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Beena Antony et al , J Biosci Tech, Vol 1 (2),2010, 59-63. SEMIQUANTITATION AND CHARACTERIZATION OF STREPTOCOCCUS MUTANS FROM PATIENTS UNDER GOING ORTHODONTIC TREATMENT Beena Antony *, Rekha.B, Anup Kumar Shetty, Thomas Kuruvilla, Ramanathan.K Dept. of Microbiology, Fr. Muller Medical College, Kankanady, Mangalore - Karnataka -575 002 *email: beenafmmc @ gmail. com Abstract Streptococcus mutans (S.mutans) is a primary aetiological agent in dental caries and is reported to colonize orthodontic patients in significant numbers. The aim of the study was to characterize and semiquantitate S.mutans from subjects undergoing active orthodontic treatment in comparison with an age matched healthy control group. Salivary samples from 75 orthodontic patients and controls were diluted ,dispersed, inoculated in appropriate media and incubated in CO2. An attempt was made to evaluate the efficacy of a Chlorhexidine mouth wash in reducing the count of S.mutans, by testing samples on two more visits with a gap of 3 weeks each. Significant growth of S.mutans was noticed in 56 out of 75 subjects in the test group, but none in the control group. Though reduction in the count was noticed in the test group, S.mutans could not be completely eliminated after using Chlorhexidine mouth wash. Due attention should be given to control this organism reinforcing oral hygiene, especially during orthodontic treatment. Key Words: Chlorhexidine, S.mutans, S.viridans 1. INTRODUCTION: S. mutans is a significant pathogen of oral cavity and initiates dental caries. This organism was isolated for the first time from the dental plaque by Clarke in 1924. The name ‘mutans’ was chosen because of its tendency to exhibit both coccal and rod shaped (mutant) cell [1] morphology . S.mutans belongs to the S viridans group, which is a poorly defined, heterologous group of the Genus Streptococcus. Members of this group are indigenous to the oral cavity. Though the classification of this particular group is oversimplified, it includes a variety of strains with different physiological and serological characteristics comprising S. mutans group, S. sanguis group, S. mitis group, S. salivarius group and S. milleri group [2]. rapid generation time, fermentation of wide range of carbohydrates, ability to withstand low pH, presence of enzyme glucosyl transferase that converts sucrose to glucose to enhance adhesion.[1] S. mutans is reported to cause systemic infections like endocarditis and Increased intravascular infections.. proportion of S. mutans is also reported in patients undergoing active orthodontic treatment [3] Altered oral ecological changes during orthodontic therapy leads to the elevated level of this organism [3]. According to the present trend, all the Streptococci which exhibit α haemolysis (other than pneumococci) are assigned to the S. viridans group. Hence, it is advisable to make Microbiologists familiarize with the identity of this oral pathogen. Many factors contribute to the cariogenicity of this organism such as The present study involves the semiquantitative isolation and detailed 59 Beena Antony et al , J Biosci Tech, Vol 1 (2),2010, 59-63. characterization of S. mutans from patients undergoing active orthodontic treatment in comparison with an age matched control group. An attempt is also made to study the efficacy of a Mouthwash Clohex plus (chlorhexidine gluconate -0.2%) in controlling S. mutans. 2. MATERIALS AND METHODS: This cross sectional study was done for a period of 1 year at the dept. of Microbiology of a tertiary care hospital in Mangalore. Test group constituted 75 healthy subjects between the age group of 13-25 years, being 19 males and 56 females undergoing active orthodontic treatment at the department of Orthodontics in a Dental College in Mangalore. For comparative evaluation, an age matched control group with good oral hygiene is also included. The criteria for patient selection included: • 20 permanent teeth that were either banded or bonded. • Subjects were free of oral and systemic disease. • Had periodontal packets not greater than 5 mm. • Not on antibiotic therapy for at least 3 months before the commencement of the study. 2.1. Collection of sample and processing: Salivary samples were obtained from these subjects (after chewing sterile paraffin to stimulate salivary flow) into wide mouthed vials and transported to the Microbiology laboratory without delay. 0.5 ml of saliva was transferred to a sterile screw capped tube that contained 4.5 ml of Reduced Transport Fluid (RTF) [4]. With aseptic precautions, vortex mixed for 1minute, to disperse the bacteria. A loopful of dispersed salivary samples was inoculated on various media using a standard calibrated loop (26 swg.4mm. diameter that can hold 0.01ml of the sample). 2.2. Media: 1. Brain Heart Infusion Blood Agar (Non selective media) 2. Gold’s Medium [5] – (Mitis Salivarius Sucrose Bacitracin Agar - selective media for S. mutans) [Figure-1] 3. Sucrose Agar & Sucrose broth (To detect extracellular glucan production) [Figure-2] 4. Carbohydrate Fermentation Media [Figure-3] The plates were streaked in 4 quadrants without heating to facilitate a rough quantitation as 1+ to 4+ according to the 60 Beena Antony et al , J Biosci Tech, Vol 1 (2),2010, 59-63. Wadsworth Laboratory Manual [6]. All the plates were incubated at CO2 rich environment for 24-48 hrs. Colonies of S.mutans appeared as dark blue, 1-2 mm in diameter, with smooth surface and an entire edge, dry and adherent in the Gold’s medium, dry and adherent refractile colonies on Sucrose agar & formation of a complete or partial gel in Sucrose broth as reported by Emilson & Bratthal [7]. Colonies morphologically resembling S.mutans in the Gold’s medium were quantitated and characterized according to the identification criteria described by Facklam [8] & Coykendall [9]. Effect of Chlorhexidine on S. mutans is given in the Table 2. In the second visit, 17 had 4+ growth and 35 had 3+ growth (significant in 52) but in the third visit only 4 had 4+ growth and 12 had 3+ growth (i.e. significant growth in 16) and no significant growth in rest of the 59. S mutans could not be completely eliminated even after using mouthwash for 6 weeks. 2.3. Evaluation of Chlorhexidine in controlling S. mutans: In order to evaluate the efficacy of Chlorhexidine mouthwash in controlling the count of S. mutans, the test group was advised to use 10 ml quantity of the above said mouth wash for rinsing the mouth twice daily. A second sample of the saliva was collected after 3 weeks and a third sample after 3 more weeks and processed like the first sample. 3. RESULTS: Significant growth of S. mutans (3+ or 4+) was noticed in 56 out of 75 subjects in the test group, but none in the control group as shown in the Table 1. Biochemical characterization employed for S.mutans is given in Table 3 4. DISCUSSION: S. mutans is the major component of oral streptococci which are indigenous to oral cavity. It can produce large amounts of extracellular glucan from sucrose by the enzyme glucosyl transferase. The production of glucan and large amounts of lactic acid by fermentation of carbohydrates constitutes major virulence factors in the causation of dental caries which is proved beyond doubts. [1] The usual trend of assigning all αhaemolytic Streptococci (other than 61 Beena Antony et al , J Biosci Tech, Vol 1 (2),2010, 59-63. pneumococci) as S. viridans group has limited its detailed characterization. Oral Streptococci can easily be differentiated by characteristic growth on selective media like Mitis salivarius agar or Gold’s medium and by simple biochemical analysis.[8,9]. A detailed account of differential growth characteristics on various media was described by Emilson & Bratthal [7]. Rosenbloom et.al [3], reported high incidence of S. mutans in subjects undergoing active orthodontic treatment. Altered oral ecological changes like low pH, increased retentive sites and increased retention of food particles are contributing factors. The present study is also in correlation with these findings. In the present study, significant growth of S. mutans (3+or 4+) was noticed in 74.7% (56/75) of subjects, compared to the age matched control group. Streptococcus viridans accounts for more than 50% of all cases of infectious endocarditis [10], is a matter of concern. The predilection to the damaged heart tissue plays a major role in causing endocarditis [10]. S.viridans group is also reported to cause other systemic infections like meningitis, lung abscess, osteomyelitis [11-15]. An Indian study reported 25.93% of viridans Streptococci from cases of bacteraemia following dental procedures [16]. Transient bacteraemia after dental manipulation may have long term sequelae, which warrants regular, follow up and prophylactic antibiotic therapy. In the present study, an attempt was made to evaluate a recently introduced antiplaque mouth wash Clohex plus (which contains 0.2% chlorhexidine gluconate, 0.05% sodium fluoride and 0.09% Zn chloride). Even though effective suppression was obtained, S. mutans could not be completely eliminated even after using this mouth wash for 6 weeks. A recent study from Madras reported that 0.4% chlorhexidine gluconate was found to be effective in inhibiting S.mutans by broth dilution technique, in vitro. [17] Orthodontic treatment lasting for 1-2 years has become the part and parcel of the beauty conscious teenagers and young adults. An increased association of S. mutans has been noticed in the present study as well as in the existing literature. As it is a primary cariogenic organism and an opportunistic pathogen in periodontal and systemic infections, due attention should be given to control this organism reinforcing oral hygiene during orthodontic treatment. Chlorhexidine gluconate is helpful in suppressing S. mutans to a great extent and maintaining oral hygiene. 5. CONCLUSIONS S.mutans , a primary aetiological agent in dental caries is reported to colonize orthodontic patients in siginificant numbers. Present study investigate the semiquantitation and characterization of S.mutans from healthy subjects undergoing active orthodontic treatment in comparison with an age matched healthy control group. The efficacy of a mouthwash, Chlorhexidine was evaluated in reducing the count of S.mutans. Even after using this mouth wash for 6 weeks, complete elimination of S.mutans was not possible. As it is a primary cariogenic organism that can disseminate to systemic infections, control measures should be adopted reinforcing oral hygiene. 62 Beena Antony et al , J Biosci Tech, Vol 1 (2),2010, 59-63. 6. REFERENCES: [1] Loesche, W. J., [2] [3] [4] [5] [6] [7] Microbiol Rev 1986, 353-80. 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