| Dental caries is a common disease endangering the health of human oral cavity, Streptococcus mutans (S.mutans) is an important aetiological agent in dental caries. Studies on S.mutans go deep into molecular level nowadays. Most of the early researches were focused on planktonic S.mutans cells in the culture medium, however, S.mutans is living in the form of typical biofilm structure of dental plaque biofilm in oral under natural conditions. After the tooth eruption, S.mutans begins to adhere and aggregate on its surface, eventually forming a complex biofilm structure to adapt to the fluctuant oral environment and lead to caries when the conditions are permit. As a result, studies of cariogenicity of biofilm cells of S.mutans are significant for ecological control, assessment of disease risk and clarification of the pathogenesis of dental caries. Furthermore, with the advent of the confocal laser scanning microscopy (CLSM) and the development of science and technology, it is possible to observe the biofilm in real-time, completely and dynamically recently.On the other hand, as the extensive, in-depth study of biological properties of cariogenic of S.mutans, researchers found that the number of S.mutans is not the only determining factor in the process of caries, but the different cariogenic ability of strains is important as well. It is clear that studying the difference of the cariogenic ability between S.mutans strains may improve the accuracy of caries risk prediction. To study the differences of the cariogenic ability of different S.mutans strains and theirs reasons, our issue team had successfully separated the S.mutans clinical strains 593 and 18 from caries-active and caries-free individuals, these two strains not only show the differences of cariogenicity in vivo, and their expression of functional protein which had been confirmed through strategies for proteome analysis, genotype through genotype analysis and the cariogenic ability which confirmed through a series of in-vitro experiments are different. Based on the results of preliminary studies, the Cariogenecity of the biofilm cells of standard strain of S.mutans ATCC 25175, clinical strains 593 and 18 are quantitative, located, and qualitative compared by the use of confocal laser scanning microscopy with fluorescent staining and scanning electron microscope. Four parts are included in this study:Part I: the differences of the ability of biofilm formation of S. mutansObjective: To know biofilm dynamic and continuous formation law and its differences of standard strain of S.mutans ATCC 25175, clinical strains 593 and 18 in vitro. Methods: S. mutans biofilms were formed on polystyrene plastic sheets in 0.5, 1, 1.5, 2, 4, 6, 12, 16, 18, 20 and 72 hours respectively. Biofilms were observed in real-time, intact by confocal laser scanning microscopy with proidium iodide and SYTO9 staining, and then the three-dimensional of biofilm pictures were obtained by the reconstruction of scanning data of biofilms, the depth was measured, the bacterial density and the percentage of vital bacteria were calculated. Results: It was shown that standard strain of S.mutans ATCC 25175, clinical strains 593 and 18 all can adhere to the surface of polystyrene plastic sheets and form dense biofilms, which were in three-dimensional spatial architecture with various forms, and were mainly composed of non-homogeneous distribution of dead and live bacteria, the density of bacteria and the percentages of vital bacteria were different at different layers and in different culture times of the same strain. Furthermore, biofilms of different strains varied with thickness, bacterial density and the percentages of viable cells. Conclusion: S.mutans biofilm is composed of a large number of micro-colonies, and had a certain degree thickness. Its structure was three-dimensioned, heterogeneous and of various forms. Strain 593 possessed much greater ability of biofilm formation than strain 18 and standard strain of S.mutans ATCC 25175, and there was no statistical difference between strain 18 and standard strain of S.mutans ATCC 25175.PartⅡ: the differences of adherence properties of biofilm S.mutansObjective: To know the process of biofilm formation of adherence and aggregation of standard strain of S.mutans ATCC 25175, clinical strains 593 and 18 in vitro, and the surface morphology of bilfilms and the differences between the three strains. Methods: S.mutans biofilms were formed on polystyrene plastic sheets in 2, 3, 4, 6, 12 and 20 hours respectively, and then the surface morphology of bilfilms and adherence of S.mutans were observed by scanning electron microscope at each time point. Results: There was a significant distinction of the ability of adherence and the surface morphology of bilfilms of three strains at each time point. Conclusion: It is indicated that strain 593 in biofilm possessed much stronger adherence properties than that of strain 18 and standard strain of S.mutans ATCC 25175 in biofilm, and there was no significant difference between strain 18 and standard strain of S.mutans ATCC 25175.PartⅢ: the differences of aciduricity of biofilm S.mutansObjective: To know the aciduricity of standard strain of S.mutans ATCC 25175, clinical strains 593 and 18 and the differences between the three strains. Methods: (1) 3, 20hours S.mutans biofilms which formed on polystyrene plastic sheets, were divided into pre-acidification and non-pre-acidification group randomly. The biofilms of pre-acidification group were immersed in TPY medium at pH 5.5 for 3 hours and then immersed in TPY medium at pH 3.0 for 30 minutes. While the biofilms of non-pre-acidification group were immersed in TPY medium at pH 3.0 for 30 minutes directly. Live and dead organisms in bioflms were differentiated by confocal laser scanning microscopy with proidium iodide and SYTO9 staining. (2) 20 hours S.mutans biofilms which formed on polystyrene plastic sheets, were divided into non-starved and starved group randomly, the biofilms of starved group were exposed to PBS for 24 hours and then immersed in TPY medium at pH 3.0 for 30 minutes, while the biofilms of non-starved group were incubated to 20ml 37℃TPY medium for 24 hours and immersed in TPY medium at pH 3.0 for 30 minutes, live and dead organisms in bioflm were differentiated by confocal laser scanning microscopy with proidium iodide and SYTO9 staining. Results: (1) It is suggested that pre-acidification increases viability both 3 and 20 hours biofilms. The percentages of vital bacteria of strain 593 were higher than two other strains significantly. (2) Whether pre-acidification or not, the decrease of survivors of 3 hours biofilms was more than 20 hours. There is a significantly statistical difference in decreasing ratio among standard strain of S.mutans ATCC 25175, clinical strains 593 and 18. The decreasing ratio of strain 593 was lower than that of strain 18 and standard strains of S.mutans ATCC 25175. (3) Starved biofilms cells showed more viable bacteria than non-starved biofilms cells, the percentages of vital bacteria of strain 593 was higher than two other strains significantly. Conclusion: S.mutans in pre-acidification biofilm show stronger aciduricity than S.mutans in non-pre-acidification biofilm; S.mutans in 20 hours biofilm show greater aciduricity than 3 hours biofilm; In addition, it is suggested that starvation protects biofilm S.mutans from acid shock. Strain 593 in biofilm displayed greater aciduricity, or ability to tolerate low pH than strain18 and standard strain of S.mutans ATCC 25175 in biofilm, and there was no statistical difference between strain 18 and standard strain of S.mutans ATCC 25175.PartⅣ: the differences of the ability to synthesize exopolysaccharides of biofilm S.mutansObjective: To know the changes of exopolysaccharides produced by biofilm cells of standard strain of S.mutans ATCC 25175, clinical strains 593 and 18 and the differences between the three strains. Methods: (1) S.mutans biofilms which were formed on polystyrene plastic sheets in 3, 12 and 20 hours respectively, exopolysaccharides distribution were visualized by fluorescent FITC-ConA stained with confocal laser scanning microscopy. (2) The amounts of exopolysaccharides by adhesive S.mutans in different culture times were determined by the anthrone method. Results: There was a significant distinction of the amounts and distribution of exopolysaccharides at different points of the same strain, furthermore, and varied considerably between the three strains. Conclusion: The ability to synthesize exopolysaccharides of biofilm S.mutans are different at different points during the formation of biofilm, strain 593 in biofilm possessed much greater ability to synthesize exopolysaccharides than strain 18 and standard strain of S.mutans ATCC 25175 in biofilm, and there was no statistical difference between strain 18 and standard strain of S.mutans ATCC 25175.In Conclusion, it was indicated that strain 593 in biofim possessed greater ability of biofilm formation, ability to synthesize exopolysaccharides, adherence properties and aciduricity than those of strain 18 and standard strain of S.mutans ATCC 25175 in biofilm. These differences may contribute to the high cariogenicity potential of strains 593.
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