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Table of Contents
ORIGINAL ARTICLE
Year : 2020  |  Volume : 4  |  Issue : 3  |  Page : 88-92

The effect of tea tree oil in inhibiting the adhesion of pathogenic periodontal biofilms in vitro


1 Department of Periodontic, Faculty of Dentistry, Trisakti University, Jakarta, Indonesia
2 Undergraduate Student, Faculty of Dentistry, Trisakti University, Jakarta, Indonesia
3 Department of Periodontic, Faculty of Medicine, Jendral Achmad Yani University, West Java, Indonesia
4 Department of Microbiology, Faculty of Dentistry, Trisakti University, Jakarta, Indonesia

Date of Submission20-Jun-2020
Date of Decision24-Aug-2020
Date of Acceptance14-Sep-2020
Date of Web Publication17-Oct-2020

Correspondence Address:
Abdul Gani Soulissa
Department of Periodontology, Faculty of Dentistry, Trisakti University, Jakarta
Indonesia
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/SDJ.SDJ_33_20

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  Abstract 


Background: Tea tree (Melaleuca alternifolia) oil (TTO) is known to have anti-inflammatory, antibacterial, antifungal, and antiviral properties. Objectives: The aim of this study was to determine the effects of TTO on the ability of Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans biofilms to adhere to enamel surfaces in vitro. Methods: P. gingivalis and A. actinomycetemcomitans were cultured in brain heart infusion (BHI) broth at 37°C for 24 h in anaerobic conditions. Eighteen premolar teeth were inoculated and incubated for 48 h to form biofilms on enamel surfaces. Subsequently, TTO in 6.25%, 12.5%, 25%, and 50% concentrations was added and incubated for 1 and 3 h. Chlorhexidine (0.2%) and BHI broth were used as positive and negative controls, respectively. The remaining biofilm colonies were counted using an enzyme-linked immunosorbent assay reader (490 nm). The teeth were placed in microtubes containing phosphate-buffered saline and vortexed for 20 s. Subsequently, biofilms were cultured in BHI agar for 24 h. The colonies in each concentration were estimated as colony-forming units per milliliter. Statistical analysis was performed using one-way analysis of variance. The level of statistical significance was set to P < 0.05. Results: Treatment with all concentrations of TTO significantly reduces biofilm adhesion compared to the negative control after both incubation periods (P < 0.05). The concentration that most effectively inhibited the adhesion of P. gingivalis was 12.5% after 1 h incubation. The concentration that most effectively inhibited the adhesion of A. actinomycetemcomitans was 25% after 1 h incubation. Conclusion: TTO inhibits the adhesion of P. gingivalis and A. actinomycetemcomitans biofilms to enamel surfaces and may be useful as a treatment for oral diseases. Further studies should examine its efficacy in vivo.

Keywords: Aggregatibacter actinomycetemcomitans, biofilm, Porphyromonas gingivalis, tea tree oil


How to cite this article:
Soulissa AG, Afifah J, Herryawan, Widyarman AS. The effect of tea tree oil in inhibiting the adhesion of pathogenic periodontal biofilms in vitro. Sci Dent J 2020;4:88-92

How to cite this URL:
Soulissa AG, Afifah J, Herryawan, Widyarman AS. The effect of tea tree oil in inhibiting the adhesion of pathogenic periodontal biofilms in vitro. Sci Dent J [serial online] 2020 [cited 2020 Oct 22];4:88-92. Available from: https://www.scidentj.com/text.asp?2020/4/3/88/298450




  Background Top


Periodontal disease is the most common dental and oral disease after dental caries both in Indonesia and worldwide. According to the 2013 Indonesian Oral Health Research (Riskesdas), the prevalence of periodontitis among respondents aged ≥15 years in Indonesia was 95.12%.[1] Severe periodontal disease was the 11th most prevalent disease globally in 2016.[2] Periodontitis is an inflammation of the tissues that support the teeth including the gingiva, alveolar bone, periodontal ligament, and cementum. Untreated periodontitis can lead to tooth loss.[3],[4]

Bacterial colonization forming biofilms that adhere to tooth surfaces and gingiva is the main etiology of periodontitis.[5] Bacteria that play a role in the pathogenesis of the periodontal disease are Gram-negative anaerobic bacteria such as Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Fusobacterium nucleatum, and Treponema denticola[6] Biofilms are aggregations of microorganisms that attach to one another or to a surface and are enclosed in extracellular polymeric substances that are self-produced and sustained by fluids – in the case of the oral cavity, saliva flowing over dental plaque on the tooth surface.[7]

P. gingivalis, a black-pigmented anaerobic Gram-negative bacterium, is the main pathogenic agent of chronic periodontitis. It can destroy the periodontal ligaments directly or indirectly and modulate the host's inflammatory response.[4],[8]A. actinomycetemcomitans, another Gram-negative bacteria that colonize the oral cavity, is the main pathogenic agent of localized aggressive periodontitis, an aggressive form of periodontitis in adolescents.[9] It can survive in periodontal pockets, evade the host's defense system, destroy hard and soft tissues supporting the teeth, and inhibit their repair.[10]

Studies have shown that traditional herbal medicines,[11],[12] and fruits can be used in the prevention or treatment of oral disease due to their ability to inhibit the adhesion of pathogenic biofilms in the oral cavity.[13],[14],[15],[16] According to the World Health Organization, traditional medicines have been proven to be effective, safe, and can contribute to the goal of accessibility to treatments and affordable care for everyone.[17] Traditional medicines are natural, are generally considered safe and nontoxic, and have fewer side effects than chemical medicines.[18]

Tea tree oil (TTO) is an essential oil extracted from the leaves of Melaleuca alternifolia, which belongs to the family Myrtaceae and is widespread in Australia.[19] Many studies have reported that TTO exerts strong antibacterial, antifungal, antiviral, and anti-inflammatory activities.[19] It contains α-terpineol and terpinen-4-ol, which have been shown to inhibit the growth of Staphylococcus aureus and Escherichia coli.[20] Terpinen-4-ol and 1,8-cineol have also been shown to inhibit the adhesion of P. gingivalis and reduce inflammation in oral tissue. Thus, TTO has the potential to treat gingivitis.[21],[22]

Despite its known beneficial effects, no previous study has examined the activity of TTO against P. gingivalis and A. actinomycetemcomitans. Therefore, thisin vitro study aimed to investigate the efficacy of TTO in impairing the ability of P. gingivalis and A. actinomycetemcomitans biofilm adherence to enamel surfaces.


  Materials and Methods Top


Eighteen caries free permanent premolar extracted teeth were used in this experimental laboratory study. This study has been approved by Ethic Commission, Faculty of Dentistry, Trisakti University with approval number : 192/S1/KEPK/FKG/9/2018 prior to the study.

Tree tea oil preparation

TTO extracted by maceration technique was obtained from the Indonesian Spice and Medicinal Crops Research Institute, Bogor, West Java, Indonesia. The sample was diluted into four different concentrations (6.25%, 12.5%, 25%, and 50%) using 5% dimethyl sulfoxide.

Tooth preparation

Eighteen extracted premolar teeth from Dharma Nugraha Hospital were used in this study. The first step, free caries premolar was cleaned and divided on the cervical of the tooth. They were then sterilized in an autoclave for 30 min at 121°C.

Bacterial culture

P. gingivalis (ATCC 33277) and A. actinomycetemcomitans (ATCC 29522) from the laboratory stock were cultured in a blood agar medium using the stroking method, placed in anaerobic jars, and incubated at 37°C for 24 h. After colonization, the bacteria were immersed in 25 mL of sterile brain heart infusion (BHI) broth. The tubes were closed, and the contents were homogenized with a vortex mixer. They were subsequently incubated at 37°C for 24 h. The colonies were marked by the medium's turbidity. BHI broth was added to dilute the colonies and homogenized with a vortex mixer. The growth of the bacterial suspensions was measured using a spectrophotometer at a wavelength of 600 nm until OD600= 0.1 = 0.5 McFarland = 1.5 × 10[8] CFU/mL.

Enamel surface biofilm assays

Biofilms were formed on the enamel surfaces after coating with artificial saliva. The teeth were placed on 24-well culture plates at 37°C for 30 min and then rinsed once with phosphate-buffered saline (PBS). They were subsequently inoculated with 2 mL P. gingivalis or A. actinomycetemcomitans suspensions and incubated at 37°C for 48 h to form biofilms on the enamel surfaces. The teeth were then rinsed with PBS to remove unattached cells. To perform biofilm assays on the enamel surfaces, TTO in 6.25%, 12.5%, 25%, and 50% concentrations was added into 24-well culture plates with P. gingivalis or A. actinomycetemcomitans biofilms and incubated at 37°C for 1 and 3 h. Chlorhexidine (0.2%) was used as a positive control, and BHI broth was used as a negative control. The teeth were rinsed once with PBS, placed in microtubes, and homogenized with a vortex mixer for 20 s. The medium was incubated at 37°C for 24 h. The colonies in each concentration were estimated as colony-forming units per milliliter. This study was conducted in triplicate.

Data analysis

The normality of data distribution was evaluated with the Shapiro–Wilk test. Nonnormally distributed data were analyzed with one-way analysis of variance and Fisher's least significant difference post hoc test. The level of statistical significance was set to P < 0.05. The statistical analyses were performed using IBM SPSS Statistics version 23. (IBM, Armonk, NY, USA).


  Results Top


The P. gingivalis and A. actinomycetemcomitans colony counts on enamel surfaces treated with all concentrations of TTO were lower than those in the negative control [Figure 1], [Figure 2], [Figure 3], [Figure 4]. These results indicated that TTO inhibited the adhesion of P. gingivalis and A. actinomycetemcomitans biofilms to enamel surfaces significantly (P < 0.05). The concentration that most effectively inhibited the adhesion of P. gingivalis was 12.5% after 1-h incubation. The concentration that most effectively inhibited the adhesion of A. actinomycetemcomitans was 25% after 1 h incubation.
Figure 1: Results of biofilm assays of Porphyromonas gingivalis on enamel surfaces treated with tea tree oil after 1 h incubation

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Figure 2: Results of biofilm assays of Porphyromonas gingivalis on enamel surfaces treated with tea tree oil after 3 h incubation

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Figure 3: Results of biofilm assays of Aggregatibacter actinomycetemcomitans on enamel surfaces treated with tea tree oil after 1 h incubation

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Figure 4: Results of biofilm assays of Aggregatibacter actinomycetemcomitans on enamel surfaces treated with tea tree oil after 3 h incubation

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  Discussion Top


In this study, the antibiofilm activity of TTO was assessed against P. gingivalis and A. actinomycetemcomitans on enamel surface to represent clinical conditions. The lower colony counts of P. gingivalis and A. actinomycetemcomitans on enamel surfaces treated with TTO can be attributed to its α-terpinol, terpinen-4-ol, and 1,8-cineole contents, which are known to exert antibacterial activity.[23] Previous studies have shown that TTO exerts activity against S. aureus, E. coli, and Pseudomonas aeruginosa by compromising the bacterial membrane's integrity and inhibiting respiration.[21]

Terpinene-4-ol, the main component of M. alternifolia, has been reported to have bactericidal and bacteriostatic effects in low concentrations.[24] Another component, 1,8-cineole, intensifies the activity of terpinene and may contribute to increasing the permeability of the bacterial membrane, allowing terpinene to penetrate. Like terpinene-4-ol, monoterpene has lipophilic properties that inhibit cellular respiration, modify the hydrophilicity of the bacterial membrane, and increase fluidity and membrane permeability.[25]

The expansion of membrane phospholipids can increase fluidity, leading to ion leakage and increasing membrane permeability.[24] The loss of intracellular material after TTO treatment disturbs bacterial cell homeostasis and inhibits respiration. Moreover, TTO impairs bacterial membrane function.[26]

Previous studies show that TTO could be toxic if it is ingested in certain amount. One study showed that poisoning occurs if swallowed as much as 10–25 mL; this causes muscle weakness and depression of the central nervous system (CNS). However, if treated immediately, the symptoms will disappear in <36 h, without any recurrent symptoms, and there are no human deaths caused by TTO.[22] The other study reported that, when taken orally in doses of 0.8 mL/kg or more, the equivalent of more than 50 mL in adults, TTO has important CNS effects. After swallowing a half of teacup (approximately 0.5–1.0 mL/kg) of TTO, an adult was comatose for 12 h and semiconscious for 36 h after swallowing. However, the 2.5 mL oral dose did not induce CNS toxicity in adult men, and this is above the normal oral dose range of 0.3–1.3 mL for essential oils.[27]

A previous study showed that TTO is effective against Gram-positive bacteria such as S. aureus and Gram-negative bacteria such as E. coli and P. aeruginosa.[28] Its mechanism against E. coli lies in damaging homeostatic K+, inhibiting respiration, altering the cell morphology, and lysing the bacterial membrane.[29]P. aeruginosa is vulnerable to TTO due to its outer membrane permeability.[19],[30] Sambyal et al. showed that TTO exerts activity against P. aeruginosa and S. aureus biofilms.[26] Increased tolerance to TTO in P. aeruginosa is directly related to the barrier and energy functions of the outer membrane and may involve efflux systems.[31] These findings suggest that, in low concentrations, TTO can contribute to antibiofilm treatment.


  Conclusion Top


This study shows that tea tree (M. alternifolia) oil can inhibit the adhesion of P. gingivalis and A. actinomycetemcomitans biofilms on enamel surfaces and may be useful as an alternative treatment for periodontal diseases. However, further studies are needed to ascertain its efficacy in vivo.

Acknowledgment

The authors would like to thank the support from the Microbiology Center of Research and Education (MiCORE Laboratory) for assistance to the author in completing the research.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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