Comparative effect of photodynamic therapy on separated or mixed cultures of Streptococcus mutans and Streptococcus sanguinis

Accepted Manuscript Title: Comparative effect of photodynamic therapy on separated or mixed cultures of Streptococcus mutans and Streptococcus sanguinis Authors: Vanesa P´erez-Laguna, Luna P´erez-Artiaga, Ver´onica Lampaya-P´erez, Santiago Camacho L´opez, Isabel Garc´ıa-Luque, Mar´ıa Jos´e Revillo, Santi Nonell, Yolanda Gilaberte, Antonio Rezusta PII: DOI: Reference:

S1572-1000(17)30234-X http://dx.doi.org/doi:10.1016/j.pdpdt.2017.05.017 PDPDT 969

To appear in:

Photodiagnosis and Photodynamic Therapy

Received date: Revised date: Accepted date:

2-3-2017 28-4-2017 20-5-2017

Please cite this article as: P´erez-Laguna Vanesa, P´erez-Artiaga Luna, LampayaP´erez Ver´onica, L´opez Santiago Camacho, Garc´ıa-Luque Isabel, Revillo Mar´ıa Jos´e, Nonell Santi, Gilaberte Yolanda, Rezusta Antonio.Comparative effect of photodynamic therapy on separated or mixed cultures of Streptococcus mutans and Streptococcus sanguinis.Photodiagnosis and Photodynamic Therapy http://dx.doi.org/10.1016/j.pdpdt.2017.05.017 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. 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.

Comparative effect of photodynamic therapy on separated or mixed cultures of Streptococcus mutans and Streptococcus sanguinis Vanesa Pérez-Laguna1,2, Luna Pérez-Artiaga1, Verónica Lampaya-Pérez1, Santiago Camacho López3, Isabel García-Luque4, María José Revillo1,5, Santi Nonell6,*Yolanda Gilaberte5,7, *Antonio Rezusta1,2,5 *These authors contribute equally to this work. 1 Department of Microbiology, Hospital Universitario Miguel Servet, Zaragoza, Spain 2 University of Zaragoza, Zaragoza, Spain 3 Department of Optic, Ensenada Center for Scientific Research and Higher Education (CICESE), Baja California, México. 4 Department of Microbiology, University of Sevilla, Sevilla, Spain. 5 IIS Aragón, Zaragoza, Spain 6 Institut Químic de Sarrià, Universitat Ramon Llull, Barcelona, Spain 7 Department of Dermatology, Hospital San Jorge, Huesca, Spain. Corresponding Author: Vanesa Pérez-Laguna, [email protected] Department of Microbiology, Miguel Servet University Hospital. C/ Padre Arrupe s/n 50009 Zaragoza, Spain S. mutans; S. sanguinis; Reactive oxygen species HIGHLIGHTS   

-aPDT with RB and MB using a specific LED lamp has a significant bactericidal effect on S. mutans and S. sanguinis strains. -RB is slightly more efficient than MB. -Effects are the same in vitro either for separate bacteria or on the samples constituted by both bacteria.

ABSTRACT Antimicrobial photodynamic therapy (aPDT) has shown to exert a bactericidal effect Page 1 of 18

against Streptococcus sanguinis and Streptococcus mutans. However, this efficacy has been reported for either type of bacteria separately. Bacterial suspensions of both strains, separately or together, were treated with concentrations of methylene blue (MB) and rose bengal (RB). Suspensions were irradiated with a light– 2

. RB-aPDT

at concentrations of 0.16-0.62 and 0.16-0.31 μg/mL, and MB-aPDT at concentrations of 0.62-1.25 and 0.31-1.25 μg/mL inhibited the growth of S. mutans and S. sanguinis respectively by 6 log10. In suspensions of both strains together, the same 6 log10 reduction in bacterial growth was achieved using the same concentrations of each photosensiziser. Keywords: Caries; In conclusion, RB-aPDT and MB-aPDT appear to exert the same bactericidal effect against suspensions of S. sanguinis and S. mutans either for single strain treatment or for samples constituted by both bacteria mixed together. RB shows to be slightly more efficient than MB. INTRODUCTION Dental caries is a chronic and invasive disease that includes demineralization of the tooth followed by destruction of the organic phase of the dentine [1]. It is caused by the interaction between oral microbiota, diet, dentition and oral environment [2]. A highly diverse community of bacteria colonizes the oral surfaces [3]. The majority belongs to the genus Streptococcus and includes the 70% of the cultivable bacteria existing in the human dental plaque [4]. S. mutans is the most prevalent microorganism of the plaque and the main pathogenic agent responsible for caries disease [5]. On the other hand, S. sanguinis, as well called S. sanguis, is considered a benign or even a beneficial microorganism of the oral cavity [6], although its association with bacterial endocarditis is well described in the literature [7]. For that reason Page 2 of 18

its presence can be potentially dangerous particularly when it can reach the bloodstream like in dental interventions [8]. In the present investigation we study both streptococci either separately or mixed together in order to get close to what happens in real life/the clinic. Photodynamic therapy (PDT) has been advocated as an alternative to antimicrobial agents to suppress subgingival bacteria species [9-11] due to the extensive and inappropriate use of antimicrobial agents, which gradually led to the development of pervasive resistance [12, 13]. The advantages of PDT also include reductions in the occurrence of side effects, which are frequently observed after the systemic administration of antibiotics, and the convenient cost of treatment [14]. The antimicrobial PDT (aPDT) is a technique that utilizes reactive oxygen species (ROS) produced by non-toxic dye or photosensitizer (PS) molecules exposed to low intensity visible light to kill a target microorganism [15]. Methylene blue (MB), a well-known phenothiazine dye whose absorption peaks at 665 nm, and Rose Bengal (RB), a xanthene dye characterized by an absorption peak at the 557 nm, have shown a good profile to photoinactivate microorganisms [16, 17]. Recently, our group has demonstrated the utility of these PSs to inactivate cariogenic microorganisms using a white light lamp that covers the absorption spectra of both of them [11]. However, a light source with an emission spectrum matching the absorption bands in the spectrum of each PS would enhance the light-PS interaction therefore increasing its efficacy [18]. The aim of this study was a straight comparison of the photoinactivation effect on S. mutans and S. sanguinis, when they were cultivated either separately or mixed together, under MB and RB treatment by using LED lamps emitting at the matching wavelength of the optical absorption bands of the chosen PS. MATERIALS AND METHODS Microorganisms and growth conditions S. mutans ATCC 35668 and S. sanguinis ATCC 10556 strains were obtained from the Page 3 of 18

American Type Culture Collection (ATCC; Rockville, MD). Columbia Blood Agar (BA) was purchased from Oxoid (Madrid; Spain). Microorganisms were grown aerobically in BA medium at 35ºC for 24 h for the case of S. sanguinis and 48 h for S. mutans. Photosensitizer solutions Methylene blue (MB) was purchased from Sigma-Aldrich (Madrid; Spain) and rose bengal (RB) from Fluka-Sigma-Aldrich (Madrid; Spain). MB and RB stock solutions were prepared in bidistilled water and diluted, also using bidistilled water, to the desired concentration immediately prior to use. The concentrations used ranged from 0.01 to 640 μg/mL. We did experiments with this range and then, based on the observed results (unpublished), we limited the range to do the experiments presented in this work to the range 0.03 to 2.5 µg/ml. All the solutions were prepared and handled under light-restricted conditions. Light source According to the absorption features of each PS [11], which show maximum optical absorption at 665 nm for MB and 557 nm for RB, we used red and green LEDs to excite the MB and RB photosensitizers, respectively. Irradiation was performed at a fluence of 18 J/cm2 with the red LED lamp (center at 625 nm, 0.007 W/cm2) for MB, and the green LED lamp (center at 515 nm, 0.0058 W/cm2) for RB (Showtec LED Par 64 Short) (Figure 1). Notice that the used wavelengths 625 nm and 515 nm fall short to the maximum absorption peaks for both PS, however both wavelengths are well within the absorption band of both PS (Fig. 1). Photodynamic treatment of microorganisms Bacterial suspensions were prepared in bidistilled water and adjusted to optical densities corresponding to 0.5 McFarland containing > 107 cell/mL. McFarland scale is recommended for performance of susceptibility testing by CLSI [19] and EUCAST [20]. 90 μL of either S.

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sanguinis or S. mutans suspension was dropped into a microtiter plate and 10 μL of the different concentrations of PS, ranged from 0.01 to 640 μg/mL were added. In experiments mixing both streptococci, 90 μL of each strain suspension was dropped and 20 μL of the different PS solutions was added. No incubation with the PS was performed before irradiation based on the best bactericidal effect achieved in a previous study [11] and because in the clinic, especially in the mouth, it is preferable to use short incubation times. Microorganism suspensions with the different PSs prepared and poured into a 96 well microtiter plate (6 mm well diameter) were LED irradiated placing the lamps at a distance of 17 cm above the plate. 17 cm of distance was used because the illumination is homogeneous. The exposures were 42 minutes and 52 seconds long for the red LED and 51 minutes and 43 seconds long for the green LED lamp (fluence 18 J/cm2). Bacterial cultures under the same conditions with and without PS, either kept in the dark or illuminated, served as controls. Samples without adding PS and non-irradiated were used as initial control. The resultant bacterial suspensions were spread in BA. After photodynamic treatment, the samples and controls were incubated at 35 ºC for 24 h for the case of S. sanguinis experiments, and for 48 h for the case of S. mutans. Viable bacteria, colony-forming unit (CFU), were determined by colony counting in BA plates. A criterion of 6 log10 unit decrease from the starting inoculum was adopted to define bactericidal activity. All experiments were carried out at least five times. Statistical Analysis S. sanguinis and/or S. mutans log10 reductions were expressed by means and standard deviation (DS). Differences between two means were analyzed using the Kruskall-Wallis test. Statistical significance was assumed at a p value of <0.05. RESULTS Photoinactivation of bacteria suspensions

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Under the experimental conditions, PDT with MB and RB at a fluence of 18 J/cm2 using the specific wavelength LED lamp inhibited the growth of both strains by 6 log10 therefore reaching a bactericidal effect. However, a significant lower concentration of RB as compared to MB was needed to kill Streptococcus spp. Whereas this bactericidal effect was achieved for S. mutans with a concentration of RB of 0.16-0.62 μg/mL, a higher MB concentration (0.62-1.25 μg/mL) was required to reach the same reduction (Figure 2). In the case of S. sanguinis, the RB and MB concentrations needed to obtain the same bactericidal effect were quite similar to those used for S. mutans: 0.16-0.31 μg/mL and 0.31-1.25 μg/mL, respectively (Fig. 2). In mixed suspension of both Streptococcus the required concentrations of each PS to obtain a bactericidal effect remains the same as in the case of separate bacteria reduction, for the same LED irradiation and growth conditions (Figure 3). There are no significant differences between the concentrations required to achieve the same log reduction between suspensions of a single bacteria and a mixture of both. Both PSs in the range of concentrations used, under the same conditions but keeping the samples in darkness, did not reduce the number of CFU/mL of the initial inoculum. Neither the LED lamps at 18 J/cm2 reduced the amount of microorganisms significantly when PS were not added to suspensions. DISCUSSION PDT may be well suited to photoinactivation of oral surfaces microorganisms [1] [21]. Our investigation showed that PDT using MB or RB and a LED lamp emitting at a specific wavelength close to the optical absorption peak of each PS can kill cariogenic microorganisms, such as S. mutans, and systemic potentially dangerous microorganisms hosted on the oral cavity, such as S. sanguinis. According to our results, RB showed slightly higher antimicrobial photodynamic effect for

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Streptococcus spp. than MB. These results are similar to those previously observed by our group using white light [11]. Both studies reached an inhibition of 99.9999 % in S. mutans and S. sanguinis, however the 6 log10 reduction in the number of CFU/mL has been achieved in the present study with PS concentrations equal or slightly lower, but using almost half of the fluence (18 J/cm2 vs 37 J/cm2). In this sense, the use of a white light lamp that covers the absorption spectrum of most PSs can be used for PDT, although the use of LED lamps emitting at a narrow bandwidth within the optical absorption band of each PS is significantly more efficient. Table 1 summarizes the in vitro PDT studies using MB and RB on S. mutans and S. sanguinis and it allows comparing our results with those of other authors. Araujo et al. [22] needed 25 μg/mL MB to reach 73% inhibition on S. mutans using red laser light, which corresponds with the maximum spectrum absorbance of MB but the fluence and the power density used is not detailed. Chan et al. [23] demonstrated that aPDT with MB was able to produce a reduction of 99-100% on cultures of S. sanguinis. However, they used both a higher concentration (100 μg/mL) of the PS and a higher fluence (21.2 J/cm2) of a laser diode (632.8 nm) as compared to what we used. The lower range reduction obtained in this study may be due to the fact of a shorter time of photodynamic treatment (60 seconds) based on a higher light energy density, which drops down the efficiency because lack of oxygenation [24]. On the contrary, in our study we exposed the bacteria to a lower fluence light source but for a longer time, i. e. a higher integrated fluence, this promotes a better oxygenation, giving as a result an enhanced oxidative damage. Regarding RB, studies carried out by Costa et al. [27] show that the concentration needed to attain 6.86 log10 CFU/mL reduction of S. mutans was 2.02 μg/mL, using a LED lamp with wavelength in the range 440-460 nm and a fluence of 95 J/cm2. Comparing to our results, they needed higher concentrations of PS and five times the fluence we used to get Page 7 of 18

a similar effect; this can be explained mainly by the very low optical absorption of RB at the exciting wavelengths. Notice also that we use distilled water as solvent, whereas Costa et al. [27] used phosphate-buffered saline; according to Nuñez et al. [25], a significant difference for aPDT under the same experimental conditions can be promoted by the use of different solvents, showing that saline solutions present less oxygen availability than water. Rolim et al. [26] reported a bactericidal effect of RB on S. mutans without need of exposure to light. Nevertheless, in all the experiments carried out not only by our group [11] but also by Costa et al. [27] no antimicrobial effects were observed at all when the strains were exposed either to the dyes or the light source, separately. Regarding aPDT-RB on S. sanguinis, Pereira et al. [28] obtained a very low reduction of 9.9 % using 5 μg/mL RB and a higher fluence (95 J/cm2) working on S. sanguinis biofilm. This very low reduction rate could be attributed to the fact that biofilm cells are at least 500 times more resistant to antibacterial agents [29]. Also, as in the case of Costa et al. [31], the exciting wavelength (435-475 nm) falls at the very tail of the absorption band, providing poor excitation. To the best of our knowledge, Nedeljkovic et al. [30] and Huanga et al. [6] studied the effect of pH on mixes of both streptococci, but there are not any more published reports for treating both microorganisms together in solution, and there are none with aPDT. Although we did not find significant differences between the photodynamic dose needed to photoinactivate either mixed bacteria or separate ones in solution, mixed cultures need to be further investigated for a better understanding approach to the in vivo process, where dental caries result from interactions among different cariogenic microorganisms. The next step in the line of this research would be the biofilm study and the study of this infection with bioluminescent microorganism in animals models [31]. The study of the potentiation Page 8 of 18

of efficacy when using inorganic salts combined with aPDT could also be explored [31]. Our present study opens the scenarios to perform aPDT against cariogenic bacteria, where RB with green light sources (500-560 nm) seems to be a very convenient PS to use in the clinic, first because its effectiveness and second because it has a more friendly color to be used in the mouth than MB, one of the most frequently employed by dentists in the clinic. Nevertheless aPDT-MB with red light sources (630-700 nm) it could be a better option to treat deep caries due to their relatively long wavelengths, which can effectively penetrate biological tissues. One limitation of our aPDT method is the long time required to reach the desired fluence with our LED lamps, more than 40 minutes. This very long exposure can make treatment uncomfortable for patients and less convenient than lasers for doctors. Perhaps the design of portable LED devices adapted for the oral cavity could reduce the irradiation time and may be a good option to treat or prevent caries in the future, and to avoid the risk of bacterial endocarditis after clinical intervention in oral cavity.

CONCLUSIONS

The use of LED lamps emitting at a specific wavelength close or matching with the absorption peak of MB or RB are effective to photoinactivate S. mutans and S. sanguinis either separately or mixed together in suspensions. The good profile of efficiency, cosmetics, and security of RB in combination with green light supports to explore its clinical application to prevent and/or treat dental decay being especially useful in situations in which antimicrobial therapy is ineffective or not recommended.

COMPETING INTERESTS

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The authors declare that they have no competing interests.

ETHICS COMMITTEE/INSTITUTE THAT APPROVED OUR STUDY Our study does not require any ethical approval.

ACKNOWLEDGEMENTS

This work was supported by grant CTQ2013-48767-C3-2-R from the Spanish Ministry of Science and Innovation, Spain and European Regional Development Fund.

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REFERENCES [1] Lima JP, Sampaio de Melo MA, Borges FM, Teixeira AH, Steiner-Oliveira C, Nobre Dos Santos M, Rodrigues LK, Zanin IC. Evaluation of the antimicrobial effect of photodynamic antimicrobial therapy in an in situ model of dentine caries. Eur J Oral Sci. 2009; 117(5):568-74. [2] Marsh PD. Microbiologic aspects of dental plaque and dental caries. Dent Clin North Am. 1999; 43(4):599-614, V-VI. [3] Marcotte H, Lavoie MC. Oral microbial ecology and the role of salivary immunoglobulin A. Microbiol Mol Biol Rev 1998; 62:71-109. [4] Jenkinson HF. Adherence and accumulation of oral streptococci. Trends Microbiol. 1994; 2(6):209-12. [5] Orasmo E MW, Otani C, Khouri S. In vitro AFM evaluation of Streptococcus mutans membrane exposed to two mouthwashes. JAPS. 2013; 3:024-8. [6] Huanga X, Palmera SR, Ahna SJ, Richardsb VP, Williamsa ML, Nascimentoc MM. and Burnea RA. A Highly Arginolytic Streptococcus Species That Potently Antagonizes Streptococcus mutans. Appl. Environ. Microbiol. 2016; 82.7 2187-2201. [7] Li X, Kolltveit KM, Tronstad L , Olsen I. Systemic diseases caused by oralinfection. Clin Microbiol Rev. 2000; 13:547-58. [8] Caufield PW, Dasanayake AP, Li Y, Pan Y, Hsu J, Hardin JM. Natural history of Streptococcus sanguinis in the oral cavity of infants: evidence for a discrete window of infectivity. Infect Immun. 2000; 68(7):4018-23. [9] Fontana CR, Abernethy AD, Som S, Ruggiero K, Doucette S, Marcantonio RC, Boussios CI, Kent R, Goodson JM, Tanner AC, Soukos NS. The antibacterial effect of photodynamic therapy in dental plaque-derived biofilms. J Periodontal Res. 2009; 44(6):751-9. [10] Jori G, Fabris C, Soncin M, Ferro S, Coppellotti O, Dei D, Fantetti L, Chiti G, Roncucci G. Photodynamic therapy in the treatment of microbial infections: basic principles and perspective applications. Lasers Surg Med. 2006; 38(5):468-81. [11] Soria-Lozano P, Gilaberte Y, Paz-Cristobal MP, Pérez-Artiaga L, LampayaPérez V, Aporta J, Pérez-Laguna V, García-Luque I, Revillo MJ, Rezusta A. In vitro effect photodynamic therapy with differents photosensitizers on cariogenic microorganisms. BMC Microbiology. 2015; 15:187. [12] Ball AR, Tego GP. Emerging Antimicrobial Drug-discovery Strategies: an Evolving Necessity. 1st ed. Cambridge: Cabi; 2012. Chapter 2. [13] Liu PF, Zhu WH, Huang CM. Vaccines and photodynamic therapies for oral microbial-related diseases. Curr Drug Metab 2009; 10:90-4. [14] O'Riordan K, Akilov OE, Hasan T. The potential for photodynamic therapy in the treatment of localized infections. Photodiagnosis Photodyn Ther. 2005; 2(4):247-62.

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[15] Lambrechts SA, Aalders MC, Van Marle J. Mechanistic study of the photodynamic inactivation of Candida albicans by a cationic porphyrin. Antimicrob Agents Chemother. 2005; 49(5):2026-34. [16] Tardivo JP, Del Giglio A, de Oliveira CS, Gabrielli DS, Junqueira HC, Tada DB, Severino D, de Fátima Turchiello R, Baptista MS. Methylene blue in photodynamic therapy: From basic mechanisms to clinical applications. Photodiagnosis Photodyn Ther. 2005 Sep;2(3):175-91. [17] Leite HL, Cavalcante SI, Sousa EM, Gonçalves LM, Paschoal MA. Streptococcus mutans photoinactivation using a combination of a high potency photopolymerizer and rose bengal. Photodiagnosis Photodyn Ther. 2016; 15: 11-12. [18] Calzavara-Pinton PG, Venturini M, Sala R. Photodynamic therapy: update 2006. Part 1: Photochemistry and photobiology. J Eur Acad Dermatol Venereol. 2007; 21(3):293-302. [19] CLSI. Performance Standards for Antimicrobial Susceptibility Testing: TwentyFourth Informational Supplement. CLSI document M 100-S24. Wayne PCaLSI, 2014. [20] The European Committee on Antimicrobial Susceptibility Testing. Breakpoint tables for interpretation of MICs and zone diameters. Version 4.0. 2014. http://www.eucast.org. [21] Wilson M. Lethal photosensitisation of oral bacteria and its potential application in the photodynamic therapy of oral infections. Photochem Photobiol Sci. 2004; 3(5):4128. [22] Araújo PV, Teixeira KI, Lanza LD, Cortes ME, Poletto LT. In vitro lethal photosensitization of S. mutans using methylene blue and toluidine blue O as photosensitizers.Acta Odontol Latinoam. 2009; 22(2):93-7. [23] Chan Y, Lai CH. Bactericidal effects of different laser wavelengths on periodontopathic germs in photodynamic therapy. Lasers Med Sci. 2003; 18(1):51-5. [24] Strasswimmer J, and Grande DJ. Do pulsed lasers produce an effective photodynamic therapy response?. Lasers Surg Med 2006; 38:22-5. [25] Núñez SC, Garcez AS, Kato IT, Yoshimura TM, Gomes L, Baptista MS, Ribeiro MS. Effects of ionic strength on the antimicrobial photodynamic efficiency of methylene blue. Photochem Photobiol Sci. 2014;13(3):595-602. [26] Rolim JP, de-Melo MA, Guedes SF, Albuquerque-Filho FB, de Souza JR, Nogueira NA, Zanin IC, Rodrigues LK. The antimicrobial activity of photodynamic therapy against Streptococcus mutans using different photosensitizers. J Photochem Photobiol B. 2012;106:40-6. [27] Costa AC, Chibebe Junior J, Pereira CA, Machado AK, Beltrame Junior M, Junqueira JC, Jorge AO. Susceptibility of planktonic cultures of Streptococcus mutans to photodynamic therapy with a light-emitting diode. Braz Oral Res. 2010; 24(4):413-8.

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[28] Pereira CA, Costa AC, Carreira CM, Junqueira JC, Jorge AO. Photodynamic inactivation of Streptococcus mutans and Streptococcus sanguinis biofilms in vitro. Lasers Med Sci. 2013;28(3):859-64. [29] J W Costerton, Z Lewandowski, D E Caldwell, D R Korber, and H M LappinScott. Microbial Biofilms. Annual Review of Microbiology. 1995; Vol. 49: 711-745. [30] Nedeljkovic I, De Munck J, Slomka V, Van Meerbeek B, Teughels W, Van Landuyt KL3. Lack of Buffering by Composites Promotes Shift to More Cariogenic Bacteria. J Dent Res. 2016; 4. [31] Hamblin MR. Antimicrobial photodynamic inactivation: a bright new technique to kill resistant microbes. Curr Opin Microbiol. 2016 Oct;33:67-73.

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FIGURES AND TABLES LEGENDS Figure 1: Absorption spectra of rose bengal (a-left) and methylene blue (b-right). Emission spectra of the LED lamps (c); green LED lamp center at 515 nm and red LED lamp center at 625 nm. The center of the emission spectra of each lamp is indicated in the respective figures of absorption spectra (a and b).

Figure 2: Photoinactivation of S. mutans (left) and S. sanguinis (right) with different concentrations of RB and MB at a constant fluence of 18 J/cm2 using a green-LED lamp (515 nm, 0.0058 W/cm2) and a red-LED lamp (625 nm, 0.0070 W/cm2) respectively for each photosensitizer.

Figure 3: Reduction dependence on the concentrations (μg/mL): MB using red-LED lamp (625 nm, 0.0070 W/cm2) (left) and RB using a green-LED lamp (515 nm, 0.0058 W/cm2) (right) at a constant fluence of 18 J/cm2 to photoeliminate S. mutans and S. sanguinis in mixed solution.

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Figr-1

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Figr-2

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Figr-3

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Table 1: Summary of the in vitro aPDT studies using MB or RB on S. mutans and S. sanguinis.

Bibliography

Streptococci

PS

Concentration

Preincubation

Inhibition

(μg/mL)

(min)

(%)

λ (nm)

Fluence

Power

(J/cm2)

density (W/cm2)

Araújo et al. [22]

S. mutans

MB

25

ND

73

638

ND

ND

Soria-Lozano et

S. mutans

MB

2.5

<1

99.9999

420–700

37

0.09

Our study

S. mutans

MB

0.62-1.25

<1

99.9999

600-650

18

0.007

Our study

S.

MB

0.62-1.25

<1

99.9999

600-650

18

0.007

al. [11]

mutans

in

mixed Costa et al. [27]

S. mutans

RB

2.02

5

99.9999

440–460

95

0.526

Soria-Lozano et

S. mutans

RB

0.62

<1

99.9999

420–700

37

0.09

Our study

S. mutans

RB

0.16-0.62

<1

99.9999

480-560

18

0.0058

Our study

S.

RB

0.16-0.62

<1

99.9999

480-560

18

0.0058

al. [11]

mutans

in

mixed Chan et al. [23]

S. sanguinis.

MB

100

<1

99–100

632.8

21.2

0.030

Soria-Lozano et

S. sanguinis.

MB

2.5

<1

999.999

420–700

37

0.09

Our study

S. sanguinis.

MB

0.31-1.25

<1

99.9999

600-650

18

0.007

Our study

S.

MB

0.31-1.25

<1

99.9999

600-650

18

0.007

al. [11]

sanguinis.in

mixed Pereira et al. [28]

S. sanguinis.

RB

5

5

9.9

435-475

95

0.526

Soria-Lozano et

S. sanguinis.

RB

0.62

<1

999.999

420–700

37

0.09

Our study

S. sanguinis.

RB

0.16-0.31

<1

99.9999

480-560

18

0.0058

Our study

S.

RB

0.16-0.31

<1

99.9999

480-560

18

0.0058

al. [11].

sanguinis.in

mixed

ND = no data

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