不同生存状态下变形链球菌合成胞外多糖的研究

目的 :比较变形链球菌合成水溶性、水不溶性胞外多糖的能力在生物膜黏附状态与浮游状态下是否存在量的差异及其可能原因。方法 :在多种蔗糖培养浓度和不同培养时间 ,对变形链球菌分别进行静置培养和摇动培养 ,以模拟生物膜黏附状态和浮游状态下生长的变形链球菌 ,蒽酮法测定细菌合成的水溶性胞外多糖和水不溶性胞外多糖的含量。结果 :相同培养时间每种蔗糖浓度条件下 ,生物膜黏附状态下变形链球菌合成的水溶性、水不溶性胞外多糖均要多于浮游状态下的细菌 (P <0 .0 5 ) ,生物膜黏附状态与浮游状态下变形链球菌的数量和合成胞外多糖的量均随着时间的增加而增加 ,不同培养时间同一蔗糖浓度条件下 ,生物膜状态下变形链球菌合成的水溶性、水不溶性胞外多糖也多于浮游状态下的细菌 (P <0 .0 5 )。结论 :生物膜黏附条件下 ,变形链球菌合成水溶性、水不溶性胞外多糖的能力均明显增强 ,这可能是生物膜中的细菌有更强致龋性的原因之一。     

高果糖玉米糖浆对变形链球菌粘附能力的影响

目的：以目前食品加工业中广泛使用的高果糖玉米糖浆（HFCS）为研究对象,通过与蔗糖、葡萄糖和果糖的比较,研究高果糖玉米糖浆对变形链球菌粘附能力的影响。方法：将变形链球菌uA159分别接种于0．25％、0．5％、1％、3％、5％五组不同浓度TSB糖培养基培养18h后,用磷酸盐缓冲液洗脱2次并采用紫外分光光度计540nm检测各试管中的A值,计算粘附比,行方差分析,比较粘附力。结果：4种饮食糖培养基的粘附比存在统计学差异（P〈0．01）,HFCS组的变形链球菌粘附比显著低于蔗糖组,与葡萄糖和果糖组之间无显著差异;5组浓度糖培养基的变形链球菌粘附比之间的差异也具有统计学意义（P〈0．01）。结论：HFCS对变形链球菌粘附力的促进弱于蔗糖,与葡萄糖和果糖无明显差异。     

远缘链球菌黏附与游离状态下合成胞外多糖的比较

目的：比较远缘链球菌在游离和黏附状态下合成水溶性、水不溶性多糖的能力并探讨其原因。方法：在不同培养时间和多种蔗糖浓度条件下,对远缘链球菌进行摇动培养和静止培养,以模拟该菌在游离和黏附的状态下生长。采用蒽酮法测定细菌合成的水溶性和水不溶性多糖的含量,实验数据采用SPSS10.0软件包分析,选用单因素方差分析的Dunnett双侧检验比较组间的差异有无统计学意义。结果：生物膜形成发展期,游离菌合成的水溶性多糖随培养时间的延长而增多,黏附菌合成的水溶性多糖则随时间呈下降趋势;在生物膜成熟期,黏附菌合成的水不溶性多糖多于游离菌（P〈0．05）,而两者合成的水溶性多糖量的差异无显著性（P〉0．05）。细菌合成的水溶性、水不溶性多糖随蔗糖浓度的增加而增加;在各个蔗糖浓度条件下,黏附菌合成的水不溶性胞外多糖量显著多于游离菌（P〈0.05）,而水溶性胞外多糖的量差异不大（P〉0．05）。结论：培养时间和蔗糖浓度增加,导致远缘链球菌合成的水不溶性胞外多糖较游离状态明显增加;生物膜特殊的生长环境,是造成这种差异的可能原因。     

高龋及无龋者变形链球菌临床分离株致龋性研究Ⅱ合成细胞外多糖能力的实验研究

目的：探讨来自不同龋敏感者的变形链球菌（血清型ｃ）临床分离株合成细胞外多糖的能力。方法：采用红外光谱分析对葡萄聚糖样本作定性分析,用蒽酮法分别定量测定水溶性和水不溶性的葡聚糖含量。结果：同一个体所带不同基因型变形链菌菌株合成细胞外多糖能力具有差异;高龋组个体定植的合成水溶性及水不溶性葡聚糖能力强的菌株所占比较显著高于无龋组。结论：高龋组变形链球菌（血清型ｃ）临床菌株的高致龋力与其携带有合成细胞外多糖能力强的菌株密切相关。     

两种糖对变异链球菌产酸能力影响的比较研究

目的:通过测量变异链菌代谢高果糖玉米糖浆(high-fructose corn syrup,HFCS)和蔗糖后△pH值的变化,比较两种糖对变异链球菌产酸能力的影响。方法:配制质量浓度为0.25%、0.5%、1%、3%和5%的高果糖玉米糖浆和蔗糖培养基,将变异链球菌UA159于上述各培养基37℃微需氧培养,分别于培养1、4、8、24、48 h各个时间点,用酸度计测量培养前后的pH值,计算ΔpH值,代表变异链球菌的产酸能力。结果:在本研究设定的糖浓度范围和培养时间段内,高果糖玉米糖浆(HFCS)和蔗糖培养基中的ΔpH值均随时间的延长而增大,在培养4～8 h内,HFCS培养基内pH值下降速度快于蔗糖培养基。在培养4、8、24 h时,5种浓度的HFCS培养基中ΔpH值均明显大于蔗糖培养基,差异均有统计学意义(P<0.05),而在培养1 h和48 h时,两种糖培养基中ΔpH值的变化无统计学意义(P>0.05)。结论:在培养4～8 h内,HFCS利于变异链球菌代谢产酸,而在24～48 h内,蔗糖则显示出较强的持续产酸能力。     

淀粉与食用糖交互作用的致龋性初探

目的　初步探讨淀粉与蔗糖、高果糖玉米糖浆（high- fructose corn syrup ,HFCS）的交互作用对变形链球菌致龋毒力的影响。方法　变形链球菌UA159培养于质量浓度均为1%的蔗糖、淀粉和HFCS组成的双糖和三糖BHI培养基中,三种单一糖源作为对照,于培养的6个时间点测量每组的pH值,计算ΔpH;96孔微孔板培养20h后采用结晶紫比色法比较7组的粘附力。结果 蔗糖+HFCS和三糖组产酸速度较快,ΔpH明显大于蔗糖+淀粉和淀粉+HFCS组（P<0.05）。4组实验组的粘附性存在统计学差异（P<0.01）,蔗糖+淀粉组的粘附性最强,三糖组的粘附性强于蔗糖+HFCS组,淀粉+HFCS组最弱。结论　淀粉和蔗糖、HFCS间的交互作用会影响变形链球菌产酸、粘附和生物膜的形成,从而导致其致龋性的改变。     

变链菌细胞外多糖合成与乳牙高龋的关系研究

目的:比较高龋和无龋幼儿变链菌临床分离株在体外利用蔗糖合成水溶性、水不溶性葡聚糖的能力。方法:蒽酮法定量测定从高龋(dmft≥5)和无龋幼儿(dmft=0)牙菌斑分离的变形链球菌临床分离株水溶性和水不溶性葡聚糖产量。结果:高龋组菌株合成水溶性与水不溶性葡聚糖量平均分别为0.0332mg/ml和0.0357mg/ml,高于无龋组菌株的0.0215mg/ml和0.0192mg/ml;高龋幼儿定植的合成水溶性及水不溶性葡聚糖能力强的菌株所占比例也高于无龋幼儿;变链菌临床菌株水不溶性葡聚糖产量平均为0.0301mg/ml,高于水溶性葡聚糖产量(0.0267mg/ml);差异有统计学意义。结论:高龋幼儿的龋活跃性与其口腔中变形链球菌临床菌株合成细胞外多糖能力强有关。     

白屈菜红碱对变形链球菌葡糖基转移酶和细胞外水不溶性多糖的影响

目的：研究白屈菜红碱对变形链球菌葡糖基转移酶和细胞外水不溶性多糖合成的影响。方法：采用二倍稀释法,用含2％蔗糖的脑心浸萃液肉汤琼脂（BHI）液体培养基,将白屈菜红碱稀释为从390．6μg／ml到24．4μg／ml共计5个浓度的溶液．以BHI液体培养基作为阴性对照组。加入变形链球菌菌液,厌氧培养24h后,将培养物离心,收集上清液．分为2份。一份提取葡糖基转移酶,采用考马斯亮蓝法和Somogyi法分别测定总蛋白和还原糖含量,计算酶活性和比活力大小;另一份上清液采用蒽酮法测定水不溶性多糖的含量。实验结果采用SPSS13．0软件包进行数据处理,总体均数之间的比较采用单因素方差分析,采用Pearson相关检验进行两组样本之间的相关分析。结果：随着白屈菜红碱溶液浓度的升高,葡糖基转移酶活性和水不溶性多糖含量逐渐降低,各组总体均数间均具有高度显著性差异（P〈0．01）,葡糖基转移酶各实验组与对照组酶活性及比活力之间均有高度显著性差异（P〈0．01）;除24．4μg／ml浓度组外．各实验组与对照组水不溶性多糖含量也均有高度显著性差异（P〈0．01）。葡糖基转移酶活性和水不溶性多糖含量之间呈正相关关系（r=0．883．P〈0．01）。结论：白屈菜红碱对变形链球菌葡糖基转移酶和细胞外水不溶性多糖的合成具有显著抑制作用。     

赤芍粗提物对变形链球菌影响的实验研究

目的研究赤芍粗提物对变形链球菌的生长、产酸、粘附及合成胞外多糖的影响。方法采用倍比稀释法测定不同浓度赤芍粗提物抑制变形链球菌生长的情况,通过统计学方法确定最低抑菌浓度（minimal inhibitory concentration,MIC）;再以MIC及低于MIC的4个倍比稀释浓度配制含药的胰酶解酪蛋白-植物蛋白胨-酵母提取物（trypticase-phytone-yeast extract medium,TPY）液体培养基,测定赤芍粗提物对变形链球菌产酸、粘附及合成胞外多糖能力的影响。结果赤芍粗提物抑制变形链球菌体外生长的MIC为12.5g/L;当赤芍粗提物浓度≥3.13g/L时有较明显地抑制变形链球菌产酸、粘附、合成水溶性胞外多糖的作用;当赤芍粗提物浓度≥6.25g/L时能明显抑制变形链球菌合成水不溶性胞外多糖。结论达到一定浓度的赤芍粗提物对变形链球菌的生长、产酸、粘附及合成胞外多糖均有一定的抑制作用。     

茶多酚对口腔细菌致龋力影响的实验研究

目的：通过研究茶多酚对致龋菌生长，产酸及产胞外多糖的影响。探讨茶多酚是否能有效调节口腔菌群生态平衡，方法：测定茶多酚对三种主要致龋菌－变形链球菌，粘性放线菌和血链球菌的最低抑菌浓度（MIC），再测定低于MIC的4个浓度的茶多酚对三种细菌产酸及产生水溶性和水不溶性多糖能力的影响。结果：茶多配合酚对三种细菌的生长，产酸均有一定的抑制作用。茶多酚能够有效抑制变形链球菌产生水溶性葡聚糖，但对变形链球菌产生水溶性葡聚糖以及粘性放线菌和血链球菌合成胞外多糖无明显的抑制作用。结论：茶多酚能有效抑制变形;链球菌，粘性放射菌和血链球菌的生长，产酸及变形链球菌产生水不溶性葡聚糖。     

Evaluation of Streptococcus mutans biofilms formed on fluoride releasing and non fluoride releasing resin composites

Objectives

The aim of this study was to evaluate the acid production, acid tolerance and composition of Streptococcus mutans biofilms formed on fluoride releasing and non fluoride releasing resin composites.

Methods

S. mutans biofilms were formed on saliva-coated discs prepared from fluoride releasing (Unifil Flow and F2000) or non fluoride releasing materials (Filtek Z350, GRADIA DIRECT and hydroxyapatite). To assess the level of acid production and acid tolerance, glycolytic pH drop and proton permeability assays were performed using 94 h old S. mutans biofilms. To evaluate the biofilm composition, the biomass (total dry-weight), colony forming unit (CFU), water-insoluble extracellular polysaccharides (EPS), water-soluble EPS and intracellular iodophilic polysaccharides (IPS) of 94 h old S. mutans biofilms were analysed. The amount of fluoride of old culture medium released from the materials during the experimental period was also determined. Each assay was performed in duplicate in at least four different experiments (n = 8).

Results

All biofilms showed similar initial rates of acid production (0.083–0.089 pH drop/min) and proton permeability (0.025–0.036 pH increase/min), irrespective of fluoride release from the materials. On the other hand, the amount of biomass, water-insoluble EPS and IPS of the biofilms on Unifil Flow, which releases a larger amount of fluoride in the early stages of biofilm formation, were significantly lower than those on the other materials (up to 27%, 38% and 36% reduction in biomass, water-insoluble and IPS, respectively).

Conclusions

Our finding suggests that fluoride releasing resin composites might contribute to the decrease in cariogenic composition of S. mutans biofilms if an appropriate amount of fluoride is released in the early stages of biofilm formation.     

The effects of orthodontic bonding steps on biofilm formation of Streptococcus mutans in the presence of saliva

Abstract

Objective. To investigate the effects of various orthodontic bonding steps on biofilm formation of Streptococcus mutans in the presence of saliva. Materials and methods: Hydroxyapatite (HA) and orthodontic adhesive (AD) disks were prepared to a uniform size. HA disks were etched with 37% phosphoric acid gel in the etched group (HE). In the primed group (HP), Transbond XT primer was applied to the etched HA surface and light-cured. For biofilm formation, Streptococcus mutans was grown on each specimen in a biofilm medium with either glucose or sucrose in the presence of fluid-phase UWS (F-UWS) or surface adsorbed saliva (S-UWS). The adherent bacteria were quantified by enumeration of the total viable counts of bacteria. Biofilms formed on each surface were examined by scanning electron microscopy. Results. When glucose was used, both F-UWS and S-UWS suppressed biofilm formation of S. mutans. Compared to HA and HE, biofilm formation was significantly inhibited on HP and AD in the presence of glucose. Biofilm-forming patterns that were inhibited by saliva were restored in a sucrose-containing medium. F-UWS promoted biofilm formation on HA and HE, while S-UWS significantly promoted biofilm formation on HP. S. mutans developed biofilm better on HA and HE than on AD when sucrose was used as the sole carbohydrate source. Conclusions. This study suggests that the biofilm development by S. mutans is significantly influenced by the orthodontic bonding procedure. Biofilm formation of S. mutans was inhibited on AD more than other surfaces, irrespective of the presence of saliva or a carbohydrate source.     

Effect of sodium fluoride on the virulence factors and composition of Streptococcus mutans biofilms

Objective

This study aimed to evaluate the influence of NaF (2, 10, 50 and 125 ppm F⁻) on the virulence factors and composition of Streptococcus mutans biofilms.

Methods

S. mutans UA159 biofilms were formed on saliva-coated hydroxyapatite discs. To assess the influence of NaF on the virulence factors of S. mutans biofilm cells, glycolytic pH drop, proton-permeability and F-ATPase activity assay were performed using 74 h old S. mutans biofilms. Glucosyltransferase (GTF) activity assay in suspension was also performed. To examine the influence of NaF on S. mutans biofilm composition, the biofilms were treated twice daily (5 min exposure/treatment) a total of five times during biofilm formation. After a total of 5 treatments, the biomass, colony forming unit (CFU) and polysaccharide composition of the treated 74 h old S. mutans biofilms were analysed by microbiological and biochemical methods, and scanning electron microscopy.

Results

NaF showed inhibitory effects on the acid production and acid tolerance of S. mutans biofilm cells at 10, 50 and 125 ppm F⁻, compared to the vehicle control (P < 0.05) and the treatments at these concentrations also affected the biomass, water-insoluble extracellular polysaccharides and intracellular iodophilic polysaccharides of the biofilms, compared to the vehicle control (P < 0.05).

Conclusions

These results indicate that NaF (10, 50 and 125 ppm F⁻) has inhibitory effects on the virulence factors and composition of S. mutans biofilms, suggesting the potential use of these concentrations as an effective measure for controlling dental biofilms.     

Inhibitory effects of the phenolic fraction from the pomace of Vitis coignetiae on biofilm formation by Streptococcus mutans

ObjectivesThe anti-cariogenic properties of the phenolic fraction from the pomace of Vitis coignetiae (VcPP) were evaluated by in vitro assays and compared with fruit juices from V. coignetiae and common grapes and with other phenolic fractions. The effects of VcPP against the biofilm of Streptococcus mutans were investigated.DesignSucrose-dependent biofilm formation by S. mutans cultured in the presence of VcPP was measured by crystal violet dye uptake. Inhibition of adhesion to the saliva-coated hydroxyapatite (sHA) beads was quantified using fluorescent-labelled cells. The MIC for S. mutans was determined by colony counting on agar plates containing VcPP. The ability of VcPP to inhibit glucan synthesis by three distinct recombinant glucosyltransferases (Gtfs) was assessed by quantifying the production of water-soluble and -insoluble polysaccharides in bacterial cultures. In addition, the buffering effect of VcPP in cultures of S. mutans was evaluated.ResultsVcPP reduced adhesion of S. mutans to sHA and biofilm formation in a dose-dependent manner. The MIC of VcPP was 7.50 mg/ml. VcPP inhibited GtfB activity associated with the synthesis of water-insoluble glucans. It also inhibited GtfD activity associated with the synthesis of water-soluble glucans at a concentration which was lower than that used for inhibition of GtfB. VcPP had no effect on acidification associated with glucose utilization by S. mutans.ConclusionsThe current study supports the potential of VcPP as a food additive for reducing caries by inhibiting adhesion to the tooth surface and GtfD-mediated soluble glucan synthesis.     

Effect of the association of maltodextrin and sucrose on the acidogenicity and adherence of cariogenic bacteria

ObjectiveThe aim was to investigate the effect of maltodextrin and sucrose association on the acidogenic and adherence profiles of cariogenic bacteria.DesignStreptococcus mutans (S. mutans) and Lactobacillus casei (L. casei) were cultivated in culture medium containing maltodextrin, sucrose, maltodextrin–sucrose mixture or glucose. Analyses of the acidogenicity and microbial adherence were conducted in triplicate for each microorganism and tested carbohydrate.ResultsFor L. casei, maltodextrin, sucrose and maltodextrin–sucrose mixture showed lower acidogenic potential compared to glucose. When the microorganism was S. mutans, sucrose and maltodextrin–sucrose mixture presented higher acidogenic potential compared to maltodextrin and glucose. Microbial adherence analysis revealed higher adherence for S. mutans in presence of sucrose and maltodextrin–sucrose mixture compared to maltodextrin and glucose. For L. casei, all the carbohydrates showed similar adherence percentages.ConclusionThe addition of maltodextrin to sucrose does not increase the cariogenicity of sucrose in terms of acidogenicity and adherence of the cariogenic bacteria.     

Extracellular polysaccharides of smooth and rough variants of Streptococcus salivarius

Abstract – Some, but not all strains of Streptococcus salivarius were demonstrated to occur in smooth (S) and rough (R) variants, growing in distinctly different colonies on sucrose-containing agar plates. Sucrosederived extracellular polysaccharides (EP) of NCTC 8666, ATCC9759S and R, ATCC13419S and R, IAS And R, and Tove S and R were isolated, puridied, and chemically studied. Extracellular enzymes of R variants yielded more water-insoluble than soluble material, while the opposite was true for 8 variants. The insoluble material consisted mainly of glucan, the soluble mainly of levan. Enzymatic hydrolysis suggested a predominance of α-1, 3linkages in the water-insoluble glucan. Cell-associated enzymes gave rise to cell-associated and -free EP. The cell-associated EP of sS variants was insoluble, while that of R variants contained water-insoluble glucan and water-soluble fructan. Cells coated with cell-associated EP flocculated due to interaction of the Ep, mainly by hydrogen bonding, in part by divalent cation bridging. The sucrose-derived EP gave rise to plaque deposit formation in sucrose broth cultures, S variant deposits being thin and firmly adherent to glass, R variant deposits being thick, rough, coherent, but only weakly adherent. The variant types were not altered by the curing agents ethidium bromide and acridine orange.     

Effects of the natural compound,oxyresveratrol, on the growth of Streptococcus mutans,and on biofilm formation,acid production,and virulence gene expression

Streptococcus mutans is one of the major pathogens of dental caries. Oxyresveratrol, a natural compound found in plants, exerts inhibitory effects on many bacterial species but its effect on S. mutans is unknown. The objective of this study was to clarify the antibacterial effect of oxyresveratrol on S. mutans, including effects on basic viability, acidogenicity, acidurity, and extracellular polysaccharide synthesis. The expression of nine genes that encode virulence and protective factors in S. mutans was measured by qRT-PCR. Oxyresveratrol showed a dose-dependent inhibitory effect on survival of S. mutans. At 250 μg ml⁻¹, oxyresveratrol reduced the S. mutans survival rate, inhibited synthesis of water-insoluble glucans, compromised biofilm formation, and significantly down-regulated the expression of glucosyltransferase-I (gtfB) and glucosyltransferase-SI (gtfC). However, the enzymatic activity of lactate dehydrogenase protein was increased and the expression of lactate dehydrogenase (ldh) and ATP synthase subunit beta (atpD) genes were also up-regulated. Besides, glucosyltransferase S (gtfD) up-regulation indicated that water-soluble glucan synthesis was promoted. The vicR, liaR, and comDE genes, which exert a self-protective function in response to external stress, were also up-regulated. In conclusion, oxyresveratrol inhibited the growth of S. mutans and also reduced biofilm formation, acid production, and synthesis of water-insoluble glucans by this organism. In addition, oxyresveratrol also activated a series of S. mutans self-protection mechanisms.     

Evaluation of Streptococcus mutans adhesion to fluoride varnishes and subsequent change in biofilm accumulation and acidogenicity

Objectives

The aim of this study was to evaluate Streptococcus mutans adhesion to fluoride varnishes and subsequent change in biofilm accumulation and acidogenicity.

Methods

After producing fluoride varnish-coated hydroxyapatite discs using Fluor Protector (FP), Bifluorid 12 (BIF), Cavity Shield (CASH), or Flor-Opal Varnish White (FO), S. mutans biofilms were formed on the discs. To assess S. mutans adhesion to the discs, 4-h-old biofilms were analysed. To investigate the change in biofilm accumulation during subsequent biofilm formation, the biomass, colony forming units (CFU), and water-insoluble extracellular polysaccharides (EP) of 46-, 70-, and 94-h-old biofilms were analysed. To investigate the change in acidogenicity, pH values of the culture medium were determined during the experimental period. The amount of fluoride in the culture medium was also determined during the experimental period.

Results

BIF, CASH, and FO affected S. mutans adhesion (67–98% reduction) and subsequent biofilm accumulation in 46-, 70-, and 94-h-old biofilms. However, the reducing effect of the fluoride varnishes on the biomass, CFU count, water-insoluble EP amount, and acid production rate of the biofilms decreased as the biofilm age increased. These results may be related to the fluoride-release pattern of the fluoride varnishes. Of the fluoride varnishes tested, FO showed the highest reducing effect against the bacterial adhesion and subsequent biofilm accumulation.

Conclusions

Our findings suggest that if the results of these experiments are extrapolable to the in vivo situation, then reduced clinical benefit of using fluoride varnishes may occur with time.

Clinical significance

Fluoride varnish application can affect cariogenic biofilm formation but the anti-biofilm activity may be reduced with time.