丹皮酚对变形链球菌生物膜影响的体外研究

目的 测定丹皮酚对变形链球菌的抑菌作用;共聚焦显微镜观察丹皮酚对变形链球菌生物膜结构和活性的影响。方法 梯度法测定丹皮酚对变形链球菌的MIC（最小抑菌浓度）、MBC（最小杀菌浓度）;体外构建变形链球菌生物膜模型,共聚焦显微镜观察不同浓度丹皮酚对变形链球菌生物膜作用后形态结构的影响并进行红绿荧光定量分析其活性变化。结果 丹皮酚对变形链球菌的MIC为6.25 mg/mL,MBC为25 mg/mL;激光共聚焦显微镜观察丹皮酚对变形链球菌生物膜作用后其生物膜结构变稀疏,细菌链变短,生物膜活性也随丹皮酚浓度的提高而逐渐降低。结论 丹皮酚对变形链球菌和变形链球菌生物膜结构及其活性均具抑制作用。     

大蒜素抗变异链球菌的致龋效果

目的 研究大蒜素对口腔变异链球菌生长及其菌斑生物膜粘附的抑制作用。方法 二倍稀释法梯度稀释测最小抑菌浓度（minimum inhibitory concentration,MIC）,将MIC以上2个梯度浓度对应的培养物涂布于BHI培养基上进行次代培养获得最低杀菌浓度（minimum bactericidal concentration,MBC）;酶标仪测A值观察不同浓度大蒜素抑菌效应;抑制产酸试验观察抑制细菌产酸效应;结晶紫法研究亚抑菌浓度提取物对变异链球菌粘附能力及生物膜总量的影响;采用激光共聚焦荧光显微镜（laser scanning confocal microscopy,LSCM）观察常态牙菌斑生物膜生长过程中及药物处理后牙菌斑生物膜中死菌和活菌的构成,研究其对牙菌斑生物膜结构和活性的影响。结果 抑菌试验中,得到大蒜素MIC为12.8 mg/L,MBC为25.8 mg/L。MIC及亚抑菌浓度抑菌试验显示均有一定的抑菌性,抑制率为2.17%~67.12%,并且抑菌性与浓度梯度成正相关。产酸试验显示24 h内大蒜素明显抑制细菌产酸（P<0.01）,细菌粘附试验结果显示大蒜素在MIC时生物膜的生成速度最慢,生物膜的总量最低（P<0.01）。共聚焦荧光显微镜可见大蒜素组随药物浓度增加,菌斑生物膜较薄,绿色的活菌及团块明显减少,抑制生物膜的生长。结论 大蒜素对变异链球菌生长、产酸与粘附有一定抑制作用。     

Acid tolerance response of biofilm cells of Streptococcus mutans

Streptococcus mutans, a major etiological agent of dental caries, is a component of the dental plaque biofilm and functions during caries progression in acidic lesions that may be at or below pH 4. In this study, we were interested in determining the acid tolerance of 1-7-day chemostat-grown biofilm cells of S. mutans BM71 growing in a semi-defined medium at a rate consistent with that of cells in dental plaque (dilution rate=0.1 h(-1)), as well as, assessing the capacity of 2- and 5-day biofilms to induce an acid tolerance response that would enhance survival at a killing pH (3.5). As expected, biofilm cell growth increased (2.5-fold) from day 1 to day 7 (10.6-25.7 x 10(6) cells cm(-)(2)) with the percentage live cells over that period averaging 79.4%, slightly higher than that of planktonic cells (77.4%). Biofilms were highly resistant to acid killing at pH 3.5 for 2 h with survival ranging from 41.8 (1 day) to 63.9% (7 day), while the percentage of live cells averaged 43.4%. Planktonic and dispersed biofilm cells were very acid-sensitive with only 0.0009%- and 0.0002-0.2% survivors, respectively. Unlike the planktonic cells, the incubation of 2- and 5-day biofilms at pH 5.5 for periods of up to 6 h induced strong acid tolerance responses that enhanced survival during a subsequent exposure to acid killing at pH 3.5.     

Streptococcus mutans glucan-binding protein-A affects Streptococcus gordonii biofilm architecture

The glucan-binding protein-A (GbpA) of Streptococcus mutans has been shown to contribute to the architecture of glucan-dependent biofilms formed by this species and influence virulence in a rat model. As S. mutans synthesizes multiple glucosyltransferases and nonglucosyltransferase glucan-binding proteins (GBPs), it is possible that there is functional redundancy that overshadows the full extent of GbpA contributions to S. mutans biology. Glucan-associated properties such as adhesion, aggregation, and biofilm formation were examined independently of other S. mutans GBPs by cloning the gbpA gene into a heterologous host, Streptococcus gordonii, and derivatives with altered or diminished glucosyltransferase activity. The presence of GbpA did not alter dextran-dependent aggregation nor the initial sucrose-dependent adhesion of S. gordonii. However, expression of GbpA altered the biofilm formed by wild-type S. gordonii as well as the biofilm formed by strain CH107 that produced primarily alpha-1,6-linked glucan. Expression of gbpA did not alter the biofilm formed by strain DS512, which produced significantly lower quantities of parental glucan. These data are consistent with a role for GbpA in facilitating the development of biofilms that harbor taller microcolonies via binding to alpha-1,6-linkages within glucan. The magnitude of the GbpA effect appears to be dependent on the quantity and linkage of available glucan.     

Sulfated vizantin inhibits biofilm maturation by Streptococcus mutans

Streptococcus mutans is the main pathogen of dental caries and adheres to the tooth surface via soluble and insoluble glucans produced by the bacterial glucosyltransferase enzyme. Thus, the S. mutans glucosyltransferase is an important virulence factor for this cariogenic bacterium. Sulfated vizantin effectively inhibits biofilm formation by S. mutans without affecting its growth. In this study, less S. mutans biofilm formation occurred on hydroxyapatite discs coated with sulfated vizantin than on noncoated discs. Sulfated vizantin showed no cytotoxicity against the human gingival cell line Ca9-22. Sulfated vizantin dose-dependently inhibited the extracellular release of cell-free glucosyltransferase from S. mutans and enhanced the accumulation of cell-associated glucosyltransferase, compared with that observed with untreated bacteria. Sulfated vizantin disrupted the localization balance between cell-associated glucosyltransferase and cell-free glucosyltransferase, resulting in inhibited biofilm maturation. These results indicate that sulfated vizantin can potentially serve as a novel agent for preventing dental caries.     

Glucan-binding proteins are essential for shaping Streptococcus mutans biofilm architecture

Glucan plays a central role in sucrose-dependent biofilm formation by the dental pathogen Streptococcus mutans. This organism synthesizes several proteins capable of binding glucan. These are divided into the glucosyltransferases that catalyze the synthesis of glucan and the nonglucosyltransferase glucan-binding proteins (Gbps). The biological significance of the Gbps has not been thoroughly defined, but studies suggest that these proteins influence virulence and play a role in maintaining biofilm architecture by linking bacteria and extracellular molecules of glucan. We engineered a panel of Gbp mutants, targeting GbpA, GbpC, and GbpD, in which each gene encoding a Gbp was deleted individually and in combination. These strains were then analyzed by confocal microscopy and the biofilm properties were quantified by the biofilm quantification software comstat. All biofilms produced by mutant strains lost significant depth, but the basis for the reduction in height depended on which particular Gbp was missing. The loss of the cell-bound GbpC appeared dominant as might be expected based on losing the principal receptor for glucan. The loss of an extracellular Gbp, either GbpA or GbpD, also profoundly changed the biofilm architecture, each in a unique manner.     

Protein interactions of SGP, an essential Streptococcus mutans GTPase, revealed by biochemical and yeast two-hybrid system analyses

SGP, a Streptococcus mutans essential GTPase, plays a role in the stress response of the organism. Recently, we proposed that one of the physiological functions of the SGP is the modulation of the GTP/GDP ratio under different growth conditions. In order to further determine the functions of SGP and its possible interactions with other molecules, we carried out immunoprecipitation, SGP binding, and the yeast two-hybrid system analyses. These approaches suggest that SGP may oligomerize and such interactions could be important for the function of this regulatory protein.     

Tea extracts differentially inhibit Streptococcus mutans and Streptococcus sobrinus biofilm colonization depending on the steeping temperature

Abstract

This study aimed to evaluate the effects of tea extracts on oral biofilm colonization depending on steeping temperature. S. mutans and S. sobrinus were cultured and treated with green or black tea extracts prepared under different steeping conditions. Biofilm formation, glucosyltransferase (GTF) levels, bacterial growth, and acidogenicity were evaluated. Biofilms were also assessed by gas chromatography-mass spectrometry and confocal laser scanning microscopy. All extracts with hot steeping showed higher inhibitory effects on biofilm formation and cell viability and lower GTF levels compared with those with cold steeping (p?<?0.05). Hot steeping significantly reduced bacterial growth (p?<?0.05) and maintained the pH. Catechins were only identified from hot steeping extracts. Within the limits of this study, extracts with cold steeping showed lower inhibitory effects on oral biofilms. The different effects between steeping extracts may be attributed to the difference in catechins released from tea extracts under the different steep conditions.     

Influence of fluoride on the bacterial composition of a dual-species biofilm composed of Streptococcus mutans and Streptococcus oralis

Despite the widespread use of fluoride for the prevention of dental caries, few studies have demonstrated the effects of fluoride on the bacterial composition of dental biofilms. This study investigated whether fluoride affects the proportion of Streptococcus mutans and S. oralis in mono- and dual-species biofilm models, via microbiological, biochemical, and confocal fluorescence microscope studies. Fluoride did not affect the bacterial count and bio-volume of S. mutans and S. oralis in mono-species biofilms, except for the 24-h-old S. mutans biofilms. However, fluoride reduced the proportion and bio-volume of S. mutans but did not decrease those of S. oralis during both S. oralis and S. mutans dual-species biofilm formation, which may be related to the decrease in extracellular polysaccharide formation by fluoride. These results suggest that fluoride may prevent the shift in the microbial proportion to cariogenic bacteria in dental biofilms, subsequently inhibiting the cariogenic bacteria dominant biofilm formation.     

LuxS基因缺陷对变形链球菌生物膜形成的影响

目的观察LuxS基因缺失后变形链球菌生物膜成熟初期的变化情况。方法通过扫描电镜观察标准菌和缺陷菌在不同营养环境中生物膜成熟初期的形成情况。结果对不同营养环境中形成的生物膜观察,发现在富含蔗糖的环境中,缺陷菌成熟初期的生物膜形成能力较标准菌弱。结论 LuxS基因缺失后变形链球菌在蔗糖环境中生物膜形成的能力减弱。     

椰子油对变形链球菌抑菌作用的体外研究

目的 研究椰子油对变形链球菌的生长抑制作用,通过观察其对生物膜活性、产酸及粘附的影响,探讨其在口腔中防龋的作用。方法 采用96孔微量板液体稀释法进行抑菌试验,并测得最低抑菌浓度（MIC）。体外建立变形链球菌生物膜模型,通过激光共聚焦显微镜（CLSM）扫描生物膜,观察不同浓度药物作用24 h后对生物膜活性的影响。其次测定处理后各组培养基上清液的终末pH值。最后通过玻璃棒粘附试验计算出不同浓度药物作用48 h后对生物膜粘附的影响。结果 椰子油对变形链球菌的生长有抑制作用,其对变形链球菌的MIC为3.13%。CLSM观察24 h后生物膜内活菌比例逐渐下降,死菌逐渐增多。培养基上清液的终末pH值随椰子油浓度的增大而升高,且均高于阴性对照组,差异具有统计学意义（P<0.05）。实验组变形链球菌的粘附率随椰子油浓度增高而降低,与阴性对照组相比差异有统计学意义(P<0.05)。结论 椰子油对变形链球菌有抑制作用,并能抑制其生物膜的活性、产酸及粘附等作用。     

Quasi-irreversible inhibition of enolase of Streptococcus mutans by flouride

Abstract Fluoride at concentrations greater than 0.01 mM was found to be a quasi-irreversible inhibitor of enolase of permeabilized cells of Streptococcus mutans GS-5 and also of isolated yeast enolase. The inhibition appeared to be of the type that has been described for P-ATPases, but was not dependent on added Al³⁺ or Be²⁺ ions. Fluoride inhibition of enolase was not reversed by repeatedly washing the permeabilized cells in chilled fluoride-free medium but could be reversed by the product, phosphoenolpyruvate, or by very high levels of the substrate, 2-phosphoglycerate. Irreversible inhibition of glycolysis was not evident after fluoride treatment of intact cells, washing to remove unbound or loosely bound fluoride and addition of glucose, presumably because intracellular levels of phosphoenolpyruvate were sufficiently high to preclude irreversible fluoride inhibition of enolase.     

变形链球菌对不同牙科充填材料的粘附及早期生物膜的形成

目的探讨变形链球菌对不同牙科充填材料的粘附和早期生物膜的形成.方法比较经放射性同位素3H-TDR(3H-胸腺嘧啶核苷)标记的变形链球菌对3种唾液包被的充填材料的粘附.采用蛋白质测量试剂盒定量分析其对唾液蛋白的吸附量;采用凝胶电泳和图像分析系统定量分析其对唾液白蛋白和α-淀粉酶的吸收率.结果各种材料对变形链球菌的粘附能力,对唾液蛋白的吸附能力均随着材料的不同而不同.Fuji IX对细菌的粘附量很高,但是对蛋白的吸附量却很低;而F2000对细菌的粘附量很低,对蛋白的吸附量却很高.结论在不同充填材料表面形成的生物膜是不同的,提示早期生物膜的形成具有一定的特异性.这种生物膜的差异对口腔微生态环境及龋病和/或牙周病的发展具有重要意义.     

The regulation of Streptococcus mutans glucan-binding protein A expression

The S. mutans GBP-A is hypothesized to be constitutively expressed and to contribute to the sucrose-dependent colonization of S. mutans. To investigate GBP-A expression, a reporter gene encoding chloramphenicol acetyltransferase (CAT) was placed downstream of the gbpA promoter and CAT activity was measured under conditions that would be associated with the sucrose-dependent colonization of S. mutans. Expression of GBP-A was optimal under anaerobiosis and neutral pH conditions, and correlated with optimal growth. The addition of sucrose to the growth medium did not elevate the expression of GBP-A.     

Inhibitory effects of 7-epiclusianone on glucan synthesis, acidogenicity and biofilm formation by Streptococcus mutans

The aim of this study was to examine the effects of 7-epiclusianone, a new prenylated benzophenone isolated from the plant Rheedia gardneriana, on some of the virulence properties of Streptococcus mutans associated with biofilm development and acidogenicity. The synthesis of glucans by glucosyltransferases B (GTF B) and C (GTF C) was markedly reduced by 7-epiclusianone showing more than 80% inhibition of enzymatic activity at a concentration of 100 microg mL(-1). Double-reciprocal analysis (Lineweaver-Burk plots) revealed that the inhibition of GTF B activity was noncompetitive (mixed) while GTF C was inhibited uncompetitively. The glycolytic pH drop by S. mutans cells was also disrupted by 7-epiclusianone without affecting the bacterial viability, an effect that can be attributed, in part, to inhibition of F-ATPase activity (61.1+/-3.0% inhibition at 100 microg mL(-1)). Furthermore, topical applications (1-min exposure, twice daily) of 7-epiclusianone (at 250 microg mL(-1)) disrupted biofilm formation and physiology. The biomass (dry-weight), extracellular insoluble polysaccharide concentration and acidogenicity of the biofilms were significantly reduced by the test agent (P<0.05). The data show that 7-epiclusianone disrupts the extracellular and intracellular sugar metabolism of S. mutans, and holds promise as a novel, naturally occurring compound to prevent biofilm-related oral diseases.     

Incorporation of fatty acids by Streptococcus mutans

In a series of investigations into the cariogenicity of Streptococcus mutans, we studied the incorporation of exogenous fatty acids with reference to glucosyltransferase secretion and membrane fatty acid changes. When cells were grown with different fatty acids, both saturated and unsaturated fatty acids were readily incorporated into the membrane lipids and were biotransformed and elongated preferentially to the longer 16- and 18-carbon-chain fatty acids. This incorporation and chain-elongation led to significant changes in fatty acids composition. By adding fatty acids to the medium, it was possible to appropriately modify the degree of unsaturation and the relative ratio between specific fatty acids in the membrane lipids of S. mutans.     

变异链球菌和血链球菌全代谢途径比对分析

为了比较变异链球菌和血链球菌全代谢途径,依据KEGG数据库(http://www.genome.ad.jp/kegg)对变异链球菌和血链球菌的全部代谢途径作逐项比对。结果显示,二者参与了85个代谢途径,包括多数以相同的酶参与的中央代谢途径,即糖酵解、三羧酸循环、磷酸戊糖途径等,和多数以不同的酶参与的双组分感应系统等。通过变异链球菌和血链球菌整体代谢网络对比,了解了变异链球菌和血链球菌理论上的全部代谢途径,为全面揭示二者代谢交流研究奠定了基础。     

Coaggregation between freshwater bacteria within biofilm and planktonic communities

The coaggregation ability of bacteria isolated from a freshwater biofilm was compared to those derived from the coexisting planktonic population. Twenty-nine morphologically distinct bacterial strains were isolated from a 6-month-old biofilm, established in a glass tank under high-shear conditions, and 15 distinct strains were isolated from the associated re-circulating water. All 44 strains were identified to genus or species level by 16S rDNA sequencing. The 29 biofilm strains belonged to 14 genera and 23.4% of all the possible pair-wise combinations coaggregated. The 15 planktonic strains belonged to seven genera and only 5.8% of all the possible pair-wise combinations coaggregated. Therefore, compared to the planktonic population, a greater proportion of the biofilm strains coaggregated. It is proposed that coaggregation influences biofilm formation and species diversity in freshwater under high shear.     

变形链球菌中的次级代谢产物及其在口腔生物被膜中的生态功能

口腔生物被膜是由数百种微生物构成的复杂微生物群体。变形链球菌作为其中的重要一员,被认为是引起龋病的主要病原菌。变形链球菌在牙齿表面以生物被膜形式生长的能力和它产酸耐酸的特点赋予其致龋性。不同的变形链球菌菌株之间保留了多样的次级代谢形式,这是与宿主长期共进化的结果。迄今为止,变形链球菌中报道的次级代谢产物包括10种细菌素(又称变链素)和一种聚酮/非核糖体肽类化合物。这些化合物多样的活性形式暗示了它们参与口腔生物被膜中跨种间和跨界间的相互作用。未来随着变形链球菌菌株数目的增加和更多菌株全基因组序列的完成,可以预见会有更多新颖活性次级代谢产物的出现。对变形链球菌次级代谢的研究不仅有助于治疗和预防口腔疾病,而且新颖活性次级代谢产物的发现对新药的研发也具有重要意义。