c血清型变形链球菌致龋相关基因/DNA片段的批量克隆与高通量筛选

目的筛选含致龋相关基因／DNA片段的阳性克隆，为探究变形链球茵致龋的分子机制奠定基础。方法将抑制消减杂交方法获得的高毒力株特异的消减PCR产物与T／A栽体pCR2．1连接，将连接产物转化E．coli TOP0F’感受态细胞，进行蓝白筛选。对96个转化子进行Colony PCR，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茵高、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茵落，经过Colony PCR，其中有1个转化子的扩增产物为双带，其余均为0．2～2kb之间的单一条带。经过杂交，以与高毒力株基因组有杂交信号，而与低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50％左右，阳性克隆中含有c血清型变形链球茵致龋相关的基因／片段。结论对抑制消减杂交方法获得的变形链球茵高毒力株特异的消减PCR产物进行批量克隆和高通量筛选，获得了致龋相关的基因／DNA片段。     

链球菌gordonii FSS2 N-乙酰氨基葡糖苷酶基因的克隆和表达

目的 :N -乙酰氨基葡糖苷酶 (N -acetylglucosaminidase)是链球菌gordonii六种胞外糖苷酶之一。为研究该酶 ,克隆和表达该基因片段。方法 :将链球菌gordoniiFSS2染色体DNA分离提纯 ,用限制性内切酶Sau 3AI不完全酶切 ,产生长度不等的随机片段 ,琼脂糖凝胶电泳分离和回收 2 - 8kpb的随机片段 ,然后与BamHI线性化及 5’端脱磷处理的载体pUC 18连接 ,转化大肠杆菌XL1-Blue;β—半乳糖苷酶筛选系统及酶切 ,检测插入片段。根据N -乙酰氨基葡糖苷酶与特异人工底物X -GlucNac反应 ,有色物质X被释放原理 ,筛选表达克隆。结果 :共有 6×10 6个转化子 ,约 5 0 %转化子含有插入片段 ;检测到 8个N—乙酰氨基葡糖苷酶表达克隆。结论 :所构建的基因组文库具有完整性 ,该基因在大肠杆菌XL1-Blue中成功表达。     

Streptococcus mutans binding to solid phase dextran mediated by the glucan‐binding protein C

Streptococcus mutans GbpC is a wall‐anchored surface protein which is involved in dextran‐dependent aggregation. The GbpC phenotype is observed only in cells grown under stress conditions. In order to detect the GbpC protein of S. mutans, we isolated the wall fraction following digestion of the cell wall of this organism by N‐acetylmuramidase, and detected the GbpC protein from S. mutans cells by western analysis with anti‐GbpC serum. Interestingly, S. mutans cells exhibiting the negative dextran(α‐1,6 glucan)‐dependent aggregation (ddag) phenotype expressed the protein and could bind to immobilized dextran.     

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.     

Identification of mutans streptococci by restriction fragment length polymorphism analysis of polymerase chain reaction‐amplified 16S ribosomal RNA genes

Mutans streptococci are frequently isolated from dental plaque and carious lesions. These bacteria have been identified by conventional methods such as biochemical and serologic tests followed by the isolation of colonies on the mitis‐salivarius agar, which are sometimes inconsistent. Recently, species‐specific polymerase chain reaction (PCR) has been reported to rapidly identify Streptococcus mutans and Streptococcus sobrinus. However, in the case of identification and classification into several species, e.g. within the group of mutans streptococci consisting of seven species, the identification using species‐specific PCR seems somewhat inefficient because of need for the development and preparation of specific primers for each species. Therefore, in this study we developed a simple method using restriction fragment length polymorphism analysis of PCR‐amplified 16S ribosomal RNA genes (16S rRNA genes PCR‐RFLP) for the identification of seven different species included in the group of mutans streptococci. We amplified 16S rRNA gene sequences from genomic DNA samples by PCR using universal primers and digested the PCR products with the restriction endonucleases, HpaII and HaeIII. HpaII produced six RFLP patterns for eight reference strains, since the patterns for S. sobrinus, Streptococcus downei and Streptococcus ferus were similar. RFLP patterns produced with HaeIII could separate these three species. Furthermore, the RFLP patterns predicted from the 16S rRNA gene sequences in the GenBank database agreed with the actual RFLP patterns produced in the present study. The 16S rRNA sequence comparisons can be used to identify oral mutans streptococci; however, the identification by sequencing is sometimes difficult in large‐scale studies and for small laboratories. Therefore, 16S rRNA genes PCR‐RFLP, using HpaII and HaeIII, could be an alternative method for the identification of mutans streptococci, and may be applicable for large‐scale studies on the cariogenicity of mutans streptococci.     

儿童口腔中变形链球菌传播方式的研究

目的通过对婴儿院儿童及其母亲之间、全托儿童之间口腔中变形链球菌（简称变链菌）传播方式的研究,为儿童龋病预防提供新的思路.方法选取四川省实验婴儿院3～4岁的20名非全托儿童及其母亲和24名全托儿童作为研究对象,无菌牙签收集其菌斑样本,MSB培养基中培养48 h,每种表型菌落挑取一个代表株在TPY平板上次代纯培养,经形态学和微量生化鉴定后对分离的变链菌株行AP-PCR扩增,对不同个体出现相似基因型的变链菌株进行分析.结果44名儿童中,65.9%的儿童口腔内检出变链菌,20对母子中有10对均检出变链菌,44名儿童及20名母亲口腔中共分离出98株变链菌;均有变链菌检出的10对母子口腔中分辨出32个不同的基因型,其中7对母子有相似基因型出现;24名全托儿童口腔中共分辨出29个不同的基因型,有2种基因型在13名全托儿童口腔中有重复检出.结论变链菌在婴儿院儿童中存在水平传播及垂直传播两种传播方式.     

Efficacy of sodium hypochlorite and coconut soap used as disinfecting agents in the reduction of denture stomatitis, Streptococcus mutans and Candida albicans

This study evaluated the reduction of denture stomatitis and the antimicrobial activity of 0.05% sodium hypochlorite opposed to Candida albicans and Streptococcus mutans (SGM) when associated with brushing complete dentures with coconut soap. The mucosal characteristics were evaluated according to Newton's classification at baseline, after cleansing the dentures with coconut soap for 15 days in group 1 (nine patients). In the other group (19 patients) the analysis were made before and after cleansing the dentures with coconut soap and with disinfection in a soak solution of 0.05% sodium hypochlorite for 10 min during 15 days. Microbiological tests were used to isolate C. albicans and SGM. Mann-Whitney and Wilcoxon tests were used to compare the mucosal characteristics and Fisher test and McNemar test to compare C. albicans and SGM levels. Statistical analysis at the 95% confidence level (P < 0.05) showed that: (i) the association of coconut soap and 0.05% sodium hypochlorite significantly reduced clinical signs of denture stomatitis, (ii) C. albicans did not reduce in counts, (iii) SGM were reduced but not significantly and (iv) the association of coconut soap and 0.5% sodium hypochlorite was effective in controlling denture biofilm.     

Comparison of glucan-binding proteins in cariogenicity of Streptococcus mutans

Streptococcus mutans has been implicated as a primary causative agent of dental caries in humans. Bacterial components associated with the adhesion phase of S. mutans include cell-associated and cell-free glucosyltransferases (GTFs), as well as protein antigen c and proteins that bind glucan. At least four types of S. mutans glucan-binding protein (Gbp) have been identified; GbpA, GbpB, GbpC and GbpD. In the present study, GbpA-, GbpB- and GbpC-deficient mutants (AD1, BD1 and CD1, respectively) were constructed, and their cariogenic properties were evaluated by comparing them to those of their parent strain MT8148. All of the Gbp mutants showed lower levels of dextran binding, while the sucrose-dependent adhesion levels of AD1 and CD1 were lower than in the parental strain. The expression of each GTF was detected in the Gbp mutants, however, they had lower levels of cell-free-GTF activity than the parental strain. On the other hand, in acid tolerance assays, BD1 was the most sensitive among all of the tested strains. These results suggest that GbpA and GbpC in S. mutans have strong relationships with cariogenicity, while GbpB may have another biological function.     

Real-time quantitative polymerase chain reaction for enumeration of Streptococcus mutans from oral samples

This study compared SYBR Green real-time quantitative PCR (qPCR) with standard plate counting for the enumeration of Streptococcus mutans in oral samples. Oral samples (n = 710) were collected from high-caries-risk children for quantification of S. mutans by qPCR using primer pairs. The S. mutans copy number was calculated with reference to a qPCR quantification cycle (Cq) standard curve and compared with the absorbance value at 600 nm of a standard suspension of S. mutans UA159. The S. mutans copy number results were evaluated in relation to standard plate count (SPC) results obtained from each sample following culture on Petri plates containing S. mutans selective media and reported as colony-forming units (CFUs). The mean S. mutans copy number calculated from qPCR was higher than the SPC CFUs (1.3 × 10(6) and 1.5 × 10(5) CFUs, respectively). The qPCR values were usually higher in individual samples and qPCR detected the presence of S. mutans 84% (231/276) of the time that the SPC did not, compared with 33% (4/12) of the time when qPCR failed to detect S. mutans and the SPC did. The qPCR technique was found to be more sensitive for detection of S. mutans from oral samples, a method that is not dependent on the viability of the sample taken and therefore is proposed as a more reliable and efficient means of quantification of S. mutans.     

Role of gene E2f1 in susceptibility to bacterial adherence of oral streptococci to tooth surfaces in mice

Dental plaque is composed of a biofilm community of microorganisms on teeth that coats the oral cavity, including attaching to the teeth, and provides a protective reservoir for oral microbial pathogens, which are the primary cause of persistent and chronic infectious diseases. Oral streptococci are pioneering organisms that play an important role in biofilm formation on tooth surfaces as well as being primary causative agents of dental caries. The purpose of this study was to clarify the role of the E2f1 gene in susceptibility to dry mouth and bacterial adherence of oral streptococci to tooth surfaces in animal model experiments. A mutation of the E2f1 gene in mice is known to cause enhanced T-lymphocyte proliferation, leading to testicular atrophy, splenomegaly, salivary gland dysplasia, and other systemic and organ-specific autoimmunity. We found a decreased volume of saliva production and protein production rate, along with increased amylase activity, IgA concentration, and mucin 1 concentration in E2F-1(-/-) mice as compared with the control C57BL/6 mice. Further, we quantified the recolonization of oral streptococci in E2F-1(-/-) mice and found that a higher number of some oral streptococci were colonized on the teeth of these mice. In particular, following oral ingestion of 1% sucrose in water, the colonization of Streptococcus mutans increased in comparison with other streptococci. Our results suggest that the E2f1 gene may affect susceptibility for oral biofilm formation by streptococci in humans with dry mouth.