Estimation of the salivary levels of this organism may be useful for assessing caries risk in patients and for monitoring their response to preventive measures (1). Streptococcus mutans is an important component of the biofilms on teeth (dental plaque) associated with many forms of dental caries. Steptococcus mutans adheres firmly to the smooth tooth surfaces and produces sticky water insoluble dextran from dietary sucrose, forming plaque, which facilitates the accumulation of microorganisms. Streptococcus mutans and other organisms in the plaque produce organic acids such as lactic acid that gradually destroy the enamel and form a cavity (2).
The caries-inducing properties of S.mutans depend on its adhesion ability (3), and also of its acid-producing and glucan synthesis activities. Bacterial adhesion or attachment to the tooth surface involves specific molecular interactions between complementary molecules on the microbial and host surface, which require the participation of two factors; a receptor and adhesion. Hydrophobic forces or cell surface hydrophobicity also play an important role in the initial adhesion of bacteria to tooth surfaces (4). Among the oral streptococci, S. mutans has a higher degree of surface hydrophobicity (5), and its adhesion to saliva-coated hydroxyapatite depends on hydrophobic interaction (6).
S.mutans produces glycosyltransferase (GTF), which allows the production of soluble and insoluble glucan. Glycosyltransferase from S. mutans is the most significant virulent factor in dental caries, where sucrose plays an important role as a natural source of energy and it is converted to long chain polysaccharides (7).
Evolutionary relationship of GtfB transfarase gene of Streptococcus mutans
3D crystal structure of glucosyltransferaseThis study represents an evolutionary relationship of GtfB transfarase gene of Streptococcus mutans. Taxonomical information of bacteria is: Bacteria; Firmicutes; Lactobacillales; Steptococcaceae; Steptococcus. GtfB gene is basically responsible for production of 'glucosyltransferase-I'. Firmicutes basically involve Bacillales, Clostridia, Lactobacillales, and Mollicutes.
A crystal structure of Udp-Glucosyltransferase GtfB can be collected from MMID 16966 and PDB ID 1IIR. Glucosyltransferase has two domain structures (Mulichak et. al, 2001).
Figur 1. A: 3D structure of glucosyltransferase gene B: Domain information of glucosyltransferase superfamily
Saliva is a complex mixture of several components (8). Whole saliva (oral fluid) is formed primarily from salivary gland secretions, but also contains gingival fluid, desquamated epithelial cells, bacteria, leucocytes, and possibly food residues, blood and viruses (9). Saliva is essential for maintenance of healthy oral tissues; it coats the oral mucosa and protects against irritation, forms an ion reservoir for tooth remineralization, functions as a buffer, aids in swallowing, exerts antimicrobial action, participates in pellicle formation and enzymic digestion of starch with amylase, and also participates in taste sensation by acting as a solvent (8). The bacterial content of saliva is estimated to approach 109 bacteria per ml (10). Saliva helps to control invasion of the mouth by microorganisms, and lack of saliva results in increased numbers of bacteria in the mouth. Saliva can act as a selective medium for bacterial growth, but continuously repeated swallowing results in clearing of bacteria (10).
Materials and Methods
Experimental site and duration of study
This study was conducted in Biological Product Laboratory, Department of Botany, University of Allahabad, during the period extending from August 2005 to May 2008.
Study group
These samples were judged on the basis of patients brushing their teeth, economic status, sex and age groups.
Site of collection
180 samples were collected from different Dental hospitals, Clinics, Schools and Colleges located at Allahabad city (North central part of India) (Fig. 2). These include.
Saumya Dental Clinic Taigore Town, Allahabad.
Sangam Dental Clinic Allahapur, Allahabad.
Naseem Dental Care Rajapur, Allahabad.
Kendriya Vidyalaya Naini, Allahabad
YMCA School, Allahabad
Pragasi Prathamic Vidyalaya Rajapur, Allahabad.
International Academy Naini, Allahabad.
Dev Prayag Phaphamau, Allahabad.
Sample collection
Saliva was collected from decayed tooth portions in sterilized test tube with the help of sterilized cotton swabs and then swabs were streaked aseptically on synder agar slant for determination of caries susceptibility test (Fig. 2). One uninoculated tube serves as a control at 37°C for 48 hours (11).
Figure 2. Collection of saliva from school childrens and various age group people at Dental Clinic of Allahabad under the supervision of dental surgeon Dr. Praveen Tiwari
Determination of dental caries susceptibility by Synder agar test
Determination of susceptibility to dental caries was performed by the synder test (11). It is the measurement of the rate of acid production from the metabolism of glucose by Lactobacilli and Streptococci. This method utilized synder agar (pH=4.7) that contains glucose as the carbohydrate and bromocresol green as the pH indicator. As the Lactobacilli present in the saliva grow in the synder agar, they utilize glucose of the medium converting it to organic acids and thereby lowering the pH to 4.4 or lower at which the caries process begins. At this pH, bromocresol green turns yellow. The susceptibility of individual to dental caries is determined by the time it takes for change of colour from green to yellow (Fig. 3). If the medium turns yellow within 24 to 48 hours the individual was said to be susceptible to dental caries.
Figure 3. Dental caries susceptibility on Synder agar slants (A) Collection of Saliva (B) Synder agar slants (C) Dental caries susceptibility on Synder agar slants
Results
180 samples were collected from different Dental hospitals, Clinics, Schools and Colleges located at Allahabad city (North central part of India). Out of 180 saliva samples collected from different age group peoples, 67% were found to be positive on the basis of dental caries susceptibility test. The Statistical analysis of data using (Chi-square)-test, showed that the most susceptible (83.3%) was found in age group 11-20 years followed by age group of >51 (73.3%), <10 (66.6%), 21-30 (63.3%), 41-50 (63.3%) and the lowest susceptible (53.3%) was noticed in age group of 31-40 (Table 1, Fig. 4).
Table 1. Incidence of dental caries susceptibility by synder agar test on the basis of different age group
Age
Group
(Years)
No. of
saliva sample collected
No. of dental caries susceptibility
% of dental caries susceptibility by synder agar test
<10
30
20
66.6
11-20
30
25
83.3
21-30
30
19
63.3
31-40
30
16
53.3
41-50
30
19
63.3
>51
30
22
73.3
Total
180
121
67
Statistical analysis of Table 1
(Chi-square)-test (5%)
Where, = sum of , Oi = observed value, ei = estimated value
Table value: 10, Calculated value: 20.9, Result: Significant
Since, the calculated value of was greater than table value at 5% level of significance. So it was concluded that dental caries susceptibility varied with age factor.
Figure 4. Incidence of dental caries susceptibility by synder agar test on the basis of different age group
The collected saliva samples from different socioeconomic status, 67% were found to be positive. The Statistical analysis of data using (Chi-square)-test showed that the maximum dental caries susceptibility (86.66%) was noticed in lower class socioeconomic people followed by Higher-class socioeconomic status (68.33%). The least proned towords dental caries with only 46.66% affected people was noticed in middle class socioeconomic status (Table 2; Fig. 5).
Table 2. Incidence of dental caries susceptibility on the basis of socioeconomic status
Socioeconomic status
No. of saliva sample collected
No. of dental caries susceptibility
% of dental caries susceptibility
Higher
60
41
68.33
Middle
60
28
46.66
Lower
60
52
86.66
180
121
67
Since, the calculated value of was greater than table value of 5% level of significance. So it was concluded that the dental caries susceptibility varied with socioeconomic status.
Figure 5. Incidence of dental caries susceptibility on the basis of socioeconomic status
Out of above mentioned saliva samples collected from different sex group, 67% were found to be positive. The Statistical analysis of data using Z - test, showed that the maximum susceptibility to dental caries was found to be 61.98% in male as compared to female which were noticed only 38.02% susceptible cases (Table 3).
Table 3. Incidence of dental caries susceptibility on the basis of Sex
Sex
No. of saliva sample collected
% of sample collected
No. of dental caries susceptibility
% of dental caries susceptibility
Male
102
56.66
75
61.98
Female
78
43.34
46
38.02
180
100
121
100
Statistical analysis of Table 3
Z-test (5%)
Where, P1 = Incidence in 1st group / total no. of case in 1st group, P2=Incidence in 2nd group / total / total no. of case in 2nd group, q1=1- p1, q2=1- p2, N1 = Total no. of cases in 1st group, N2 = Total no. of cases in 2nd group
Table value: 1.96, Calculated value: 50, Result: Significant
Since, the calculated value of Z was greater than table value at 5% level of significance. So, it was concluded that the dental caries susceptibility also depends upon the sex.
The 67% were found to be positive in case of different brushing schedule, The Statistical analysis of data using (Chi-square)-test, showed that the maximum susceptibility to dental caries was found to be 78.57% in patient whose brushed their teeth once in day, followed by patient whose brushed their teeth twice in day (63.46%), the minimum (36.66%) susceptibility to dental caries was observed in patient whose brushed their teeth thrice in day (Table 4; Fig. 6).
Table 4. Incidence of dental caries on the basis of patient brushing their teeth per day
Brushing teeth /day
Total saliva sample collected
No. of dental caries susceptibility
% of dental caries susceptibility
Once
98
77
78.57
Twice
52
33
63.46
Thrice
30
11
36.66
180
121
67
Since, the calculated value of was greater than table value at 5% level of significance. So it was concluded that caries susceptibility shows dependency on sheudule of brushing.
Figure 6. Incidence of dental caries on the basis of patient brushing their teeth
per day
Discussion
The fact that dental caries is a world-wide disease requiring vast economic resources and causing a great deal of discomfort has called upon attempts aimed at developing an accurate screening method for detection of the 5-20% of subjects comprising the high-caries-risk group (12). Because mutans streptococci are considered to be the predominant pathogens of dental caries disease, individuals heavily colonized by mutans streptococci were thought to automatically be at high risk for caries. Indeed, in young children, early mutans streptococcal colonization on tooth surfaces has been recognized as an indicator of later high scores of decayed, missing and filled surfaces in deciduous teeth (dmfs index) (13). However, it has become evident that, although prevalence of the infection is indicative of the disease status on a population level, when it comes to an older child or adult, on the individual level, the caries risk rate cannot be accurately predicted on the basis of how heavily the subject is colonized by mutans streptococci (14).
In the present study, total 180 saliva samples were collected from different age group, socioeconomic status, sex as well as different brushing schedule of peoples.
From different age group peoples, 67% were found to be positive dental caries susceptibility on the basis of Synder agar test. Ali et al., (15) was also using similar type of study for caries suspectibility, the high frequency of caries causing bacteria (83.3%) in the saliva of children 11 to 22 years aged. However, Grábris et al., (16) have also reported a high prevalence of Streptococcus mutans in adolescents aged 14 to 16 years. Roters et al., (17) reported that at 2 years of age, teeth up to the deciduous molars have erupted in most children and subsequent changes in development did have no major effects on the oral microbiota.
According to Rogers et al., (17), this factor may reflect changes in the oral microbial population. Children in the age ranges of below 10 years and 10 to 20 years presented this species. Edwardson et al., (18) detected Streptococcus sanguis, Streptococcus mutans and Streptococcus salivarius in the saliva of children aged 9 to 11 years old observing that this last specie corresponded to 34% of the total Streptococci count. Children aged 11 to 20 years show that in the present study Streptococcus mutans also presented a higher frequency in the same age range, corresponding to 83.30% by the standard method.
Grindefjord et al., (19), in Sweden, observed that of 692 saliva samples from children aged 1 to 2.5 years showed Streptococcus mutans and 21% carried lactobacilli, differing of the 116 samples containing Streptococcus mutans observed in the present study among a total of 262 saliva samples analyzed (29.51%). These differences in colonization by Streptococcus mutans may be due to the different methods employed and also to the dietary habits and dental anatomy in the populations studied. Recently, the polymerase chain reaction (PCR) has been introduced to identify the mutans streptococci (20), and several techniques may be used together in order to obtain a reliable identification. However, it requires considerable time consuming and may not provide satisfactory reliability (20).
In the present study, maximum dental caries susceptibility (86.66%) was noticed in lower class followed by Higher class (68.33%) where as the least with only 46.66% affected people was noticed in middle class. The significantly higher susceptibility to dental caries was seen 61.98% in male as compared to female 38.02%. The higher susceptibility to dental caries 78.57% in patient who brushed their teeth once in day, followed by whose brushed their teeth twice in day (63.46%), the minimum (36.66%) observed in patient who brushed their teeth thrice in day.
Conclusions
Dental caries may even lead to life threatening infections, and the costs for operative dental treatment are significant both for individuals and society. Today, mutans streptococci are considered to be the main aetiological microorganisms in caries disease, with lactobacilli and other microorganisms participating in the disease progression. Occasionally, some other microorganisms have been traced as initiator microorganisms. Acidogenesis at a low pH has also been reported for a group of non-mutans streptococci. That's why the estimation of slaiva and inhancincing the quality of saliva is one of the important criteria to avoid dental caries.
Acknowledgements
The authors are very grateful to Department of Science and Technology (DST) for financial assistance. Thanks to the Head, Department of Botany, University of Allahabad and Dr. Shalini Dikshit, Principal Kendriya Vidyalaya, Naini, Allahabad, India for providing necessary facilities.
Declaration of Interest
The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the manuscript.
