Research Article ANTIBACTERIAL ACTIVITY OF BEE PROPOLIS AGAINST CLINICAL STRAINS OF STREPTOCOCCUS MUTANS AND SYNERGISM WITH CHLORHEXIDINE Arul Selvan K 1, Rajendra Singh.C 2 and Dr.Prabhu T 3 Address for Correspondence 1 Asst. Professor of Microbiology, Krishnadevaraya College of Dental Sciences & Hospital Bangalore, Ph.D Scholar, Dr. MGR University, Chennai. 2 Lecturer in Bio-Technology, Sir.M. Visvesvaraya Institute of Technology, Bangalore 3 Professor of Microbiology, formerly Principal, Bangalore Medical College & PhD Supervisor, Dr. MGR University, Chennai Email: arulselvan30@gmail.com ABSTRACT Dental caries is a common oral infection prevalent in developing countries. Streptococcus mutans, a commensal bacteria of the oral cavity, plays a major role in the etiology of caries. Dentists around the world use fluoride varnishes and pastes in the prophylaxis and treatment of caries. Mouthrinses containing Chlorhexidine are also being used in the prevention of caries. Fluoride is a highly toxic substance and has been observed to cause several side effects. Chlorhexidine has been recorded to be less effective in controlling caries. Hence natural substances like Propolis, which possesses high anti-bacterial activity, have attracted the attention of researchers in the prophylaxis of dental caries. The inhibitory effect of ethanolic extract of Propolis on Streptococcus mutans has been well established in previous studies. In our study we prepared the ethanolic extract of Propolis (EEP) in our laboratory and examined the inhibitory effect of EEP, Chlorhexidine and 1:1(v / v) combination of EEP & Chlorhexidine against 50 strains of Streptococcus mutans isolated from patients with dental caries attending our dental hospital. The disc diffusion method (Kirby-Bauer) was used to detect the inhibition of the growth of mutans streptococci. We have observed that all the clinical strains were inhibited by EEP, the mean zone size of inhibition of growth being 26mm. Chlorhexidine also inhibited all the strains, the mean zone size being 22.56mm. the combination of EEP & Chlorhexidine was observed to be the most effective in the inhibition Strep. mutams, the mean zone size of inhibiton being 32.22mm. We are reporting that Propolis exhibits synergistic activity with chlorhexidine in the inhibition of mutans streptococci. KEY WORDS: Propolis, Chlorhexidine, Streptococcus mutans, Dental caries. INTRODUCTION Streptococcus mutans is considered one of the most important cariogenic species of the human oral microbial flora (8). There is ample evidence for the association between S. mutants and dental caries (15). The suppression of S. mutans by antimicrobial agents, especially locally administered chlorhexidine, is consequently of clinical importance (13, 14). Dental plaque, a film of microorganisms on the tooth surface, plays an important part in the development of caries and periodontal diseases (9). Some of the oral commensal bacteria such as the mutans streptococci can colonize the tooth surface and initiate plaque formation by their ability to synthesize extracellular polysaccharides from sucrose, mainly water-insoluble glucan, using the enzyme glucosyltransferase (3). Inhibiting the colonization of mutans Streptococci on the tooth surface is believed to prevent the formation of dental plaque and development of dental caries. The use of mouth rinses containing Chlorhexidine has been recommended by many dentists to prevent caries. Chlorhexidine has been reported to reduce the accumulation of plaque. Mouthrinses are widely used as adjuncts to oral hygiene and in the delivery of active agents to the teeth and gums. Many recent studies have demonstrated antimicrobial activity against selected oral pathogens from natural sources. Natural products have been used for folk medicine purposes throughout the world for thousands of years. Many of them have demonstrable pharmacological properties, such as antimicrobial, anti-inflammatory and cytostatic, among others and more recently propolis has been recognized as useful for human and veterinary medicine. Recently, Propolis has been attracting the attention of researchers due to its various biological activities and therapeutic properties. Propolis is a complex resinous material collected by Apis mellifera honey bees from various plant sources and mixed with secreted beeswax. It is non-toxic and possesses multiple pharmacological effects and a complex chemical composition (2). Propolis has been shown to reduce the incidence of dental caries in rats.(5). The mechanism of antimicrobial activity of propolis is complex and could be attributed to the synergistic activity between phenolic and other compounds (7). The combination of propolis with antibiotics can reduce drug dosages, minimize drug side effects, and decrease chances of drug resistance. The synergism
between EEP and antimicrobial drugs, especially those agents that interfere on bacterial protein synthesis such as choramphenicol, gentamicin, netilmicin, tetracycline,and vancomycin has been demonstrated. (4). In our study we have examined the antimicrobial activity of ethanol extract of Propolis, Chlorhexidine, a combination of Propolis and Chlorhexidine against clinical strains of Streptococcus mutans. MATERIALS AND METHODS PROPOLIS SAMPLES Beehives were collected from different places in Hunasamaranahalli village, Bangalore. The samples were kept in a deep freezer at -20 0 C for 3 days. The hardened material was ground by a grinder and ethanolic extraction was done as per the procedure described by Bianchi, 1995(1). 30 grams of the ground propolis was dissolved in 100 ml of 95% Ethanol (A.R. grade, S.D. Fine Chemicals, Mumbai) (Figure 1). This mixture was preserved for 2 days in a bottle corked tightly and kept in an incubator at 30 0 C. After dissolving, it was filtered twice with Whatman No.4 and No.1 filter papers. This solution was evaporated by negative pressure at 50 0 C to get a yellowish solid. This was called the ethanol extract of propolis (EEP) and was preserved at +4 0 C Chlorhexidine diacetate was obtained from a commercial source (Hi-Media, Mumbai). specimens. This medium contains the selective agents - crystal violet, potassium tellurite and Bacitracin. These agents inhibit most gram-negative bacilli and most gram-positive bacteria except streptococci. Sucrose is incorporated in a 5% concentration which is utilized as an energy source by the streptococci. The specimens were streaked onto a dry MSB agar medium for isolation and incubated in a candle extinction jar for 24 hours at 37 0 C. Streptococcus mutans forms raised, convex, undulate, opaque, pale blue colonies, granular "frosted glass" appearance, sometimes exhibiting a glistening bubble on surface of colony due to excessive synthesis of glucan from sucrose. (Figure 2). Figure 2: Colonies of Streptococcus mutans on Mitis-Salivarius-Bacitracin Agar Identification of mutans streptococci Strep. mutans was identified by Gram stain morphology, culture morphology and biochemical tests (mannitol / sorbitol fermentation positive, esculin hydrolysis positive and arginine hydrolysis negative) Figure 1: Ethanol extraction of Propolis Clinical isolates Dimineralized portions of the decayed tooth were obtained from 50 patients with caries attending the out patient clinic of the Krishnadevaraya College of Dental Sciences & Hospital, Bangalore. The patients had dental caries at various stages. The specimens were collected in 2 ml of sterile thioglycollate broth in screw capped vials and incubated for 30 minutes at 37 0 C. Primary isolation medium Mitis Salivarius Bacitracin agar (MSB agar) with 0.1 % Potassium tellurite was used for the primary isolation of mutans streptococci from the clinical Figure 3: Gram Stain appearance of S. mutans Anti-microbial Susceptibility testing Anti bacterial susceptibility testing for propolis was done by the disk diffusion method as per the National Council for Clinical Laboratory Standards guidelines (NCCLS). Disk diffusion test: (Kirby-Bauer method) Reagents for the Disk Diffusion Test 1) Brain Heart Infusion agar (BHI agar) BHI agar is a highly enriched medium for the growth of fastidious bacteria, useful in the cultivation of streptococci. The medium has been found effective in the recovery of dental pathogens from clinical
specimens. The medium contains pancreatic digest of casein,brain heart infusion, peptic digest of animal tissue, sodium chloride, disodium phosphate, dextrose and agar. The brain heart infusion and peptone provide the nutrients, organic nitrogen, carbon, sulfur, vitamins and trace substances. Dextrose provides the carbohydrate source for fermentative microorganisms. Disodium phosphate is added to the medium in order to maintain an optimal ph. BHI agar was prepared in our laboratory from a commercially available dehydrated base (Hi Media, Mumbai) according to the manufacturer's instructions. Immediately after autoclaving, the medium was allowed to cool in a 45 to 50 C water bath. The freshly prepared and cooled medium was poured into glass flat-bottomed petri dishes on a level, horizontal surface to give a uniform depth of approximately 4 mm. This corresponds to 25 to 30 ml for plates with a diameter of 100 mm. The agar medium was allowed to cool to room temperature and unless the plates were used the same day, they were stored in a refrigerator (2 to 8 C). Plates were used within seven days after preparation. A representative sample of each batch of plates was examined for sterility by incubating at 30 to 35 C for 48 hours. Preparation of anti-microbial agents 1) 0.2 % chlorhexidine digluconate solution was used as the positive control (soln A) 2) 50 mg of the dried ethanolic extract of Propolis was dissolved in 10 ml of sterile dimethyl sulphoxide (DMSO) and was used as the test antimicrobial agent. (soln B) 3) A mixture of 1:1 (v / v) of solution A & B (soln C) 4) Sterile dimethyl sulphoxide was used as the negative control (soln D) Preparation of filter paper discs Whatman filter paper No. 1 was used to prepare discs approximately 6 mm in diameter, which are placed in a petri dish and sterilized in a hot air oven at 160 0 C for 2 hours. Turbidity standard for inoculum preparation To standardize the inoculum density for the antimicrobial susceptibility test, a BaSO 4 turbidity standard, equivalent to a 0.5 McFarland standard was used. Procedure for Performing the Disc Diffusion Test Inoculum Preparation Growth Method The growth method was performed as follows 1. At least three to five well-isolated colonies of the same morphological type were selected from an Blood agar plate culture. The top of each colony was touched with a loop, and the growth was transferred into a tube containing 5 ml of BHI broth. 2. The broth culture was incubated at 37 0 C for 4 hours. 3. The turbidity of the actively growing broth culture was adjusted with sterile saline to obtain turbidity optically comparable to that of the 0.5 McFarland standards. This results in a suspension containing approximately 1.5 x 10 8 CFU/ml for strep mutans ATCC 25175 4. Within 15 minutes of adjusting the turbidity of the broth 50 µl of the broth was transferred to the middle of a dry BHI agar and spread uniformly over the entire agar surface using a sterile L spreader. 5. 4 sterile filter paper discs were placed at equal distances on the inoculated medium using sterile forceps. 6. The discs were impregnated with 1) 5µl of 0.2% chlorhexidine digluconate solution (soln A) - Positive control 2) 5µl of ethanol extract of Propolis dissolved in DMSO (soln B) 3) 5µl of combination of Propolis & Chlorhexidine (soln C) 4) 5µl of sterile DMSOsolution (Soln D) Negative control 7. The plates were incubated in a candle extinction jar for hours at 37 0 C. 8. After 18 hours of incubation the plates were observed for uniform lawn culture growth of the bacteria and the zone of inhibition of growth around the discs was measured. 9. The tests were performed in duplicate. 1 Figure 4: Zone of inhibition of growth of Streptococcus mutans against 1) 0.2% Chlorhexidine diacetate (disc 1) 2) 0.5% Ethanol extract of Propolis EEP (disc 2) 3) Mixture of Chlorhexidine & EEP (disc 3) 3 2
RESULTS All the strains of Streptococcus mutans isolated from the patients (n = 50) were inhibited by the ethanolic extract of Propolis, (Figure 1) the mean zone size of inhibition of growth being 26.0 mm. (Graph 1) All the strains were also inhibited by Chlorhexidine, the mean zone size being 22.6 mm. The highest antimicrobial activity was observed in the combination of Propolis & Chlorhexidine which inhibited the growth of Strep mutans, with the mean zone size of inhibiton being 32.22 mm. DMSO did not inhibit the growth of any strain of Streptococci. The actual zone sizes of inhibition of growth Strep mutans against all tha reagents used is presented in Table 1. Chi square for trend was calculated to see the trend in the anti-microbial activity of the different antimicrobials used in the study and found significant (P < 0.001). Graph 1 Synergistic activity of Propolis & Chlorhexidine Zone size of Inhibition (mm) 35 30 25 20 15 10 5 0 32.22 26 22.56 1 2 3 Anti-microbial agents Mean zone size of Inhibition LEGEND: 1. Mean zone size of inhibition (mm) of growth for 0.2% chlorhexidine (Solution A) 2. Mean zone size of inhibition (mm) of growth for 0.5% Propolis (Solution A) 3. Mean zone size of inhibition (mm) of growth for 1:1 Propolis & Chlorhexidine Table 1: Results of the antimicrobial susceptibility testing (Disc diffusion method) showing Size of the zone of inhibition of growth Clinical Strain 0.2 % Chlorhexidine (Soln A) 0.5 % Propolis (Soln B) Combination (Soln C) DMSO (Soln D) 1 26 mm 28 mm 36 mm 0 mm 2 23 mm 31mm 41 mm 0 mm 3 22 mm 25 mm 34 mm 0 mm 4 21 mm 25 mm 32 mm 0 mm 5 26 mm 28 mm 34mm 0 mm 6 24 mm 27 mm 34 mm 0 mm 7 22 mm 24 mm 28 mm 0 mm 8 25 mm 28 mm 33 mm 0 mm 9 21 mm 24 mm 31 mm 0 mm 10 22 mm 25 mm 31 mm 0 mm
Clinical Strain 0.2 % Chlorhexidine (Soln A) 0.5 % Propolis (Soln B) Combination (Soln C) DMSO (Soln D) 11 24 mm 26 mm 30 mm 0 mm 12 20 mm 22 mm 29 mm 0 mm 13 23 mm 25 mm 31 mm 0 mm 14 24 mm 26 mm 31 mm 0 mm 15 24 mm 29 mm 32 mm 0 mm 16 19 mm 22 mm 28 mm 0 mm 17 23 mm 27 mm 31 mm 0 mm 18 20 mm 23 mm 29 mm 0 mm 19 22 mm 27 mm 34 mm 0 mm 20 24 mm 26 mm 30 mm 0 mm 21 21 mm 26 mm 32 mm 0 mm 22 18 mm 23 mm 26 mm 0 mm 23 19 mm 24 mm 30 mm 0 mm 24 23 mm 24 mm 29 mm 0 mm 25 25 mm 29 mm 32 mm 0 mm 26 26 mm 28 mm 33 mm 0 mm 27 21 mm 24 mm 31 mm 0 mm 28 25 mm 28 mm 34 mm 0 mm 29 17 mm 19 mm 24 mm 0 mm 30 19 mm 23 mm 27 mm 0 mm 31 23 mm 28 mm 35 mm 0 mm 32 18 mm 21 mm 39 mm 0 mm 33 22 mm 24 mm 36 mm 0 mm 34 21 mm 22 mm 28 mm 0 mm 35 24 mm 27 mm 32 mm 0 mm 36 24 mm 28 mm 36 mm 0 mm 37 21 mm 25 mm 31 mm 0 mm 38 26 mm 28 mm 33 mm 0 mm 39 25 mm 28 mm 35 mm 0 mm 40 24 mm 27 mm 31 mm 0 mm 41 22 mm 27 mm 34 mm 0 mm 42 24 mm 28 mm 34 mm 0 mm 43 20 mm 23 mm 27 mm 0 mm 44 21 mm 24 mm 26 mm 0 mm 45 23 mm 27 mm 31 mm 0 mm 46 24 mm 31 mm 40 mm 0 mm 47 26 mm 34 mm 42 mm 0 mm 48 24 mm 29 mm 38mm 0 mm 49 23 mm 26 mm 34 mm 0 mm 50 24 mm 27 mm 32 mm 0 mm DISCUSSION Dental caries is one of the common oral infections and inflicts a huge burden on the population and economy of developing countries like India. Unlike other infections, dental caries continues throughout life, with new lesions in older people, an observation consistent with the prevalence of caries in adults where up to 95% have experienced one or more carious lesions.(10). Currently, fluoride in various preparations is the mainstay for caries prevention. Fluoride exerts its major effects by reducing enameldentine demineralization and enhancing remineralization of early caries lesions. Rated by degree of toxicity, fluorine would be placed somewhere between lead and arsenic (6). Research indicates that fluorides may cause allergies, osteoporosis, and dental fluorosis, as well as neurological damage (11). Nevertheless, as currently used, fluoride offers incomplete protection against dental caries. Previous studies have substantiated that Propolis is highly active against pathogenic bacteria. The minimal inhibitory concentrations (MICs) of EEPs for C. jejuni isolates have been determined by the agar dilution method with EEP showing MIC
values of 0.3125 0.156 mg/ml (12). Propolis being non toxic has enormous medicinal scope. In our study we have observed that bee propolis in combination with chlorhexidine possess high antimicrobial activity against Streptococcus mutans. Ethanol extract of Propolis has synergistic activity with chlorhexidine in inhibiting the growth of Strep mutans. Propolis in combination with chlorhexidine can suppress the pathogenic potential of dental plaque by inhibiting the adherence and accumulation of cariogenic streptococci on the tooth surface. The inhibition of growth of the clinical strains by trace quantities of propolis in our study suggests that it can be used in treatment of dental caries. Further, the concentration of chlorhexidine in mouthwashes can be reduced without lowering the anti-bacterial potential. ACKNOWLEDGEMENT The authors are grateful to Sri Krishnadevaraya Educational Trust for permitting to carry out this study through departmental grants. We are also thankful to Mr. Arun Kumar, Biostatiatician for providing statistical help. REFERENCES 1) Bianchi, G. In Waxes: Chemistry, Molecular Biology andfunctions; Hamilton, R. J., Ed.; The Oily Press; Dundee, 1995, p 175. 2) Burdock, G. A. 1998. Review of the biological properties and toxicity of propolis. Food Chem. Toxicol. 36:341-363. 3) Gibbons, R. J., and J. van Houte. 1975. Bacterial adherence in oral microbial ecology. Annu. Rev. Microbiol. 29:19 44. 4) Fernandes Junior A, Elaine Cristina etal. 2005 Propolis: anti-staphylococcus aureus activity and synergism with antimicrobial drugs. Mem inst Oswalde Cruz vol 100(5) 563 566 August 2005 5) Ikeno K, Ikeno T Miya etal Effect of Propolis on dental caries in rats Caries Res 1991;25(5):347-51 6) Jan Sallstrom, 1993 Fluoride in dentistry is a cell poison. The Art Bin lectures and essays 7) Krol W, Scheller S, Shani J, etal. 1993. Synergistic effect of ethanolic extract of propolis and antibiotics on the growth of Staphylococcus aureus Arzneimittel-forsch 43: 607-609. 8) Loesche, W. J. 1986. Role of Streptococcus mutans in human dental decay. Microbiol. Rev. 50:353-380. 9) Marsh, P. D. 1992. Microbiological aspects of the chemical control of plaque and gingivitis. J. Dent. Res. 71:1431 1438. 10) National Institutes of Health. (2001). Diagnosis and Management of Dental CariesThroughout Life. NIH Consensus Statement 18 (1), pp. 1 30. National Institutes of Health, Bethesda, MD, USA. 11) Phyllis Mullenix et al.1995: Neurotoxicity of sodium fluoride in rats. Neurotoxicol Teratol. 17(2), 12) Raffaella Campana, Vania Patrone, etal Antimicrobial activity of two Propolis samples against human Campylobacter jejuni Journal of Medicinal Food. 12(5): 1050-1056. (2009) 13) Rask, P.-I., C. G. Emilson, B. Krasse etal 1988: Effect of preventive measures in 50-60-year-olds with a high risk of dental caries. Scand. J. Dent. Res. 96:500-504. 14) Tenovuo, J., P. Hfikkinen, P. Paunio etal 1992: Effects of chlorhexidine-fluoride gel treatments in mothers on the establishment of mutans streptococci in primary teeth and the development of dental caries in children. Caries Res. 26:275-280. 15) Zickert, I., C. G. Emilson, and B. Krasse. 1982. Effect of caries preventive measures in children highly infected with the bacterium Streptococcus mutans. Arch. Oral Biol. 27:861-868.