012） in which an experimental laser device equipped with the second harmonic of Nd-YAG laser（wavelength: 532 nm, PAX Co. Ltd., Sendai, Japan）was used. Output power of the laser was set at 60 mW. The diameter of the irradiation field was set to be equal to that of a well （6.4 mm）of a 96-well microplate so that almost all of the light could pass through the test solution. Thus, the irradiance was calculated to be 187 mW/cm2. NaCl solution（0.075%（w/v））was electrolyzed for 15 min using a batch-type electrolyzed water generator （ALTRON MINI AL-700A; Altec Corporation, Nagano, Japan）at a regular AC voltage of 100 V and a rated current of 0.6 A. The characteristic values of the resultant AcEW and AlEW were determined using a pH/ORP meter（SG2; Mettler-Toledo, LLC, Columbus, OH, USA）for pH and ORP, and a residual chloride meter （HI196771C; Hanna Instruments Japan, Tokyo, Japan） for residual chloride concentrations. The pH, ORP, and residual chloride concentration of the AcEW were 2.42.6, >1100 mV, and 55 ppm, respectively, and those of the AlEW were 11.4-11.7, -868 mV, and 5 ppm, respectively. Experimental biofilm formation was induced according to our previous study（Ikai et al., 2010）. A stock culture strain of S. mutans JCM 5705 was purchased from Japan Collection of Microorganisms, RIKEN BioResource Center（Wako, Japan）. The bacterial strain was cultured on Brain Heart Infusion（BHI）agar（Becton Dickinson Labware, Franklin lakes, NJ, USA）anaerobically using the Anaero Pack（Mitsubishi Gas Chemical Company, Tokyo, Japan）at 37℃ for 2 days. Then, the bacterial suspension was prepared in sterile physiological saline to contain approximately 2×10 7 colony forming units（CFU）/mL. In a well of a 96-well microplate, 20 µL of the bacterial suspension was mixed with 180 µL of BHI broth containing 1% sucrose. Then, the microplate was incubated anaerobically as described above for 24 h to form biofilm at the bottom and the side wall of the well. After incubation, the medium containing unattached bacteria was removed. The well was gently washed twice using sterile physiological saline and was filled with 180 µL of saline, AcEW, or AlEW, and 20 µL of 100 µM rose bengal, erythrosine, or phloxine（Wako Pure Chemical Industries, Osaka, Japan）. Immediately after addition of the reaction mixture, the well containing the biofilm was irradiated by laser light for 3 min. Then, the reaction mixture was removed and 200 µL of sterile physiological saline was added to the well following two washings with sterile physiological saline. The biofilm was scraped with a disposable plastic stick and was suspended in the solution. A ten-fold serial dilution of the solution was prepared
PACT WITH PLAQUE DISCLOSING AGENTS AGAINST EXPERIMENTAL BIOFILM and 10 µL of the dilution was plated on BHI agar. Agar plates were incubated anaerobically as described above to enumerate CFU/well. The condition of the biofilm was observed by scanning electron microscopy（SEM） . A plastic plate cut out from the bottom of the 96-well microplate was placed in a well of a 48-well plate. Then 100 µL of the bacterial suspension was added and mixed with 900 µL of BHI broth containing 1% sucrose followed by anaerobic incubation as described above for 24 h. The plastic plates were fixed with 2.5%（w/v）glutaraldehyde （Wako Pure Chemicals Industries）at 4℃ overnight, and post-fixed with 2%（w/v）OsO4（SERVA Electrophoresis, Heidelberg, Germany）at 4℃ for 2 h. The fixed specimens were then routinely processed for SEM, i.e., dehydrated through an ethanol series, and substituted with t-butyl alcohol for freeze-drying（JFD-320, JEOL, Tokyo, Japan） . Each dried specimen was coated with platinum using an auto fine coater（JFC-1600, JEOL）and observed by using a scanning electron microscope（JXA8500F, JEOL）at 7 kV. In the presence of AlEW, incorporation efficiency of photosensitizers into bacterial cells in the biofilm was evaluated by a method essentially identical to that in our previous study（Ishiyama et al., 2012） . As was the case with SEM, 90 µL of the bacterial suspension was mixed with 810 µL of BHI broth containing 1% sucrose in a well of a 48-well plate followed by anaerobic incubation as described above for 24 h for biofilm formation. After two washings with sterile physiological saline, an aliquot （100 µL）of each photosensitizer and 900 µL of AlEW was added to each well（final concentration of each photosensitizer was 100 µM）followed by incubation at room temperature for 3 min under a light shielding condition. After being washed twice with saline, the biofilm in the saline was scraped with a disposable plastic stick. The resultant suspension was centrifuged at 5,000 x g for 5 min, and the pellet was washed twice with the saline. For the extraction of the photosensitizer, 200 µL of 99.5% ethanol was added to the pellet followed by vigorous agitation. Following centrifugation, absorbance of the supernatant was determined at the maximal absorbance wavelength of each photosensitizer. Incorporation rates of the photosensitizers were calculated by the following equation:（incorporated amount of photosensitizer/added photosensitizer）x 100. Statistical significance in the CFU/mL obtained in the bactericidal assay and in the incorporation efficiency of photosensitizers was assessed by the Tukey-Kramer HSD multi-comparison test. The analysis for the bactericidal assay was performed following logarithmic conversion. P values of 3-log reduction of viable bacterial count, suggesting that PACT with rose bengal in combination with AlEW could be applied to treat dental biofilm. Since the present study dealt with a single-species biofilm with S. mutans, the effect on mixed-species biofilm should be examined in the near future to further ascertain the feasibility of the treatment s application in dentistry. Our previous study showed that of the three photosensitizers tested, rose bengal exerted the highest bactericidal activity against planktonic S. mutans, most likely due to its high incorporation efficiency into bacterial cells（Ishiyama et al., 2012）. Thus, in this study, we also examined the incorporation efficiency of these photosensitizers in combination with AlEW into bacterial cells in the biofilm（Fig.4）. The results clearly showed that the highest incorporation rate was obtained with rose bengal. Therefore, it is speculated that AlEW causes damage to the extracellular matrix of the experimental biofilm as reported previously（Matin et al., 2011） , which would let the rose bengal easily be attached to and
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FIG. 4．Incorporation rates of photosensitizers in combination with AlEW into the bacterial cells in the biofilm. Each value is the mean with the standard deviation（n=3）.
with higher pH can enhance the bactericidal activity in biofilm. The failure of AcEW to enhance the PACT with the three photosensitizers would be attributable to the lack of physical damage to the extracellular matrix of the biofilm even though it has a high bactericidal potential. From these results and considerations, it is strongly suggested that rose bengal is a suitable photosensitizer as a plaque disclosing agent as compared to the other two photosensitizers, erythrosine and phloxine, when used for PACT to treat dental plaque. The feasibility of PACT with rose bengal also is to be ascertained by animal and human studies. REFERENCES
FIG. 3．Bactericidal activity of photo-activated photosensitizers against experimental biofilm in combination with acid and alkaline electrolyzed water（AcEW and AIEW）. P and L stand for the photosensitizer and laser light, respectively. Each value is the mean with the standard deviation（n=6） . Significant differences（p< 0.05）within each group are denoted by different alphabetical letters（i.e., bars with the same alphabetical letter are not significantly different）.
incorporated into the bacterial cells. As was the case with planktonic bacteria, the affinity of the photosensitizer to the target bacteria would be a pivotal factor to exert bactericidal action even against the damaged biofilm. Regarding the combination effect of AlEW, it might be enhanced with pH as Fukuzaki（2006）, in a review of the removal efficiency of proteins by OH-, pointed out that at pH values above 11, the efficiency was enhanced markedly with increasing pH. Thus, we will further examine if the combination effect of AlEW
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