In vitro inhibitory effects of Polygonum cuspidatum on bacterial viability and virulence factors of Streptococcus mutans and Streptococcus sobrinus

OBJECTIVES: Polygonum cuspidatum has been used in Korean folk medicine to improve oral hygiene. This study was performed to evaluate the effects of methanol extract from root of P. cuspidatum (MEP) on bacterial viability and the virulence factors of Streptococcus mutans and Streptococcus sobrinus. METHODS: To test the effects of MEP on bacterial viability, we determined the minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) against 20 bacterial strains, including S. mutans and S. sobrinus, using a micro-dilution assay. In case of S. mutans and S. sobrinus, the assays for time-kill and bacterial growth rate at sub-MIC concentrations were also performed. To determine effects of the extract on the virulence factors of S. mutans and S. sobrinus, the assays for sucrose-dependent adherence, water-insoluble glucan formation, glycolytic acid production, and acid tolerance were performed at sub-MIC levels. Phytochemical analysis for constituents of MEP was carried out. RESULTS: MEP showed a broad antibacterial range (MIC 0.5-4 mg/ml). The MBC was two to four times higher than the MIC. The time-kill curves showed S. mutans and S. sobrinus were significantly killed after 1h of incubation. At sub-MIC levels, doubling times of S. mutans and S. sobrinus dose-dependently increased up to 211% and 123%, respectively. At sub-MIC levels, MEP also showed inhibitory effects on the virulence factors of S. mutans and S. sobrinus in a dose-dependent fashion. Phytochemical analysis revealed the presence of alkaloids, sterol/terpenes, tannins, flavonoids, and carbohydrates. CONCLUSION: These data indicate that MEP has inhibitory effects on bacterial viability at higher concentrations (> or =MIC) and the virulence factors of S. mutans and S. sobrinus at sub-MIC concentrations, suggesting that it might be useful for the control of dental plaque formation and subsequent dental caries formation.     

Salivary Streptococcus mutans and caries prevalence in Brazilian schoolchildren

The aim of the present study was to determine the levels of salivary Streptococcus mutans in Brazilian schoolchildren, and its possible correlation with caries prevalence. A positive correlation between different levels of S. mutans and caries prevalence was observed, individuals with high levels of S. mutans (greater than 100 colonies) had a DMFS significantly higher than those with low or no detectable S. mutans (0, 1-20, 21-40 colonies).     

变形链球菌葡糖基转移酶遗传多态性与龋病发生关系的研究

目的:探讨变形链球菌葡糖基转移酶遗传多态性与龋病发生的关系。方法:菌株分别选自本实验室前期工作中分离鉴定的合成水不溶性多糖能力较强和较弱的血清c型变形链球菌临床分离株，提取全菌DNA，经PCR扩增gtfBC(961 - 5574 by)后，分别采用限制性内切酶Hinf工、Mb。工和Taq工进行限制性片段长度多态性分析。结果: 不同龋敏感人群变形链球菌血清。型临床分离株经Hinf工酶切后限制性片段长度多态性分析酶谱出现了差异，有 1.9 kb长片段的菌株在高龋组中的比例高于无龋组(P < 0.05)。结论:萝基因型的不同是导致菌株合成水不溶性多糖能力差异的原因之一。     

Streptococcus mutans establishment and dental caries experience in children from 2 to 4 years old

Abstract – 39 children were studied longitudinally at the age of 2, 3, and 4 yr for the colonization of S. mutans in plaque and saliva and for caries experience. S. mutans was found in 38% of the children, and the predominant serotype group was c/e/f. A total of 16 children got caries before the age of 4. Children who harbored S. mutans in their plaque at the age of 2, appeared to be the most caries-active individuals. Their caries index values (number of decayed, missed and filled surfaces, dmfs = 10.6±5.3) at the age of 4 differed significantly from the values of children who harbored S. mutans later (dmfs = 3.4±1.8,P<0.005) or remained free from S. mutans infection (dmfs = 0.3±1.1, P<0.0003). It was thus concluded that the early establishment of S. mutans in the plaque of primary incisors indicated early and extensive caries attack in young primary dentition.     

变异链球菌蛋白质组研究进展

变异链球菌是口腔龋病的重要的病原菌之一。随着实验新技术的不断涌现,对于变异链球菌的研究,也从形态学、生化鉴定、基因等的水平,上升到蛋白质组水平。变异链球菌的蛋白组随着生长环境、生长状态和菌珠的变化而变化。本文综述了近年来变异链球菌蛋白质组的研究状况。     

Prevalence of dental caries and Streptococcus mutans in Meo of Northern Thailand

The prevalence of dental caries was studied in 505 Meo, a minority of Northern Thailand, using the DMFT index. Plaque material from all patients was examined qualitatively for S. mutans. The DMFT (dmft) index score ranged between 1.69 and 7.06 with a mean of 2.17 +/- 3.24 for men and 3.45 +/- 3.72 for women. There was a statistical difference in caries prevalence between males and females (P less than 0.05). There was no statistical correlation between miang chewing and caries prevalence. Fluoride concentration of water samples was between 0.043 and 0.077 ppm F-. Of 505 plaque samples, 13.3% were positive for S. mutans, mostly of biotype I. Compared to other Northern Thai hill tribes and the Thai rural population, the Meo appear to have a higher dental caries prevalence rate.     

葡糖基转移酶多肽疫苗HDS免疫大鼠的实验研究

目的　动物体内研究葡糖基转移酶多肽疫苗HDS免疫原性和防龋效果。方法　构建大鼠人工龋模型,分别用人工抗原HDS-KLH,多肽抗原HDS,葡糖基转移酶(GTF)免疫大鼠,酶联免疫吸附法检测大鼠血清和唾液中抗HDS、GTF抗体;体视显微镜下观察各组大鼠磨牙龋坏情况,并用Keyes计分法计分。结果　HDS-KLH免疫组大鼠唾液、血清中抗HDS IgG和IgA水平均高于对照组(P<0·05);HDS-KLH,HDS,GTF免疫组大鼠龋Keyes计分均低于对照组(P<0·01),光滑面防龋效果最为显著。结论　人工抗原HDS-KLH能刺激机体产生针对GTF的保护性免疫,并减少实验大鼠龋齿发生。     

Streptococcus mutans and dental caries in urban and rural schoolchildren in Thailand

Two groups of Thai schoolchildren aged 11-13 yr were examined for dental caries, dental fluorosis and Streptococcus mutans. One hundred children lived in an urban district, Bangkok, and 71 children in a rural district, Petchaboon. Saliva samples were analyzed for S. mutans by the spatula method. The fluoride content of the drinking waters was also determined. The prevalence of caries, diagnosed according to WHO, was 89% in Bangkok and 18% in the rural district. Mean DMFT was 3.46 and 0.38 respectively. S. mutans was found in 98% of the urban children and in 82% of the rural. The differences in distribution between S. mutans and DMFT classes were statistically significant for the total sample. Mild fluorosis was present in some of the rural children.     

The earlier the colonization by mutans streptococci, the higher the caries prevalence at 4 years of age

Seventy-eight 4-year-old children, examined earlier for the presence of mutans streptococci at 4-month intervals from 15 months of age, were recalled for recording of dental caries and salivary sampling. The saliva samples were analysed for the presence of mutans streptococci, including the species Streptococcus mutans and Streptococcus sobrinus and lactobacilli. The results showed that the earlier the mutans streptococci had been detected in the children, the higher the caries experience. Eighty-nine percent of the children colonized at 2 years of age had experienced caries and had a mean dfs of 5.0 compared with 25% of the non-colonized 4-year-olds with a mean dfs of 0.3. S. mutans was the predominant species. S. sobrinus was usually found in combination with S. mutans , except in 2 children where S. sobrinus was the only species detected. More children with multiple species had higher numbers of total mutans streptococci and a tendency to higher caries prevalence than children with only S. mutans.     

变形链球菌葡糖基转移酶缺陷突变株粘附特征的观察

为了揭示变形链球菌葡糖基转移酶（ｇｌｕｃｏｓｙｌｔｒａｎｓｆｅｒａｓｅ，ＧＴａｓｅ）在变链菌粘附中的作用，通过扫描电镜观察了其缺陷突变株对玻片的粘附。结果显示，在蔗糖存在的条件下，变链菌ＭＴ８１４８及其ＧＴａｓｅ缺陷株表现出不同的粘附模式。提出了细胞吸附型ＧＴａｓｅ（ＣＡＧＴａｓｅ）与细菌间的聚集有密切关系的观点，认为这种聚集的紧密程度高于由细胞外ＧＴａｓｅ（ＣＦＧＴａｓｅ）所介导的聚集。而ＣＦＧＴａｓｅ所合成的葡聚糖可能与细菌间某种有序的粘接有关。本研究结果对深入认识变链菌的致龋机理有参考价值。     

不同浓度的葡萄糖对变形链球菌初始粘附能力的影响

目的 研究不同浓度的葡萄糖对变形链球菌初始粘附能力的影响,同时观察高龋组、无龋组变形链球菌初始粘附能力的差别。方法 选取高龋组、无龋组变形链球菌（血清 c型）临床分离株各10株及1株参考株UA159,用唾液包被的羟基磷灰石（SHA）模拟口腔中牙面情况。各菌株分别在含3H的0.2%、1.0%、5.0%葡萄糖的液体培养基中培养,配成菌悬液。菌液与SHA作用90 min,清洗、吸干,液体闪烁计数测量各样本中粘附于SHA的细菌的量。各变形链球菌菌株在不同浓度葡萄糖条件下对SHA的粘附能力以粘附量CPM值表示。结果 变形链球菌高龋组分离株初始粘附能力明显高于无龋组分离株（P<0.05）;同时变形链球菌在不同浓度的葡萄糖营养条件下其初始
粘附能力也有统计学上的差别（P<0.05）,5.0%葡萄糖组粘附力最强,0.2%葡萄糖组粘附力最低,1.0%葡萄糖组粘附力界于两者之间。结论 ①变形链球菌的初始粘附能力可能与龋病的发生有关;②葡萄糖可能与变形链球菌的初始粘附相关,在一定范围内,葡萄糖可能会促进变形链球菌的 初始粘附。     

Immune response in humans to a nasal boost with Streptococcus mutans antigens

We previously reported that a Streptococcus mutans enriched-glucosytransferase (E-GTF) preparation induces an immune response following intranasal, but not tonsillar, immunization of humans. In this study, we determined whether intranasal immunization of these subjects 2 years later resulted in augmented immune responses compared to those seen in control subjects. Subjects previously immunized via the intranasal (IN, n = 7) or tonsillar (IT, n = 7) route and control (n = 12) subjects were immunized via the intranasal route with E-GTF. Nasal wash, saliva, and serum were collected before immunization and then weekly for 3 months after immunization. Significant (P < 0.05) mucosal and serum immunoglobulin A (IgA) anti-E-GTF responses were observed in all three groups. Nasal and serum IgA anti-E-GTF responses were significantly higher (P < 0.05) in the IN group. The salivary responses in the three groups were, in general, similar. These results indicate that intranasal immunization primes the immune system for a localized secondary response to S. mutans antigens.     

High prevalence of mutans streptococci in a population with extremely low prevalence of dental caries

The quantitative distribution of the mutans streptococci was investigated in populations with very low caries prevalence. Three hundred and seven school children from rural Sudan with an estimated age of 12 years were examined for dental status and salivary mutans streptococci. Forty-two isolates from the salivary cultures were determined to species level by studying the serogroup and biotype. Denial caries in permanent teeth was diagnosed in 12% of the children and the mean DMFT was 0.17 (range 0-6 DMFT). Caries was confined to fissures. Mutans streptococci were found in 96% of the children and high counts were obtained in 45%. Of 42 isolated strains, 37 belonged to Streptococcus mutans , 5 to Streptococcus sobrinus and none to Streptococcus rattus. The observations show that the mutans streptococci can be widespread in human populations with extremely low prevalence of dental caries and consequently that these bacteria may be readily propagated in human populations without association to a caries-promoting life style.     

变形链球菌GbpA的GBD免疫防龋实验研究

目的：观察变形链球菌GbpA的GBD融合蛋白免疫SD大鼠的防龋效果。方法：用纯化的变形链球菌GbpA的GBD融合蛋白皮下免疫SD大鼠,喂Keyes改良的高糖Diet 2000,第1次免疫20d时,连续3d大鼠口腔中接种S．mutans Ingbritt,在接种S．mutans后第77天处死大鼠,收集大鼠颌骨标本,用于龋齿记分分析,用t检验进行统计学分析。结果：GBD融合蛋白免疫大鼠,实验组龋损范围及龋坏程度均显著低于对照组,P＜0．01。结论：变形链球菌GbpA的GBD融合蛋白疫苗可有效降低龋齿的发生。     

变异链球菌毒力相关转录调节因子的研究进展

研究发现众多转录调节因子参与调节变异链球菌耐酸性等关键致龋毒力因子的表达,虽然仍有许多转录调节因子的作用靶点和调控机制尚不明确,但已有学者针对变异链球菌个别宏观转录调控因子进行单基因突变研究。本文就已发现的影响变异链球菌致龋毒力因子表达的调节因子及其功能和可能的作用机制进行综述。     

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.     

The immunogenicity of various peptide antigens inducing cross-reacting antibodies to a cell surface protein antigen of Streptococcus mutans

A cell surface protein antigen (PAc) of Streptococcus mutans may be involved in the binding of bacteria to the tooth surface, and has long been focused upon as a candidate for a preventive vaccine of dental caries. Previously the peptide PAc (365-377) was shown to raise an antibody in B10.D2 mice which inhibited the binding of salivary components to the PAc molecule. Using this peptide as a unit peptide, two constructs based on multiple antigenic peptides, and several types of tandem repeats of two or three copies were synthesized to estimate the immunogenicity of these peptides. Increase in the immunogenicity was observed for all constructs with the use of an adjuvant compared to the unit peptide alone. However, the tandem repeat constructs generally induced antibody production in the absence of adjuvant, while the multiple antigenic peptide constructs did not induce antibody production under the same condition. Although such a phenomenon may be restricted to this particular peptide sequence, these results may influence the strategy for the design of peptide vaccines.     

树脂充填前后唾液中变异链球菌的变化

目的 观察使用树脂进行龋齿治疗前后唾液中变异链球菌数量的变化.方法 招募志愿者20名,对其口腔内所有龋齿去净龋坏组织后使用不含氟树脂材料进行充填,分别收集充填前及充填后第1、2、3、4周的非刺激性唾液样本,使用轻唾-杆菌肽琼脂培养基厌氧培养48 h后检测变异链球菌数量,比较其在龋齿充填治疗前后的变化.结果 龋齿行树脂材料充填后1周唾液中变异链球菌数量较充填前下降明显(P＜0.05),充填后2、3、4周较充填前无明显变化(P＞0.05).结论 对患龋个体进行龋齿备洞后的树脂充填能在短期内降低唾液中变异链球菌的含量,但随时间推移,唾液中变异链球菌数量会逐渐恢复到治疗前的状态.单纯依靠龋齿充填治疗并不能改变唾液中变异链球菌的数量.     

聚合酶链反应检测致龋性变形链球菌

本研究旨在应用聚合酶链反应检测致龋性变形链球菌。针对血清C型变形链球菌的spaP基因序列合成特异性引物,用聚合酶链反应(PCR)扩增spaP基因片段的方法检测变形链球菌各菌株和口腔多种常居菌。结果只有变形链球菌血清C型产生了扩增产物,呈现单一的192bpDNA条带。这种方法使过去不能检出的微量靶序列(3个cfu)得以检出,具有高度的特异性和敏感性。用本方法检测50例口腔菌斑标本,有44例呈阳性结果。提示该检测技术是一种快速、准确检测变形链球菌的新方法。