高龋者口腔中c血清型变形链球菌的基因组指纹分析

目的：确定高龋者口腔中c血清型变形链球菌的基因型分布。方法：从20例高龋者口腔中分离出c血清型变形链球菌，采用chelex法提取细菌染色体DNA，通过AP—PCR进行临床分离株的基因组指纹分析。结果：共分离获得了87株c血清型变形链球菌，不同个体所携带的变形链球菌临床分离株的基因型不同，同一个体可携带不同基因型的c血清型变形链球菌，26．7％的高龋者携带1种基因型，60．0％携带2种基因型，13．3％携带3种基因型。结论：大多数高龋者口腔中定植有2种或2种以上基因型c血清型变形链球菌。     

老年人高龋患者牙菌斑中变形链球菌临床分离株耐酸能力的研究

目的:分析老年人高龋患者牙菌斑中,变形链球菌临床分离株(血清型C)在酸性条件下生存的耐酸能力。方法:体外培养从老年人高龋患者、无龋者牙菌斑中分离的101种不同基因型变形链球菌株临床分离株(血清型C),检测菌株在不同的pH条件下的耐酸能力。结果:老年人高龋患者口腔中分离出的变形链球菌临床分离株耐酸能力明显高于无龋组,与国际参考菌株无明显的统计学差异。同时研究发现,在老年人高龋患者同一个体的口腔中,变形链球菌临床分离株耐酸能力存在明显差异。结论:老年人高龋患者牙菌斑中变形链球菌临床分离株(血清型C)的高致龋性,与其携带有耐酸能力强的菌株关系密切。     

老年人高龋患者牙菌斑变形链球菌临床分离株产酸能力的研究

目的：探讨老年人不同龋敏感者口腔中变形链球菌（血清C型）的临床分离株产酸能力的差异。方法：收集老年人高龋患者、无龋者牙菌斑变形链球菌临床分离株（血清C型），比较细菌产酸能力，并与国际参考菌株比较。结果：老年人高龋患者口腔中分离出的变形链球菌临床分离株产酸能力明显高于无龋者口腔中分离出的变形链球菌临床分离株，与国际参考菌株却无明显地统计学差异。同时我们研究也发现，在老年人高龋患者同一个体的口腔中变形链球菌的临床分离株产酸能力存在明显差异。结论：老年人高龋者口腔中变形链球菌（血清C型）临床分离株的高致龋性与其携带有产酸能力强的菌株有关。     

变形链球菌体外耐酸能力的实验研究

目的 :探讨来自不同龋敏感者变形链球菌 (血清型c)临床分离株耐酸能力的差异。方法 :配制相同浓度的各变形链球菌临床分离株菌悬液 ,分别在不同pH浓度 (pH 4 .0～ 7.0 )的TPY液体培养基中培养相同时间后 ,用紫外分光光度计测定吸光度 ,比较细菌的生长情况。结果 :同一个体所带不同基因型变形链球菌菌株耐酸能力具有差异 ;高龋组个体定植的耐酸能力强的菌株所占比例显著高于无龋组 (P <0 .0 5 )。结论 :高龋组变形链球菌 (血清型c)临床株的高致龋力与其耐酸能力强密切相关。     

高龋及无龋者变形链球菌临床分离株致龋性研究Ⅰ对唾液包被羟磷灰石的粘附实验

目的：探讨来自不同龋敏感者的变形链球菌（血清型ｃ）临床分离株的粘附能力。方法：采用液体闪烁计测法测量＾３Ｈ－ＴＤＲ标记的变形链球菌临床分离株对唾液包被羟磷灰石（ＳＨＡ）的粘附。结果：同一个体所带不同基因型菌株对ＳＨＡ粘附量差异较大;高龋组个体定植的粘附能力强的菌株所占比较显著高于无龋组。结论：高龋组变形链球菌（血清型ｃ）临床菌株的高致龋力与其携带有粘附能力强的菌株密切相关。     

c血清型变形链球菌抑龋相关基因文库的构建

目的: 构建c血清型变形链球菌抑龋相关基因的抑制消减文库,为变形链球菌抑龋基因的筛选奠定基础。方法:从一对c血清型变形链球菌高、低毒力株中提取基因组DNA,AluⅠ酶切,以低毒力株的DNA酶切产物为testerDNA,高毒力株的DNA酶切产物为driverDNA,testerDNA连接接头后与driverDNA进行抑制消减杂交,并检测连接效率与消减效率。将获得的消减PCR产物,即差异基因 /差异DNA片段,与pCR2.1载体连接,转化E.coliTOP10F′感受态细胞,进行蓝白筛选。结果:AluⅠ酶切高、低毒力株基因组DNA,产生的酶切产物位于 0.1~2kb之间。testerDNA与接头的连接效率>25%,抑制消减杂交后消减效率检测显示,同时存在于tester与driverDNA中的 23SrRNA基因在消减组中出现时间较未消减组晚 12个循环,将消减PCR产物克隆后,挑取96个转化子,构建抑龋相关基因的抑制消减文库。结论:利用抑制消减杂交技术进行c血清型变形链球菌高、低毒力株之间基因组DNA的比较,初次构建了抑龋相关基因的抑制消减文库。     

c血清型变形链球菌抑龋相关基因/DNA片段的高通量筛选

目的　筛选含抑龋相关基因 /DNA片段的阳性克隆 ,为探究变形链球菌高、低毒力株致龋能力的差异奠定基础。方法　将抑制消减杂交方法获得的低毒力株特异的消减PCR产物与T/A载体pCR2 .1连接 ,将连接产物转化E .coliTOP10F′感受态细胞 ,进行蓝白筛选。对 96个转化子进行ClonyPCR ,将PCR产物点样于同一尼龙膜上 ,分别与地高辛标记的变形链球菌高、低毒力株基因组DNA -AluI酶切产物杂交 ,高通量筛选阳性克隆。结果　随机挑取了 96个白色或白色中央有蓝色的菌落 ,经过ClonyPCR ,其中有 4个转化子的扩增产物为双带 ,其余均为 0 .2～ 2 .0kb之间的单一条带 ,由原转化平板上另外挑取 4个白色菌落替代 ,其中 1个未扩增出产物。经过杂交 ,以与低毒力株基因组有杂交信号 ,而与高毒力株基因组无杂交信号或信号明显弱的转化子为阳性克隆 ,筛选的阳性率为 5 0 %左右 ,阳性克隆中含有c血清型变形链球菌抑龋相关的基因 /片段。结论　对抑制消减杂交方法获得的变形链球菌低毒力株特异的消减PCR产物进行高通量筛选 ,获得了抑龋相关基因 /DNA片段     

高龋及无龋者变形链球菌临床分离株致龋性研究Ⅱ合成细胞外多糖能力的实验研究

目的：探讨来自不同龋敏感者的变形链球菌（血清型ｃ）临床分离株合成细胞外多糖的能力。方法：采用红外光谱分析对葡萄聚糖样本作定性分析,用蒽酮法分别定量测定水溶性和水不溶性的葡聚糖含量。结果：同一个体所带不同基因型变形链菌菌株合成细胞外多糖能力具有差异;高龋组个体定植的合成水溶性及水不溶性葡聚糖能力强的菌株所占比较显著高于无龋组。结论：高龋组变形链球菌（血清型ｃ）临床菌株的高致龋力与其携带有合成细胞外多糖能力强的菌株密切相关。     

分离自日本儿童的变异链球菌临床株诱发的实验龋

为探索用动物模型研究变形链球菌临床株致龋的可能性,作者将从日本儿童分离的变形链球菌用抗链霉素标记后,植入特殊的无病原的Sprague-Dawley大鼠口腔内,并判定其致龋活性。结果发现,所试验的7株变形链球菌均易植入口腔,并在牙齿上持续存在和增殖。在大鼠磨牙上接种从牙斑分离的血清型c、d、e、f的变形链球菌临床株,并给予致龋饮     

老年人高龋患者牙菌斑中变形链球菌临床分离株合成胞外多糖能力的研究

目的；探讨老年人高龋患者牙菌斑中变形链球菌（血清C型）临床分离株合成胞外多糖能力。方法：收集老年人高龋患者、无龋者牙菌斑中变形链球菌（血清C型），检测合成胞外多糖能力，并与国际参考菌株比较。结果：老年人高龋患者牙菌斑中分离出的变形链球菌临床分离株合成胞外多糖能力明显高于无龋组。同时研究发现，在老年人高龋患者同一个体牙菌斑中，不同基因型变形链球菌临床分离株合成胞外多糖的能力仔任差异，在无龋者牙菌斑中发现没有这一现象。结论：老年人高龋患者牙菌斑中变形链球菌（血清C型）临床分离株合成胞外多糖能力强的菌株与龋病的发生密切有关。     

Binding of streptococci of the "mutans" group to type 1 collagen associated with apatitic surfaces

The present study surveyed the ability of 21 strains of cariogenic streptococci of the "mutans" group to bind to human type 1 collagen adsorbed on hydroxyapatite (HA) surfaces. All strains of Streptococcus cricetus (serotype a) and Streptococcus rattus (serotype b) tested attached strongly to collagen-treated HA (C-HA). Streptococcus mutans strains of serotype c and Streptococcus sobrinus strains of serotype d or g attached poorly; or not at all, to C-HA. S. mutans strains OMZ-175 (serotype f) and B14 (serotype e) were exceptions which exhibited significant binding to C-HA. Binding of S. cricetus AHT and S. rattus LB-1 to C-HA occurred in a dose-dependent manner, and was specific since it was inhibited by the presence of soluble collagen, but not by egg albumin, or by human fibrinogen or fibronectin. Binding occurred maximally between pH 6 and 8 and was not affected by any of several sugars tested. Trypsin treatment the streptococci had little affect, but heating at 100 degrees C reduced their ability to bind to C-HA.     

Effect of inoculum size and frequency on the establishment of Streptococcus mutans in the oral cavities of experimental animals

SPF Sprague-Dawley rats and ICR mice were inoculated with either Streptococcus mutans MT8148R (serotype c) or 6715 (g), and the influence of inoculum size, inoculum frequency, and sucrose on the establishment of S. mutans in the oral cavity was examined. Successful colonization of S. mutans in the experimental animals was absolutely dependent on the number of the cells introduced orally. Furthermore, inoculum frequency and sucrose seemed to act as secondary factors to modify the establishment of S. mutans, and it is suggested that high inoculum frequency may decrease the inoculum size necessary for the colonization of S. mutans in the oral cavity.     

The influence of saliva on infection of the human mouth by mutans streptococci

The relationship between oral implantation of Streptococcus mutans IB1600 (serotype c) and Streptococcus sobrinus OMZ65 (serotype g), the aggregating activity of saliva, and its influence on the adherence of these bacterial strains in vitro was examined in seven human subjects. All the saliva samples aggregated strain IB1600 but not strain OMZ65 cells. Whole saliva from subjects with low levels of infection by Strep. mutans aggregated strain IB1600 to a greater degree than did whole saliva from those who were readily infected. Whole saliva from subjects most resistant to infection supported the adsorption of the highest number of either strain IB1600 or OMZ65 to hydroxyapatite surfaces. Thus the ability of whole saliva to aggregate Strep. mutans may influence the ability of these microorganisms to infect the mouth.     

Detection of serotype k Streptococcus mutans in Thai subjects

Introduction:  Streptococcus mutans , known to be a pathogen of dental caries as well as bacteremia and infective endocarditis, is classified into four serotypes, c , e , f and k , based on the structures of serotype-specific polysaccharides. Serotype k was recently designated using blood isolates from Japanese subjects and such strains are considered to be virulent in the bloodstream. The purpose of the present study was to analyse the serotype distribution of strains isolated from Thai subjects and determine whether serotype k strains were present.
Methods:  A total of 250 S. mutans strains were isolated from 50 Thai subjects, and serotypes of all strains were determined. Then, molecular and biological analyses were carried out for serotype k strains.
Results:  Immunodiffusion and polymerase chain reaction analyses showed that serotype c was the most prevalent (70%), followed by serotypes e (22.8%), f (4.4%) and k (2.8%), which indicated that serotype k S. mutans strains occurred in Thai individuals at a similar rate to that previously reported for Japanese and Finnish populations. Molecular analyses of the seven serotype k strains showed extremely low expression of rgpE , which is related to glucose side-chain formation in serotype-specific rhamnose-glucose polymers, similar to previous reports for those other populations. In addition, analysis of the biological properties of the seven serotype k strains demonstrated low levels of sucrose-dependent adhesion, cellular hydrophobicity, dextran-binding activity and phagocytosis susceptibility by human polymorphonuclear leukocytes, which are characteristics similar to those of serotype k strains previously isolated in Japan.
Conclusion:  Our results indicate the possibility of a worldwide prevalence of serotype k strains with properties in common with those of previously reported strains.     

Role of glucose side chains with serotype-specific polysaccharide in the cariogenicity of Streptococcus mutans

Previously, we isolated and characterized a new Streptococcus mutans strain (serotype k) from human blood and oral cavity samples, and found that the serological properties of serotype k strains were similar to those of a gluA-inactivated mutant strain of MT8148 (MT8148GD). MT8148GD showed significantly lower sucrose-dependent adhesion to glass surfaces, sucrose-independent adhesion to saliva-coated hydroxyapatite, dextran-binding activity, and cell-associated glucosyltransferase (GTF) activity than the parent strain. Further, Western blot analysis revealed reduced GTFB and GTFC expression in serotype k strains as compared to MT8148, though the caries-inducing activities of MT8148GD and a serotype k oral isolate in rats were similar to that of MT8148. We conclude that a glucose side-chain defect in the serotype-specific polysaccharide of S. mutans may be associated with its cariogenicity, though to a lesser extent than its other major surface proteins.