ORIGINAL RESEARCH

https://doi.org/10.5005/jp-journals-10005-2662
International Journal of Clinical Pediatric Dentistry
Volume 16 | Issue S-2 | Year 2023

Antibacterial Efficacy of Dual-dye and Dual Laser Photodynamic Therapy on Oral Biofilms of Enterococcus faecalis, Streptococcus mutans, and Prevotella intermedia: An In Vitro Study

Chandrashekar Yavagal¹ https://orcid.org/0000-0002-0564-5612, Puja C Yavagal², Nikhil Marwah³, Sachin B Mangalekar⁴, Vikaraman K Sekar⁵, Meenal S Sahu⁶

¹Department of Laser Dentistry, Maratha Mandal’s Nathajirao G. Halgekar Institute of Dental Sciences & Research Centre, Belagavi, Karnataka, India

²Department of Public Health Dentistry, Bapuji Dental College & Hospital, Davangere, Karnataka, India

³Department of Pediatric Dentistry, Mahatma Gandhi Dental College, Mahatma Gandhi University Medical Sciences and Technology, Jaipur, Rajasthan, India

⁴Department of Periodontology, Bharati Vidyapeeth (Deemed to be University) Dental College and Hospital, Wanlesswadi, Sangli, Maharashtra, India

⁵Department of Pedodontics and Preventive Dentistry, Maratha Mandal’s Nathajirao G. Halgekar Institute of Dental Sciences & Research Centre, Belagavi, Karnataka, India

⁶Oral Dental Care and Research Centre, Bhilai, Chhattisgarh, India

Corresponding Author: Puja C Yavagal, Department of Public Health Dentistry, Bapuji Dental College & Hospital, Davangere, Karnataka, India, Phone: +91 9972946664, e-mail: pujacyavagal@gmail.com

ABSTRACT

Aim: To assess and compare the antibacterial efficacy of methylene blue (MB) and red laser (660 nm) antimicrobial photodynamic therapy (aPDT), indocyanine green (ICG) and infrared laser (810 nm) aPDT, and dual-dye (MB and ICG) and dual light (red and infrared) aPDT on oral biofilms of Enterococcus faecalis (E. faecalis), Prevotella intermedia (P. intermedia), and Streptococcus mutans (S. mutans).

Materials and methods: Biofilms of E. faecalis, S. mutans, and P. intermedia were grown at 36°C and 5% CO₂ for 7 days in a 96-well plate in a brain heart infusion (BHI) growth medium. Before aPDT, a total of 27 inoculums were collected from culture wells and grown on culture plates to assess baseline colony forming units (CFU). The microbial wells were treated with MBaPDT (group I), ICGaPDT (group II), and MBICGaPDT (group III). Post-aPDT, inoculums were collected from wells to be cultured to assess CFU. One-way analysis of variance (ANOVA) and student paired t-tests were used for statistical analysis. The significance level was fixed at p ≤ 0.05.

Results: Methylene blue antimicrobial photodynamic therapy (MBaPDT) caused a significant reduction in E. faecalis counts compared to other groups (f = 11.15, p = 0.01). aPDT on S. mutans resulted in a significant (p = 0.04) reduction of bacterial counts in the ICGaPDT group. aPDT on P. intermedia resulted in a significant reduction in bacterial counts (p ≤ 0.05) in MBaPDT and ICGaPDT groups.

Conclusion: Dual-dye and dual light aPDT showed an antibacterial effect against E. faecalis. It was ineffective against S. mutans and P. intermedia.

Clinical significance: Dual-dye aPDT may effectively reduce E. faecalis counts in infected root canals and improve the outcomes of root canal treatment.

How to cite this article: Yavagal C, Yavagal PC, Marwah N, et al. Antibacterial Efficacy of Dual-dye and Dual Laser Photodynamic Therapy on Oral Biofilms of Enterococcus faecalis, Streptococcus mutans, and Prevotella intermedia: An In Vitro Study. Int J Clin Pediatr Dent 2023;16(S-2):S128–S132.

Source of support: Nil

Conflict of interest: Dr Nikhil Marwah is associated as Editor-in- Chief of this journal and this manuscript was subjected to this journal’s standard review procedures, with this peer review handled independently of the Editor-in-Chief and his research group.

Keywords: Enterococcus faecalis, Indocyanine green, Methylene blue, Photodynamic therapy, Prevotella intermedia, Streptococcus mutans

INTRODUCTION

Oral pathologies like dental caries, periodontal disease, and endodontic infection have been attributed to the proliferation of oral bacteria and their ability to form stable polymicrobial biofilms.¹Enterococcus faecalis (E. faecalis) has been associated with root canal treatment failure. The reason for this is the incomplete eradication of bacterial biofilms from the root canal.² Similarly, periodontal pathogens like Prevotella intermedia (P. intermedia) form biofilms, which have a high level of organization represented by microcolonies. They have a protection system called a polysaccharidic envelope, which exhibits increased resistance to the host immune defenses and antibiotic therapy.³Streptococcus mutans (S. mutans) has a unique ability in plaque biofilm formation, leading to dental caries and periodontal diseases. Specific antimicrobial interventions targeted at the inhibition of the growth and formation of plaque microorganisms lead to effective management and treatment of plaque-induced oral diseases.⁴ “Antimicrobial photodynamic therapy (aPDT)” is a novel photochemical technique for the elimination of pathogenic plaque biofilm microorganisms. It consists of three components: a light-sensitive substance (photosensitizer), tissue oxygen, and light. After administration of the photosensitizer (e.g., by capsules, infusion, creams, or liquid solutions), the affected tissue is irradiated with light wavelengths according to the absorption spectrum of the photosensitizer. This photophysical activation process produces substances in the corresponding body region, primarily reactive oxygen species, which damage viruses, bacteria, or tumor cells and prevent them from replicating.⁵

Methylene blue (MB), a methylthioninium chloride hydrophilic phenothiazine photosensitizer, absorbs light at 640 nm. It is used for aPDT as it has a potent antimicrobial effect against a broad spectrum of bacteria, fungi, and viruses.⁶

Indocyanine green (ICG) photosensitizer has wide applications in dentistry as it is less toxic with nonionizing properties. It is water soluble and absorbs light of near-infrared wavelengths, exhibiting good tissue penetration. Several studies have shown the efficacy of near-infrared 810 nm/ICG aPDT as an adjunctive periodontal treatment.⁷ The antimicrobial efficacy of aPDT is evident for planktonic bacteria, whereas in biofilms, bacteria are more resistant to any antibacterial treatment. Furthermore, data on the effect of dual-dye photodynamic therapy on oral bacterial biofilms is sparse. Hence an in vitro study was planned to assess and compare the antibacterial efficacy of MB and red laser (640 nm) aPDT, ICG and infrared laser (810 nm) aPDT, and dual-dye (MB and ICG) and dual laser (red and infrared) MBICGaPDT on oral biofilms of E. faecalis, P. intermedia, and S. mutans.

MATERIALS AND METHODS

The Institutional Ethical Committee Board of the college where the study was conducted provided ethical clearance after the study protocol review before the start of the study (Ref no 1377; dated 1-2-22).

Preparation and inoculation of Biofilm Organisms

Enterococcus faecalis (E. faecalis), American type culture collection 29212, P. intermedia (ATCC256II), and S. mutans (ATCC 25175) bacteria were grown at 36^°C, 5% CO₂ in brain heart infusion (BHI) broth in an incubator. The bacterial suspension was diluted with 0.9% NaCl solution to an optical density (OD) of 0.46 and McFarland standard number 1. Biofilms were cultured in flat-bottom 96-well plates with 100 μL of 0.46 OD E. faecalis, P. intermedia, and S. mutans suspension in each separate well, containing 100 μL of BHI-broth growth medium at 36°C and 5% CO₂ for 7 days.

Photodynamic Treatment Procedure

Before laser PDT, 27 samples of 10 µL of bacterial inoculum (nine inoculums of each organism) were collected from culture wells and incubated for 24–48 hours at 37°C on culture plates and were checked for the colony count to get colony forming units (CFU) of all the three microorganisms. After baseline microbial count assessment, each microbial well in the plate was soaked with photosensitizer for 4 minutes, followed by laser irradiation (Fig. 1). The laser and dye parameters used in each group are described in Table 1 and Figure 1. After photodynamic therapy, bacterial inoculums were collected from each of the wells for posttreatment microbial count assessment.

**Table 1:** Laser and dye parameters used in the study
Interventional group	Laser parameters	Dye used
Group I: red laser (MBaPDT)	Device: Novolase Gold Manufacture: Novolase Technologies, India; wavelength: 640 nm, power—200 mW, irradiation distance from the culture plate—1 cm; irradiation time: 60 seconds	Novo Blue dye (MB: 100 µg/mL)
Group II: infrared laser (ICGaPDT)	Device: Novolase Gold Manufacture: Novolase Technologies, India; wavelength: 810 nm, power—300 mW, irradiation distance from the culture plate—1 cm; irradiation time: 60 seconds	Novo Green dye (ICG: 100 µg/mL)
Group III dual laser (MBaPDT + (ICGaPDT)	Device: Novolase Gold Manufacture: Novolase Technologies, India; wavelength: 640 nm, power = 100 mV, wavelength: 810 nm, power = 100 mV, irradiation distance from the culture plate—1 cm; irradiation time: 120 seconds	Dual-Dye (combination of 50% MB and 50% ICG)

Fig. 1: Laser and dye parameters used in the study

Statistical Analysis

IBM Statistical Package for the Social Sciences Statistics for Windows, version 21 (IBM Corporation, Armonk, New York, United States of America) was utilized for data analysis. The mean number of CFU of tested organisms pre and postintervention in all the groups were assessed. Data followed normal distribution; hence, one-way analysis of variance (ANOVA) followed by Tukey’s honestly significant difference post hoc test and students paired t-tests were used for inter and intragroup comparisons, respectively.

RESULTS

Photodynamic effects on E. faecalis indicated a significant reduction in bacterial counts (p ≤ 0.05) in all three groups postintervention (Fig. 2). On intergroup comparison, the photodynamic antimicrobial effect of MBaPDT (group I) was more potent than other groups [f = 11.15, p = 0.01, degree of freedom (df) = 2] (Table 2).

**Table 2:** Inter and intragroup comparison of *E. faecalis* counts (CFU × 10⁶)
PDT group	Group I			Group II			Group III
PDT group	Pretest	Posttest	Difference in bacterial counts	Pretest	Posttest	Difference in bacterial counts	Pretest	Posttest	Difference in bacterial counts
Sample 1	364	59	305	286	157	125	329	131	198
Sample 2	341	24	317	252	192	60	292	154	138
Sample 3	324	112	212	305	205	100	283	118	165
Mean microbial count (mean ± SD)	343 ± 20.07	65 ± 44.30	278 ± 57.47^aAB	281 ± 26.85	184.66 ± 24.82	95 ± 32.78^bA	301.33 ± 24.37	134.33 ± 18.23	169 ± 30.04^cB

^ADenotes a significant difference postintervention between groups I and II (p = 0.004); ^BDenotes a significant difference postintervention between groups I and III (p = 0.04) with one-way ANOVA test (df = 2, f = 14.48, and p = 0.005) followed by Tukey post hoc test; small case letters denote significant difference within groups (p < 0.05) with student’s paired t-test (p-values: a: 0.01, b: 0.04, and c: 0.01); group I: red laser, group II: infrared laser, and group III: dual laser

Fig. 2: Enterococcus faecalis (E. faecalis) growth

Photodynamic effects on S. mutans resulted in significant (p = 0.04) reduction of bacterial counts in ICGaPDT (group II). However, the intergroup comparison revealed no significant difference in the reduction of bacterial counts between groups (f = 4.10, p = 0.07, df = 2) (Table 3) (Fig. 3).

**Table 3:** Inter and intragroup comparison of *S. mutans* counts (CFU × 10⁶)
PDT group	Group I			Group II			Group III
PDT group	Pretest	Posttest	Difference in bacterial counts	Pretest	Posttest	Difference in bacterial counts	Pretest	Posttest	Difference in bacterial counts
Sample 1	325	110	215	157	145	12	144	120	24
Sample 2	384	84	300	210	204	6	197	143	54
Sample 3	164	132	32	220	207	13	163	139	24
Mean microbial count (Mean ±SD)	291 ± 113.87	108.66 ± 24.02	182.33 ± 136.95	195.66 ± 33.85	185.33 ± 34.96	10.33 ± 3.76^*	168 ± 26.85	134 ± 12.28	34.00 ± 17.32

*Denotes significant difference within groups (p < 0.05) with student’s paired t-test (p-value: 0.04; group I: red laser, group II: infrared laser, group III: dual laser

Fig. 3: Growth of S. mutans

Photodynamic effects on P. intermedia resulted in a significant reduction in bacterial counts in MBaPDT (group I) as well as ICGaPDT (group II) (Fig. 4). However, the reduction of bacterial counts was similar in all the groups (f = 1.95, p = 0.22, df = 2) (Table 4).

**Table 4:** Inter and intragroup comparison of *P. intermedia* counts (CFU × 10⁶)
PDT group	Group I			Group II			Group III
PDT group	Pretest	Posttest	Difference in bacterial counts	Pretest	Posttest	Difference in bacterial counts	Pretest	Posttest	Difference in bacterial counts
Sample 1	265	56	209	384	132	252	277	158	119
Sample 2	244	122	122	242	132	110	281	132	149
Sample 3	362	101	261	379	105	274	232	188	44
Mean microbial count (mean ± SD)	290.33 ± 62.94	93 ± 33.71	197.33 ± 70.28^a	335 ± 80.57	123 ± 15.58	212 ± 89.01^b	263.33 ± 27.20	159.33 ± 28.02	104 ± 54.08

Small case letters denote significant differences within groups (p ≤ 0.05) with student’s paired t-test (p-values: a: 0.04, b: 0.05); group I, red laser; group II, infrared laser; group II, dual laser

Fig. 4: Growth of P. intermedia

DISCUSSION

The simultaneous, synchronized application of an ICG/810 nm aPDT and MB/660 nm aPDT (dual light and dual-dye) resulted in a significant reduction in E. Faecalis counts. However, dual light aPDT did not lead to a significant reduction in P. intermedia and S. mutans counts.

Antibacterial Efficacy of aPDT on E. faecalis

Photodynamic effects on E. faecalis indicated a significant reduction in bacterial counts in all three groups postintervention. However, MBaPDT demonstrated a more potent antibacterial effect compared to other groups. A similar result was observed in a study done by Lopez-Jimenez et al., where E. faecalis biofilms treated with MB/670 nm aPDT demonstrated significant antibacterial. Beltes et al. demonstrated potent activity of ICG/810 nm aPDT against E. faecalis in their study.⁸ Akbari et al. showed that the antimicrobial property of ICG photosensitizer dye was improved by ionization of ICG into nanographene oxide (nGO) as a new photosensitizer, which showed a significant reduction in E. faecalis counts with ICGnGO/808 nm aPDT.⁹

Antibacterial Efficacy of aPDT on S. mutans

Antimicrobial photodynamic therapy (aPDT) on S. mutans indicated a reduction in bacterial counts in all three groups postintervention. However, ICGaPDT demonstrated a more potent antibacterial effect compared to other groups. A study by Nikinmaa et al. demonstrated improved and sustained antibacterial efficacy of dual light aPDT with ICG/810 nm and 405 nm against S. mutans.⁷ A study by Azizi et al. showed potent antibacterial properties against S. mutans using aPDT with curcumin and MB/660 nm.¹⁰ Nemezio et al. in their study demonstrated that twice daily treatment with MBaPDT effectively decreased S. mutans viability in biofilms which was comparable to antimicrobial activity of chlorhexidine.¹¹ In a study by Liang et al., aPDT with MB/650 nm exhibited more potent antibacterial effect against S. mutans compared to hematoporphyrin monomethylether/532 nm aPDT.¹²

Antibacterial Efficacy of aPDT on P. intermedia

Antimicrobial photodynamic therapy (aPDT) on P. intermedia indicated a reduction in bacterial counts in all three groups postintervention. However, MBaPDT and ICGaPDT groups demonstrated a more potent antibacterial effect compared to the dual light group. Studies by Fimple et al. and Theodoro et al. showed potent antibacterial activity of MB/665–660 nm against P. intermedia and other multispecies root canal and periodontal pathogens.¹³^,¹⁴

Methylene blue (MB), a hydrophilic photosensitizer, is potent bactericidal as it can bind to the cell wall.¹⁵ Aqueous MB reduces the quantum yield of reactive oxygen species (ROS) due to the abolition of excited states between the individual-dye molecules, inhibiting its photodynamic activity.¹⁶ ICG has wide clinical applications and is a Food and Drug Administration approved dye. It has temperature-raising and antibacterial properties within a biofilm. Three distinct energy-releasing mechanisms allow the ICG molecule to transit to its ground state. Firstly, the energy can be transformed into a fluorescence emission with a wavelength between 750 and 950 nm.

Secondly, part of the energy is transferred to an ICG triplet state via intersystem crossing, producing reactive oxygen species yielding triplet formation of ICG. The quantum yield of triplet ICG is sufficient to release reactive oxygen species. Thirdly, internal conversion occurs within the ICG molecule, converting the energy to heat. It has been found that ICG can produce heat from 85% of the energy it absorbs.¹⁷ ICG’s capacity to exert antibacterial action via a variety of pathways offers an alluring safety feature, particularly if aPDT were to be regularly administered. Since the present study is the first to test a combination of MB with ICG dye activated by dual-wavelength light, the results of the study could not be compared with similar studies.

CONCLUSION

Dual-dye aPDT with MB and ICG sensitized by dual light of wavelengths 640 and 810 nm exhibited significant antibacterial properties against E. faecalis. However, it was not effective against S. mutans and P. intermedia. The findings of the current study highlight new possibilities for generating hypotheses and trying new methods of aPDT technologies and their applications in antimicrobial therapies targeted at preventing oral diseases.

ORCID

Chandrashekar Yavagal https://orcid.org/0000-0002-0564-5612

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