Effectiveness of erythrosine-mediated photodynamic antimicrobial chemotherapy on dental plaque aerobic microorganisms: A randomized controlled trial.

BACKGROUND: Dental plaque is one of the predominant causes of major oral diseases. Although mechanical and chemical methods are extensively followed to control the development of plaque, plaque-related diseases still persist. Therefore, this necessitates for alternative measures of plaque control, one such alternative is photodynamic antimicrobial chemotherapy (PACT).
MATERIALS AND METHODS: Split mouth randomized clinical trial (CTRI/2017/03/008239) was conducted on 30 participants who reported to the hospital. Participants were asked to rinse their mouth for 1 min using 10 ml of 25 μM erythrosine solutions. Same tooth on both quadrants of the same jaw are selected as the test and control. Intervention used was halogen-based composite curing light with wavelength of 500-590 nm. Plaque sample from the control tooth and test tooth was collected before and after exposure, respectively, and sent to microbiological laboratory for colony count.
RESULTS: Logarithmic mean and standard deviation of control group with 102 dilutions of aerobic microbial count were found to be 5.34 ± 0.94, and for experimental group, it was 4.47 ± 1.37. The statistical difference between mean CFU values between aerobic bacterial counts was significant (P = 0.006).
CONCLUSIONS: Erythrosine-mediated PACT reduces the extent of dental plaque microbial count and has a potential preventive and therapeutic use in day-to-day life and dental clinics.

INTRODUCTION

Dental plaque is demarcated clinically as a structured, resilient, yellow-greyish substance that adheres tenaciously to the intraoral hard surfaces, including removable or fixed restorations.[12] It embraces of microorganisms and intercellular matrix which are allied with a range of oral diseases including dental caries and periodontal diseases, root canal failures, halitosis, denture stomatitis, moniliasis, and dental implant failures.[3] Elimination of dental plaque is considered as basic necessity in preventing these diseases.[3456]

Existing therapeutic procedures involve either episodic mechanical disruption of oral microbial biofilms as in mechanical plaque control or preserving therapeutic concentrations of antimicrobials in the oral cavity as in chemical plaque control.[3] However, mechanical plaque control can be the subject to meager patient compliance[78] and may be unsuitable for some patients with the mechano-blistering disease, for example, epidermolysis bullosa, where mechanical strength can damage the oral mucosa and cause intolerable pain and distress.[8] Antibacterial agents are also extensively used either in local or systemic formats, but it has a risk of development of bacterial resistance on long-time usage.[8] Although these plaque control methods are used every day, oral problems related to biofilm still exist. Hence, the development of alternative antibacterial therapeutic strategies becomes important in the evolution of methods to control microbial growth in the oral cavity.

The practice of photodynamic therapy for inactivating microorganisms was initially validated more than 100 years ago.[3] Photodynamic antimicrobial chemotherapy (PACT) is a therapeutic procedure that utilizes light energy to activate a photosensitizing agent (photosensitizer) in the presence of oxygen. The principle of the PACT is that the photosensitizer undergoes a transition to a higher energy state, generating an extremely reactive state of oxygen,[9] and this singlet oxygen causes a venomous effect on microorganisms. It shows minimum resistance because it inhibits the plasmid interchange involved in the transfer of antibiotic resistance. This gives more impact on the treatment against resistant microorganisms.

Most of the oral infections are biofilm linked and localized in nature. These diseases are well studied in PACT against systemic antimicrobials.[10] However, more instant advantage could be derived from photosensitizers already permitted for oral use. One such photosensitizer is erythrosine, which is an organic iodine compound that has photodegradable property when introduced to a light of 530–590 nm. It is primarily used for food coloring and also as dental plaque disclosing agent. It is economical and least harmful to the body.[9]

Hence, this study was undertaken to evaluate the effect of erythrosine-mediated PACT on plaque microorganisms.

MATERIALS AND METHODS

The present research was two-arm simple randomized controlled trial using split-mouth design conducted over 2 weeks [Figure 1]. Before the start of the main study, a pilot study was conducted on 10 participants. Intra-examiner calibration on plaque and gingival score was done among 10 participants before the start of the research; intra-examiner reliability came around 90%. Estimation of sample size was done under 5% error and 95% power of the test. The required sample size came up to 29 which were rounded up to 30 in each group. Ethical clearance was obtained former to the start of the study (IRB. NO.2011/P/COM/2).

Figure 1

Flowchart

Prospective adult participants who indicated willingness to participate in the study were scheduled for screening visit by the examiner. All enrolled participants were examined by the examiner, who conducted an oral examination. All the participants were asked to rinse their mouth for 1 min using 10 ml of 25 μM erythrosine solution (Alpha Plac red no. 3, DPI). Participants who had similar plaque scores on the contralateral tooth of the same arch were included and participants with orthodontic appliances or more than one incisor, with prosthetic crown, those who required immediate care, destructive periodontal disease, pregnant and breastfeeding women, chronic systemic conditions (heart, kidneys, liver, or infectious diseases including AIDS), those undergoing antibiotic or steroid therapy in the preceding month were excluded from this study.

Clinical procedures

To avoid selection bias, randomization was performed with lottery method. In the present study, standardization of dental plaque was done by considering the same tooth on both quadrants of the same jaw as experimental and control tooth [Figure 2]. First, plaque sample from the control tooth was collected separately under sterile condition. After collecting the control sample, a composite curing tungsten filament light source with 500–6000 nm wavelength was directed over the experimental tooth for 60 s. Plaque sample from the experimental tooth was collected separately under sterile condition and sent for microbiological analysis. Here, control samples were collected first to eliminate the spill-over effect of light application into the control area [Figure 3].

Figure 2

Quantification of plaque using plaque index

Figure 3

Application of light to experimental area using halogen-based composite curing light

Microbiological procedure

Collection and transport

The dental plaque samples were collected by a sterile Columbia scaler from the tooth surface and immediately transferred to a sterile plastic tube containing 4 ml transport media (Thioglycollate Broth, TG broth) seeded with a few sterile 0.5-mm glass beads. The plastic tubes were vortexed at high setting to disperse the plaque, so as to obtain homogenous suspension.

Dilution

All the dilutions were performed in a sterile biosafety cabinet (2AII); the cabinet was treated with ultraviolet light for 30 min before the beginning of the procedure for the sterilization of the cabinet. Dilution of the plaque solutions was done by adding 1 ml plaque solution into a sterile tube containing 9 ml of saline seeded with glass beads. These tubes were vortexed at high settings so that the solutions form a uniform 101 fold dilution. 102 fold dilutions were performed by adding 1 ml of 101 fold diluted solution into 9 ml of saline and vortexed at high settings. Similarly, serial dilutions were performed till 105 dilutions. Based on the pilot study results, the dilutions to be used for aerobic and anaerobic bacterial colony counting was determined. The dilutions found to be 101, 102, and 103 for aerobic bacterial colony culture.

Inoculation

The 5% brain–heart infusion (BHI) agar (HiMedia laboratories, Mumbai) plates were kept under sterile condition. The plates were incubated for 24 h before the inoculation procedure to ensure sterility.

About 100 μl from abovementioned dilutions, aerobic, and anaerobic bacterial cultures were inoculated onto 5% BHI agar plates. A sterile “L”-shaped spreader and a turntable were used to ensure the uniform spreading of diluted dental plaque sample. Inoculated plates with aerobic bacterial culture were marked with subject and sample characteristics. Then, the plates were incubated in air at 37° centigrade for 24 h.

Colony counting

All plates with well-dispersed colonies were read for the number of colony-forming units using the digital colony counter [Figure 4].

Figure 4

Microbiological colony counting using digital colony counter

Statistical analysis

The collected data were entered into the computer (MS-Office, Excel 2010) and subjected to statistical analysis using the statistical package – SPSS version 20 (IBM Statistical Package for Social Sciences Version 20; IBM corporation). Normal distribution was determined by Kolmogorov–Smirnov and Shapiro-Wilk test after logarithmic transformation of the mean values of colony forming units. Independent sample t-test was used to know the statistical significant differences between the two groups.

RESULTS

A total of 30 participants with 22 males of mean age 31.9 years and 8 females with mean age of 29.75 years were included in the present study.

Aerobic colony counts

Control and experimental group with dilution 102 showed 290.50 ± 216.53. After logarithmic transformation, the mean and standard deviation of control group of the same dilution showed 5.34 ± 0.94. Similarly, mean and standard deviation of experimental group with 102 dilutions and its logarithmic transformation showed 155.9 ± 141.69 and 4.47 ± 1.37, respectively [Table 1 and Figure 5].

Table 1

Mean and standard deviation and their logarithmic transformation of aerobic colony counts

Figure 5

Logarithmic mean of aerobic colony count in control group and plaque treated with the erythrosine and halogen-based composite curing light (experimental)

Comparison of mean difference between control and experimental groups using independent sample t-test for aerobic bacterial count with 102 dilutions were performed. Levene's tests of equality of variance in different samples were used to determine the assumption of equal variances or not. Here, t-test assumes the variance of the population from which different samples were drawn were equal. It tests the null hypothesis that the population variances were equal. The P value of Levene's test for Equality of Variances here is 0.123 which was more than that of 0.05. Hence, the null hypothesis of equal variances was accepted. Based on this, the P value of independent sample t-test with equal variance was assumed. Independent sample t-test with equal variance showed that there was highly statistical significant difference among the control and experimental group (P = 0.006) for aerobic bacterial count with 102 dilutions [Table 2].

Table 2

Comparison of mean difference between control and test groups for aerobic bacterial colony count

DISCUSSION

Oral health is an essential portion of overall health; it has been rightly said that one is not healthy without good oral health.[1112] One of the key factors which play a major role in the oral health is dental plaque. Controlling the dental plaque will help us in controlling most of the oral diseases including caries, endodontic and periodontal diseases, halitosis, candidiasis, and dental implant failures.[13] Present therapeutic techniques comprise either intermittent mechanical elimination of oral microbial biofilms or maintaining the antimicrobial concentrations in the oral cavity, both of which are with limitations.[13] The problem here is the progress of bacterial resistance to antimicrobial agents. It is commonly recognized that the development of bacteria in biofilms indicates a substantial decrease in susceptibility to antimicrobial agents matched with cultures grown in suspension.[1314] The development of alternative antibacterial therapeutic strategies which do not develop bacterial resistance, therefore, becomes important in the development of methods to regulate microbial growth in the oral cavity.[13]

In the present study, we targeted plaque microorganisms using the photodynamic antimicrobial chemotherapeutic principle. The singlet oxygen (Nascent oxygen) used in PACT, which has a diffusion distance of approximately 100 nm (137) and a half-life of <0.04 l s[1314] shows no resistance observed by any bacteria as it directly targets on cell wall and DNA of the bacteria.[13151617]

Erythrosine is an organoiodine compound also known as Red No. 3. It is fluorine derivative, cherry-pink in color, chiefly used for food coloring. Its maximum light-absorbance is at 530 nm in an aqueous solution, and it is subjected to photodegradation. Dentists currently use erythrosine as dental plaque disclosing solution or tablets. Erythrosine retains a positively charged surface, thereby it can directly aim on both Gram-positive and Gram-negative bacteria.[1013181920] The positive charge of photosensitizer shows high affinity toward the negatively charged outer bacterial membrane. This primarily induces localized damage, which helps its penetration[1321] and the ability of erythrosine to initiate photochemical degradation reactions is well explained.[72223] The killing efficacy of photoactivated erythrosine is also well documented.[724] Evidently, erythrosine has a gain over other photosensitizers, as it already aims dental plaque and has full agreement for use in the oral cavity. In the present study, we used erythrosine of 25 μM concentrations which is 1000 folds less concentrated than that of plaque disclosing solution. In many studies, this concentration showed effective antimicrobial property.[725]

The light source utilized in the present research was Quartz–halogen-based composite curing gun with wavelength of light ranging from 500 to 600 nm which is parallel to the range of light source required for activation of Erythrosine.

The power output from 75 watts halogen dental curing unit was 663.72 mW/cm2 which was calculated with the formula I=P/A,[30] where I is intensity of light, P is power of the light in Watts, A is surface area of the globe of light distribution, and A = 4pr2 where r is the radius in meter.

The present study is two-arm simple randomized controlled trial using split mouth designs. Because the patients serve as their own control, which can intensify the statistical efficiency, on an average, fewer patients are needed.[26] Carryover effect was eliminated by collecting the control sample before the light exposure (intervention) on the experimental group.

Light application was performed only for 60 s on the test side. Even though approximately 98% of the photocytolysis action was revealed by erythrosine in 5 min,[727] in the present study, we exposed for only 1 min (60 s). The time limitation of 60 s was maintained for two reasons – Manufactures of curing gun have mentioned to use 1 min in each 5 min gap, and beyond 60 s the lamp would get heated up, causing discomfort to both subject and operator.

Light dose was calculated by multiplying the output power by the irradiation time as given in the succeeding equation:

On calculation, the light dose or fluence in the present study is around 39.82 J/cm2 which is very less compared to the light dose used for the killing of premalignant and malignant cells.[28] The light dosage in the present study was determined because of the following reason – it should not cause any collateral damage as the phototoxic dose for normal cell is higher than that of microorganisms, potentially malignant, and malignant cells[28] and it should show effective destruction of plaque microorganism. The safety of PACT in the host tissues has been established by numerous animal and clinical studies and these studies advocate that the adverse-effects on host tissues may not be problematic because the photosensitizer concentrations and light energy doses needed to kill the infecting microorganism have little effect on adjacent host tissues.[2930313233] There are still concerns about short-term and long-term effect of PACT on biological tissues. Luan et al. conducted a research on safety of toluidine blue facilitated photosensitization to periodontal tissues in mice and stated that no necrotic or inflammatory changes were found in periodontal tissues following photodynamic therapy. This shows that PACT is a harmless treatment, that does not damage the adjacent normal tissues.[31]

It was observed that there was highly significant difference (P = 0.006) among control and experimental group for aerobic bacterial count with 102 dilutions. It showed statistical and laboratorial significant reduction in microbial count in the experimental group. This reduction in bacterial count was in line with the other studies done in vitro and in situ.[71025272834] Some research has shown the utilization of PACT in the therapy of a number of pathogenic bacterial, fungal, and viral infections.[2935363738]

CONCLUSION

Therefore, it can be concluded that erythrosine-mediated PACT reduces the extent of dental plaque microbial count and might have a potential preventive and therapeutic use in day-to-day life and in dental clinics.

Declaration of patient consent

The authors certify that they have obtained all appropriate patient consent forms. In the form the patient(s) has/have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.