变形链球菌在日托儿童口腔中水平传播的初探

目的 通过对日托儿童口腔中变形链球菌（S.mutans）水平传播的研究，为儿童龋的早期预防提供新思路。方法 选择32名3～4岁日托儿童，根据是否患龋分为有龋组和无龋组。用无菌牙签采集牙面菌斑，MSB琼脂培养，微量生化反应鉴定分离S.mutans菌落，纯培养后提取基因组DNA，用AP-PCR检测其基因多态性，对来自不同个体的具有相似AP-PCR扩增图谱的菌株再进行DNA指纹分析。结果 32名受检儿童中，变形链球菌的检出率为78.1%；有龋组和无龋组的变形链球菌检出率分别为100%和69.6%。在检出变形链球菌的25名儿童中，携带有2种及以上基因型的儿童占88%；有龋组为88.9%，无龋组为87.5%，OR值为1.143，二者之间的差异无统计学意义。DNA指纹分析表明，有2种基因型变形链球菌分别在2名儿童口腔中重复检出。结论 变形链球菌在日托儿童中可能存在水平传播，但其基因型检出数目与龋的发生关系不明显。     

Salivary and serum antibody response to Streptococcus mutans antigens in humans

Humoral immunity against Streptococcus mutans infection was analyzed in caries-active and caries-free young adults by immunoblotting. All volunteers from both groups had detectable salivary immunoglobulin A (IgA) and serum IgG antibodies, with similar profiles. They could be classified on the basis of relative intensity of the immunoblot bands into categories of high or low responders. Common protein antigens with molecular weight ranging from approximately 45 to 190 kDa could be found either extracellularly or associated with the cell wall of S. mutans cultured in vitro. The predominant reactive antigens recognized by both IgA and IgG were of proteins around 63 and 60 kDa. Detection of IgA antibodies to the various antigens of S. mutans in individual saliva samples did not always correlate with serum IgG antibody profiles. In addition, distinct bands, which reacted preferentially with either IgA or IgG, could be detected by antibodies from specific subjects. Differential reactivities of salivary IgA and serum IgG antibodies to two, cell-wall associated protein antigens around 33 and 36 kDa were found in caries-active and caries-free young adults; 30.8% of caries-free subjects and 12% of caries-active subjects (P < 0.01) exhibited detectable antibody response to these antigens. This difference was not attributable to variations in antibody levels, since antibody response to these proteins were still detectable in some caries-free but not caries-active individuals whose levels of antibodies to other antigens were low. Thus, a new antibody profile which correlates with dental caries disease activity has been identified in a selected population. Differences in mucosal and systemic immune responses to S. mutans seem to be both antigen and host dependent.     

Effects of apigenin and tt‐farnesol on glucosyltransferase activity,biofilm viability and caries development in rats

Propolis, a resinous hive product secreted by Apis mellifera bees, has been shown to reduce the incidence of dental caries in rats. Several compounds, mainly polyphenolics, have been identified in propolis. Apigenin and tt‐farnesol demonstrated biological activity against mutans streptococci. We determined here their effects, alone or in combination, on glucosyltransferase activity, biofilm viability, and development of caries in rats. Sprague‐Dawley rats were infected with Streptococcus sobrinus 6715 and treated topically twice daily as follows: (1) tt‐farnesol, (2) apigenin, (3) vehicle control, (4) fluoride, (5) apigenin +tt‐farnesol, and (6) chlorhexidine. Apigenin (1.33 mM) inhibited the activity of glucosyltransferases in solution (90–95%) and on the surface of saliva‐coated hydroxyapatite beads (35–58%); it was devoid of antibacterial activity. tt‐Farnesol (1.33 mM) showed modest antibacterial activity against biofilms and its effects on glucosyltransferases were minimal. The incidence of smooth‐surface caries was significantly reduced by apigenin +tt‐farnesol (60%), fluoride (70%), and chlorhexidine (72%) treatments compared to control (P < 0.05).     

Isolation and characterization of Lactobacillus species inhibiting the formation of Streptococcus mutans biofilm

Oral bacterium S11 was isolated from the saliva of young children without dental caries and with little or no visible supragingival plaque. The S11 strain showed 99.5% similarity with Lactobacillus fermentum, and was identified on the basis of biochemical characteristics and a 16S rDNA sequence. S11 strain and its culture supernatant significantly inhibited the formation of the insoluble glucan produced by Streptococcus mutans Ingbritt. S11 did not affect the multiplication of S. mutans Ingbritt, but the adherence of S. mutans Ingbritt onto cuvette walls was inhibited completely.     

变形链球菌表面蛋白和葡糖基转移酶基因疫苗免疫防龋动物实验:体质量影响研究

目的 :了解变形链球菌基因疫苗单独及联合免疫对定菌鼠体质量的影响。方法 :2 8d龄Wistar大鼠 3 6只 ,随机分为pcDNA3 pac组、pcDNA3 gtfB组、pcDNA3 pac联合pcDNA3 gtfB组、变形链球菌灭活全菌阳性对照组、pcDNA3空载体阴性对照组和PBS液空白对照组 ,进行 3次双侧颌下腺腺周注射免疫 ,建立定菌鼠模型及诱龋实验 3个月 ,于实验开始和结束时测定体质量。结果 :基因疫苗免疫组体质量与 pcD NA3及PBS组无显著差异 (P >0 .0 5) ,而灭活全菌细胞免疫组体质量显著下降 (P <0 .0 1)。结论 :pcD NA3 gtfB和pcDNA3 pac对大鼠体质量无不良影响 ,较灭活全菌疫苗安全     

Effects of Nidus Vespae extract and chemical fractions on glucosyltransferases, adherence and biofilm formation of Streptococcus mutans

Nidus Vespae (the honeycomb of Polistes olivaceous, P. japonicus Saussure and Parapolybiavaria fabricius) have been extensively used in traditional Chinese medicine, given their multiple pharmacological activities, including antimicrobial, anti-inflammatory, anti-virus, anti-tumor and anesthetic properties. The present study evaluated the anti-glucosyltransferases (GTFs) activity, anti-adherence and anti-biofilm properties of 95% ethanol/water extract, cyclohexane/ethyl acetate, petroleum ether/ethyl acetate and chloroform/methanol fractions of Nidus Vespae. Chloroform/methanol fraction showed a remarkable capacity for inhibiting the adherence of Streptococcus mutans ATCC 25175 to saliva-coated hydroxyapatite disc (S-HA) at sub-MC concentrations. In addition, the Nidus Vespae extract and chemical fractions significantly inhibited the activity of cell-associated and extracellular GTFs at sub-MIC concentrations, and the chloroform/methanol fraction was the most effective one. For the anti-biofilm activity assays, minimum biofilm inhibition concentrations (MBIC50) and minimum biofilm reduction concentrations (MBRC50) were determined using the microdilution method. The chloroform/methanol fraction showed the highest anti-biofilm activities with a MBIC50 of 8mg/ml and a MBRC(50) of 16mg/ml against Streptococcus mutans ATCC 25175. The significant inhibition of GTFs activity and biofilm formation demonstrated by Nidus Vespae shows it to be a promising natural product for the prevention of dental caries.     

Initial biofilm formation of Streptococcus sobrinus on various orthodontics appliances

Biofilms accumulate on hard and soft surface in the oral cavity. Accumulation of biofilms on orthodontic appliance bear scientific and clinical interest. The objection of this study was to examine the formation of dental biofilm by Streptococcus sobrinus on different types of orthodontics appliances, using a model consisting of host and bacterial constituents. The adsorption pattern of saliva to the orthodontics appliances was determined by means of gel electrophoresis coupled with computerized densitometry techniques. The amount of salivary proteins adsorbed onto the surfaces was measured using the Bradford method. Sucrose-dependent bacterial adhesion to the saliva-coated orthodontics appliances was tested by radioactive-labelled S. sobrinus. Our results show different adsorption patterns of salivary proteins to the various orthodontic appliances as modules, brackets, springs and intra oral elastics. Modules and brackets demonstrated the most affinity to salivary proteins. A surface dependent adhesion profile was recorded, showing a high affinity of albumin and amylase to modules. Bacterial accumulation was the highest on modules compared with springs which demonstrated the least bacterial adhesion. Our study demonstrates the specificity of biofilm formation on the different orthodontic appliances. Formation of a variety of dental biofilms has a significant impact on the progression of dental diseases associated with orthodontic treatment.     

口腔链球菌丙酮酸氧化酶基因缺陷型变异株的构建

目的:利用口腔链球菌丙酮酸氧化酶基因(sopox) 的克隆序列,重组构建新的不能产生H2O2 的口腔链球菌变异株。方法:口腔链球菌株ATCC10557 经培养后用酚-氯仿法抽提细菌染色体基因组DNA ,经PCR 扩增sopox 基因,用BamHI 进行限制酶切;参照Chris 方法进行电转化,挑选阳性菌落测定其上清液中H2O2 的含量;将细菌传3～4 代后再次重复上述检测。结果:转化子经筛选后得到1 株阳性菌落,测定上清液中H2O2 含量,第1 次检测表明变异株产生H2O2 的量仅有所下降(介于阳性对照ATCC 10557 和阴性对照大肠杆菌JM109 之间) ,经3～4 次传代后变异株中上清液H2O2 量已经明显低于阴性对照。结论:成功构建了口腔链球菌丙酮酸氧化酶基因缺陷型变异株。     

变形链球菌防龋基因疫苗pcDNA3-gtfB免疫途径筛选的实验研究

目的:利用含有葡糖基转移酶抗原基因的重组质粒pcDNA3-gtfB作为基因疫苗,筛选有效的免疫途径。方法:重组质粒pcDNA3-gtfB通过股四头肌注射、鼻腔灌注和颌下腺周注射免疫Wistar大鼠,采用ELISA法测定血清 IgG、唾液IgA的动态变化。结果:经股四头肌注射免疫后产生的血清IgG抗体水平明显高于其它两组血清IgG抗体水平(P<0101),且鼻腔灌注组和颌下腺周注射组间的血清IgG抗体水平无显著性差异(P>0105)。各组唾液S-IgA 抗体水平均存在显著差异(P<0101),腺周注射组产生的唾液S-IgA抗体水平最高(P<0101)。结论:基因疫苗通过腺周注射免疫途径能最有效激发特异性唾液S-IgA抗体的产生,可望成为一种有效的防龋基因疫苗免疫途径,为下一步抗龋动物实验提供了实验基础。     

Short-term consumption of probiotic-containing cheese and its effect on dental caries risk factors

Cheese is known to contain compounds that reduce the risk of dental caries. The long-term consumption of milk containing Lactobacillus rhamnosus GG, ATCC 53103 (LGG), has been shown to reduce caries risk in children. The aim of the present study was to examine whether short-term consumption of cheese containing LGG and Lactobacillus rhamnosus LC 705 would diminish caries-associated salivary microbial counts in young adults. Altogether, 74 18-35 year-old subjects completed this double-blinded, randomised, placebo-controlled study. During the 3 week intervention, the subjects ate 5 x 15 g cheese per day. Oral examinations were made before and after the study. Stimulated salivary secretion rates, buffer capacity and counts of salivary Streptococcus mutans, yeast and lactobacilli were evaluated before and after the intervention and after a 3 week post-treatment period. The results showed no statistically significant difference between the groups in Streptococcus mutans counts after the intervention, but during the post-treatment period there was a significantly greater reduction in these counts in the intervention group compared to the control group (P=0.05). However, Streptococcus mutans counts decreased in 20% (P=0.01) and yeast counts in 27% (P=0.005) of all the subjects, regardless of the intervention group. Results from logistic regression showed a trend indicating that probiotic intervention might reduce the risk of the highest level of Streptococcus mutans (OR=0.37, 95% CI 0.08-1.75, P=0.21) and salivary yeasts (OR=0.40, 0.09-1.71, P=0.22).