Efficacy and effect of Salvadora persica (Miswak), Toothbrush and Toothpaste on colonization of Oral Microbial Flora

Oral hygiene is one of the most important daily routine practices, it is necessary for keeping the mouth and teeth clean and preventing many health problems [1]. Different clinical studies have demonstrated the effects of microbial species in the oral cavity. Oral microbial communities are some of the most complex microbial floras in the human body, consisting of thousands of bacterial and hundreds of fungal species [2,3]. Only 54% of these species are cultivable and identified, 14% are cultivable but not identified, the 32% are not even cultivated [4]. The combined activities of microorganisms within the oral and the host responses to them, lead to the progression of the disease and tissue damage [5]. Modern dental care tools are designed to provide both mechanical and chemical means of removing plaque and food residues from the surface and spaces between the teeth. Throughout history, people have been using different tools and chemicals to maintain their oral health, such as chewing sticks, toothbrushes, gum, mouthwashes, toothpaste, floss, and traditional toothbrush [6,7]. Miswak is known as the “toothbrush tree” Salvadora persica L, named chewing sticks, belongs to the family known as Salvadoraceae [8]. Miswak sticks have been used for oral hygiene since ancient times [9]. Fibrous branches of S. persica have been approved by the World Health Organization for use as oral hygiene due to their protective effect on some oral pathogens [1]. The unique complexity of the Miswak phytochemicals and minerals, along with its long fibers, gives it an advantage as a tool for oral and dental health care through providing all of the necessary means of mechanical and chemical cleaning and maintaining healthy teeth and gums. The released chemicals, minerals and some of these biologically active constituents such as Sterol, Phenol, Palmitic Acid, Carvacrol, Eugenol, and Oleic Acids [10-12]. Miswak at the time of usage stimulates saliva production and buffer sits pH [13,14]. These ingredients have the potency to heal the inflamed gums, stimulatory effect on the gingiva, remove tartar, re-mineralize dental hard tissue, whitens teeth, provide enamel barrier, chewing sticks also contains volatile oils, tannic acid, sulphur, and sterols which contribute to anti-septic, astringent and bactericidal properties. It helps in reducing plaque formation, provides anti-carious effects, eliminates bad odor, and improves taste sensation [15], and a strong antimicrobial activity [16]. As well as protecting from pathogens that enters the body through the mouth [17].

Study Objectives

The aim of this research was:
1. To evaluate the efficacy of Salvadora persica (Miswak) products on oral bacteria in comparison with toothbrushes and toothpaste.
2. To assess the efficacy of Salvadora persica (Miswak) products on oral fungi in comparison with toothbrushes and toothpaste.

Study design

This is a study with a cross-sectional design. It was conducted on 60 volunteers, divided into two groups. Group Miswak uses, while the other group uses toothbrush & toothpaste (3 times a day for a week). This study was conducted between the Department of Medical Laboratory Sciences, Faculty of Medical Technology, and the Department of Medical Microbiology and Immunology, College of Human Medicine, from March to August 2021.

Study population

Sixty subjects aged 10- 65 y (39 females and 21males) consented to participate and were enrolled in the study. The selected subjects had not used any antibiotics or antiseptic mouthwash during the week. Smokers were not included in the study; none of the participants had used Miswak previously. The volunteers were divided into two groups with 30 in each group. Data were collected by questionnaire, were including age, sex, oral condition health. Sample collection culture and identifications Sampling was carried out two times:
1) Salvadora persica (Miswak) group: In two stages: The first sample was before use Miswak, and the second sample was after a week of use Miswak. Subjects were instructed to Miswak using, advised to use it three times daily, and shown how to keep it fresh by cutting off the edge of the Miswak every day and storing it in the refrigerator at night.
2) Toothbrush & toothpaste group: The first sample was before use toothbrush & toothpaste, and the second sample was after a week of use toothbrush & toothpaste, instructed to use ordinary toothpaste three times daily for a week.
Samples were obtained by an oral concentrated rinse in which the mouth was washed for 30 seconds with 10ml of sterile water. The mouthwash liquid is then deposited in tubes until microbiological processing, 100μl aliquots were inoculated by a spiral plating system onto the surface nutrient agar, blood agar, and mannitol salt agar, and Sabouraud’s dextrose agar with antibiotics. Culture plates were incubated for up to 24 h at 37 °C and at 30 ºC for 24-72h for yeast The identification of these isolates was performed using convention and standard biochemical tests including Gram stain, catalase, coagulase, and oxidase tests as described [18]. Growth assessed by enumeration of colonies and expressed as candida colony forming units per mL (cfu/mL-1) of rinse [19]. Yeasts species identified by germ-tube production, and chlamydospores production on cornmeal agar plus 1% Tween 80. All isolates show germ tube test and chlamydospores test positive on cornmeal agar and Tween 80 was identified as C. albicans or C. dubliniensis. Sunflower seed agar and xylose hypertonic media were used to differentiate both species [20].

Statistical analysis

The raw data were entered into excel spreadsheets and later imported to SPSS software version 26 (IBM Corp., Armonk, N.Y., USA). Descriptive statistics were used to calculate the frequency distribution, mean, standard deviation, and median. For the total number of microorganisms in the two groups, a T-test was used. The level of statistically significant difference was set at P<0001.

Of the 60 study volunteers, 39 (65%) were female, and 21 (35%) were male. The mean age of the participants was 34.23 years ± 17.428 SD (range: 10-65). On two groups, 30 were Miswak users, and 30 were toothbrush & toothpaste users. This study also indicated biochemical tests adopted for identification of isolated strains and their results were tabulated in Table 1 & 2. The results show that there was a statistically significant difference from the collected samples after a week using Miswak, compere toothbrush with toothpaste, according to the microbial growth on a nutrient agar plate, and Sabouraud’s dextrose agar, found that Miswak more effective than toothbrush & toothpaste, showed a significant decrease in the total number of colonies for each sample with a correlation coefficient. These indicate that a positive relationship between the Miswak and the total number of the bacteria, and candida with a P < 0.0001. Tables 3 & 4 as show the effect of Miswak on the bacterial strains, and candida, were total bacterial count (Mean±SD 22.633±17.88948, IQR=20, P-value=0.000) from Miswak group, and in [Mean±SD 149.767±26.27006, IQR =39, P-value=0.237] toothbrush & toothpaste group. Total candida count [Mean ± SD 27.5±18.48422, IQR=30, P-value=0.000] from Miswak group, and in [Mean±SD 247.0±90.13976, IQR =110, P-value=0.979] toothbrush & toothpaste group. Table 5 Shows the pattern of all the organisms isolated from Miswak group & toothbrush & toothpaste group, amongst the bacterial isolates Streptococci mutans had the highest occurrence with 16 (53.33%) from Miswak group, and in 17 (56.67%) of toothbrush & toothpaste, followed by Lactobacillus 12 (54%), and in Miswak group, 10 (33.33%) toothbrush & toothpaste group. Staphylococci aureus was found in Miswak group 2 (6.67%) and 3 (10%) toothbrush & toothpaste groups. The most frequent Candida isolates organisms were C. albicans 11 (36.67%) from Miswak group, and in 12(40%) of toothbrush & toothpaste group, the second isolate was C. dubliniensis 2 (6.687%) in Miswak group, and in 6 (20%) toothbrush & toothpaste group, followed by C. glabrata 1 (3.33%) from Miswak group. However, the isolated organisms can become pathogenic if the conditions become congenial.

Table 1: Tests used for the diagnosis of bacterial isolates.

Table 2: Phenotypic and microscopic characteristics of isolated Candida species.

Table 3a: Bacterial count in the different brushing groups.

Table 3b: Candida count in the different brushing groups.

Table 4: Comparison between Miswak and toothbrush & toothpaste through cultural growth before and after use.

Table 5: Microorganisms isolated from the mouth of test group, n=30.

Several studies have shown, Miswak has been reported to impart an essential antibacterial role, and antifungal [21,22]. where explained that Miswak has a more effective antimicrobial effect against S. aureus and C. Albicans compared to ordinary toothpaste, thereby preventing oral candidiasis [21,23,24]. The Miswak has been contain several medicinal properties; it has been scientifically proved to be very helpful in preventing tooth decay, even when used alone and without other methods of cleaning teeth. Several studies have assessed Miswak and its effect on oral health [25,26]. The potential of Miswak chemical components releasing during chewing may reduce the potential of contamination of Miswak [27]. As a consequence, the antimicrobial activity of the released phytochemicals reduces the total number of bacteria [28]. In this study, an assay after week was conducted to illustrate the difference between Miswak use, and toothbrush with toothpaste. The results showed that Miswak had an effect on all bacteria and candida in all. Miswak is effective against various types of oral bacteria which are involved in caries or periodontal diseases [29]. Miswak was significantly more effective in reducing S. mutans than ordinary toothpaste both immediately and after two weeks of use. This is also attributed to the antibacterial effect of Miswak. The importance of this study is based on being in vivo assessment of Miswak on oral flora followed up for the week. Despite that, it is highly recommended to extend the period of follow-up in future studies to measure the long-term effects on Miswak.

A. For the first use, the person should soak the tip of the Miswak for several hours, likely about 24hrs.
B. Before the use of Miswak, the tip meant for brushing must be washed with water.
C. The Miswak, which will be used, should be immersed in water for a few minutes (between 2 and 5 min) before using.
D. It is necessary to cut the tip of the Miswak every time.
E. When Miswak not used, it should be advisable to be stored in a humid place.
F. Must be dried after use in order not to cause mold due to the remaining water inside the used part.
G. It is recommended that further research should be carried out to study the role of Miswak on oral infections including, oral ulcers and other lesions in the oral cavity.

Our study results indicated that the total oral bacterial and oral candida carriage, on Miswak use was significantly reduced as compared with a toothbrush with toothpaste, thus the use of Miswak can limit the risk for oral bacterial contamination and translocation.

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