Effect Of Domestic Water-soluble Propolis On The Cariogenicity Of The Dominant Cariogenic Streptococcus Together With Their Fluoride-resistant Strains

Background:As a chronic untransmissible disease, dental caries is one of the most common and frequently occurring disease, which greatly jeopardizing to human health. Dental caries has been defined as a disease of chronic and progressive destruction on the dental hard tissue initiated by multiple factors, especially the bacteria. Bacteria is the dominant factor during caries process, in which Streptococcus mutans (S.mutans) Serotype c and S. sobrinus (S.sobrinus) Serotype g are closely related to dental caries in human beings and characterized as the dominant cariogenic Streptococcus. As one of the major virulence factors of Streptococcus mutans group,Glucosyltransferase (GTF) plays an important role in the adhesion of dental bacteria and the formation of dental plaque, which acts as a prerequisite and substance basis of the cariogenicity of S. mutans.Several aspects, such as adhesion,accumulation and proliferation of Streptococcus mutans group, have been involved in the current researches caries prevention and cure. Furthermore, researches on inhibition of the proliferation of bacteria are focused mainly on the effect of antibacterial agents. There were many domestic and foreign studies on the anti-cariies medicine. Although the anticaries effect of fluoride was proved, the difficulty for anticaries increased with the generally application of fluoride which may produce fluoride-resistant strains.Therefore, a new effective and safe anti-cariies medicine need to be found ,and this has become the focused research.Propolis is a kind of sticky and aromatic solid colloidal substance which has strong bacteriostasis effect on oral pathogens. Although considerable work has been done on the propolis, few study about the effect of domestic water-soluble propolis on the growth of the dominant cariogenic Streptococcus mutans together with their fluoride-resistant strains and on the enzyme activity of GTF has been reported. Moreover, a great variation on the composition and antimicrobial activity is demonstrated among the propolis of different sources and manufacturers, or extracted by different techniques.Due to the important role of GTF plays on the adhesion of dental bacteria, the domestic water-soluble propolis was chosed in the present study to evaluate the effect of which on the growth inhibition of Streptococcus mutans, Streptococcus sobrinus fluoride-resistant strains and their parental strains. the effect of the domestic propolis on the enzyme activity of GTF was also assessed. Objective:To study the effect of the domestic propolis on the growth inhibition of Streptococcus mutans, Streptococcus sobrinus fluoride-resistant strains together with their parental strains, and on the enzyme activity of GTF, thus to further confirm the effectiveness of propolis as a natural medicine on cariostasis and to provided theoretical foundation for the development and utilization of domestic propolis.Material & Methods:1. S.mutans (S.m) and S. sobrinus(S.s) were induced with sodium fluoride by minimum inhibitory concentration(MIC) method to induce fluoride-resistant strains(S.m-FR,S.s-FR)Freeze-dried bacteria S.mutans (S.m) and S. sobrinus (S.s) were inoculated into BHI blood agar medium, 37℃micro-aerobic activated cultured 48h (900ml/L N2, 100ml/L CO2). After Gram staining and biochemical identification, Fluoride sensitivity of the pure culture was determined by dilution test. Single colonies of S.m and S.s were inoculated into TSA at 37℃micro-aerobic activated cultured 48h, bacteria lawn was scraped and coated on the TSA containing NaF with fluoride contents were 600, 650, 700, 750, 800, 850, 900, 950, 1000mg/L(S.m) and 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000mg/L(S.s) respectively for gradually inducing fluoride-resistant strains. After serial passage 20 times on the TSA agar without NaF, the culture was inoculated on TSA medium with 1000 mg/L NaF at 37℃, resulting in S.m and S.s fluoride-resistant strains (S.m-FR, S.s-FR). Cryopreserve the strains after microscope examination of Gram-stained smear and biochemical identification.2. Experiment of propolis to inhibit growth of Streptococcus mutans, Streptococcus sobrinus and their fluoride-resistant strains.Filtered sterilized 100g/L propolis solution was made into 25g/L TSA propolis liquid, nine different concentrations of propolis solution were formulated by means of liquid doubling dilution with the maximum concentration of 25g/L and the lowest concentration of 0.10g/L, each of them was split 1ml into test-tubes. 0.05 ml bacteria suspension of 1.5×106 CFU/ml were adding into 1 ml TSA containing different propolis concentrations respectively, 37℃micro-aerobic cultured 48h (900ml/L N2, 100 ml/L CO2) to observe MIC results. Test tube clarity after shaking shown that no bacterial growth, the lowest drug concentration in the clarity tube was the minimum inhibitory concentration of propolis.(MIC). The smallest drug concentration corresponding that no colony growth on the plate was the minimum bactericidal concentration (MBC). Determination of each strain were repeated three times, taking MIC, MBC mean. The experiment was repeated three times and positive control group and negative control group were set up every time.3.Experiment of propolis to detect the influence on the GTF of Streptococcus mutans, Streptococcus sobrinus and their fluoride-resistant strains.Freeze-dried S.m, S.m-FR, S.s, S.s-FR bacteria strains activated 48h were identified, four kinds of bacteria suspension of 1.5×108 CFU/ml were then prepared respectively using Maxwell turbidity. 5ml each suspensions was incubated in 50ml TSB containing 10g/L saccharose, 37℃micro-aerobic cultured 18h (900ml / L N2, 100 ml / L CO2). Centrifuged the above bacterial culture (3 300×g, 20min, 4℃) to collect the supernatant, then added solid ammonium sulfate up to 60% saturation, stored at 4℃overnight. Precipitation collected after centrifuged (10 000×g, 30min, 4℃) were dissolved with 0.01 mol/ L phosphate buffer (pH 6.4) containing 0.01% NaNO3 and then were dialysed with the same buffer for 48h, with medium change after every 12 hours. At last the precipitation were concentrated by polyethylene glycol (relative molecular mass 100 000) to 5ml to get crude GTF enzymes. After incubation of 0.05ml crude enzyme solution, 0.1ml 0.5mol/L phosphate buffer (pH 6.0), 0.1ml 0.5mol/L sucrose, 0.4ml double distilled water, and 0.05ml propolis solution with different concentrations at 37℃for 1h , 0.1ml 0.24mol / L HCL was added to terminate reaction, the solution mixing with 2.5ml double distilled water was then centrifuged 2 000×g for 5min,while reducing sugar content in the supernatant was measured by Neson-Somogyi method. As empty tube zero, optical density was read at a wavelength of 620nm [D (620)]. The experiment was divided into 7 groups, including five experimental group with different concentrations and two control groups. There were six parallel tubes in each group, each tube was measured three times and the results whichever is mean. The negative control group and positive control group were established.Results:1.The fluoride-resistant strains still grew in the medium of the TSA containing 1000 mg/L NaF after continuous passage of TSA without NaF on 20 times, with a slightly enlarged morphology compared with parental strains under a microscope. The main biological features of Sm, Sm-FR were: mannitol(+), sorbitol(+), raffinose(+), Melibiose(+), arginine(-),while those of Ss, Ss-FR were mannitol(+), sorbitol(+), raffinose(-), Melibiose(-), arginine(-). However, the appearance time of positive reactions of fluoride-resistant strains was later than those of the parent strains.2. MIC of propolis for S.m,S.m-FR,S.s,S.s-FR were respectively 0.39, 0.78, 0.20, 0.39g/L . MBC of propolis for them were respectively 0.78, 1.56, 1.56, 1.56g/L.3.. 6.25,3.13,1.56,0.78,0.39 g/L propolis can inhibited the GTF of S.m, S.m-FR, S.s, S.s-FR. There were significant statistically difference between the concentration of the propolis group and the positive control group (P < 0.05).There were statistically difference between 6.25,3.13,1.56 g/L groups and 0.78,0.39 g/L group (P <0.05). Conclusion:1. At the concentration of 0.78g/L of propolis, an entire growth inhibition of Streptococcus mutans, Streptococcus sobrinus and their fluoride-resistant strains was demonstrated . At the concentration of 1.56g/L of propolis, a complete sterilization Streptococcus mutans, Streptococcus sobrinus and their fluoride-resistant strains was shown.2.Propolis is able to inhibit or kill fluoride-resistant strains, which suggests that the demestic water-soluble propolis can inhibit or kill S. mutans and S. sobrinus and their fluoride-resistant strains.3. Propolis can inhibit the activity of GTF. The GTF of S.m, S.m-FR, S.s, S.s-FR decreased gradually with the increasing concentration of propolis, however the effect was no longer increased when propolis'concentration was up to 1.56g /L, which provided some reference for future clinical trials.4. There are certain research values on application of propolis in the treatment of dental caries.