Antimicrobial photodynamic therapy efficacy against specific pathogenic periodontitis bacterial species

Journal Pre-proof Antimicrobial photodynamic therapy efficacy specific against periodontitis pathogen bacterial species Danbi Park, Min kim, Jin Woo Choi, Jeong-Hwa Baek, Seoung Hoon Lee, Kyunghwa Baek

PII:

S1572-1000(20)30041-7

DOI:

https://doi.org/10.1016/j.pdpdt.2020.101688

Reference:

PDPDT 101688

To appear in:

Photodiagnosis and Photodynamic Therapy

Received Date:

18 November 2019

Revised Date:

29 January 2020

Accepted Date:

18 February 2020

Please cite this article as: Park D, kim M, Woo Choi J, Baek J-Hwa, Lee SH, Baek K, Antimicrobial photodynamic therapy efficacy specific against periodontitis pathogen bacterial species, Photodiagnosis and Photodynamic Therapy (2020), doi: https://doi.org/10.1016/j.pdpdt.2020.101688

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier.

Antimicrobial photodynamic therapy efficacy specific against periodontitis pathogen bacterial species Danbi Park a,†, Min kim a,†, Jin Woo Choi b, Jeong-Hwa Baek c, Seoung Hoon Lee d,*, Kyunghwa Baek a,* a

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Department of Pharmacology, College of Dentistry and Research Institute of Oral Science, Gangneung-Wonju National University, Gangwon-do, 25457 Republic of Korea b Department of Pharmacology, College of Pharmacy, Kyung Hee University, Seoul 02453, Republic of Korea c Department of Molecular Genetics, School of Dentistry and Dental Research Institute, Seoul National University, Seoul 110-749, Korea d School of Dentistry and Dental Research Institute, Wonkwang University, Iksan, Choenbuk, 54538, Republic of Korea

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Danbi Park: [email protected] Min Kim: [email protected] Jin Woo Choi: [email protected] Jeong-Hwa Baek: [email protected]

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* Correspondence to S.H.L ([email protected]) and K.B ([email protected])

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*Corresponding Author: Seoung Hoon Lee, School of Dentistry and Dental Research Institute, Wonkwang University, Seoul, 54538, Republic of Korea, Tel: +82-63-850-6981, Fax: +82-63-850-7313; E-mail: [email protected] Kyunghwa Baek, Department of Pharmacology, College of Dentistry, Gangneung-Wonju National University, Gangwondo, 210-702, Republic of Korea. Tel: +82-33-640-2462; Fax: +82-33-642-6410; E-mail: [email protected] †D. P. , M.K contributed equally to this work.

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Highlights  PDT combination did not alter the viability of the common resident bacterial species in oral flora.

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PDT reduced oral cells’ viability but cytotoxic level was comparable to standard oral antiseptics.

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PDT combination showed equivalent bactericidal rate to the Amoxicilln + Metronidazole.

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Abstract Background: To determine the safety and efficacy of antimicrobial photodynamic therapy (aPDT) combination of 0.33 mM Toluidine Blue O (TBO) with 60 mW/cm2 LED irradiation for 5 min that we had established, this study investigated the cytotoxic effect of aPDT combination on mammalian oral cells (gingival fibroblast and periodontal ligament cells) and compared the antimicrobial efficacy of antibiotics (the combination of amoxicillin (AMX) and metronidazole (MTZ)) against representative periodontitis pathogenic bacteria (Porphyromonas gingivalis, Fusobacterium nucleatum, and Aggregatibacter actinomycetemcomitans) versus our aPDT combination. Result: aPDT combination did not show any detectable effect on the viability of Streptococcus sanguinis or Streptococcus mitis, the most common resident species in the oral flora. However, it significantly reduced CFU values of P. gingivalis, F. nucleatum, and A. actinomycetemcomitans. The cytotoxicity of the present aPDT combination to mammalian oral cells was comparable to that of standard antiseptics used in oral cavity. In antimicrobial efficacy test, the present aPDT combination showed equivalent bactericidal rate compared to the combination of AMX + MTZ, the

most widely used antibiotics in the periodontitis treatment. The bactericidal ability of the AMX + MTZ combination was effective against all five bacteria tested regardless of the bacterial species, whereas the bactericidal ability of the aPDT combination was effective only against P. gingivalis, F. nucleatum, and A. actinomycetemcomitans, the representative periodontitis pathogenic bacterial species. Conclusion: The present study demonstrated the safety and efficacy of the present aPDT combination in periodontitis treatment. TBO-mediated aPDT with LED irradiation has the potential to serve as a safe single or adjunctive antimicrobial procedure for nonsurgical periodontal treatment without damaging adjacent normal oral tissue or resident flora.

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Keywords; Periodontitis, Toluidine blue O, photodynamic therapy, Photosensitizer

Introduction

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Periodontitis is a complex inflammatory disease characterized by pathologic loss of the periodontal ligament and alveolar bone as a result of dynamic interactions among bacterial pathogens, cell populations, and mediators. [1]. The outgrowth of multiple opportunistic microorganisms in oral cavity is considered as one of the critical factors of inflammation that induces periodontitis. These microorganisms induce various inflammatory cytokines, release endotoxins, and finally form a dental plaque biofilm, which is the aggregates of bacteria where cells embedded within a self-generated matrix of extracellular polymeric substance can adhere to each other [2, 3]. The basis of photodynamic therapy (PDT) is the activation of a nontoxic photosensitizing chemical substance by light of a specific wavelength which produces reactive oxygen species (ROS) that can damage DNA and cell membranes to elicit a cytocidal effect [4, 5]. In addition, antimicrobial photodynamic therapy (aPDT) has the efficacy to destruct biofilm which confers resistance against antibiotics treatment [6]. Previous studies have demonstrated the bactericidal effect of a photosensitizing dye, Toluidine Blue O (TBO) which is delivered using a blue light emitting diode (LED) on periodontitis pathogenic bacteria including Porphyromonas gingivalis (P. gingivalis), Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans) and Porphyromonas intermedia (P. intermedia), suggesting the potential of TBO as a treatment for periodontal disease [7-10]. We have recently reported the efficacy of TBO-mediated aPDT with LED irradiation to treat periodontitis. The penetrability of LED light at a wavelength of 650 nm through 3-mm-thick skin tissue and its activation of the antibacterial and anti-biofilm effect of TBO against P. gingivalis and F. nucleatum were demonstrated. Oral ointment formulation was optimized to enhance the delivery and oral targeting of TBO to the periodontal lesion. The antibacterial efficacy of this TBO formulation mediated aPDT was demonstrated in both in vitro and in vivo models [11]. Prior to the clinical trial of aPDT in periodontitis treatment, it is critical to determine whether host tissues and resident oral flora would be affected by aPDT that are effective against periodontal pathogenic bacteria. In the present study, we investigated the effect of aPDT combination that we had established on mammalian oral cells, e.g. Gingival fibroblast, Periodontal ligament cells and odontoblast. In addition, the effect of aPDT combination that we had established on Streptococcus sanguinis (S. sanguinis) and Streptococcus mitis (S. mitis), the two most common resident species in oral flora, was tested. The use of antibiotics aiming at controlling microorganisms is the conventional approach that is still widely used in periodontitis treatment. However, the long-term use of systemic antibiotics could lead to the development of antibiotics-resistant strains and superimposed infections in patients [12, 13]. Moreover, it is difficult to deliver antibiotics to the periodontal lesions because of the anatomical features of surrounding tissues and biofilms [14]. The clinical efficacy of the combination of amoxicillin (AMX) and metronidazole (MTZ) has been extensively studied and well documented [15]. Thus, we compared the efficacy of antibiotics (the combination of AMX and MTZ) against representative periodontitis pathogenic bacteria, P. gingivalis, F. nucleatum, and A. actinomycetemcomitans with our aPDT combination. We also compared the antimicrobial

efficacy of antibiotics against S. sanguinis and S. mitis with our aPDT combination to test if aPDT therapy might show bactericidal specificity for periodontitis pathogenic bacterial species. 2.

Materials & Methods

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2.1. Bacterial strains and culture conditions Streptococcus sanguinis (S. sanguinis; ATCC10556), Streptococcus mitis (S. mitis; ATCC49456), Porphyromonas gingivalis (P. gingivalis; ATCC33277), Fusobacterium nucleatum (F. nucleatum; ATCC25586), and Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans; ATCC33384) were obtained from the Korean Collection for Type Culture (KCTC, Daejeon, Korea). S. mitis, S. sanguinis, and A. actinomycetemcomitans were incubated on agar plates containing Brain Heart Infusion (Becton, Dickinson, and Company, MD, USA). P. gingivalis and F. nucleatum were incubated on agar plate containing tryptic soy broth (Becton, Dickinson, and Company, MD, USA), 5 μg/mL hemin (Sigma Aldrich, MO, USA), 5 % sheep’s blood (Komed, Seongnam-si, Korea), and 0.1 μg/mL menadione (vitamin K1) (Sigma Aldrich, MO, USA). Five types of bacteria were incubated at 37℃ for 48-72 hr in an anaerobic workstation. The density of the bacterial suspension was adjusted using a spectrophotometer (Ultrospec 3000pro, Amersham-Pharmacia, Uppsala, Sweden) at a wavelength of 540 nm (S. sanguinis: 1.0, S. mitis: 0.5, P. gingivalis: 0.8, F. nucleatum: 0.5, A. actinomycetemcomitans: 0.5).

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2.2. Cell culture and preparation Gingival fibroblasts (GF, ATCC PCS-201-018) cells were maintained in Alpha-modified Eagles medium (αMEM; Hyclone, TX, USA) supplemented with 10% Fetal Bovine Serum (FBS; Hyclone, TX, USA). Periodontal ligament (PDL, ATCC CRL-1882) cells were maintained in Dulbecco’s Modified Eagles Medium (DMEM; Hyclone; Logan, UT, USA) supplemented with 10% FBS. These cells were incubated at 37°C with 5% CO2 in a controlled humidified incubator. For aPDT activation experiments, cells were cultured in 4-well plates (SPL Life Sciences, Gyeonggi-do, Korea). After 24 hr, cells were treated with 0.33, 1.63, 3.33, and 16.34 mM of TBO dissolved in growth culture media and then exposed to the LED irradiation for 5min. Cells were then exposed to the LED irradiation for 5 min.

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2.3. Photodynamic activation studies All experiments were performed in a dark room. TBO in powder form (Sigma Aldrich, MO, USA) was dissolved in 1 X phosphate-based saline (1X PBS) and used freshly for every experiment. A 100 µL sample of adjusted bacterial suspension was placed into wells of a 96-well strip immunoplate (SPL Life Sciences, Gyeonggi-do, Korea) for photodynamic activation. TBO solutions were added to each bacterial suspension to obtain final concentrations of 0.33, 1.36, 3.33, and 16.34 mM TBO. As TBO-negative control groups, an equal volume of 1X PBS was added. Immediately after adding the TBO solution, wells of TBO-containing bacterial suspension were exposed to a LED (MK3003D, MKPOWER, Seoul, Korea: power output of 90 mW) at a wavelength of 650 nm for 5 min. The distance between the plate and the LED was 1 cm. After LED irradiation, all tested bacterial suspensions (total volume 100 µL) were spread onto agar plates and incubated in accordance with the incubation conditions for 24 hr. The number of bacterial colonies was counted and converted to logarithmic scale. 2.4. Antimicrobial analysis of antibiotics To reach final concentration of 2 mg/L Amoxicillin (AMX; Sigma Aldrich, MO, USA) + Metronidazole (MTZ; Sigma Aldrich, MO, USA), the same amount of AMX and MTZ were dissolved in bacterial culture media. The bacterial suspension was incubated for 24 hr in the presence or absence of a combination of AMX + MTZ. Then 100 ul of tested bacterial suspension was spread onto an agar plate and incubated for 24 hr. The number of viable bacterial colonies was then counted.

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2.5. Cell viability assay Mitochondrial-dependent reduction of MTT on formazan was used as an indicator of cell viability. For MTT assay, cells were seeded at the density of 5 × 10 4 cells/well and incubated for 24 hr. Immediately after the aPDT experiment, 0.5 mg/ml Thiazolyl Blue Tetrazolium Bromide (Sigma Aldrich, MO, USA) solution was added to adhered cells and incubated for 4 hr. The solution was then removed, and the formazan precipitate was solubilized in DMSO. The dissolved solution was transferred to a 96-well plate to measure absorbance at wavelength of 540 nm using a microplate reader (BioTek, St. Winooski, VT, USA). For LIVE/DEAD cell staining, cells were seeded at the density of 3 × 104 cells/well and incubated for 24 hr. Experiments were performed using a LIVE/DEAD® Viability/Cytotoxicity Kit (Invitrogen, CA, USA) according to the manufacturer's instructions. A combination of calcein AM and ethidium homodimer -1 solution were diluted with 1X PBS. Adhered cells were stained for 30 min at room temperature. To obtain fluorescence images, the green fluorescence of live cells was detected at 520 nm and the red fluorescence of dead cells was detected at 495 nm using an Axio Imager.A2 microscope (Zeiss, Oberkochen, Germany). Overlay of green and red fluorescence images was performed using Image J program.

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2.6. Comparison with antiseptics Cells were seeded at the density of 5 × 104 cells/well and incubated for 24 hr. Adhered cells were treated with 3.02 % hydrogen peroxide (H2O2, DUKSAN, Seoul, Korea), 0.025 % benzalkonium chloride (DUKSAN, Seoul, Korea), and 0.12 % chlorhexidine gluconate (Sigma Aldrich, MO, USA). After 5 min, cells were washed with 1x PBS and subjected to MTT assay according to the method described in the previous section.

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2.7. Statistical analysis. The results were expressed as mean values ± standard deviations (mean ± SD). A two-way analysis of variance (ANOVA) was performed with post hoc testing (Tukeys’ test) as appropriate to determine whether there were significant differences among the test conditions. A p-value < 0.05 was considered statistically significant. Results

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3.1. aPDT shows bactericidal effect against P. gingivalis, F. nucleatum, and A. actinomycetemcomitans, but not against S. sanguinis or S. mitis First, the effect of the present aPDT combination on S. sanguinis and S. mitis, the two most common resident species in oral flora, was tested. Based on study results obtained previously, a combination of 0.33 mM TBO with 60 mW/cm2 LED irradiation for 5 min was used to evaluate their in vitro antibacterial activity. Mean CFU values of colonies exposed to TBO-mediated LED photoactivation were analyzed. Incubation of S. sanguinis and S. mitis with TBO in the dark showed no effect on bacterial growth. The present aPDT combination did not show any detectable effect on the viability of S. sanguinis or S. mitis (Fig. 1A, B and S1A, B). As demonstrated in our previous study, the present aPDT combination induced ~99% bactericidal rate for P. gingivalis and F. nucleatum (Fig. 1C, D and S1C, D). In addition, the therapeutic potential of the present aPDT combination on another bacterial species, A. actinomycetemcomitans, a representative localized aggressive periodontitis (LAP) causing bacterium, was assessed. The present aPDT combination induced ~90% bactericidal rate for A. actinomycetemcomitans (Fig. 1E and S1E).

3.2. aPDT shows cytotoxic effect on gingival fibroblasts and periodontal ligament cells but the reduction in cell viability is within conventional levels observed with antiseptics To evaluate whether the present aPDT combination could affect mammalian oral cell survival, we initially performed Live/Dead staining, an assay in which the levels of cell death (red ethidium homodimer staining) and cell viability (green calcein AM staining) could be qualitatively compared. This analysis revealed that 5X higher (1.63

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mM) TBO concentration than that in present aPDT combinations started to lead death of cells (Fig. 2A, B). The application of LED did not significantly increase the cytotoxic effect of TBO. Next, we performed quantitative MTT assays to assess mitochondrial function and cell survival after PDT treatment. Fig. 2C, D show cytotoxic effects of aPDT on three types of oral cells based on MTT assay. The present aPDT combination significantly reduced viabilities of GF and PDL cells by 49%, and 69%, respectively (all p < 0.05). The application of LED did not enhance the reduction in cell viability by any concentration of TBO. We next tested whether the cytotoxicity observed with our aPDT combination was comparable to that of standard antiseptics used in oral cavity. As shown in Fig. 2C, D, the viability of cells treated with TBO (0.33 mM to 16.34 mM) and LED irradiation was higher than or not significantly different from the viability of cells treated with 3.02% H2O2, 0.025% benzalkonium chloride, or 0.12% chlorhexidine, demonstrating that the reduction in cell viability under the present aPDT therapy (0.33 mM * 5 min LED) was within conventional levels.

Discussion

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3.3. Antibiotic efficacy of AMX + MTZ combination is equivalent to that of aPDT and AMX + MTZ combination shows bactericidal ability against S. sanguinis and S. mitis as well as P. gingivalis, F. nucleatum, and A. actinomycetemcomitans Because the clinical efficacy of the combination of amoxicillin (AMX) and metronidazole (MTZ) has been extensively studied and well documented in the periodontitis treatment [15], we next compared the antibiotic efficacy of AMX + MTZ and aPDT treatment to the bacterial species we tested above. Bacterial suspensions were treated with a combination of 1 mg/L AMX and 1 mg/L MTZ and incubated for 24 hours. Results showed that the antibiotic efficacy of AMX + MTZ and aPDT were not significantly different with regard to their impact on CFU values compared to results shown in Fig. 1 (C-E). Figure 3A-E and S3A-E shows overall results of the antibiotic effect of AMX + MTZ combination. The combination of AMX + MTZ showed bactericidal ability against all five bacteria tested, regardless of bacterial species. The bactericidal rate was ~ 99% compared to the CON group.

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As a follow-up to the previous study demonstrating that TBO-mediated aPDT using LED irradiation has potential for periodontitis treatment, the purpose of the present study was to evaluate the safety and the efficacy of aPDT therapy that we established. In the present study, we demonstrated that aPDT combination did not show any detectable effect on the viability of S. sanguinis or S. mitis, the most common resident species in oral flora. The cytotoxicity observed with the present aPDT combination (0.33 mM TBO with 5 min LED irradiation) to mammalian oral cells was comparable to that of standard antiseptics used in oral cavity. In antimicrobial efficacy test, the present aPDT combination showed equivalent bactericidal rate to the combination of AMX + MTZ, the most widely used antibiotics in the periodontitis treatment. Bactericidal ability of the AMX + MTZ combination was effective against all five bacteria we tested, regardless of the bacterial species, whereas the aPDT combination was only effective against P. gingivalis, F. nucleatum, and A. actinomycetemcomitans, the representative periodontitis pathogenic bacterial species. In the present study, aPDT showed selective cytotoxic effect only to periodontitis causing bacteria (P. gingivalis, F. nucleatum, and A. actinomycetemcomitans). aPDT did not show any detectable effect on the viability of S. sanguinis or S. mitis, the representative resident microorganisms in the oral cavity. When a nontoxic photosensitizing dye is activated by light of a specific wavelength, it produces reactive oxygen species (ROS) that can damage DNA and cell membranes to elicit a microbial killing effect. Given that the bacteria we tested as periodontal causative bacteria are gram-negative anaerobic species but the bacteria tested as resident microbes in oral cavity are gram-positive anaerobic species, it could be speculated that periodontal causative bacteria are more susceptible to aPDT combination due to

their structural characteristics without solid cell walls. This finding provides a clue about which microbial species or microorganism could be specifically targeted by TBO-mediated aPDT with LED irradiation in the periodontitis treatment..

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Ethidium homodimer-1 / calcein AM staining results showed that our aPDT combination does not alter the viability of gingival fibroblasts and periodontal ligament cells. Cytotoxic effect was not observed until the use of 5X higher TBO concentration (1.63 mM). However, viabilities of gingival fibroblasts and periodontal ligament cells using MTT assay revealed that our aPDT combination induced an increase in the relative proportion of dead cells. TBO alone negatively influenced the viability of oral cells. The application of LED did not enhance the cytotoxic effect of TBO. But, the in vitro reduction of cell viability under the present aPDT condition did not exceed or was even lower than that observed with antiseptics widely used in dental clinics. This means that the cytotoxicity of aPDT, if it exists, is within conventional levels. This result is in line with a previous report demonstrating the safety of aPDT for clinical application in the periodontitis treatment. Ichinose-Tsuno et al. have reported that a combination of 500 or 1000 ug/ml TBO and LED irradiation for 20 sec can significantly reduce the CFU number of S. oralis and that the cytotoxicity of aPDT is comparable to that of standard antiseptics used in oral cavity[16]. Additionally, given that previous reports have shown resistance of bacteria to antiseptics such as benzalkonium chloride, reducing bacterial resistance against antiseptics would be another benefit of introducing the present aPDT combination in clinical application [17-19]. Regarding the safety of aPDT, the present aPDT combination in periodontitis treatment could be clinically applied without damaging adjacent normal tissue or resident flora.

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In recent decades, metronidazole (MTZ) and amoxicillin (AMX) have been selected as effective adjuncts in the treatment of severe periodontitis in adults [20-25]. In the present study, the combination of AMX + MTZ was unequivocally shown to have ~99.9% antimicrobial efficacy in vitro. The present aPDT combination also showed equivalent antimicrobial efficacy with regard to its impact on CFU values of P. gingivalis and F. nucleatum. Prolonged, long-term use of systemic antibiotics exposes patients to the risk of developing antibiotics-resistant strains and superimposed infections [12, 13]. If a combination of aPDT with conventional antibiotics therapy could result in greater reduction of the risk of developing antibiotics-resistant strains, it would help establish effective strategies, especially for severe periodontitis subjects with impaired immune system such as those with diabetes.

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In addition, we demonstrated that aPDT treatment in A. actinomycetemcomitans suspension achieved ~90 % photo-killing rate (Fig. 3C, E). A. actinomycetemcomitans is known to be a main etiologic agent in Localized Aggressive Periodontitis (LAP) [26]. LAP is a rare form of inflammatory periodontal disease characterized by a rapid progression with dramatic attachment and bone loss, specifically on first molars and incisors [27]. Mechanical removal of bacteria and calculus through scaling and root planing coupled by use of systemic antibiotics is the current therapy for LAP treatment. Considering the aPDT antimicrobial efficacy against A. actinomycetemcomitans demonstrated in the present study, a combination of aPDT with conventional antibiotics therapy could be an effective strategy for LAP subjects. Moreover, bactericidal ability of the combination of AMX + MTZ was effective against all five bacteria we tested regardless the bacterial species, whereas bactericidal ability of the aPDT combination was effective only against P. gingivalis, F. nucleatum, and A. actinomycetemcomitans, the gram-negative species. This finding suggests that aPDT therapy could be an efficient strategy in the treatment of periodontitis without damaging resident oral flora. Taken together, the present study demonstrated the safety and efficacy of the present aPDT combination in periodontitis treatment. TBO-mediated aPDT with LED irradiation has potential to serve as a safe single or adjunctive antimicrobial procedure for nonsurgical periodontal treatment without damaging adjacent normal tissues or resident flora. Acknowledgements

This research was supported by a grant from the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health and Welfare, the Republic of Korea (HI16C0501).

Author Contributions

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J.C. and K.B. conceived the study design. D.P. and M.K. conducted the experiments. D.P., M.K., J.C., J.B., S.L. and K.B. interpreted the data. D.P., M.K. and K.B. wrote the draft manuscript. S.L., J.C. and K.B. revised and approved final version of manuscript. All authors reviewed the manuscript.

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[19] M. Tandukar, S. Oh, U. Tezel, K.T. Konstantinidis, S.G. Pavlostathis, Long-term exposure to benzalkonium chloride disinfectants results in change of microbial community structure and increased antimicrobial resistance, Environ Sci Technol 47(17) (2013) 9730-8. [20] I. Borges, M. Faveri, L.C. Figueiredo, P.M. Duarte, B. Retamal‐ Valdes, S.C.L. Montenegro, M. Feres, Different antibiotic protocols in the treatment of severe chronic periodontitis: A 1‐ year randomized trial, Journal of clinical periodontology 44(8) (2017) 822-832. [21] N. Cionca, C. Giannopoulou, G. Ugolotti, A. Mombelli, Amoxicillin and metronidazole as an adjunct to full‐ mouth scaling and root planing of chronic periodontitis, Journal of periodontology 80(3) (2009) 364-371. [22] N. Cionca, C. Giannopoulou, G. Ugolotti, A. Mombelli, Microbiologic testing and outcomes of full‐ mouth scaling and root planing with or without amoxicillin/metronidazole in chronic periodontitis, Journal of periodontology 81(1) (2010) 15-23. [23] M. Feres, G.M.S. Soares, J.A.V. Mendes, M.P. Silva, M. Faveri, R. Teles, S.S. Socransky, L.C. Figueiredo, Metronidazole alone or with amoxicillin as adjuncts to non‐ surgical treatment of chronic periodontitis: a 1‐ year double‐ blinded, placebo‐ controlled, randomized clinical trial, Journal of clinical periodontology 39(12) (2012) 1149-1158. [24] J.M. Goodson, A.D. Haffajee, S.S. Socransky, R. Kent, R. Teles, H. Hasturk, A. Bogren, T. Van Dyke, J. Wennstrom, J. Lindhe, Control of periodontal infections: a randomized controlled trial I. The primary outcome attachment gain and pocket depth reduction at treated sites, Journal of clinical periodontology 39(6) (2012) 526-536. [25] F. Sgolastra, R. Gatto, A. Petrucci, A. Monaco, Effectiveness of systemic amoxicillin/metronidazole as adjunctive therapy to scaling and root planing in the treatment of chronic periodontitis: A systematic review and meta‐ analysis, Journal of periodontology 83(10) (2012) 1257-1269. [26] D. Burgess, H. Huang, P. Harrison, I. Aukhil, L. Shaddox, Aggregatibacter actinomycetemcomitans in African Americans with localized aggressive periodontitis, JDR Clinical & Translational Research 2(3) (2017) 249-257. [27] T.J. Oh, R. Eber, H.L. Wang, Periodontal diseases in the child and adolescent, J Clin Periodontol 29(5) (2002) 400-10.

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Fig. 1. Bactericidal effect of TBO with LED irradiation on S. sanguinis, S. mitis, P. gingivalis, F. nucleatum, and A. actinomycetemcomitans strains. Experiments were performed in the dark. TBO in powder form was dissolved in 1X PBS. Bacterial suspensions of (A) S. sanguinis, (B) S. mitis, (C) P. gingivalis, (D) F. nucleatum, and (E) A. actinomycetemcomitans were directly exposed 60 mW/ cm 2 of 650 nm wavelength LED for 5 min in the presence or absence of 0.33 mM TBO. TBO-negative control groups were added with an equal volume of PBS. (Fig. S1A-E) After LED irradiation, all tested bacterial suspensions (total volume: 100 µL) were spread onto agar plates. The number of viable bacterial colonies was counted and converted to logarithmic scale. Data represent the mean ± the standard deviation of triplication. *p < 0.05 vs. CON w/o LED irradiation. CFU, colony forming unit.

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Fig. 2. Effect of aPDT on viability of cells from periodontal tissue. (A,C) GF, and (B,D) PDL cells were cultured in 4-well plate. TBO in powder form was dissolved in growth culture media. (A,B) These cells were exposed to a combination of 0, 0.33, 1.63 or 3.33 mM TBO with LED irradiation for 5 min. The number of cell Live / Dead was monitored using a calcein AM (green, live cell) / ethidium homodimer – 1 (red, dead cell) double staining. The green fluorescence of live cell was detected at 520 nm and the red fluorescence of dead cell was detected at 495 nm. Overlay of the green and red fluorescence images was performed using Image J program. Proportions of dead cells were quantified as percentage of live cells (gre en fluorescence) in total number of cells (green + red fluorescence) for each condition. (C,D) The effect of aPDT on cell viability was analyzed by MTT assay. Cells were exposed to a combination of TBO + LED irradiation or treated with 3.02 % H2O2, 0.025 benzalkonium chloride, 0.12 % chlorhexidine for 5 min. Samples were incubated with 0.5 mg/ml thiazolyl blue tetrazolium bromide for 4 hr. Formazan precipitate was solubilized in DMSO and measured at 540 nm absorbance using a microplate reader. Absorbance values of measured samples were converted into percentages. Data

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represent the mean ± the standard deviation of duplicates. * p < 0.05 vs. 0 mM w/o LED irradiation, # p < 0.05 vs. TBO w/ LED irradiation groups.

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Fig. 3. Efficacy of antibiotics against S. sanguinis, S. mitis, P. gingivalis, F. nucleatum, and A. actinomycetemcomitans strains. Bacterial suspensions of (A) P. gingivalis, (B) F. nucleatum, (C) A. actinomycetemcomitans, (D) S. sanguinis, and (E) S. mitis were incubated in the presence or absence of a combination drug of 1 mg/L amoxicillin (AMX) and 1 mg/L metronidazole (MTZ) (total concentration 2 mg/L) for 24 hr. (Fig. S2A-E) All tested bacterial suspensions were spread onto agar plates. The number of viable bacterial colonies was then counted and converted to logarithmic scale. Data represent the mean ± the standard deviation of triplication. *p < 0.05 vs. control. CFU, colony forming unit.