Screening And Identification Of Different Cariogenic Character Strains Of Streptococcus Mutans

The mutans streptococci is the major cariogenic bacteria of human beings and animals. However, the MS detected from human oral cavity is streptococcus mutans primarily. The identification of MS is the basis of the research of caries. Starting from the isolation and purification of streptococcus mutans with the method of morphology, biochemistry and Molecular-biology, etc, and this research has conducted in vitro cariogenicity experiment to the acquired isolates, The different causing caries strains are selected to provide experimental material for research on the cariogenic characteristics of caries disease and clinical treatment.Part One Isolation and Identification of Streptococcus Mutans in Different Caries Susceptible PersonsObjective:To isolate and identify the streptococcus mutans from carious free and carious active persons, and genotyping of bacterial strains is made to provide experimental bacterial strains for further research. Methods:40 subjects were divided into two groups, while there were 14 persons in caries free group (DMFT=0) and 26 persons in caries- active group (DMFT≥6). Dental plaque, which was collected from the subjects'oral cavity, was conducted isolation of bacteria, primary culture and sub-purification culture. The purified streptococcus mutans were identified by morphology, micro biochemical tube, API 20 Strep and VITEK full automatical biochemical analyzer. Besides, as for the intrinsic genes of streptococcus mutans, such as surface protein antigenⅠ/Ⅱ(SpaP) gene, GtfB gene and DexA gene, the 192bp,517bp and 1272bp gene segments were separately added according to PCR. Based on the electrophoresis results, the accurateness of streptococcus mutans isolates was further detected. Genotyping of clinical isolated strains of 46 streptococcus mutans is made adopting way of AP-PCR. Results:40 subjects participated in the experiment, and from 32 subjects'bacterial plaque in oral cavity,46 clinical isolates of streptococcus mutan were separated and identified. The microscopic examination of bacteria morphology could observe its Obvious characteristics. The identification results of bacterial micro tube biochemical reactions, API 20 Strep and Vitek biochemical analyzer were correct. When identifyingthe genes of 46 isolates of streptococcus mutan, the electro phoreto grams of intrinsic sequences which were respectively corresponding to SpaP gene, GtfB gene and DexA gene. AP-PCR genotyping was done to 46 isolates, and then 41 isolates of different genotypes were acquired. Conclusion:Genotyping is high efficient and accurate, and AP-PCR could obtain streptococcus mutans in different genotypes, so as to provide suitable isolates for further research.Part Two Detection of in-vitro cariogenicity ability of streptococcus mutans clinical isolatesObjective:Through in-vitro experiments of adherence, acidogenicity, acidodurance and detecting their ability to synthesize glucans on the clinical isolated strains of streptococcus mutans, which isolated and identified previously, its purpose is to obtain high and low cariogenic character strains of streptococcus mutans, the relationship with strains and different caries susceptible persons was explained initially. It provides train of thought for further research on pathogenesis and characteristics of causing caries. Methods:The experiment of adherence capability of sucrose, acidogenicity, capability and their ability to synthesize glucans were made in clinical isolated strains of streptococcus mutans of 41 bacteria strains with different genotype. The detailed experiment methods can be seen in the flow chart. The datas of all bacterial strains in four in-vitro cariogenicity experiment were measured. Consequently, the result of each kind of experimental data was ranked in accordance with order of from the larges to the smalls. The larger values in all four aspects were considered as high cariogenic character strains. Otherwise, it was considered as low cariogenic character strains. Thus, suitable bacterial strains were screened. Combining ssDNA aptamer H19 gained from the previous experiment of clinical isolated strains of strepto- coccus mutans with sensitivity of different caries, the Combining intensity status of aptamer H19 and bacterial strains screened by the approach of flow cytometric analysis was texted. Consequently, accuracy of high and low cariogenic character strains screened was verified. Results:All four experimental cariogenicity data of 41 clinical isolates of streptococcus mutans were listed (the datas were arranged from high to low). Based on the comprehensive analysis of the cariogenicity experimental data of 41 clinical isolates of streptococcus mutans, having stronger capacity in four experiment detection were high cariogenic character strains (12-1,16-2,27-1 isolated strains) otherwise, they were low cariogenic character strains (5-1,8-1,9-1 isolated strains). The results of 41 streptococcus mutan isolates displayed that they all possessed certain in vitro cariogenic ability. In this experiment, free-cariogenic isolates did not detected. The results of flow cytometry show that after combining with ssDNA aptamer H19, high cariogenic character strains fluorescence intensity increases more significantly than low/free strains, The right shift of the graph was comparatively clear. Conclusion:the results of flow cytometry correspond with the combination features of ssDNA aptamer and high, low cariogenic character strains, which verifies the accurateness of the screening strains. The high cariogenic character strains obtained by screening, were with strong comprehensive cariogenic ability, while low/free strains were with weak comprehensive cariogenic ability. There is an uncertainty between some isolates' in-vitro cariogenic ability and patients'cariogenicity, which is worth further research.