The Effect of Miswak and Toothbrush on Saliva Total Bacterial Count and Cariogenic Bacteria

Miswak, a chewing stick prepared from the roots, twigs or stems of Salvadora persica. The beneficial effects of miswak in respect of oral hygiene and dental health are partly due to its mechanical action and partly due to its pharmacological actions. The aim of this study was to evaluate the effects of miswak sticks, miswak extract and toothbrush/toothpaste as antimicrobial agents on total bacterial count in the saliva, and to study the effect of miswak stick, toothbrush/toothpaste and saline on salivary Streptococcus mutans (S. mutans) and lactobacilli in vivo. The study clarified that there was a marked reduction in the total bacterial count among all groups. The reduction was 100%, 100%, 97.5% and 67.5% among users of miswak stick, miswak extract, toothbrush/toothpaste and saline (control), respectively. The results of the current study also showed marked reduction of S. mutans in 66.7% of the individuals using miswak and among individuals using toothbrush/toothpaste, whereas the reduction was only 29.2% among saline users. The reduction of lactobacilli was 62.5%, 58.3% and 54.2% by using miswak stick, toothbrush/toothpaste and saline, respectively. The difference was not statistically significant. It can be concluded that miswak has an antimicrobial effect comparable to the use of fluorinated toothpaste. It was clear from the current study that the Caries Risk Test (CRT) kit is a rapid and a simple, semiquantitative method for counting lactobacilli and S. mutans, which can be used in dental clinics without the need for trained technicians. It is recommended that further studies can be performed on larger sample size of patients, at high risk of developing dental caries. INTRODUCTION Dental caries and periodontal diseases are the two main afflictions to mankind. Bacterial plaque is solely responsible for the initiation and progression of periodontal diseases. (1,2) Several microorganisms have been characterized as having high cariogenic potential, but two classes of microorganisms, Streptococcus mutans (S. 580 Bull High Inst Public Health Vol.38 No.3 [2008] mutans) and lactobacilli have been exclusively studied. (3)S. mutans has been strongly associated with the initiation of caries, while lactobacilli have been mainly connected with the further development of caries lesion. (4) The methods available for maintenance of oral health are mainly mechanical and chemical. Toothbrushes and dentrifices are widely used for cleaning teeth. Miswak , a traditional toothbrush or chewing stick, is deeply rooted in Islamic culture.(2,5) It is a chewing stick prepared from the roots, twigs or stems of Salvadora persica (S. persica) which is is an upright evergreen small tree or shrub. The leaves of S. persica are small, oval, thick and succulent with a strong smell of cress or mustard. The fresh leaves are eaten as salad and are used in traditional medicine for cough, asthma, scurvy, rheumatism, piles and other diseases. The flowers are small and fragrant and are used as a stimulant and are mildly purgative. (2) The beneficial effect of miswak in respect of oral hygiene and dental health are partly due to its mechanical action and partly due to its pharmacological actions. (1) It has been shown that extracts of miswak posses various biological properties including significant antibacterial and anti-fungal effects. Extracts of S. persica and other related plants may be effective against the bacteria that are important for the development of dental plaque.(6,7) Therefore, it has been claimed that, miswak sticks may have antiplaque effects and may also affect the pathogenesis of periodontal diseases by reducing the virulence of periodontopathogenic bacteria. (8) The anti-microbial and cleaning effects of miswak have been attributed to various chemicals detectable in its extracts. These effects are believed to be due to its high content of sodium chloride and potassium chloride as well as salvadourea and saponins, tannins, vitamin C, silica, and


INTRODUCTION
Dental caries and periodontal diseases are the two main afflictions to mankind.Bacterial plaque is solely responsible for the initiation and progression of periodontal diseases. (1,2)Several microorganisms have been characterized as having high cariogenic potential, but two classes of microorganisms, Streptococcus mutans (S. Bull High Inst Public Health Vol.38 No. 3 [2008]   mutans) and lactobacilli have been exclusively studied. (3)S. mutans has been strongly associated with the initiation of caries, while lactobacilli have been mainly connected with the further development of caries lesion. (4)e methods available for maintenance of oral health are mainly mechanical and chemical.Toothbrushes and dentrifices are widely used for cleaning teeth.Miswak , a traditional toothbrush or chewing stick, is deeply rooted in Islamic culture. (2,5)It is a chewing stick prepared from the roots, twigs or stems of Salvadora persica (S. persica) which is is an upright evergreen small tree or shrub.The leaves of S. persica are small, oval, thick and succulent with a strong smell of cress or mustard.
The fresh leaves are eaten as salad and are used in traditional medicine for cough, asthma, scurvy, rheumatism, piles and other diseases.The flowers are small and fragrant and are used as a stimulant and are mildly purgative. (2)The beneficial effect of miswak in respect of oral hygiene and dental health are partly due to its mechanical action and partly due to its pharmacological actions. (1) has been shown that extracts of miswak posses various biological properties including significant antibacterial and anti-fungal effects.Extracts of S.
persica and other related plants may be effective against the bacteria that are important for the development of dental plaque. (6,7)Therefore, it has been claimed that, miswak sticks may have antiplaque effects and may also affect the pathogenesis of periodontal diseases by reducing the virulence of periodontopathogenic bacteria. (8)e anti-microbial and cleaning effects of miswak have been attributed to various chemicals detectable in its extracts.These effects are believed to be due to its high content of sodium chloride and potassium chloride as well as salvadourea and saponins, tannins, vitamin C, silica, and Wasfi et al.,

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resin, in addition to cyanogenic glycoside and benzylisothio-cyanate. (7,9) has also been claimed that the vitamin C and sitosterol content of this plant have great roles in strengthening the gum capillaries and preventing gum inflammation. (9,10)Sulfated compounds and isothiocyanate are known to be responsible for antibacterial effects of the plant, while fluoride and calcium salts are quite effective in preventing dental caries. (11,12)reover, the silica and calcium salts in the plant act as grinder and detergent. (13)imethylamine is known to be effective in reducing surface adhesion and also in decreasing plaque accumulation.Tannins, tannic acid and benzyl isothiocyanate, are reported to have antimicrobial effects and help the healing of gum inflammation. (12,13)e World Health Organization (WHO) has recommended and encouraged the use of these sticks as an effective tool for oral hygiene, because of tradition, availability and low cost, (1,5) however, its antibacterial value has not been fully substantiated.Therefore, this study was carried out.

AIM OF THE WORK
The aim of this research is to evaluate the effects of miswak sticks, miswak concentrate , and toothbrush/toothpaste as antimicrobial agents on total bacterial count in the saliva as well as on salivary S.mutans and lactobacilli in vivo.

MATERIAL AND METHODS
A randomized controlled trial was carried out in Ajman University for Science and Technology.

SUBJECTS:
The study subjects were divided into two groups to be studied simultaneously with our aim.c.Toothbrush with toothpaste Group: Forty subjects were asked to brush their teeth for 6 minutes.
d. Saline (control) Group: Forty subjects were asked to rinse their mouth with saline for 6 minutes.

Group B:
Seventy-two healthy individuals (criteria as for Group A) divided into three groups of twenty-four each, were chosen randomly from the staff and the students working at Ajman university.The same procedure was carried on the three groups (the use of miswak stick, toothbrush/toothpaste and saline).

SAMPLES:
From all studied subjects, a saliva sample (2ml) was collected in a sterile container before and after the use of the allotted method.
Stimulated saliva was collected from each individual, by giving each a small piece of paraffin wax (1cm long) and was asked to chew for a period of 30 seconds, then, to swallow any saliva but not the paraffin wax.Thereafter, the patients continued to chew the paraffin wax and saliva was collected at 2 minutes intervals for a period of six minutes.

METHODS
1. Group A: 10µl of saliva of each sample was evenly distributed on Brain heart infusion agar with 5% sheep blood.The plates were incubated at 37ºC for 24 hours.Following incubation, the number of the isolated colonies present was determined and multiplied by a factor that converts the volume of saliva to one ml.The final calculation was then equal to the number of organisms per ml of sample. (10)ull Liechlenstein/Europe).

•
The kit comprised a slide attached to the cover of the vial.

•
The commercial product had one side of the slide coated with a solid selective culture medium (mitis salivarius agar enriched with sucrose) for the cultivation of S .mutans, while the medium on the other side of the slide (Rogosa agar) was for the cultivation of lactobacilli.

•
The salivary samples were used as per instructions of the manufacturer.
The samples were incubated at 37ºC for 48 hours.
• Growth density of the bacteria was evaluated under good lighting condition by naked eyes and as per manufacturer's instructors.(Figure 1)

Statistical analysis
The data were entered and analyzed using computer program SPSS version "13".The following was performed: (14) • Descriptive statistics as mean and standard deviation for the age was carried out.
• Kolmogorov-Smirnov test was used to test the distribution of the data.
The data were not normally distributed so the nonparametric statistics was used.bacterial counts after use of various agents.
• Chi-square test was used for categorical data.
• Z test was used for comparison of two proportions.
• P <0.05 was set as the cut of level of significance

Total bacterial counts among users of various agents
The

Streptococcal and lactobacilli counts among users of various agents
Three groups of 24 subjects each were the sample used to test the effect of using toothbrush, miswak and saline on Streptococci mutans and lactobacilli counts.
Among the toothbrush users, the mean age was 27.5 ±8.10 years and 66.7 % were females, For the miswak group , there was 75 % females , mean age was 29.17 ± 6.51 years.Regarding the saline users group , mean age was 31.17 ±6.45 years and more than 90 % were females (91.7 %).
Table (3) revealed that there was one log reduction of mutans streptococci (median 90 % of change) after using different agents as toothbrush, miswak stick and saline.It was also clear that among the toothbrush or miswak users, 66.7 % showed reduction in Streptococcus mutans counts while 33.3 % did not show any change.Among the saline users the reduction was only noticed among 29.2 %.This difference was statistically significant (X 2 2 = 9.06,P<0.05)Reduction of lactobacilli is presented in table (4).The one log reduction was also 90 % of change).It was noticed that, 62.5 % of miswak users showed change in antibacterial activity (reduction) compared to toothbrush or saline (58.3% and 54.2%, respectively), but the difference was not statistically significant, P>0.05.

Table (3) : Frequency distribution of reduction in mutans streptococcal counts among users of various agents
oth brushing can reduce the likelihood of caries by reducing number of cariogenic organisms and removing the substrate.

Table (4): Frequency distribution of reduction in lactobacilli counts among users of various agents
Tooth brushing is the most common method used to remove plaque.However, resources for oral health care are limited in many developing countries and the need to explore and test easily available and inexpensive traditional preventive measures is strongly needed. (16)e present study revealed a one log reduction in the total bacterial counts among all groups and as clear from table  (15) Darout and Skaug also reported that the periodontal status of miswak users was better than that of toothbrush users. (7)Also, our results are strongly supported by that reported by Socransky et al that lower caries is experienced among miswak users. (17)In addition, this is also in agreement with previous epidemiological studies. (18,19,20) O the other hand, some studies found that there were more plaque  (21) The present study results are in agreement with Darout and Skaug results regarding lactobacilli, that Miswak users had lower results of cariogenic bacteria than toothbrush users. (7).Almas and Al Zeid reported similar findings to our results, in which the results showed a marked reduction of S.mutans among all groups, but there was no significant difference for lactobacilli.
They explained that this may be due to the amount of lactobacilli present in saliva at the time of collection. (9)though saliva is thought to possess no distinctive microbiota of its own, it harbors as much as 10 8 or 10 8 bacteria/ml.These high numbers reflect micro-organisms derived from oral biofilms adhering to teeth and oral mucosal surfaces, gingival crevices,and periodontal pockets (22) .In More studies can also be performed on larger samples of patients that are diagnosed to be a high risk group of developing dental caries.

1 .
Group A: One hundred and sixty subjects were chosen randomly from the staff members working at Ajman University, and who accepted to volunteer to participate in this study.The individuals selected were medically healthy with no systemic diseases and did not use any antibiotic or mouth gargle for the past two weeks.They were further subdivided into 4 subgroups of 40 individuals each.Subjects who used more than one Wasfi et al., 583 method were excluded.a. Miswak stick Group: Forty subjects were asked to use the miswak stick for 6 minutes.b.Miswak extract Group: Forty subjects were asked to rinse their mouth with the miswak extract for 6 minutes.
factors are necessary for caries to occur.These factors include the interaction of the susceptible host, oral flora, and the substrate which is primarily sucrose which should be present for a sufficient length of time.Removal of dental Plaque is effective in treating gingivitis, preventing periodontal disease and dental caries.Plaque covering early caries lesions usually contains high levels of S. mutans and lactobacilli.
addition to the previous reason, saliva was used for the bacterial count instead of plaque samples because saliva represents the oral load of the microorganism as well as their average colonization in the dentition.In addition, plaque samples represent only a single site, and there is a wide variation in bacterial numbers among different tooth surfaces.Moreover, saliva samples are less time consuming and easier to collect outside dental clinics.(9)It was also reported byDarout et al, that S. mutans and lactobacilli occurred significantly more frequently in saliva than in subgingival plaque. (22)CONCLUSION & RECOMMENDATIONS It can be concluded from the present study that Miswak has an antimicrobial and prophylactic effect comparable to the use of toothbrushing with fluorinated toothpaste.Therefore, miswak can be a good alternative to the toothbrush since it is inexpensive, and readily available.Miswak contains many medicinal properties, and is available in most rural areas of the poor countries.In addition, miswak does not need expertise or expensive resources to manufacture it.Thus it is recommended as an important and effective tool for oral hygiene.It was also clear from the current study that the CRT kit is a rapid and a simple, semi-quantitative method for counting lactobacilli and S. mutans, which can be used in dental clinics without the need for trained technicians.It is recommended that further studies can be performed by the use of serial dilution of the saliva samples to get accurate counts of cariogenic bacteria.