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Table of Contents
ORIGINAL ARTICLE
Year : 2019  |  Volume : 3  |  Issue : 3  |  Page : 85-89

Efficacy of disinfectants on microbial contaminated toothbrushes


1 Department of Microbiology, Faculty of Dentistry, Trisakti University, Jakarta, Indonesia
2 Department of Conservative Dentistry, Faculty of Dentistry, Hasanuddin University, Makassar, Indonesia

Date of Submission30-Jun-2019
Date of Decision21-Aug-2019
Date of Acceptance04-Sep-2019
Date of Web Publication14-Oct-2019

Correspondence Address:
Dr. Armelia Sari Widyarman
Department of Microbiology, Faculty of Dentistry, Trisakti University, Jakarta
Indonesia
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/SDJ.SDJ_23_19

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  Abstract 


Background: The storage condition of a toothbrush can influence the growth of bacteria. Indonesia as a developing country has a low awareness of the importance of toothbrush hygiene. Thus, an efficient and economic toothbrush storage solution is needed. Objective: The aim of this study was to evaluate the effect of soaking toothbrushes in mouthwash solution on the total number of Streptococcus mutans and Fusobacterium on the toothbrush. Methods: Eighteen toothbrushes were provided to healthy individuals aged 19–23-years. For 1 month, each toothbrush was soaked in disinfectant solution (25 mL) for 20 min after brushing, with Group 1 using mouthwash and Group 2 using sterile tap water. The real-time polymerase chain reaction (PCR) method was used to evaluate the number of microorganisms on each toothbrush. The total number of DNA targets was identified using real-time PCR followed by SYBR Green reagents and 16S rRNA-gene specific primers for S. mutans and Fusobacterium. A paired sample t-test was used for statistical analysis, and the level of significance was set at P < 0.05. Results: There were significant differences in the total bacterial numbers of S. mutans and Fusobacterium on toothbrushes after soaking with mouthwash solution. The average scores (Log10-CFU/mL ± standard deviation) of the total bacteria (2.66 ± 0.39), S. mutans (1.21 ± 0.18), and Fusobacterium (10.35 ± 6.02) on toothbrushes in Group 1 were significantly decreased compared to the average scores of the total bacterial load (5.19 ± 0.41), S. mutans (2.71 ± 1.59), and Fusobacterium (18.96 ± 4.26) in Group 2. Statistical evaluation brought statistically significant difference of total bacteria numbers and S. mutans between Group 1 and Group 2 (P < 0.05). Conclusion: Soaking toothbrushes in mouthrinse solution reduce the total number of bacterial load, S. mutans, and Fusobacterium. Further studies are needed to explore the effects of mouthwash solution against other oral pathogens.

Keywords: Disinfectant, Fusobacterium, mouthwash, Streptococcus mutans, toothbrush


How to cite this article:
Tiara A, Widyarman AS, Rovani CA. Efficacy of disinfectants on microbial contaminated toothbrushes. Sci Dent J 2019;3:85-9

How to cite this URL:
Tiara A, Widyarman AS, Rovani CA. Efficacy of disinfectants on microbial contaminated toothbrushes. Sci Dent J [serial online] 2019 [cited 2019 Dec 6];3:85-9. Available from: http://www.scidentj.com/text.asp?2019/3/3/85/269001




  Background Top


Oral health is crucial to promote the general health of population.[1] Toothbrush is one of the most-used instruments to clean the teeth and the oral cavity. However, awareness of the importance of toothbrush hygiene is still low in Indonesia. After use, there is residue left on the toothbrush bristles.[2] This residue, which is usually in the form of bacteria, can survive and multiply on the toothbrush bristles. In result, these bacteria can cause the toothbrush's user to fall ill. The conditions of the toothbrush's storage can influence the growth of bacteria.[3] Bacteria grow well in high-humidity environments, such as a bathroom. The bacteria usually came from the human feces, saliva, mucus, and nasal secretions that contain pathogens.[4]

The conventional method of covering the toothbrush will prolong its drying, which supports the growth of bacteria.[5] Rinsing the toothbrush with tap water can also lead to further bacterial contamination.[6] Bactericidal agents can be used to reduce the possibility of the bacterial contamination of toothbrushes. Chlorhexidine, Brushtox, and some toothpastes were used as bactericidal agents.[7] Some research suggests that the use of microwaves and ultraviolet light are the most effective methods to clean toothbrushes. However, these methods are too expensive for household use.[8] The aim of this research was to propose another method to clean toothbrushes. The effects of toothbrush soaking in mouthwash solution on the total number of microorganisms (Streptococcus mutans and Fusobacterium) on the toothbrush were observed.


  Methods Top


This was an experimental study to determine the influence of toothbrush soaking in a mouthwash solution on the number of microorganisms. This research was performed in the Microbiology Center of Research and Education Laboratory, Faculty of Dentistry, Trisakti University, and PT GeneCraft Labs. The individuals of this research were 19–23-year-old Trisakti University students who live in Tawakal area, West Jakarta, Indonesia. The Lemeshow equation was used to determine the size of the sample in this research. The ethical clearance was granted from the Ethical Committee of Faculty of Dentistry, Trisakti University (No: 319/KE/FKG/8/2016).

The inclusion criteria 2 of this research were students with no systemic disease, diabetes mellitus, or consuming antibiotics. The toothbrushes were placed in a 2 m2 bathroom in the same manner and position. The research began with collecting samples from participants. Eighteen toothbrushes were provided to healthy individuals. For 1 month, the toothbrushes were soaked in disinfectant solution (25 mL) for 20 min after brushing, with Group 1 using Listerine® mouthwash and Group 2 using sterile tap water.

The real-time polymerase chain reaction (PCR) method was used to evaluate the number of microorganisms on each toothbrush. The toothbrushes were collected in a tube containing medium broth, vortexed for 20 s, and incubated in brain–heart infusion (BHI) broth and BHI agar at 37°C for 72 h. After incubation, the colony-forming unit (CFU) numbers in the agar were counted, and the bacterial cells in the BHI broth were harvested for DNA extraction. DNA was extracted using QiaAmp DNA extraction kit. The total number of DNA targets was identified using real-time PCR followed by SYBR green reagents and 16S rRNA-gene specific primers for S. mutans and Fusobacterium. Primers for S. mutans were forward 5'-AGCCATGCGCAATCAACAGGTT-3' and reverse 3'-CGCAACGCGAACATCTTGATCAG-5',[9] and those for Fusobacterium were forward 5'-CGCAGAAGGTGAAAGTCCTGTAT-3' and reverse 3'-TGGTCCTCACTGATTCACACAGA-5'.[10] S. mutans and Fusobacterium were cultured using BHI media for 2 × 24 h in anaerobic conditions. After 48-h incubation, the numbers of colonies on each dilution were used to extrapolate a standard curve.

Statistical analysis

The data were analyzed using a parametric statistical test. The normality test and paired sample t-test used for statistical analysis and level of significance were set at P < 0.05.


  Results Top


There were significant differences in the total bacterium numbers, S. mutans, and Fusobacterium on toothbrushes after soaking with mouthwash solution. The average scores (Log10 CFU/mL ± standard deviation) of the total bacterial load (2.66 ± 0.39), S. mutans (1.21 ± 0.18), and Fusobacterium (10.35 ± 6.02) on toothbrushes in Group 1 were significantly decreased compared to the average scores of the total bacterial load (5.19 ± 0.41), S. mutans (2.71 ± 1.59), and Fusobacterium (18.96 ± 4.26) in Group 2 [Figure 1]. There were statistically significant differences in total bacterium numbers, S. mutans, and Fusobacterium between Group 1 and Group 2 (P< 0.05).
Figure 1: The total bacterial numbers on the both nonsoaked (Group 1) and soaked (Group 2) toothbrushes (Log10 CFU/mL)

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The total numbers of bacteria on both nonsoaked and soaked toothbrushes are shown in [Figure 1]. The soaked toothbrushes had a lower bacterium count than the nonsoaked toothbrushes. It is also noticeable that some samples gave 0 CFU/ml bacteria after the soaking. The paired t-test indicated that there was a significant difference between the total bacteria and S. mutans counts on nonsoaked and soaked toothbrushes. On the other hand, the amount of Fusobacterium in both samples was not significantly different.

The results for S. mutans and Fusobacterium are shown in [Figure 2], [Figure 3], [Figure 4]. For both bacteria types, the soaked toothbrushes had a lower bacterium count than the nonsoaked toothbrushes. The mouthwash solution acted as an antibacterial agent that reduced the numbers of both bacteria.
Figure 2: The Streptococcus mutans numbers on both nonsoaked (Group 1) and soaked (Group 1) toothbrushes (Log10 CFU/mL)

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Figure 3: Fusobacterium numbers on both nonsoaked (Group 1) and soaked (group 2) toothbrushes (Log10 CFU/mL)

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Figure 4: Number of bacteria in pie diagrams. Group 1: Toothbrush soaked with Listerine® mouthwash, Group 2: Toothbrush soaked with sterile tap water

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  Discussion Top


All negative control toothbrush samples had a higher number of total microorganisms compared to the mouthrinse-soaked toothbrush. The difference between the numbers of microorganisms in both samples was significant. The results indicated that there was a reduction in the total number of microorganisms after the soaking treatment using nonchlorhexidine mouthrinse solution that contained effective essential oils (EOs).

In this study, the mouth rinse contained EOs-based mouthwash from herbal. Plant species can synthesize an abundant variety of organic compounds that act as secondary metabolites to protect themselves from herbivores, including arthropods and vertebrates, and provide protection against a wide variety of microorganisms, including viruses, bacteria, and fungi.[11] Natural products, such as plant EOs obtained from plant secondary metabolite compounds, display antimicrobial activity (against viruses, fungi, and Gram-positive and Gram-negative bacteria), antioxidant properties, antimutagenic activities, anti-inflammatory properties, and many potential health benefits,[12] have been widely used to treat various diseases since ancient times.[13]

Based on the knowledge of the antimicrobial and healing properties of natural EOs, some pharmaceutical companies have successfully mixed EOs into antiseptics and medicinal products, including mouthrinse products. In this study, the EOs-based mouthwash as chosen to eradicate the growth of microorganisms on the toothbrush bristles. This mouthwash consists of 21.6%–26.9% aqueous-ethanolic solution, which acts as a vehicle solution for four different EOs – thymol (0.06%), eucalyptol (0.09%), menthol (0.04%), and methyl salicylate (0.05%). The alcohol in this formula acts not only as a means to stabilize the main active ingredients of the product but is also believed to display antimicrobial activity.[14]

Most EOs belong to the terpenoid group in the form of monoterpenes, sesquiterpenes, hemiterpenes, and their derivatives, with a broad spectrum of antimicrobial properties. This antimicrobial activity arises from toxic effects on microbial membrane structures that can cause the disruption of membrane stability, which may lead to intracellular leakage, inhibition of cellular respiration, and alteration of ion transport mechanisms, leading to cell death.[15]

Thymol and its isomer carvacrol are phenol derivatives of terpenes isolated from thyme (Thymus vulgaris L.) and Lippia gracilis leaves. Thymol displays a broad spectrum of antimicrobial activity against Gram-positive bacteria (including Micrococcus spp., Staphylococcus aureus, and Bacillus spp.), Gram-negative bacteria (including  Escherichia More Details coli, O157:H7, Proteus mirabilis,  Salmonella More Details typhimurium, Serratia marcescens, and Pseudomonas spp.), and several species of fungi (including Candida albicans) due to its mechanism of disruption of the microbial cell membrane integrity.[16] Eucalyptol (1,8-cineole), which is extracted from the genus Eucalyptus, has been reported to possess strong antimicrobial properties against many pathogens and spoilage organisms, including E. coli, Bacillus subtilis, S. aureus, Staphylococcus epidermidis, Klebsiella pneumonia, Pseudomonas aeruginosa, C. albicans, and Aspergillus niger.[17] Gram-positive bacteria are more susceptible to eucalyptol than Gram-negative bacteria. These broad-spectrum antimicrobial properties are related to the lipophilic nature of the compounds, which can cause disruption of the membranes and accumulation inside the membranes, causing energy depletion and cell death.[18]

Menthol (derived from Mentha arvensis) and methyl salicylate (derived from genus Gaultheria and/or Betulla), in the form of natural EOs and synthetic derivatives, have been widely used in pharmaceutical industries as antiseptics and as ingredients for mouthrinse products to combat halitosis and other oral diseases. The main mechanism of their antimicrobial properties is their hydrophobicity, which can increase the permeability of microbial membranes and cellular leakage.[19] Methyl salicylate had also been reported to exhibit therapeutic properties, including anti-inflammatory properties, due to its ability to alter host cell immune mechanisms, and it also displays a wide range of antimicrobial activities, mainly through its ability to directly bind to proteins, both in the membranes and inside microbial cells, thus altering cellular signaling processes.[20] The combination of several EOs can act synergistically to target multiple biochemical processes in microorganisms, thus increasing the antimicrobial activity of the product.[12]

Other than the four EOs that have been mentioned, there are other natural products that have been widely used in dentistry, including eugenol. Eugenol (Eugenia caryophyllus), which is derived from the clove bud (Syzygium aromaticum), is commonly used by dentists as a restorative/cementation material and a desensitization agent. Eugenol also exhibits antibacterial properties. Tjandrawinata et al. were reported that eugenol in the form of toothpaste successfully reduced the total microbial load (including S. mutans and Lactobacillus spp.) in the saliva of healthy individuals in Indonesia.[21] In addition to EOs, crude plant extracts also have antimicrobial activities. Crude extracts of the strawberry fruit have been reported to significantly reduce the number of Enterococcus faecalis and Porphyromonas gingivalis and prevented biofilm formation in vitro.[22] Bromelain isolated from the Indonesian pineapple was also effective in eradicating E. faecalis in vitro through enzymatic processes.[23] In Widyarman et al., pomegranate juice was found to be effective against periodontal pathogens, such as Treponema denticola, P. gingivalis, and Aggregatibacter actinomycetemcomitans in vitro due to the presence of its major components, flavonoids, which can inactivate the processes required by microbial cytoplasmic membranes, thus leading to cell death.[24]

The composition of EOs in Listerine is based on a formula of four EOs – 0.064% thymol, 0.092% eucalyptol, 0.06% methyl salicylate, and 0.042% menthol. The antimicrobial mechanisms in these EOs function by penetrating microbial biofilms and killing microorganisms by damaging their cell walls and inhibiting their enzymes. Eucalyptol is an antibacterial, antifungal, and antiseptic agent. Menthol is an antiseptic. Thymol is a powerful antibacterial, antifungal, antiviral, and antiparasitic agent, and methyl salicylate is an anti-irritant.[25]

From this research, it can be concluded that soaking a toothbrush in a mouthwash solution can be used to reduce bacteria count. This is important, as literature shows that toothbrush can be a perfect place for the multiplication of microorganisms. Previous research pointed out that disinfection of toothbrushes could be safely and economically feasible using 0.12% chlorhexidine gluconate or 1% sodium hypochlorite solution.[26] Those previous studies support the results of this current research, which shows a reduction in the total amount of bacteria after soaking the toothbrush in a mouthwash solution.[26],[27]


  Conclusion Top


Mouthwash solution has the ability to significantly reduce the number of total bacterial load, and S. mutans, in particular, in toothbrush bristles. Further studies are needed to explore the effects of mouthwash solution against other oral pathogens.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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