Interactions between and within Streptococcus mutans and Streptococcus sobrinus isolated from humans harboring both species.

The prevalence of Streptococcus mutans and Streptococcus sobrinus was examined in plaque samples from small discrete areas of the buccal tooth surfaces of seven subjects. Strains of S. mutans and S. sobrinus were isolated and tested for bacteriocin-mediated interactions between and within the two species, using the stab inoculation technique. S. mutans and S. sobrinus did not colonize each tooth surface uniformly and, in plaque from small discrete sites, S. mutans and S. sobrinus were either undetected or present in different interspecies proportions. Within the same subject, there were no bacteriocin-mediated interactions between strains of the same mutans species and no difference in bacteriocin activity was found between the strains of S. mutans and S. sobrinus from different sites. When bacteriocin interactions were tested between isolated strains from all seven subjects a somewhat higher inhibition ability was found for producer strains isolated from plaque compared with those isolated from saliva. S. mutans appeared to be more bacteriocinogenic than S. sobrinus. Replacing the glucose in the medium with sucrose enhanced the bacteriocin activity of S. mutans towards other S. mutans strains but reduced the inhibitory interaction towards strains of S. sobrinus.     

Differentiation of Streptococcus mutans and Streptococcus sobrinus via genotypic and phenotypic profiles from three different populations

Routine identification of Streptococcus mutans and Streptococcus sobrinus is generally based upon growth on various selective media, colony morphology and biochemical characteristics. We examined various approaches of differentiating these two species through a combination of the conventional phenotypic methodology with chromosomal DNA fingerprint (CDF) and arbitrarily primed polymerase chain reaction (AP-PCR) methods. Initially, ten ATCC type strains and 20 randomly selected clinical isolates of mutans streptococci (MS) were characterized and grouped into two major types based on patterns generated by the CDF using HaeIII digestion. The CDF's patterns with restriction fragments equal to or greater than 6.6 kb were defined as the CDF-1 group. The CDF's patterns with restriction fragments less than 6.6 kb were defined as the CDF-2 group. Both groups were then examined for biotype, serotype, and composition of DNA via thermal denaturation. AP-PCR was applied and evaluated for the capability of delineating S. mutans from S. sobrinus strains. Results of this study showed that all CDF-1 strains fit within a G+C range of 36.2% to 42.2%, whereas the CDF-2 strains had a G+C range of 45.8% to 47.0%. The serotyping assay exhibited 100% sensitivity, 90% specificity and 86.7% agreement with the CDF. The biotyping assay presented the poorest specificity (38.5%), indicating the highest variability. The capability of AP-PCR in differentiation of S. mutans from S. sobrinus was comparable to the CDF method, suggesting that either of these two approaches can and may serve as a viable alternative method to serotyping or biotyping of MS.     

Dental location of Streptococcus mutans and Streptococcus sobrinus in humans harboring both species

The distribution and prevalence of Streptococcus mutans and Streptococcus sobrinus were determined in plaque samples from the cervical areas of all buccal, lingual and approximal tooth surfaces and from the fissures of all occlusal sites in 40 subjects harboring both species. S. mutans was detected more often and in higher numbers than S. sobrinus. There were more teeth detected with S. mutans only than with S. sobrinus only. Most teeth harbored both of these mutans streptococci species, indicating a positive association. The highest numbers of CFU for both species were detected on the molars, with the lowest incidence on the anterior teeth. The presence of S. mutans was relatively similar on all teeth tested, while the presence of S. sobrinus was relatively higher on the molars compared to the anterior teeth. S. mutans and S. sobrinus were found to colonize the buccal surfaces in almost equal numbers. On all other surfaces, S. mutans was detected more frequently or in higher numbers compared to S. sobrinus. No significant differences could be found in the relative proportions of S. mutans and S. sobrinus between sound, decayed or filled tooth surfaces.     

儿童猛性龋变链菌分离株的产酸性

目的：确定儿童猛性龋变链菌和远缘链球菌临床分离株的产酸性。方法：采用酸度计和自动气相色谱仪比较儿童猛性龋、非猛性龋、无龋变链菌（各6株）和远缘链球菌（儿童猛性龋6株,非猛性龋和无龋各3株）的临床株降低环境pH值的能力（ΔpH）和乳酸产量,并以此推测其致龋力。结果：远缘链球菌ΔpH及乳酸产量均高于变链菌,特别是在低pH水平下,二者差异更为显著（P＜0．05）。儿童猛性龋远缘链球菌分离株ΔpH和乳酸产量显著大于非猛性龋和无龋分离菌株（P＜0．05）。结论：远缘链球菌的产酸能力强于变链菌;儿童猛性龋远缘链球菌分离株较非猛性龋和无龋儿童分离菌株产酸性更强。     

Antimicrobial susceptibility of 1042 strains of Streptococcus mutans and Streptococcus sobrinus: comparison from 1985 to 1989

A total of 1042 strains of Streptococcus mutans and Streptococcus sobrinus isolated between 1985 and 1989 were tested to study the evolution of their sensitivity to penicillin, amoxycillin, amoxycillin/clavulanic acid, cefuroxime, tetracycline, erythromycin, spiramycin, acetyl spiramycin, lincomycin and clindamycin. The strains were taken from stock cultures and isolated from human saliva and dental plaque. The minimal inhibitory concentration (MIC) was determined by an agar dilution method. Except for spiramycin and acetyl spiramycin, all the antibiotics inhibited 100% of the strains with concentrations less than or equal to 2 micrograms/ml. Microorganisms from both species underwent a slow progressive loss of sensitivity to all the antibiotics over a 5-year period of study, showing statistically significant results in most cases.     

人工合成变形链球菌表面蛋白多肽体外抗粘附实验

目的 :观察一种人工合成变形链球菌表面蛋白多肽体外抗粘附作用。方法 :H3标记细菌羟磷灰石法观察多肽体外抗粘附作用。结果 :对于S .mutansIngbritt和S .sobrinus 6 715 ,1pmol%D 1μmol均有抑制作用 ,抑制作用随多肽浓度增加而增加。多肽浓度在 1μmol有较强抑制作用。 结论 :人工合成变形链球菌表面蛋白多肽具有体外抗粘附 ,降低变形链球菌的粘附于羟磷灰石的作用 ,并且该多肽无直接抑菌作用 ,有可能成为一种新的生物防龋制剂。     

变形链球菌和远缘链球菌耐氟菌株超微结构观察

目的：探讨变形链球菌和远缘链球菌耐氟突变后,其超微结构变化及氟化物对细菌结构的影响。方法：将变形链球菌和远缘链球菌分为3组：A为正常对照组;B为加氟孵育组;C为耐氟菌株组。透射电镜观察3组细菌的超微结构。结果：与正常亲代菌株相比,加氟孵育后的菌株及耐氟菌株菌体出现不同程度超微结构改变,包括胞浆电子密度减低,细胞肿胀,内容物外溢,远缘链球菌链状结构消失等。结论：变形链球菌和远缘链球菌与氟共同孵育及耐氟突变后,其超微结构发生了改变,表现为自溶活动及解链活动增强。     

变形链球菌耐氟菌株、远缘链球菌耐氟菌株与亲代菌株基因组AP-PCR的比较

目的：利用随机引物聚合酶链反应(AP-PCR)技术，对变形链球菌、远缘链球菌和耐氟菌株的基因组进行比较，判定变形链球菌耐氟突变、远缘链球菌耐氟突变后，遗传型是否发生改变方法：采用AP-PCR技术，经多次实验自行确立AP-PCR反应条件，对变形链球菌、远缘链球菌及耐氟闲株的基因组进行比较结果：变形链球菌耐氟菌株、远缘链球蒲耐氟菌株的基因组已发生改变。结论：变形链球菌耐氟突变、远缘链球菌耐氟突变后，已具有自身的遗传性。     

Susceptibility of Streptococcus mutans and Streptococcus sobrinus to antimicrobial agents after short-term oral chlorhexidine treatments

Effects of three different types of short-term applications (1–3 limes during 1 week) of chlorhexidine (1 or 40%) on the susceptibility of 863 clinical isolates of Streptococcus mutans and 53 isolates of Streptococcus sobrinus from 58 subjects were studied. Chlorhexidine-resistant isolates were not found either before or after the treatment. The minimum inhibitory concentrations (MICs) to chlorhexidine of all isolates of S. mutans were ≤1 μg/ml, and of S. sobrinus ≤2 μg/ ml. S. mutans and S. sobrinus were also susceptible to ampicillin, penicillin, cefuroxime, and tetracycline. In conclusion, different short-term chlorhexidine regimens do not induce resistance in S. mutans or S. sobrinus and, furthermore, these species have so far retained their susceptibility to common antibiotics.     

Characterisation of monoclonal antibodies to common protein epitopes on the cell surface of Streptococcus mutans and Streptococcus sobrinus

Three monoclonal antibodies (MAb) were prepared against a cell surface antigen which cross-react between Streptococcus mutans (serotypes c, e and f) and Streptococcus sobrinus (serotypes d and g). Two of the MAb also recognise a determinant on the surface of Streptococcus cricetus (serotype a). The common antigen shared between S. mutans and S. sobrinus was demonstrated by Western blotting to be about 200 kD in size. This antigen is shared not only by the cell surfaces of serotypes a, c, d, e, f and g, but also by the major cell surface antigen of S. mutans of 185 kD and another of 150 kD. These MAb identify all but one mutans type of streptococci and can be utilised as analytical reagents.     

Relationship of quantitative salivary levels of Streptococcus mutans and S. sobrinus in mothers to caries status and colonization of mutans streptococci in plaque in their 2.5-year-old children

Abstract – Objectives: The aim of this study was to assess the relationships of quantitative salivary levels of Streptococcus mutans and S. sobrinus in mothers with the colonization of mutans streptococci (MS) in plaque and caries status in their 2.5‐year‐old children. Furthermore, the dynamics of caries status in the children was evaluated in a 2‐year follow‐up survey. Methods: After oral examination of 54 mother‐and‐child pairs, the saliva samples from the mothers and the plaque samples from the children were collected. The levels (log DNA copies/ml saliva) of S. mutans and S. sobrinus were quantified using real‐time polymerase chain reaction (PCR) assays, while MS in the plaque samples were detected using a cultivation method. In addition, 50 of the 54 children participated in a 2‐year follow‐up survey of caries prevalence. Results: In the 2.5‐year‐old children, the percentage of dft‐positive subjects and mean number of dft were significantly higher in the MS(+) group when compared with the MS(?) group. Findings from the 2‐year follow‐up survey indicated that MS(+) subjects had a persistently higher mean number of dft at 4.5 years. The 2.5‐year‐old children were divided into three groups based on the quantitative levels of salivary S. mutans and S. sobrinus in their mothers: those whose mothers had low levels of S. mutans (<4 log DNA copies/ml) and S. sobrinus (<2) (group 1); those whose mothers had a high level of S. mutans (≥4) and low level of S. sobrinus (<2) (group 2); and those whose mothers had high levels of both (≥4 and ≥2, respectively) (group 3). Among the three groups, the percentages of MS(+) and dft‐positive children were highest in group 3 and lowest in group 1. Furthermore, multiple logistic regression analyses revealed that grouping the mothers based on salivary level of S. mutans and S. sobrinus was an efficient means to predict both MS colonization (OR = 2.96) and prevalence of dental caries (OR = 9.39) in children at 2.5 years of age. Conlusions: In the 54 mother‐and‐child pairs tested, the maternal salivary levels of S. mutans and S. sobrinus determined by real‐time PCR were significantly related to MS colonization in plaque as well as dental caries in their children at 2.5 years of age. Thus, determination of maternal levels of both organisms using the present cut‐off values is proposed as an efficient method to indicate the risks of maternal transmission of MS and childhood dental caries.     

中药五倍子对变形链球菌及远缘链球菌抑制作用的体外研究

目的：研究五倍子及其主要成分鞣酸在体外对变链菌及远缘链球菌的抑制作用，并摸索适宜剂型和浓度。方法：本实验采用纸片扩散法研究五种浓度五倍子煎剂，五倍子浸剂，鞣酸标准品抑制变链菌及远缘链球菌生长的作用。结果：65.2mL/L以上浓度五倍子煎剂，浸剂，鞣酸对变链菌及远缘链球菌生长均有抑制作用，其中五倍子浸剂抑菌作用最强。抑菌作用随着各剂型浓度的增国而增强。结论：五倍子可以抑制变链菌及远缘链球菌生长，其中浸剂效果最优，鞣酸组效果较差，可初步推测五倍子中含有其他协同鞣酸抑菌的成分。     

鸡蛋黄防龋抗体的制备、抗体效价及持久性观察

目的：从鸡蛋中提取特异性抗体IgY,并研究鸡蛋黄抗体免疫应答的持久性,以探索一种新的被动免疫防龋方法。方法：以变形链球菌GTF高表达株S.mutans B29-33及茸毛链球菌S.sobrinus 6715免疫母鸡。采用硫酸铵沉淀法提取鸡蛋黄抗体,SDS－PAGE鉴定抗体性质,分光光度法检测蛋白质含量,间接酶联免疫吸附法（ELISA）测定鸡血清及鸡蛋黄抗体效价滴度变化。结果：SDS－PAGE鉴定证实：用硫酸铵沉淀法可提取出鸡蛋黄抗体,其性质为IgG,蛋白质含量约为每毫升7.00mg鸡蛋黄。免疫应答持续时间超过24周。结论：鸡蛋黄抗体来源充足,抗体含量高,并能在一段较长时间内从免疫母鸡鸡蛋黄中获取,有较好的应用前景。     

蜂胶涂膜对变形链球菌生长和黏附的抑制作用

目的观察蜂胶涂膜对变形链球菌生长和黏附的抑制作用。方法采用纸片琼脂扩散法观察10、25、50g/L和100g/L蜂胶防龋涂膜对变形链球菌c型和d型的抑菌作用。以蜂胶涂膜涂布黏附板,置变形链球菌培养液中培养,观察其抗黏附效果。结果各浓度蜂胶涂膜及基质都能够抑制细菌生长和黏附,且抑菌作用呈现明显的浓度依赖性,100g/L涂膜组的抑菌效果与1.6g/L洗必泰溶液无显著性差异(P>0.05)。结论25~100g/L蜂胶涂膜均可以有效抑制变形链球菌c型和d型的生长黏附。     

Simple and rapid detection of Streptococcus mutans and Streptococcus sobrinus in human saliva by polymerase chain reaction

Streptococcus mutans and Streptococcus sobrinus are major pathogens causing dental caries in humans. A simple and rapid method to detect these species in human saliva simultaneously was developed using the polymerase chain reaction (PCR). Chromosomal DNA was extracted by boiling bacterial cells in lysis solution containing 1% Triton X-100. Oligonucleotide primers specific for portions of the glucosyltransferase genes (gtfB of S. mutans and gtfI of S. sobrinus) were designed. After PCR using two sets of these primers, S. mutans and S. sobrinus were specifically identified. The method was capable of amplifying DNA fragments specific for these species from chromosomal DNA extracted from 1 x 10(3) cells, or from 10 microliters of clinical saliva samples containing 1 x 10(3) colony-forming units of either streptococcal species. A second PCR, using the first PCR product as a template with newly designed internal primers, made it possible to detect 1 x 10(2) colony-forming units of either streptococcal species in 10 microliters of saliva samples. These results indicate that the PCR method developed in this study is useful for detecting S. mutans and S. sobrinus in saliva and that it can be used in epidemiological studies to evaluate the prevalence level of these organisms.     

Rapid detection of the cariogenic pathogens Streptococcus mutans and Streptococcus sobrinus using loop-mediated isothermal amplification

INTRODUCTION: Streptococcus mutans and Streptococcus sobrinus are associated with the development of dental caries in humans. In this study, we developed a rapid, sensitive method for detecting these major cariogenic pathogens using loop-mediated isothermal amplification (LAMP). The assay procedure is quite simple: the amplification is carried out in a single tube under isothermal conditions at 63 degrees C, and the result can be obtained in less than 1 h. METHODS: Initially, a set of six primers was designed by targeting S. mutans-specific and S. sobrinus-specific regions, identified using the genomic subtractive hybridization technique. We evaluated the specificities and sensitivities of these assays. Furthermore, we detected and quantified these bacteria in saliva and carious dentin from eight children. RESULTS: The sensitivities of the S. mutans-specific and S. sobrinus-specific LAMP methods, examined using agarose gel electrophoresis, were each one cell for a 30-min reaction. The detection limits using real-time turbidimetry analysis were 1 to 10(7) cells (3.28 x 10(1) to 3.28 x 10(8) fg S. mutans template DNA) per reaction tube and 1 to 10(5) cells (2.72 x 10(3) to 2.72 x 10(8) fg S. sobrinus template DNA) per reaction tube. Using these assays, we detected and quantified these cariogenic bacteria for evaluation of the LAMP assay for clinical diagnosis. CONCLUSIONS: Our results suggest that the LAMP-based assay in combination with subtractive hybridization is valuable for preparing species-specific primers for closely related species. Furthermore, the LAMP-based assay will be a useful tool for the rapid and sensitive prediction of dental caries.     

高发龋和无龋儿童菌斑中变形链球菌及远缘链球菌的任意引物PCR检测

目的采用任意引物聚合酶链反应（arbitrarily primed-polymerase chain reaction，AP-PCR）法探讨高发龋和无龋儿童牙菌斑致龋菌的检出情况，分析变形链球菌及远缘链球菌与乳牙龋齿发生的关系。方法从20例高发龋患儿和20名无龋儿童的牙菌斑中分离、鉴定变形链球菌和远缘链球菌，以Shiroza的DNA提取方法提取细菌基因组DNA，以形态学、生化和AP-PCR的方法鉴定变形链球菌和远缘链球菌。结果AP-PCR法检测发现高发龋患儿变形链球菌和远缘链球菌的检出率分别为100％和40％，而无龋儿童两种细菌的检出率分别为75％和5％，差异有统计学意义。结论用AP-PCR法检测发现高发龋患儿变形链球菌和远缘链球菌的检出率均明显高于无龋儿童，口腔中定植的变形链球菌和远缘链球菌是乳牙龋高发的危险因素。     

变链菌分离株的形态、生理生化和遗传学鉴定

目的：探讨形态学和生理生化反应与DNA碱基含量测定鉴定变链菌和元缘链球菌临床分离株的一致性。方法：采用形态学和生理生化试验鉴定变链菌和远缘链球菌临床分离株,采用高效反相液相色谱法测定细菌DNA G＋Cmol%以检验生化鉴定的准确性。结果:形态及生化鉴定为变链菌和远缘链球菌的临床分离株,其DNA碱基含量分别为37．52和45．19,各自位于变链菌和远缘链球菌36－38和44－46的G＋Cmol％参考范围。结论：变链菌和远缘链球菌的生化鉴定结果与DNA G＋Cmol％测定结果一致。通过形态及生理生化鉴定基本可以鉴定变链菌和远缘链球菌。