Introduction:

The oral cavity is a major gateway to the human body. Microorganisms from the oral cavity have been shown to cause a number of oral infectious diseases, including caries (tooth decay), periodontitis (gum disease), endodontic (root canal) infections, alveolar osteitis (dry socket), and tonsillitis.^[1,2] Oral microbiota consists of variety of micro-organisms such as bacteria, protozoa, fungi and hardly viruses. Bacteria are the predominant population found as microbiota.^[3,4] Bacteria play a major role in the initiation of disease facilitated by different factors like quality of dietary material, pH of saliva consumption of antibiotic whenever desired for treatment of other diseases.^[5] Dental caries is one of the most common chronic infection of tooth enamel caused by the interaction of Streptococcus mutans and Streptococcus sobrinus. These bacteria break down sugars for energy, causing an acidic environment in the mouth and result in demineralization of the enamel of the teeth and develop dental caries.^[6] Streptococci are the utmost group having number of species been reported as involved in damaging different components of oral cavity. However, S. sangunis and S. mutans formed colonization on the teeth in contrast to other species are reported as restricted to the cheeks and gums. Staphylococcus and streptococcus constitutes the major portion of microflora of oral cavity.^[7] Among the oral bacteria, Mutans streptococci have been implicated as a major cariogenic bacteria. The degree of colonization of these organisms correlates with the prevalence of dental caries in children and adults. Epidemiological studies have reported that S. mutansis more prevalent than S. sobrinus in the oral cavity, but have also shown that the prevalence of S. sobrinus is more closely associated with a high caries experience.^[8] Streptococcus mutans has been in the focus of researchers and recognized as the primary contributing cause of human dental caries. The organism yields certain extrinsic factors like glucosyltransferases responsible for the production of glucans from sucrose.

The detection and identification of oral streptococci in the dental biofilm is considered to be an important step for the understanding of dental caries. Nearly all investigations into the microbial pathogenesis have been done by cultivation of bacteria. This conventional method of culturing the bacteria is more time consuming and is sometimes inaccurate. It has also been reported that MS-bacitracin inhibits the growth of S. sobrinus to a greater extent than that of S. mutans on the agar medium, thereby providing inappropriate results.[9] However, the advanced molecular methods have revealed that the bacterial involvement in the development of dental caries is more complex than previously believed. In the present study, PCR method was used for amplification of the gene sequences of S. mutans and S. sobrinus. According to Igarashi PCR is a rapid, more sensitive and simpler method for the detection of micro-organisms. They also reported that the conventional cultural methods used for the detection of micro-organisms in dental plaque takes a long time nearly 1 week whereas, PCR method reduces the time to 6-7 hours.^[10]

Biochemical analysis has proposed that the bacterium secretes two different types of gtfs. Both gtfI and gtfB act cooperatively and synthesize insoluble glucans involved in the colonization of bacteria on tooth surfaces.^[11] So, the current study was undertaken to isolate the cariogenic Streptococcus and to relate the prevalence of its subtypes S. mutans and S. sobrinus through PCR amplification using type specific oilgos in the patients with the history of orthodental complication presented at University Hospital.

Materials and Methods

Total of 130 samples were collected from dental patients attended outdoor patient department (OPD) of the University Dental Hospital, The University of Lahore, Punjab. Three ml of saliva mixed gingival fluid was collected from every patient with 2mm size of hard dental plaque and shifted to 20 ml sterile plastic container. Samples were stored at 4°C in a cool place and then transported to the laboratory. (Fig. 1)

Figure 1: Collection of Saliva mixed gingival fluid from dental patients

Sample Size

The sample size was calculated by using the formula of Kish & Lisle states that [12] n = z2 p (1-p) / d2)
Where, z = Score for 95% confidence interval = 1.96
p = Prevalence
d = Sampling error that could be tolerate= 5%.
1-p= Probability

Specified grams of mannitol salt (MS) agar, nutrient agar, blood agar were weighed using digital balance (Shimadzu Scientific Instruments-Japan) and suspended in 1000 ml glass flask containing distilled water according to manufacturer instructions (Oxoid Ltd- England). The suspension was sterilized by autoclaving at 121°C for 15 minutes. 20 ml of each autoclaved nutritional media was poured on 9mm disposable petri plate and subjected for incubation at 37°C for 48 hours. Loopful of saliva mixed gingival fluid and 100 µl caries/ sulcus normal saline washing solution was streaked on the nutrient agar and MS agar. Cultures were incubated anaerobically for 48 hours at 37°C. (Fig. 2)

Figure 2:Media Preparation

After the incubation period, the colonies were identified on the basis of colony morphology. The typical colonies from each sample plate were transferred to blood agar (Oxoid Ltd- England) for further purification and incubated anaerobically at 37°C for two days. The following steps were involved in identification and characterization of the isolates: a. Colonial shape and form on MS-agar and Blood agar.
b. Gram-staining and microscopic examination.
c. Analytical profile index analysis
d. Antibiotic Susceptibility test
e. Molecular characterization through Polymerase chain reaction (PCR).

Every pure culture grown on the surface of selective medium showing standard characteristics colony were selected for Gram Staining. It was performed according to standard procedure. Smear was established by placing a loopful of culture on microscope glass slide re-suspended with drop of distilled water in circular pattern. The smear was fixed by passing 3-4 times on flame followed by heat air drying. Straining was performed according to the method described by Bergey.^[13]

The analytical profile index of Gram positive cocci isolated from samples was determined through Remel RapIDTM STR System (Thermofisher scientific-USA), following instructions of the manufacturer. (Fig. 3)

Figure 3:Biochemical identification by using Analytical Profile Index (API)

Antibiotic susceptibility test of the clinical isolates was done using modified Kirby-Bauer disc diffusion method in accordance with the guidelines of the Clinical and Laboratory Standards Institute.[14] The antibiotics used in this study include Amoxicillin (AMX), ciprofloxacin (CIP), erythromycin (E), chloramphenicol (C), tetracycline (TE), vancomycin (VA), cefixime (CFM), ampicillin (AM), clindamycin (CD), cefuroxime (CXM), bacitracin (B), spectinomycin (SPT), and kanamycin (K). The antimicrobial combinations used were AMC and AM/sulbactam (A/S). Bacterial inoculums were prepared by suspending the freshly grown bacteria in 2 ml of sterile brain heart infusion broth with yeast extract, and the turbidity of tube was matched with 0.5 McFarland turbidity standards and then swabbed onto Mueller-Hinton agar plate. To prevent condensation, antibiotic discs were allowed to warm to ambient temperature before application. Within 15 min of inoculation, the antibiotic discs were placed at side down on the seeded agar surfaces using sterile forceps, followed by incubating agar plates for 24 hours at 37°C. The relative susceptibility of the bacterial growth to each antibiotic shown by a clear zone of inhibition was measured in mm and zone diameter criterion was used to interpret the level of susceptibility to each antibiotic.

All the samples including positive known bacteria were subjected for extraction of DNA by using QAIamp DNA Mini kit (QIAGEN-Germany) according to the manufacturer instructions. All the extracted DNA was separately stored in 1 ml labeled eppendorf tubes at -200C. These samples were subjected to molecular identification.

The extracted DNA of all samples were subjected to PCR for amplification of the desired genes gtfB and gtfI of S. mutan and S. sobrinus. The sequence of these primers were:

Figure 3:Biochemical identification by using Analytical Profile Index (API)

For the PCR reaction, 2µl of 10X PCR buffer, 1 µl of forward and reverse primer, 2 µl of 80 mM MgCl2, 2 µl of DNTPs mixture and 1 µl of Taq DNA polymerase (Perkin-Elmer Cetus, Norwalk, Conn.) were added to the reaction mixture. Reaction volume was obtained by adding 9 µl of Nuclease-free Water and 2 µl of extracted DNA sample. The PCR tubes were incubated in thermal cycler (Applied Biosystems) with an initial denaturation of 94°C for 2 minutes followed by 30 cycles of denaturation at 95°C for 30 seconds, annealing at 51°C, 560C for 30 seconds and extension at 72°C for 45 seconds. Final extension was conducted at 72°C for 5 minutes. (Fig. 4)

Figure 4:: Polymerase chain reaction (PCR) of Extracted DNA

Figure 5: Gel Electrophoresis analysis of PCR product

Results

Caries is a polymicrobial infection and every bacterial species plays a role in determining the carcinogenicity of the biofilm or dental plaque. Out of 130 samples, 117(90%) pink colonies upto 1mm diameter on mannitol salt agar recovered form saliva mixed gingival fluid of caries active patients were declared as streptococcal species. Gram staining of such colonies showed 102(78.46%) non-sporeformer, round in shape forming long chains having purple color in compound microscope. The colonies showing alpha and gamma hemolysis on blood agar were selected and streaked on Mueller-Hinton agar media for antibiotic susceptibility pattern. Analytical profile index analysis revealed 73.50% colonies are positive for mannitol, sorbitose, oxidase and raffinose. While 26.50% showed negative reaction to hydrolysis of arginine production. Molecular characterization of streptococcus species isolated from dental patient showed 60% and 36% percentage positivity of S. mutans and S. sobrinus respectively. (Fig. 6, 7)

Figure 6:Isolation and growth of bacteria on Selective Medium. A.Nutrient Agar. B. Mannitol Salt Agar. C. Blood Agar

Figure 7:Gram staining and Microscopy of Isolated Bacteria

Kirby-Bauer disc diffusion analysis of clinical isolates against 15 commercially used antimicrobial drugs in dentistry. All isolates were sensitive against chloramphenicol (C), amoxicillin (AMX), ciprofloxacin (CIP), erythromycin (E), tetracycline (TE), ampicillin (AM), whereas least activity was shown by Bacitracin (B), while cefixime (CFM) and kanamycin (K), clindamycin (CD) and spectinomycin (SPT) showed no inhibition zones and isolates were reported as resistant against them. Combinations were used with an objective to broaden the spectrum of antimicrobial action. Among the antimicrobial combinations used, the greatest potency was shown by A/S followed by AMC. Among these antibacterial drugs tested, chloramphenicol showed maximum zone of inhibition followed by amoxicillin (AMX), erythromycin (E) and ciprofloxacin (CIP). Fig. 8)

Figure 8:Antimicrobial susceptibility pattern of isolated Streptococcus from Carries active patients

In male caries active patients, API based analysis revealed that 50(76.90%) samples were positive for Streptococci subsequently to high prevalence of S. mutans 42(64.6%) followed by S. sobrinus 19(29.2%) for gftB and gftI genotype sequences respectively. Moreover, 36(55.32%) samples of female dental patients with active caries were screened for significant bacterial count and identified as Streptococci in analytical profile index analysis. The frequency of gftB and gftI genotype sequences of S. mutans and S. sobrinus was 37(56.9%) and 28(43%) respectively. Furthermore, Out of 5 samples collected from healthy caries individuals, 60% showed typical colonies. Whereas, only 2 samples were observed positive for microscopic morphology and API analysis. While none of the primer could amplify the extracted genome for a typical amplicon size. The prevalence of S. mutans and S. sobrinus in male dental patients based on age group indicates that individuals of the 8-10 years age had high incidence (100%) than age group 11-30 years (60%) and 31-75 years (66.6% and 31.1%) respectively through PCR analysis. Streptococcus mutans and Streptococcus sobrinus specific amplicon were detected in (83.33%) and (66.6%) samples from the individuals of female caries active patients of 8-10 years age group. In contrast, the percentage positivity of both the Streptococcal isolates were slightly higher in age range of 11-30 years than age group 31-75 years. (Fig. 9, 10)

Figure 9:Gel Electrophoresis analysis of S. mutans. Lane L is 1000bp Ladder, Lane -C is negative control, Lane +C is positive control Lane 1-19 are isolates (517bp product).

Figure 10:Gel Electrophoresis analysis of S. sobrinus. Lane L is 1000bp Ladder, Lane -C is negative control, Lane +C is positive control Lane 1-19 are isolates (712bp product).

Table Ⅰ: Identification of Isolates by Using RapID RemelTM STR System

Table Ⅱ:. Distribution of Percentage Positivity of S. mutans and S. sobrinus Regarding the Age groups in Gender

*Facultative Anaerobic, visible growth in 24 hours at 37 ̊C, Pink colonies up to 1mm in diameter colonies on MS agar, Gamma hemolysis on blood agar
** Gram positive, cocci in chains, non-spore former
*** The isolate of each of the typical colony showed positive reaction to mannitol, sorbitose, oxidase, Raffinose. Moreover each of the isolate showed negative reaction to hydrolysis of arginine production

Figure 11: Prevalence of S. mutans and S. sobrinus on the basis of age distribution in Males caries active patients.

Figure 12: Prevalence of S. mutans and S. sobrinus on the basis of age distribution in Females caries active patients

TableⅢ:Distribution of Percentage Positivity of S. mutans and S. sorbinius Isolates Regarding the Gender

*Facultative Anaerobic, visible growth in 24 hours at 37 ̊C, Pink colonies up to 1mm in diameter colonies on MS agar, Gamma hemolysis on blood agar
** Gram positive, cocci in chains, non-spore former
*** The isolate of each of the typical colony showed positive reaction to mannitol, sorbitose, oxidase, raffinose. Moreover each of the isolate showed negative reaction to hydrolysis of arginine production

Figure 13:Comparison between S. mutans and S. sobrinus on the basis of Gender

Discussion

Dental caries is an infectious disease in which members of the Mutans streptococci have been implicated as the etiological agents. In this study we analyzed samples of saliva mixed gingival to determine the presence of S. mutans and S. sobrinus and to relate it to the presence caries on the basis of gender and age. The saliva mixed gingival fluid was used as a source for detection of cariogenic bacterial spieces. The intention is to relate the presence of cariogenic bacteria and dental caries by establishing an effective association. Although the presence of S. mutans is high in saliva, it is lower on the surface of enamel, where the bacteria actually manifests its capacity to produce acids leading to subsequent demineralization.^[14]

The colonization by mutans streptococci in the oral cavity are important factors for dental caries.[15] Bacteria can be isolated by colony morphology, biochemical assay, DNA sequence analysis, species specific PCR, antibiotic susceptibility assay indicates some of the strains are resistant to one or more antibiotics.[16] In the present study, oral cariogenic pathogens were isolated and identified on MS and Blood agar, showed typical colonies which were further stained and microscopic observations revealed purple colored interconnected cocci forming long chains. The pure cultures grown on the selective medium were identified through commercially available API kit showed high percentage prevalence of S. mutans 67.52% followed by S. sobrinus 40.17%. Antimicrobial susceptibility was performed for all the clinical isolates using Kirby-Bauer’s disk diffusion method. Nucleic acid of the isolates was amplified using type specific primers displayed particular bands through PCR.

It was observed that conventional techniques for detection of microorganisms are economic, easier to execute and still applicable however, results could be obtained in limited period of time by using advanced technique of PCR. Observation of the current study regarding dominance of PCR over conventional methods of microbiology for the identification of S. mutans is reported in comparative order. PCR was done as the previous tests did not provide the genotypic data of the samples so it was concluded from PCR results S. mutans (60.70%) and S. sobrinous (36.10%) that PCR is more sensitive and specific genotypically as compared to previous conventional methods.

Selection of appropriate antibiotic and effective dosage for treatment after detection of dental caries is vital to limit the infection. The results are similar to the observations reported by Hirose et al., 1993 showed that all isolates were highly sensitive to Chloramphenicol (C), followed by Amoxicillin (AMX), Ciprofloxacin (CIP) and Erythromycin (E) whereas, Cefixime (CFM) and Kanamycin (K) were reported as resistant against them.^[17]

Genotypic prevalence of gtfB and gtfI sequence of S. mutans and S. sobrinus was significantly higher (100%) in male caries active patients of age group ranging 8-10 years than 31-75 years age (66.6%, 31.1%) followed by 11-30 years age group (60%). Similarly, the sample of female dental patients also showed high frequency of both isolates in 8-10 years age group (83.33%, 66.6%). The high prevalence revealed its virulence nature in the form of dental caries. The results of the current study are well supported by the observations of Carlsson et al., 1968, who reported the high frequency of S. mutans in children aged 3 to 14 years.[18] Fujiwara et al., 1991 and Roters et al., 1995 were recorded the similar observations which revealed that S. mutans was isolated more frequently from children aged 6 years or slightly older.^[19,20]

The mechanism behind all the damage caused by bacteria is cariogenic species in the biofilm is composed of streptococcus mutans. When sucrose, disaccharide sugar from food is broken down by bacteria in mouth, glucose and fructose are produced. The glucose is used to make dextran which is part of the extracellular matrix of the biofilm. Fructose is fermented producing organic acid such as lactic acid. The acids dissolve the minerals of the tooth including enamel. The acid work even more quickly on exposed dentine. Over time, the plaque biofilm can become thick and eventually calcify and become hardened in this way it is called as the dental calculus. If the pulp is affected than the root canal may be needed to completely remove the infection.^[21]

According to the current study, the overall percentage prevalence of S. mutans and S. sobrinus in caries active male patients (64.6%, 29.2%) were significant higher as compare to female caries active patients. The results collaborates with Levine et al., 2014 who reported the prevalence of S. mutans (73%) and S. sobrinus (48%) higher in male dental patients than female dental patients.^[22] Mattos-Graner et al., 2000 suggested that the ability to synthesise water-insoluble glucan is an important virulence factor in initial caries development, in that it increases mutans streptococcal adherence and accumulation in the plaque of young children.^[23] Gronroos et al., 1998 found that the mutacin activity of clinical isolates is reasonably stable, and this virulence factor seems to be of clinical importance in early colonisation by S. mutans.^[24] An understanding of the route of transmission of mutans streptococci may help clinicians to develop measures to prevent, delay, or reduce colonization, thereby decreasing caries incidence.^[25,26]

The microbes present in oral cavity can cause damage to the teeth and can also lead to the infection i.e. cariogenic bacteria causes damage to enamel, dentine and eventually inner most structures the pulp resulting in infection leading to abscess. Tooth decay results from metabolic activities of microbes that are present on tooth.[27] A layer of proteins and carbohydrates form when clean teeth come in contact with saliva. Microbes are attached to food and forms a thin biofilm on all the surfaces of the oral cavity. This thin biofilm is also known as plaque which is composed of food debris, bacteria and saliva Streptococcus mutans and Streptococcus sobrinus are the prominent member of caries causing bacterial family.^[28]

It was observed that bacteriological methods are consistent in microbial diagnosis in limited resources but it requires considerable time however, PCR is highly sensitive and more reliable technique could be used for the further genotyping.

References:

1. Colak H, Dulgergil CT, Dalli M, Hamidi MM. Early childhood caries update: a review of causes, diagnosis, and treatments. J Nat Sci Biol Med 2013; 4:29–38.
2. Seymour GJ, Ford JP, Cullinan MP, Leishman S, Yamazaki K. Relationship between periodontal infections and systemic disease. Clin. Microbiol. Infect 2007; 13(Suppl. 4):3-10.
3. Vacaru R. The efficiency of dental plaque control measures based on risk prediction, using modern prophylactic methods. OHDM 2003; 2:4.
4. de Soet JJ, van Dalen PJ, Pavicic MJAMP, de Graaff J. Enumeration of mutans streptococci in clinical samples by using monoclonal antibodies. J Clin Microbiol 1990; 28:2467-2472.
5. Tan S, Marsh P, Arteriovenous Malformation of the Oral Cavity: A Case Report. Oral Hyg Health 2015; 3:1-4.
6. Beck, JD, Offenbacher S. Systemic effects of periodontitis: epidemiology of periodontal disease and cardiovascular disease. J. Periodontol 2005; 76:2089-2100.
7. Rashkova M, et al. Secretory Immunoglobulin A (S-IgA) and the Oral Risk Markers: Quality of Saliva, Dental Biofilm, Oral Candida and Lactobacillus spp. OHDMBSC 2009; 8:3.
8. Edwardsson S, Kock G, Öbrink M. Streptococcus sanguis, Streptococcus mutans and Streptococcus salivarius in saliva. Prevalence and relation to caries increment and prophylatic measures. Odont Revy 1972; 23: 279-96.
9. Nada HAM, Essam FAJ, Nidhal AAM, Abd WS. Isolation and identification of mutan’s Streptococci bacteria from human dental plaque samples. Journal of Al-Nahrain University. 2008; 11(3):98-105.
10. Duarte S. The Challenge of Treating Oral Infections Caused by Biofilms. Oral Hyg Health. 2013; 1:2.
11. Carlsson, J. A numerical taxonomic study of human oral streptococci. Odantologist Revy. 1968; 19:137-160.
12. Redmo EI, Thorhqvist E. Genotypes of mutans streptococci tend to persist in their host for several years. Caries Res 2000; 34:133–9.
13. Kish, Leslie. (1965). Survey Sampling. New York: John Wiley and Sons, Inc.
14. Bergey, John G.; Holt, Noel R.; Krieg, Peter H.A. Sneath (1994). Bergey's Manual of Determinative Bacteriology, 9th ed. Lippincott Williams & Wilkins.
15. Bauer AW, Kirby WM, Sherris JC, Turck M. Antibiotic Susceptibility Testing by a Standardized Single Disk Method. American Journal of Clinical Pathology 1966; 45:493-496.
16. Cury JA, Francisco SB, Del Bel Cury AA, Tabchoury CP. In situ study of sucrose exposure, mutans streptococci in dental plaque and dental caries. Braz Dent J 2001; 12:101–104.
17. Hardie. Oral microbiology: current concepts in the microbiology of dental caries and periodontal disease. British Dental Journal 1992; 172(7):271-281
18. Hirose H, Hirose K, Isogai E. Miura H, Ueda I. Close association between Streptococcus sorbinus in the saliva of young children and smooth-surface caries increment. Caries Res 1993; 27:292-297.
19. Carlsson J, Sundström B. Variations in composition of early dental plaque following ingestion of sucrose and glucose. Odontol Revy 1968; 19:161–169
20. Fujiwara T, Sasada E, Mima N, Ooshima T. Caries prevalence and salivary mutans streptococci in 0–2-year-old children of Japan. Community Dent Oral Epidemiol 1991; 19:151–154.
21. Roters FJM, Van De Hoeven JS, Burgersdijk RCW, Schaeden MJM. Lactobacilli, mutans streptococci and dental caries: A longitudinal study in 2-year-old children up to the age of 5 years. Carie Res 1995; 29:272-279.
22. Lepp WP, Brinig MM, Ouverney CC, Palm K, Armitage CG, Relman AD. Methanogenic Archaea and human periodontal disease. Proceedings of the National Academy of Sciences 2004; 101(16):6176-6181.2.
23. Levine WZ, Samuels N, Williams RC. Effect of a Botanical Mouth Rinse on Dental Plaque Formation: A Randomized, Double-blinded, Placebo-controlled Trial. J Oral Hyg Health 2014; 2:150.
24. Mattos-Graner RO, Smith DJ, King WF, Mayer MPA. Waterinsoluble glucan synthesis by mutans streptococcal strains correlates with caries incidence in 12- to 30-month-old children. J Dent Res 2000; 79:1371–1377.
25. Gro¨nroos L, Saarela M, Ma¨tto¨ J, Tanner-Salo U, Vuorela A, Alaluusua S. Mutacin production by Streptococcus mutans may promote transmission of bacteria from mother to child. Infect Immun 1998; 66:2595–2600.
26. Carlsson J, Gandour I A, Olsson B, Rickardsson B, Abbas K. Hight prevalence of mutans streptococci in a population with extremely low prevalence of dental caries. Oral Microbiol Immunol 1987; 2:121-4
27. Balakrishnan M, Simmonds RS, Taggs JR. Dental caries is a preventable infectious disease. Aust Dent J 2000; 45:235-45
28. Peter T, Daniela LZ, Peter M, Andrej MK. Enamel and dentine remineralization by nano-hydroxyapatite toothpastes. Journal of dentistry 2011; 39:430-437.
29. Paes Leme AF, Koo H, Bellato CM, Bedi G, Cury JA. The role of sucrose in cariogenic dental biofilm formation--new insight. Journal of Dental Research 2006; 85(10) 878-87