Effect of Combining Erythrosine with a High-Power Dental Curing Light Appliance on the Viability of a Planktonic Culture of Streptococcus mutans

Abstract

Objective:

The aim of this in vitro study was to evaluate the effectiveness of antimicrobial photodynamic therapy (aPDT) composed of the association of the photosensitizer (PS) erythrosine irradiated by a high-intensity dental light source against a culture of Streptococcus mutans, comparing this effect with that of a 0.12% chlorhexidine solution.

Materials and methods:

For this purpose, planktonic suspensions of S. mutans were subjected to experimental conditions in which three different concentrations of erythrosine (E) (2, 4, and 8 μM) associated with three different doses emitted by the light source (L) (48, 96, and 144 J/cm²) were crossed, corresponding to the exposure times of 40, 80, and 120 sec, respectively, delivered in pulsed mode. The following experimental conditions were evaluated: G1—treatment with dye and light source (E+L+); G2—treatment with the dye only (E+L−); G3—treatment with the light source only (E−L+); G4—absence of dye and light (negative control); and G5—0.12% chlorhexidine (positive control). After treatment, aliquots of each group were plated on blood agar, then the colony forming units per milliliter (CFU/mL) later counted. The results were subjected to ANOVA and Tukey tests, considering the level of significance of 5%.

Results:

Group aPDT showed complete eradication of microorganisms as from the concentration of 4 μM irradiated for 40 sec, demonstrating statistically significant difference in comparison with the negative control group (p ≤ 0.05) and efficacy similar to that of the 0.12% chlorhexidine group (p ≥ 0.05).

Conclusions:

The authors concluded that the light-polymerizing appliance used in pulsed mode, associated with the PS erythrosine, was efficient for the control of S. mutans in a planktonic suspension in a short period of irradiation time.

Introduction

Antimicrobial Photodynamic Therapy (aPDT) has been investigated as a possible adjuvant therapeutic method for the control of microorganisms associated with oral diseases, including dental caries.^1
–3

In aPDT, photosensitizers (PSs) and light sources [laser or light emitting diode (LED)] are essential parts of this alternative antimicrobial approach. At present, among those most studied, erythrosine has been shown to be a promising PS.^4
–6 This dye forms part of the xanthene group; therefore, it is a cyclic compound that absorbs light in the visible region, very close to the irradiations from light sources used in dental clinics for light polymerization of resin composites, making its use more accessible in dental clinical practice.^7,8

Its choice as a PS agent is based on the advantage of having been approved for use in the oral cavity by the Food and Drug Administration (FDA), in addition to the promising results found in previous studies, and its more accessible cost.^7
–9 Further, a body of evidence has pointed out its pronounced photodynamic potential against planktonic forms and oral bacteria organized in biofilm.^4,5,10

Among the light sources used in aPDT, LEDs have shown high antimicrobial efficiency, and they are widely used in dentistry due to their low cost and accessibility in comparison with other appliances.^7,8 As regards light sources, the long exposure times required to attain significant inhibitory action against microorganisms related to oral diseases (caries and periodontal disease) have been considered one of the main reasons for restricting their use in aPDT practice.^5,10 Therefore, since high-intensity dental light sources are more potent than those that have previously been tested, they may present photodynamic efficacy in shorter exposure times.^11,12

Considering the foregoing, the aim of this study was to evaluate the antimicrobial photodynamic potential of erythrosine associated with a high-intensity dental light source against a planktonic culture of Streptococcus mutans.

Materials and Methods

Microorganism preparation

The strain of S. mutans UA159 (ATCC 700610) was reactivated in brain heart infusion culture medium with a final concentration of 1% glucose, in a microaerophilic atmosphere, at 37°C for 16–18 h. When growth was verified, the suspension was centrifuged at 503 g, for 5 min, washed twice with phosphate buffer saline (PBS) solution, and the supernatant was discarded afterward. The turbidity of the resultant material was adjusted with the aid of a spectrophotometer, at a wavelength of 540 nm, corresponding to a suspension for the stock solution of ≈2 × 10⁶ colony forming units per milliliter (CFU/mL).¹³

Light source

The appliance used was a high-intensity LED-based polymerizing unit (L) (3 M ESPE; St. Paul, MN), with an energy intensity of 1200 mW/cm². The light source had a visible light spectrum in blue region and low heat generation, as tested previously.¹² In this study, the pulsed irradiation mode¹⁴ was used in different doses of 48, 96, and 144 J/cm² for the light emission times evaluated corresponding to 40, 80, and 120 sec, thus receiving energy doses of 24, 48, and 72 J, respectively. The samples were irradiated at a distance of 5 mm—ideal—so that there would be no heat generation and dissipation of the light beam.¹²

Tested substances

For photosensitizing the studied strain, the PS erythrosine (E) (Sigma Aldrich, St. Louis, MI) was used. A solution of 0.031 g of erythrosine in 100 mL of sterile distilled water was prepared, resulting in a standard solution with a concentration equal to 400 μM. After initial dilution, the microtube was agitated in a vortex to completely homogenize the mixture. From this standard solution, the quantities necessary for the different concentrations of dye, 2, 4, and 8 μM, used in the study were taken. Owing to the degradation capacity of the dye, all the stages of PS preparation were carried out in an environment protected from light. As the substance of choice for positive control, a solution of 0.12% chlorhexidine digluconate (PerioGard, Colgate, NJ) was used.

aPDT application

To determine an optimal parameter of photoirradiation, three different doses of light (48, 96, and 144 J/cm²) were crossed with three different concentrations of PS (2, 4, and 8 μM). Thus, the bacterial strain was subjected to the following experimental conditions: sensitization with erythrosine and irradiation with light source (E+L+: Group PDT); sensitization with erythrosine only (E+L−: Group erythrosine); only with light source (E−L+: Group Light); 0.12% chlorhexidine (positive control); and PBS solution (negative control; E−L−).

According to the protocol established, aliquots of 100 μL of the bacterial suspension of S. mutans were transferred to the sterilized microcentrifuge covers (diameter = 8 mm; area = 50 mm²) and to these 100 μL of the previously diluted PS (final volume = 200 μL) was added. The samples were covered for 60 sec [preirradiation time (PIT)] and irradiated at a distance of 5 mm, by using the light source in pulsed mode until the tested exposure times were reached. After application of the therapy, aliquots of 100 μL were subjected to serial dilutions, and for each dilution, aliquots of 25 μL were plated on blood agar. The plates were incubated at 37°C and 5% CO₂. After 48 h, the CFUs were counted and correlated with the volume in milliliters (CFU/mL). The assays were performed in triplicate in two time intervals (n = 6)

Statistical analysis

The CFU/mL data obtained were transformed (log₁₀) and subjected to the analysis of variance (ANOVA), followed by the Tukey test, considering the level of significance equal to 5%.

Results

According to analysis of the data obtained and illustrated in Table 1, with regard to Group aPDT, bacterial growth was observed when the experimental conditions were used in the concentration of 2 μM of PS in all the studied irradiation times, showing no statistically significant difference in comparison with the other groups studied (p > 0.05). In contrast, statistically significant difference was verified in comparison with all the groups when the concentrations of 4 and 8 μM of erythrosine associated with the three LED irradiation times were used, with exception of the comparison with Group chlorhexidine (p < 0.05). Although the latter group showed statistical difference in comparison with Groups Light and PS alone, as well as with the Positive Control (p > 0.05), these did not differ statistically among them (p < 0.05).

Table 1.

Streptococcus mutans Counting After Different Treatments (CFU/mL Mean Values in Log ₁₀ )

Groups	Experimental conditions	CFU/mL (log₁₀) (mean ± standard deviation)
(aPDT) E+L+/E–2 μM	48 J/cm² (40 sec)	5.26 ± 0.41^a
	96 J/cm² (80 sec)	4.78 ± 0.31^a
	144 J/cm² (120 sec)	4.70 ± 0.24^a
(aPDT) E+L+/E–4 μM	48 J/cm² (40 sec)	0.00^b
	96 J/cm² (80 sec)	0.00^b
	144 J/cm² (120 sec)	0.00^b
(aPDT) E+L+/E–8 μM	48 J/cm² (40 sec)	0.00^b
	96 J/cm² (80 sec)	0.00^b
	144 J/cm² (120 sec)	0.00^b
E−L+	48 J/cm² (40 sec)	6.69 ± 0.27^a
	96 J/cm² (80 sec)	6.77 ± 0.16^a
	144 J/cm² (120 sec)	6.72 ± 0.10^a
E+L−	4 μM	6.77 ± 0.26^a
	8 μM	6.67 ± 0.19^a
	2 μM	6.77 ± 0.12^a
CHX	0.12%	0.00^b
E−L−	—	6.67^a

Data were analyzed by ANOVA followed by Tukey test, p-value at 5%.

Different superscript letters denote statistical significant difference.

ANOVA, analysis of variance; aPDT, antimicrobial photodynamic therapy; CFU/mL, colony forming units per milliliter; E, erythrosine; E+L−, PS (photosensitizer) group; E−L+, light group; E−L−, negative control group; E+L+, aPDT group; L, LED; LEDs, light emitting diodes.

Discussion

This study demonstrated that a high-intensity dental light source applied for a short period of time effectively caused the death of S. mutans in a planktonic model when associated with the PS erythrosine. The data demonstrated that the exposure time could be reduced to seconds, differently from the data observed in previously published studies on the same topic, with times ranging from 60 sec to 15 min of irradiation.^4,10,15,16

Caries disease is difficult to understand completely, because it is considered a pathology of a multi-factorial nature.¹⁷ However, the microorganism S. mutans is considered an important factor participating in its etiology, in addition to being responsible for the process of demineralization of the tooth surface.¹⁸ The importance of S. mutans in this process is owing to diverse virulence factors this bacteria has, such as its capacity for adherence, secretion of polysaccharides, and fermentation of organic acids from carbohydrates present in the diet.^18,19 Therefore, the quest for alternatives with the purpose of inactivating this microorganism is necessary, as a complementary means of controlling caries disease.

Photodynamic therapy has been widely disseminated in recent times and evaluated as an alternative to the existent measure in the control of cariogenic microorganisms.^20,21 The purpose of applying aPDT would be to complement the efforts already being made, such as the use of chlorhexidine (mouth wash solutions or professional applications), which have shown limitations in conditions that require their prolonged use (patients with special needs, periodontal diseases, and reduction in the microbial load),²² as well as some cases related to the resistance of oral microorganisms.²³ Interestingly, there are no reports of the development of bacterial resistance after the application of photodynamic therapy, because the singlet oxygen and free radicals formed in the process act by interacting with diverse cellular structures and metabolic pathways, making the action of this therapy unspecific and effective against different bacterial species, including S. mutans. ²⁴

The application of erythrosine as a PS in dentistry is its use as a dental biofilm stain⁹ and it has the spectrum of light absorption for photochemical reaction in the visible region (480–550 nm), complementary to the wavelength of the LED appliances used by dentists in clinical practice.^12,13 Its use for revealing dental biofilm varies according to the concentration from 9 to 25 mM,²⁵ which is 10,000 times more concentrated than the erythrosine solutions used in this study (2, 4, and 8 μM).

Experiments previously performed with erythrosine on biofilm of S. mutans have shown that it is a PS with excellent antimicrobial action, with a more efficient performance when compared with Photofrin and methylene blue.⁹ In addition to its performance in the inactivation of S. mutans, the dye erythrosine has photodynamic efficiency in vitro against species of Aggregatibacter actinomycetemcomitans,²⁶ S. sanguinis,⁵ Candida albicans,²⁷ and aerobic microorganisms in an in vivo approach.²⁸

Studies^7,8,11,12 have reported the efficacy of blue light in photodynamic therapy, and associated with this, in this study a PS was used, to which professionals have easy access, making the application of this therapy even more feasible. To prove that the efficacy of the therapy as regards bacterial death only occurred during association with the elements of light and PS, groups were formed and subjected to isolated experimental conditions, in which only light irradiation or dye alone was applied; these groups presented bacterial growth similar to that of the negative control group. Further, the authors observed that the reduced times found in the model of S. mutans in suspension indicated that even when the parameters used were increased, such as the dose of light and dye concentration, the bacterial counts would still be reduced in comparison with the findings of previously published studies.

The data obtained in this investigation pointed toward the possibility of using a low concentration of PS and a shorter application time in clinical practice. The use of the high-intensity dental light source associated with a low concentration of erythrosine was shown to be promising in photodynamic therapy, representing an encouraging proposal for studies with a biofilm model and clinical trials for later day-to-day use of this therapy by dental professionals.

Conclusions

The authors concluded that the high-intensity light-polymerizing appliance used in pulsed mode, associated with the PS erythrosine, was efficient for the control of S. mutans in a planktonic suspension in a short time of irradiation.

Footnotes

Author Disclosure Statement

No competing financial interests exist.

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