Photomedicine and Laser SurgeryVolume 25, Number 6, 2007© Mary Ann Liebert, Inc.Pp. 513–518DOI: 10.1089/pho.2007.2109

The Impact of Photodynamic Therapy on the Viability ofStreptococcus mutans in a Planktonic Culture

I.M. BEVILACQUA, D.D.S., M.Sc.,1,2 R.A. NICOLAU, D.D.S., Ph.D.,1,2 S. KHOURI, M.Sc.,1

A. BRUGNERA JR.,2 G.R. TEODORO,1 R.A. ZÂNGARO, Ph.D.1,2 and M.T.T. PACHECO, Ph.D.2

ABSTRACT

Objective: This study investigated the effect of photodynamic therapy (PDT) with toluidine blue O (TBO) anda light-emitting diode (LED) on the viability of Streptococcus mutans cells in a planktonic culture. BackgroundData: Growth of Streptococcus mutans is the first step in the development of tooth decay. The use of light anddyes promotes cellular death in a noninvasive way, reducing treatment time. Methods: The LED used in thisstudy had output power of 116 mW, its energy was 21 J, and the fluency was 2.18 J/cm2. Samples were pre-pared and divided into five groups: (1) control group (�); (2) control group (�); (3) TBO; (4) LED; and (5)LED � TBO. Results: One hundred percent of the bacteria were killed following irradiation with LED andTBO. The biofilm that formed on the glass surfaces was analyzed by SEM and colony count. Conclusions: Itwas demonstrated that PDT was efficient at killing microorganisms and preventing the formation of biofilms.

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INTRODUCTION

MANY MICROBIAL SPECIES are present in the biofilm that formson teeth. The effectiveness of antimicrobial therapy, both

topical and systemic, tends to be minimized by this biofilm, andhas led to the evaluation of new therapeutic modalities.1

Dental plaque formation is one of the initial phases of toothdecay, which is a microbial disease that affects a tooth’s calci-fied tissues. Streptococcus mutans is one of the most importantbacteria present in dental plaque, and its growth is the first stepin the development of tooth decay. It causes demineralizationof the inorganic part of the tooth, and if untreated it progressesto destruction of the organic portion.2 Thus, elimination of path-ogenic microorganisms on teeth is fundamental to preventionand control of tooth decay.3

There are limitations to removing dental plaque using man-ual instruments. For this reason, use of lasers or LEDs of dif-ferent wavelengths, in association with various photosensi-tizing dyes, can play an important role as an alternativetreatment.4–7 The dyes work as optical absorption agents, andare activated by irradiation with light of a specific wave-length, resulting in the generation of cytotoxic species, in-cluding singlet oxygen and free radicals, that exert a bacte-ricidal effect, but that are not toxic to host cells. LEDs are

an alternative source of light in PDT used to reduce the num-ber of bacteria.9 The use of the LED’s nonpolarized light hasproven to be effective therapy, since to produce an antibac-terial effect, the light source must have a wavelength in theabsorption band of the dye being used.9,10 Thus, the purposeof this study was to evaluate the effect in vitro of LED light(640 � 20 nm) in association with toluidine blue O (TBO)on the viability of Streptococcus mutans (S. mutans) cells ina planktonic culture.

MATERIALS AND METHODS

Construction of samples

The samples were made of glass capillary tubes with 1.0 mminternal diameter, 1.5 mm external diameter, and approximately2.3 cm long that were curved on one end (0.5 cm). All sampleswere sterilized in a vertical autoclave (Phoenix AV 75), andthen put into test tubes.

Photosensitising dye

The dye used in this study was toluidine blue O (TBO-Fór-mula & Ação) at a concentration of 100 �g/mL.1,2,11,12

1Health and Sciences College, and 2Dental Laser Center, Institute of Research and Development, Vale do Paraíba University (UNIVAP), Ur-banova, São José dos Campos, Brazil.

Light source

A LED (Microdont) emitting electromagnetic radiation in thewavelength range 600–670 nm, with a peak at 640 � 20 nm,was used. Before starting the experiments, the LED equipmentwas calibrated using a laser power energy monitor (2 W broad-band power/energy meter, Model 13 PEM 001/J. The Nether-lands). Irradiation of the culture medium was done as shown inFig. 1, following the protocols shown in Table 1.

Preparation of the bacterial inoculum

The bacteria used in this study was Streptococcus mutans (S.mutans) that had been incubated at 37°C for 24–48 h under mi-croaerophilic conditions to analyze its viability. The bacteria weresupplied by the Microbiology Laboratory of Universidade do Valedo Paraíba (UNIVAP) Sciences and Health College. Preparationof the S. mutans inoculum (ATCC 25175) was per the standard0.5 McFarland scale, with a final concentration of 1.5 � 108 (150million) cells13 per milliliter of saccharose broth.14

Experimental groups

Test tubes containing bacterial culture were grouped asshown in Table 2.

Group 1 (negative control): In this group, the capillary tubeshad none of the bacterial inoculum, nor did they receive irradia-tion with LED energy or phosensitization with TBO. This groupwas used as a control, without bacterial plaque formation, and wasanalyzed by the scanning electron microscope (SEM).

Group 2 (positive control): In this group, the capillary tubescontained 1 mL of bacterial inoculum and 1 mL of saline so-lution with sterile samples. They were neither irradiated with

LED energy nor photosensitized with TBO. This group wasused as a control to verify the formation of bacterial plaque,and was analyzed by the SEM.

Group 3: In this group, the capillary tubes contained 1 mLof bacterial inoculum and 1 mL of TBO. Sterile samples werealso added to the culture. The capillary tubes (bacterial cul-ture � dye � sample) were put into a kiln, where they remainedfor 5 min (pre-irradiation time; PIT) to allow the dye to absorblight. After that, they were incubated in microaerophilic condi-tions for 48 h.

Group 4: In this group, the capillary tubes had 1 mL of bac-terial inoculum and 1 mL of saline solution. The bacterial cul-ture was then transferred to Petri dishes and irradiated accord-ing to the parameters shown in Table 1. Later, sterile sampleswere added, and then transferred back to the capillary tubes,where they were incubated in microaerophilic conditions for 48 h.

Group 5: In this group, the capillary tubes had 1 mL of bac-terial inoculum and 1 mL of TBO. This solution (bacterial cul-ture � dye) was transferred to Petri dishes and put into a kiln,where it remained for 5 min (PIT) to allow the dye to absorblight. The Petri dishes were irradiated according to the param-eters shown in Table 1. After that, the solution was put backinto the test tubes, sterile samples were added, and they wereincubated in microaerophilic conditions for 48 h.

After 48 hours of incubation, one sample from each groupwas removed from the capillary tube and analyzed by SEM.This was done with three samples from each group. The qual-itative parameter of the analysis was the presence or absenceof bacterial plaque, which was analyzed after viewing them at100�, 1000�, and 5000� magnification. This was done bycounting how many colonies per milliliter were obtained fromserial dilutions of the bacterial culture and plating on blood agar.These procedures were carried out in triplicate for all groups.

Statistical analysis

Values obtained in colony forming units (CFU) are expressedin mean � standard deviation. Statistical calculations were doneusing Graph Pad Instat (version 2.0) statistical data analysissoftware. Analysis of variance testing (ANOVA) was done withthe Bonferroni post-test. Statistical differences were consideredsignificant when p � 0.05.

RESULTS

Qualitative analysis

SEM analysis showed that there was reduction or elimina-tion of bacterial plaque in the samples from group 5 (treatedwith photodynamic therapy), and in samples from group 1 (neg-ative control) as shown in Figs. 2, 3, 4, and 5.

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TABLE 1. PROTOCOL USED FOR IRRADIATION WITH THE LED

PowerWavelength Energy density Energy Power density Area Time

(nm) (J/cm2) (J) (W) (W) (W/cm2) (sec)

640 � 20 nm 2.18 21 0.116 0.01 9.62 180

FIG. 1. Schematic diagram of setup for the irradiation of thetubes in groups 4 and 5. The distance between the LED andPetri dish was kept constant to maintain the proper power den-sity. The LED was positioned 2 cm directly over the Petri dish.

Table 3 presents an analysis of the CFU count of S. mutansper milliliter in saccharose broth in the different groups tested.We found a reduction in the level of microbial growth of 28.4%in group 3 and of 23.4% in group 4 in relation to group 2 (pos-itive control) with p � 0.001. The therapy tested in this studyachieved 100% microbial reduction (group 5).

DISCUSSION

The present study used an LED with an average wavelengthof 635 nm, since the dye we studied (TBO) has its absorptionpeak between 620 and 700 nm.15 The TBO was mixed at a con-centration of 100 �g/mL, as at this concentration it does notcause damage to buccal tissues (i.e., tooth staining and cellulartoxicity).1,2,11,12

The dye’s pH must not be acidic. It was demonstrated thatat an alkaline pH there is an increase in the production of sin-glet oxygen by TBO,16 which increases the therapy’s efficacy.Komerik and Wilson17 found a decrease of antimicrobial ac-tion at an acidic pH. In our study, the pH of TBO was mea-

sured before starting the experiment, and the therapy was mostefficient when the pH was over 6. The pH is a factor of fun-damental importance, since patients who are undergoing che-motherapy and radiotherapy have more acid saliva, which maycompromise the efficacy of PDT.

TBO can diffuse through the bacterial cell membrane, be-cause is has a higher transmembrane permeability coefficientthan other dyes. It proved to be efficient in destroying bacteriabecause it can easily penetrate the bacterial cell membrane, andbecomes concentrated in the interior of the bacterial cell.18

Pre-irradiation time is important to help achieve PDT’s an-tibacterial effect, as it helps to keep the photosensitizer in-side the bacteria, allowing more light absorption.19 The re-sults obtained through SEM analysis showed that there wasa reduction in the formation of bacterial plaque in the grouptreated with PDT. In the other groups there was formation ofa microbial matrix, made of chains of S. mutans, that wasvery similar to that seen on the surface of the glass in sam-ples from group 2 (positive control), in which the samplesreceived no treatment with LED or dye. Thus it is importantto use both LED energy and TBO, to most efficiently reduce

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TABLE 2. THE EXPERIMENTAL GROUPS

CultureGroup medium TBO LED

1 Negative control2 Positive control X3 TBO X X4 LED X X5 PDT X X X

TBO, toluidine blue; LED, light-emitting diode; PDT, photodynamic therapy.

FIG. 2. SEM micrograph of the glass surface of a sample from group 1 (a) and group 5 (b) (100�). The negative control sam-ple (a) has a surface similar to that of the PDT sample from group 5 (b), and shows an absence of biofilm formation after PDTtreatment.

the numbers of bacteria, thus preventing the formation ofbiofilm.

The results obtained demonstrated that total elimination ofS. mutans only occurred when LED energy and TBO were ap-plied simultaneously (group 5), and had 100% efficacy. The useof either the dye alone (group 3) or the LED energy alone (group4) caused a reduction in the CFU count of less than 30% of theefficacy seen in group 5. We observed a 28.4% reduction ingrowth in group 3, and 23.4% reduction in group 4, with rela-

tion to group 2 (positive control) at the level of p � 0.001. Fromthe results in group 3, we can hypothesize that the more the dyecan diffuse through the membrane, the greater the degree ofbacterial destruction.18 In accordance with this, Wilson andMia20 demonstrated a microbial reduction of 20% with the dyealone. The results obtained with dye or LED energy alone, withthe protocols we used, appeared to have less microbicidal ac-tivity. From a clinical point of view, such therapy would prob-ably cause a slight reduction in the amount of bacterial plaque,

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FIG. 3. SEM micrograph of the glass surface of a sample from group 1 (a) and group 5 (b) (1000�). With higher magnifica-tion of the PDT sample it is apparent that there was no formation of a microbial matrix or chains of S. mutans after PDT treatment.

FIG. 4. SEM micrograph of the glass surface of a sample from group 1 (a) and group 5 (b) (5000�). At this magnificationthere is no biofilm present after PDT treatment (b) nor on the sample from the negative control group (a).

but there would remain many bacteria that could form newbiofilms.

In summary, our findings corroborate those of other studiesthat aimed to reduce microbial activity using PDT, which alsoemployed laser energy associated with a photosensitizer.5,12,21

However, in our study, bacterial reduction occurred with theuse of a non-coherent light source (LED) along with TBO, andwe believe that more research is needed using similar lightsources.

CONCLUSION

This study allowed us to reach two conclusions. First, thatthe therapy studied herein demonstrated that it could efficientlyprevent the generation of biofilms, and secondly that this ther-apy had a bactericidal effect, and was able to kill 100% of S.mutans organisms present in the saccharose broth.

Despite the positive results we found of therapy using LEDenergy with TBO, further in vivo studies should be carried outin order to confirm its efficacy, and to make practical its use inclinical practice. Such confirmation is important to allow theuse of this therapy to prevent a number of oral pathologies.

ACKNOWLEDGMENTS

The authors would like to thank to Doctor Josepa Rigau iMas (Unitat d’Histologia i Neurobiologia, Facultat de medic-ina i Ciències de la Salut, Universitat Rovira i Virgili, Reus,Spain) and to Fundação de Amparo à Pesquisa do Estado deSão Paulo (FAPESP, process 06/55806-6).

REFERENCES

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TABLE 3. COUNT OF S. MUTANS CFU PER MILLILITER

Group 2 Group 3 Group 4 Group 5(positive control) (TBO) (LED) (PDT)

CFU ofS. mutans/mL � 107 8.1 � 0.3a,b,c 2.3 � 0.2d 1.9 � 0.2e 0

Data expressed in mean � standard deviation (p � 0.05).aGroup 2 vs. group 3.bGroup 2 vs. group 4.cGroup 2 vs. group 5.dGroup 3 vs. group 5.eGroup 4 vs. group 5.TBO, toluidine blue O; LED, light-emitting diode; PDT, photodynamic therapy.

FIG. 5. SEM micrograph of the glass surface of samples from three different groups (5000�). (a) Positive control sample(group 2), (b) TBO sample (group 3), and (c) LED sample (group 4). In these groups a microbial matrix (dextran synthesis) madeof chains of S. mutans (bacterial plaque or biofilm) can be seen, and there was no reduction in it seen in group 2, with only aslight reduction in groups 3 and 4.

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Address reprint requests to:Dr. Isabela Medeiros Bevilacqua

Health and Sciences CollegeVale do Paraíba University (UNIVAP)

IP&D-FCS/Univap Av. Shishima Hifumi 2911Urbanova, 12244-000

São José dos Campos, SP, Brazil.

E-mail: isabevilacqua@terra.com.br

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