IJSRD - International Journal for Scientific Research & Development| Vol. 5, Issue 09, 2017 | ISSN (online): 2321-0613

Isolation and Characterization of Mutans and Streptococcus Sobrinus from Dental Caries to Determine its Inhibition by Bacteriocins of P. Thapar1 R. K. Malik2 M.K. Salooja3 1Research Scholar 2Emeritus Scientist 3Professor 1,3Indira Gandhi National Open University (IGNOU), Maidan Garhi, New Delhi- 110068, India 2National Dairy Research Institute (NDRI), Karnal-132001, India Abstract— Oral health is now recognized equally important improper growth and development which severely affects in relation to general health. Despite of numerous studies and their health [3]. improvements in dental health, major oral health problems B. Bacteriocins and their application in prevention of caries around the world are generally considered to be dental caries. It has been surveyed and concluded that the caries mainly Bacteriocins are the bacterial peptides that help in regulating occurs in children below the age group of 7-10 years. There competitive interactions in natural microbial systems. They are certain bacteriocins produced by the of lactic acid possess certain characteristics like narrow target range, bacteria. The bacteriocins like Nisin A and mutacin B-Ny266 stability, high activity and low toxicity. Increasing are active against a range of organisms, including species of antimicrobial resistance and growing awareness of Actinomyces, , , Corynebacterim, microbiome for the importance of human health, underscore , Gardnerella, Lactococcus, Streptococcus, and the need of this class of antimicrobials, as an approach for the Staphylococcus. In the present study, the bacteriocin treatment of infectious diseases [10]. These are believed to be produced by the lyophilized culture of Pediococcus safe for human consumption since they become inactive pentosaceus PP34 has been used to determine its anti- when comes in contact with digestive in the stomach microbial activity against the dental caries causing [13]. A bacteriocin called mutacin 1140 is found to prevent organisms. The caries causing organisms like Streptococcus dental caries by [4]. mutans and Streptococcus sobrinus have been isolated from dental cavities on selective agar media-TYCSB media. The II. MATERIAL AND METHODS microscopic, cultural, biochemical and molecular A. Collection of sample characterization of the isolates has been performed to confirm the isolated strains upto species level. The isolates were Samples of caries infected teeth were collected from various confirmed as Streptococcus mutans and Streptococcus dentists. The teeth were preserved in sterile phosphate sobrinus. On performing the anti-microbial activity, the buffered saline (PBS) (Hi-Media, India) until used. These bacteriocin produced by Pediococcus pentosaceus PP34 were then subjected to microbiological analysis for isolation showed inhibitory effect against the isolated strains of of Streptococcus mutans and Streptococcus sobrinus. Streptococcus mutans and Streptococcus sobrinus. Therefore, B. Isolation of Streptococcus mutans and Streptococcus the bacteriocins from can be fortified to sobrinus various food products like chocolates so that the selective Suitably diluted exudate from caries infected was food product itself becomes a weapon against dental cavities spread plated on selective media- TYCSB agar (composition in humans. by Wan et al, 2002 [1]) and the plates were incubated at 37ºC Key words: Streptococcus mutans; Streptococcus sobrinus; for 65h. They were observed periodically for microbial lactic acid bacteria; bacteriocins; Pediococcus pentosaceus growth. The pure cultures of this suspected Streptococcus spp. was streaked on the slants of the selective media so as to I. INTRODUCTION obtain pure culture of the isolates. A. Dental caries and its prevalence in India C. Microbial and Biochemical identification of isolates , also called as dental caries has been ranked at Several identification tests were performed. The microbial 3rd position by World Health Organization among all chronic 4a staining include- Negative staining and Gram staining. Some non-communicable diseases . It has been pointed out by confirmatory biochemical tests, selective for Streptococcus W.H.O., that despite of great improvements in the oral health mutans and Streptococcus sobrinus spp. were performed, of population in several countries; dental caries still persists which includes: and has become a global problem [8]. A group of caries 1) Catalase test: Few drops of hydrogen peroxide (30% causing organisms include these species of genus H2O2) were gently poured on microbial growth to Streptococcus including S. mutans, S. sobrinus, S. ratti, S. observe for presence or absence of effervescence. salivarius, S. infantarius, S. vestbularis, S. mitis, S. oralis, S. 2) Rapid differentiation test for Streptococcus mutans: Few infantis, S. perois, S. cristatus, S. sanguinis, S. parasanguinis, drops of test solution- 10% mannitol & 4% TTC-2,3,5- S. gordonii, S. anginosus, S. intermedius and S. constellatus triphenyltetrazolium chloride (from Hi-media, Mumbai, [9]. According to a study, more than 40% of the children in . India) was gently poured on the microbial growth. The India are prevalent to dental caries since past 15 years [6]. colonies were observed for the appearance of pink Children suffering with tooth decay, also suffer with colour.

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3) Utilization of different carbohydrate sources: The of isolates is mentioned in Table 1. Microscopic analysis isolates of bacteria were plated on Brain Heart infusion showed all the isolates in cocci shape. Some of the cells were agar (from Hi-media, Mumbai, India) containing 0.02% present in chains and some were scattered singly. The cells phenol red colour. Four different carbohydrates were violet in colour i.e. all the cells were Gram positive. including salicin, , inulin and sorbitol in the form The colonies of Streptococcus mutans strains of sterilized disks of 0.05 mg were placed on plates and formed mutiberry-shaped colonies growing deep into the incubated at 37ºC for 48h. The change in colour from red agar, while the very hard colonies of Streptococcus sobrinus to yellow gave positive result for carbohydrate were usually surrounded by extracellular polysaccharides. fermentation and vice-versa. Streptococcus mutans is a Gram-positive, non- motile, non- spore forming, catalase- negative, facultative anaerobic and D. Molecular identification cocci shaped bacteria [7]. To confirm the isolates to species level, the bacterial genomic DNA was isolated by the method previously described [2]. B. Biochemical Examination of Isolates The bands were measured by electrophoresis on 1.5% agarose The 60 isolates obtained were subjected for catalase test and gel and stained with ethidium bromide. The isolated DNA the results are shown in Table 1. After catalase test, the 27 was further subjected to Polymerase chain reaction to detect isolates which showed positive test for catalase, were Streptococcus mutans and Streptococcus sobrinus. The same discarded and not used for further study. The remaining 35 procedure was followed as used previously [2]. isolates were subjected to further examination and the results are shown in Table 2. After examination by sucrose E. Standard Culture fermentation, some of the isolates were discarded as they The lyophilized culture of Pediococcus pentosaceus PP34 gave negative result for sucrose fermentation. Thus, out of 35 was inoculated in MRS broth (from Hi-media Company, isolates, 14 isolates were remaining to be applied for Mumbai, India) and incubated at 37ºC for 24h. The microbial detection. Therefore, further analysis for carbohydrate growth was streaked on the plates of selective media- MRS fermentation was done for salicin, sorbitol and inulin with agar so as to obtain the pure culture. remaining isolates. Out of 14 isolates, 4 isolates gave negative F. Preparation of culture supernatant result for salicin fermentation. For further confirmation, rapid differentiation test for confirmation of Streptococcus mutans The standard culture Pediococcus pentosaceus PP34 was was done. Thus, out of 14 isolates, 7 isolates gave positive subjected to centrifugation at 10,000 rpm for 20 minutes at result for the test. 4ºC in a refrigerated centrifuge. The culture supernatants so Streptococcus mutans ferment of wide range of obtained were sterilized by passage through a 0.22 µm Millex carbohydrates sucrose, mannitol, melibiose, raffinose, GV filter (Millipore) and stored at -18ºC untill assay of cellobiose sorbitol, lactose, salicin trehalose and inulin [5]. It bacteriocin activity. is demonstrated that all the mutans streptococci group that G. Anti-microbial assay against Streptococcus mutans and were isolated from humans are capable to ferment mannitol, Streptococcus sobrinus sorbitol and inulin sugars except for Streptococcus sobrinus Spot-on-lawn assay technique was done in order to determine and Streptococcus cricetus. A rapid differentiation test the bacteriocin activity. The isolated strains of Streptococcus between colonies belonging to mutans streptococci group mutans and Streptococcus sobrinus were used as indicator from that of other streptococci was done on MS- agar media. organisms and plated on TYCSB agar media. Five µl of the Results indicated that thirty five isolates were stained with culture supernatant (crude bacteriocin solution) of the dark pink colour and considered to be positive result [11]. Samples of Designation of Catalase Pediococcus pentosaceus PP34 was put as spot on the dried S.No. agar surface of each isolate of Streptococcus mutans and Dental Plaques isolates test 1(a) - Streptococcus sobrinus. The plates were incubated at 37ºC for 1. Adult male 24h. The plates were subsequently examined for clear zones 1(b) + around the wells which indicated growth inhibition due to 2(a) - bacteriocin activity. 3(a) - 4(a) - III. RESULTS AND DISCUSSION 5(a) - 2. Adult female 2(b) - A. Cultural characterization and microscopic identification 3(b) + of isolates 4(b) + The total isolates obtained were 60 in number. In each culture 5(b) - two types of colony was observed i.e. colony (a) and (b). In 6(b) + some cultures colony (Y) and (E) were also found. Colony (a) 6(a) - had creamish colour, punctiform, entire margin and flat 7(a) + elevation; jelly like appearance. Colony (b) had white colour, 8(a) - punctiform, entire margin and raised elevation; opaque 9(a) - appearance. Colony (Y) had yellow colour, raised elevation, 3. Adult male 10(a) - entire margin, punctiform and opaque appearance. Colony (E) had creamish colour, entire margin, punctiform, opaque 11(a) + appearance and colony embedded into agar. The designation 12(a) - 13(a) -

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10(a 7(b) + 9. + + - + + + 8(b) - ) 12(a 9(b) + 10. - x x x x x ) 10(b) + 13(a 11. + + + + + + 11(b) + ) 12(b) + 14(a 12. + + + + + + 13(b) + ) 14(a) - 15(a 13. - x x x x x 15(a) - ) 16(a 16(a) - 14. - x x x x x 17(a) + ) 19(a 18(a) + 15. - x x x x x 4. Adult female ) 19(a) - 20(a 16. - x x x x x 20(a) - ) 21(a) - 21(a 17. - x x x x x 14(Y) + ) 22(a 15(Y) + 18. + - - - - - 22(a) + ) 23(a 23(a) - 19. + + + + + + ) 24(a) + 26(a 20. - x x x x x 25(a) + ) 5. Adult female 26(a) - 28(a 21. + + + + + + 27(a) + ) 29(a 28(a) + 22. + - - - - - 29(a) - ) 33(a 16(b) + 23. + + + - + - ) 30(a) + 35(a 24. + + + - + + 31(a) + ) 32(a) + 36(a 25. + + + - + + 33(a) - ) 6. Adult female 17(E) - 26. 2(b) - x x x x x 18(E) + 27. 5(b) - x x x x x 19(E) - 28. 8(b) - x x x x x 17(E 20(E) + 29. - x x x x x ) 21(b) - 19(E 30. + - - - - - 22(b) - ) 23(b) - 21(b 7. Adult male 31. - x x x x x 24(b) - ) 34(a) + 22(b 32. - x x x x x 35(a) - ) 36(a) - 23(b 8. Adult male 33. + + - + - - 25(b) - ) 24(b 37(a) + 34. + + + + - - 9. Adult male ) 26(E) - 25(b 35. + - - - - - Table 1: Catalase Test for the Isolates ) Rapid Anti- Table 2: Biochemical Examination of Isolates Carbohydrate Fermentation Different micro

Test iation bial C. Molecular identification Test assay The molecular identification was done by polymerase chain S. Isola Sucr Sali Sorb Inu reaction which confirmed the isolates as Streptococcus

No. tes ose cin itol lin mutans and Streptococcus sobrinus. Out of 14 isolates, the 1. 1(a) - x x x x x bands of 5 isolates appeared at 700 bp and the bands of 5 2. 2(a) - x x x x x isolates appeared at 900 bp respectively. The bands of 3. 3(a) - x x x x x remaining 4 isolates did not show any bands (Fig.1). 4. 4(a) - x x x x x In an experiment, with the primers used, 5. 5(a) - x x x x x Streptococcus mutans and Streptococcus sobrinus produced 6. 6(a) - x x x x x single DNA fragments of 517 and 712 bp, respectively [2]. 7. 8(a) - x x x x x 8. 9(a) - x x x x x

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motivation and guidance made this research possible. I am indebted to many of my colleagues who supported me throughout my work. It’s a pleasure to thank my parents, as without their support and patience, I would have not reached at this stage of my life.

REFERENCES [1] K. L. Wan, W. K. Seow, L. J. Walsh, & P. S. Bird, “Comparison of five selective media for the growth and enumeration of Streptococcus mutans”, J. Aus. Dent., vol. 47, pp. 21-26, 2002. [2] T. Franco, P. Amoroso, M. J. Marin, & A. F. Vila, “Detection of Streptococcus mutans and Streptococcus sobrinus in samples from Brazilian Fig. 1: PCR amplification showing Lane 1 (from left) as Preschool children by polymerase chain reaction”, J. DNA marker; lanes 2- 6 (from left) as S. mutans and lanes Braz. Dent., vol. 18, pp. 329-333, 2007. 11-15 (from left) as S. sobrinus [3] Gupta, R. K. Momin, A. Mathur, K. T. Srinivas, & A. D. Anti-microbial assay against Streptococcus mutans and Jain et al.,“Dental Caries and Their Treatment Needs in Streptococcus sobrinus 3-5 Year Old Preschool Children in a Rural District of Out of 13 isolates of Streptococcus spp., the zone of India”, North American J. Med. Sci., vol. 7, pp. 143–150, inhibition was formed against 6 isolates, indicating inhibition 2015. of Streptococcus mutans and Streptococcus sobrinus by [4] F. M. Bastos, V. M. Coelho, & O. Santos, “Resistance to bacteriocin of lactic acid bacteria i.e. Pediococcus bacteriocins produced by Gram-positive bacteria, J. pentosaceus (Table 2 and Fig. 2). Microbio., vol. 161, pp. 683-700, 2015. The bacteriocins like Nisin A and mutacin B-Ny266 [5] H. A. Nada, A. Mudallal, F. A. Essam, A. Jumaily, A. A. are active against a range of organisms, including species of Nidhal, & W. A. Shaibany, “Isolation and Identification Actinomyces, Bacillus, Clostridium, Corynebacterim, of Mutan’s Streptococci bacteria from human dental Enterococcus, Gardnerella, Lactococcus, , plaque samples”, J. Al-Naharain Uni., vol. 11, pp. 98- Micrococcus, Mycobacterium, Propionibacterium, 105, 2008. Streptococcus, and Staphylococcus [12]. [6] H. Kundu, P. Basavaraj, A. Singla, & K. Singh, “Dental Caries Scenario Among 5, 12 and 15-Year-old Children in India- A Retrospective Analysis”, J. Clinical & Diagnostic Res., vol. 9, pp. 1-5, 2015. [7] K. K. Sunder, “Prevalence of dental caries, oral hygiene status and treatment needs in physically handicapped children attending various special schools of Davangere District”, Indian J. Public Health Res. Develop., vol. 1, pp. 159-167, 2012.

(a) Control (b) Positive result [8] K. Yadav, & S. Prakash, “Dental Caries: A Review”, Fig. 2: Anti-microbial assay showing zone of inhibition Asian J. Biomed. Pharma. Sci., vol. 6, pp. 1-7, 2016. [9] K. Yadav, Dental Caries: Bacterial profile of Dental caries. Lap Lambert Academic Publishing, Germany, pp. IV. CONCLUSION 120, 2016. Salient findings of the study suggest that out of 14 isolates of [10] L. Dorit, M. S. Robert, & A. M. Riley, “The Bacteriocins Streptococcus spp., 4 isolates were confirmed as Current Knowledge and Future Prospects”, vol. 1, pp. Streptococcus sobrinus and 7 isolates as Streptococcus 168-169, 2016 www.caister.com, last accessed: 25, mutans. The bacteriocin produced by standard strain of March, 2017. Pediococcus pentosaceus PP34, showed inhibitory effect [11] M. E. G. Sherbiny, “Control of growth Streptococcus against 3 strains of Streptococcus mutans and 3 strains of mutans isolated from saliva and dental caries”, Int. J. Streptococcus sobrinus. Therefore, these bacteriocins from Current Microbiol. Applied Sci., vol. 3, pp. 1-10, 2014. lactic acid bacteria can be applied to various food products [12] M. Mota-Meira, H. Morency, & M. C. Lavoie, “In vivo which have tendancy to inhibit dental cavities in humans. activity of mutacin B-Ny266”, J. Antimicrobial Thus, these bacteriocins will help in preventing cavities in Chemother., vol. 56, pp. 869–871, 2005. consumers from the bacteriocin fortified food sources. [13] S. R. Upendra, P. Khandelwal, K. Jana, A. N. Kumar, G. Devi, & L. M. Stephaney, “Bacteriocin Production from ACKNOWLEDGEMENT Indigenous Strains of Lactic Acid Bacteria Isolated from I owe my deepest gratitude to Dr. R.K. Malik who gave me Selected Fermented Food Sources”, Int. J. Pharma. Res. an opportunity to work on this project and was responsible Health Res., vol. 4, pp. 982-990, 2016. for all the fundings of the project. His guidance and teachings at all the stages of my life, made this research possible for me. I also give my sincere thanks to Dr. M.K. Salooja, whose

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