Why is sucrose so cariogenic? The role of glucosyltransferase and polysaccharides

Abstract – Some aspects of the cariogenicity of sucrose are discussed, in particular the data indicating that glucosyltransferase (GTF) mediates the well known stickiness of sucrose exposed S. mutans and plaque in vivo. The non-sucrose dependent colonization of teeth, which has been studied extensively, is judged to be of less importance because S. mutans is only pathogenic in combination with sucrose. Strong evidence from several laboratories show that free GTF is present in saliva and that it adsorbs to the pellicle and is able to form glucan in the adsorbed state. Glucan-glucan interaction between 1–3 linked glucans gives a strong sucrose dependent interaction between glucan chains originating from the GTF adsorbed to the pellicle and from GTF on the bacterial surface.     

Why is sucrose so cariogenic? The role of glucosyltransferase and polysaccharides

Some aspects of the cariogenicity of sucrose are discussed, in particular the data indicating that glucosyltransferase (GTF) mediates the well known stickiness of sucrose exposed S. mutans and plaque in vivo. The non-sucrose dependent colonization of teeth, which has been studied extensively, is judged to be of less importance because S. mutans is only pathogenic in combination with sucrose. Strong evidence from several laboratories show that free GTF is present in saliva and that it adsorbs to the pellicle and is able to form glucan in the adsorbed state. Glucan-glucan interaction between 1-3 linked glucans gives a strong sucrose dependent interaction between glucan chains originating from the GTF adsorbed to the pellicle and from GTF on the bacterial surface.     

Effect of sucrose concentration on dental biofilm formed in situ and on enamel demineralization

The relationship between sucrose concentration and cariogenic potential was studied in situ. Adult volunteers wore intraoral palatal appliances containing human dental enamel blocks, which were extraorally submitted 8 times a day for 14 days, to the treatments: deionized distilled water and sucrose solutions from 1 to 40%. The biofilm formed was analyzed with respect to acidogenicity and biochemical composition; enamel demineralization was evaluated by microhardness. The results showed that 1% sucrose is less cariogenic than 5% or higher concentrations, although sucrose solution at 40% was still able to increase the concentration of insoluble polysaccharide in the biofilm formed. The findings suggest that the threshold of sucrose solution concentration for the formation of a cariogenic biofilm is 5%, which provided the same cariogenic potential as that observed for 10 and 20% sucrose solution.     

不同培养条件对口腔细菌生物膜生长的影响

目的：研究5种口腔细菌在体外形成生物膜的能力、形成过程及不同培养条件对生物膜形成的影响,并通过激光共聚焦扫描显微镜（CISM）观察细菌生物膜的形成及结构特征。方法：选择与龋病发生关系密切的5种口腔细菌,分别接种在含牛心脑浸液培养基（BHI）、人工唾液（BM5）以及人唾液培养基的标准96孔板中,在培养6h、8h、12h、24h、36h、48h和60h后取出相应的微孔板,11％Crystal Violet染色生物膜,95％乙醇复吸Crystal Violet,HTS7000PLUS多孔板高效分析仪测定各时段细菌生物膜的生长情况,并绘制生长曲线。CLSM观察细菌生物膜形成的结构变化。结果：在BHI及BM5中5种口腔细菌均能形成生物膜,在人唾液中没有形成生物膜。细菌生物膜形成表现为缓慢的非线性生长,出现了一个相对的生长停滞期,这个时期出现在36～48h之间,不同细菌略有差别。激光共聚焦扫描显微镜（asM）观察发现在培养6h后仅有少量细菌粘附形成散在微菌落,24h后出现生物膜的基本结构,48h形成成熟的生物膜。结论：研究发现在体外细菌形成生物膜的能力与培养基条件密切相关,细菌生物膜形成缓慢,其间有一个相对生长停滞期。细菌定植,粘附,共集聚是生物膜形成的重要步骤。     

Influence of sucrose and xylitol on an early Streptococcus mutans biofilm in a dental simulator

ObjectivesIn vitro methods to study dental biofilms are useful in finding ways to support a healthy microbial balance in the oral cavity. The effects of sucrose, xylitol, and their combination on three strains of Streptococcus mutans and one strain of Streptococcus sobrinus were studied using a dental simulator.MethodsA simulator was used to mimic the oral cavity environment. It provided a continuous-flow system using artificial saliva (AS), constant temperature, mixing, and hydroxyapatite (HA) surface in which the influence of xylitol was studied. The quantities of planktonic and adhered bacteria were measured by real-time qPCR.ResultsCompared against the untreated AS, adding 1% sucrose increased the bacterial colonization of HA (p < 0.0001) whereas 2% xylitol decreased it (p < 0.05), with the exception of clinical S. mutans isolate 117. The combination of xylitol and sucrose decreased the bacterial quantities within the AS and the colonization on the HA by clinical S. mutans isolate 2366 was reduced (p < 0.05). Increasing the concentration (2%–5%) of xylitol caused a reduction in bacterial counts even in the presence of sucrose.ConclusionsThe continuous-culture biofilm model showed that within a young biofilm, sucrose significantly promotes whereas xylitol reduces bacterial colonization and proliferation. The results indicate that xylitol affects the ability of certain S. mutans strains to adhere to the HA. Clinical studies have also shown that xylitol consumption decreases caries incidence and reduces the amount of plaque. This study contributes to the understanding of the mechanism behind these clinical observations.     

Effect of starch and sucrose on dental biofilm formation and on root dentine demineralization

The cariogenicity of starch alone or in combination with sucrose is controversial and the effect on dentine demineralization and on the dental biofilm formed has not been explored under controlled conditions. A crossover, single-blind study was conducted in four steps of 14 days each, during which 11 volunteers wore palatal appliance containing 10 slabs of root dentine to which the following treatments were applied extraorally: 2% starch gel-like solution (starch group); 10% sucrose solution (sucrose group); a solution containing 2% starch and 10% sucrose (starch + sucrose group), or 2% starch solution followed by 10% sucrose solution (starch --> sucrose group). On the 14th day of each phase the biofilms were collected for biochemical and microbiological analyses, and dentine demineralization was assessed by hardness. A higher demineralization was found in dentine exposed to sucrose and starch sucrose combinations than to starch alone (p < 0.01), but the sucrose-containing groups did not differ significantly from each other (p > 0.05). The concentrations of soluble and insoluble extracellular polysaccharides (EPS), and the proportion of insoluble EPS, were lower in the biofilm formed in presence of starch (p < 0.01) than in those formed in the presence of sucrose or sucrose/starch combinations; however, no significant difference was observed among the groups containing sucrose (p > 0.05). RNA was successfully isolated and purified from in situ biofilms and only biofilms formed in response to sucrose and starch/sucrose combinations showed detectable levels of gtfB and gtfC mRNA. The findings suggest that the combination of starch with sucrose may not be more cariogenic to dentine than sucrose alone.     

Effect of sucrose on the selection of mutans streptococci and lactobacilli in dental biofilm formedin situ

Microorganisms are selected in dental biofilm by the acidic environment created by sugar fermentation, but the effect of extracellular polysaccharide (EPS) on the counts of cariogenic bacteria is not clear. Dental biofilm was formed in situ for 13 days under exposure 8 times a day to distilled-deionized water, glucose + fructose or sucrose solutions. Mutans streptococci (MS) counts were not different among the groups, but lactobacilli (LB) were significantly higher in glucose + fructose and sucrose groups, without significant difference between them, irrespective of the higher insoluble EPS concentration in the sucrose biofilm matrix. The data suggest that exposure to sugar is more relevant for the predominance of LB in dental biofilm than for MS and that insoluble EPS does not change the counts of these microorganisms in the biofilm.     

Effect of sucrose containing iron (II) on dental biofilm and enamel demineralization in situ

Since the effect of iron (Fe) on the cariogenicity of sucrose in humans is unexplored, this study assessed in situ the effect of Fe co-crystallized with sucrose (Fe-sucrose) topically applied in vitro on the acidogenicity, biochemical and microbiological composition of the dental biofilm formed in vivo and on the demineralization of the enamel. During two phases of 14 days each, 16 volunteers wore palatal appliances containing blocks of human enamel, which were submitted to four groups of separate treatments: (1) water; (2) 20% sucrose; (3) 20% (w/v) sucrose plus 18 microg Fe/ml, and (4) 20% (w/v) sucrose plus 70 microg Fe/ml. The solutions were dripped onto the blocks 8 times per day. The biofilms formed on the blocks were analyzed with respect to acidogenicity, biochemical and microbiological composition. Mineral loss was determined on enamel by surface and cross-sectional microhardness. Lower demineralization was found in the blocks subjected to Fe-sucrose (70 microg Fe/ml) than in those treated with sucrose (p < 0.05). This concentration of Fe also reduced significantly the populations of mutans streptococci in the biofilm formed on the blocks. In conclusion, our data suggest that Fe may reduce in situ the cariogenic potential of sucrose and the effect seems to be related to the reduction in the populations of mutans streptococci in the dental biofilm formed.     

The effect of dental restorative materials on dental biofilm

To investigate the arrangement of biofilms formed in vivo, volunteers wore splints with slabs of six different dental materials inserted to collect smooth surface plaque. After 5 d of undisturbed plaque accumulation, the specimens were vital stained and analyzed by the confocal laser scanning microscopy (CLSM) to evaluate the percentage of vital biofilm microflora (VF percentage). Further parameters were the area of the specimens covered by plaque (surface coating; SC, %) and the height of the biofilms (BH, pm). The metals amalgam and gold, the compomer, as well as the glass-ionomer cement harboured an almost entirely dead biofilm (VF <8%). Resin composite led to vitality values between 4 and 21%, while a very thin biofilm on ceramic revealed the highest vitality values (34-86%). SC varied from 6% on glass-ionomer cement to 100% on amalgam. BH reached its highest value on amalgam and gold of 17 and 11 microm, respectively, while heights of between 1 and 6 microm were found on the ceramic, resin composite, compomer and the glass-ionomer cement. Within their limits, the present findings indicate that amalgam, gold, compomer and glass-ionomer cement exert an influence against the adhering biofilm. No general relationship could be established between the different parameters VF percentage, SC percentage and BH (microm).     

牙周致病菌和致龋菌间生长关系的体外动态观察

目的研究6种代表性牙周致病菌和致龋菌在菌斑生物膜和悬浮液中的消长关系。方法将双菌组（致龋菌＋牙周致病菌）接种于改良恒化器中,连续培养1h、24h、48h和96h,然后取在羟基磷灰石表面形成的生物膜和悬浮液再进行细菌培养。结果与血链球菌混合培养时,菌斑生物膜和悬浮液中牙周致病菌均增多,而血链球菌明显减少（P〈0.05）;具核梭杆菌与变形链球菌培养时,菌斑生物膜和悬浮液中具核梭杆菌明显增多,而变形链球菌减少（P〈0.05）。菌斑生物膜中致龋菌占优势,相应的悬浮液中牙周致病菌96h开始占优势。悬浮液中细菌量波动更明显。结论2类致病菌在菌斑生物膜与相应的悬浮液中的生长是不一致的。2类致病菌之间相互作用也许是决定菌斑生物膜内部生态环境发展方向的主要因素。     

Antimicrobial activity of nanoemulsion on cariogenic planktonic and biofilm organisms

IntroductionNanoemulsions (NE) are a unique class of disinfectants produced by mixing a water immiscible liquid phase into an aqueous phase under high shear forces. NE have antimicrobial properties and are also effective anti-biofilm agents.Materials and methodsThe effectiveness of nanoemulsion and its components was determined against Streptococcus mutans and Lactobacillus casei by live/dead staining. In vitro antimicrobial effectiveness of nanoemulsion against planktonic S. mutans, L. casei, Actinomyces viscosus, Candida albicans and mixed culture was determined by a serial dilution technique to obtain minimum inhibitory concentration and minimum bactericidal concentration (MIC/MBC). In addition, efficacy was investigated by kinetics of killing, adherence and biofilm assays.ResultsCompared to its components, nanoemulsion showed notable antimicrobial activity against biofilm organisms, up to 83.0% kill within 1 min. NE dilutions ranging from 243 to 19683 were effective against planktonic S. mutans, L. casei, A. viscosus, C. albicans and mixed culture of these four strains as shown through MIC/MBC assays. NE showed antimicrobial activity against planktonic cells at high dilutions, confirmed by time kill studies. The level of adhesion on glass surface was reduced by 94.2–99.5% in nanoemulsion treated groups (p < 0.001). 4-Day-old S. mutans, L. casei, A. viscosus, C. albicans and mixed cultures biofilms treated with NE showed reductions of bacterial counts with decreasing dilutions (p < 0.001).ConclusionThese results suggest that nanoemulsion has effective anti-cariogenic activity against cariogenic microorganisms and may be a useful medication in the prevention of caries.     

The cariogenic characters of xylitol-resistant and xylitol-sensitive Streptococcus mutans in biofilm formation with salivary bacteria

Streptococcus mutans metabolize carbohydrates, such as glucose and sucrose, to produce acid and enhance biofilm formation with the early colonizing bacteria to induce dental caries. Xylitol has been used as a reliable substitute for carbohydrate to inhibit the acid production of S. mutans. However, long-term xylitol consumption leads to the emergence of xylitol-resistance in S. mutans. The aim of this study was to investigate the cariogenic trait of Xylitol-resistant (X(R)) S. mutans using biofilm formation and coaggregation of xylitol-sensitive (X(S)) and X(R) S. mutans with salivary bacteria and their glucosyltransferases expression. When X(S) or X(R) S. mutans were incubated in brain heart infusion broth with bacteria from human saliva, X(R)S. mutans exhibited reduction in biofilm formation in comparison to X(S) S. mutans. The coaggregation between X(R) S. mutans and S, gordonii, S. mitis, S. oralis or S. sanguinis was less pronounced than that of X(S) S. mutans in the presence of sucrose. However, there was no difference in the coaggregation between X(R) and X(S) S. mutans in the sucrose-free condition. The level of gtfB and gtfC mRNA expression of X(R) S. mutans was lower than that of X(S) S. mutans, whilst the level of gtfD mRNA expression did not differ between the two strains. The reduction of biofilm formation in X(S) S. mutans due to decrease in glucosyltransferases expression suggests that X(R) S. mutans may be less cariogenic than X(S) S. mutans.     

Correlation of cariogenic bacteria and dental caries in adults

Many studies suggest that mutans streptococci (MS), Lactobacillus (LB), and salivary buffering capacity are important risk factors for dental caries. However, target populations for most studies were children. In adult patients, the same risk factors affect the number of fillings or prostheses or secondary caries. It is therefore important to investigate these risk factors as predictors of caries in adults. In the present study, we evaluated the oral conditions of adult subjects at private dental offices using bite-wing radiographs. Detection of salivary LB level using Dentocult LB had a statistically significant correlation with the number of flat surface caries and approximal caries (P < 0.001). Detection of salivary MS level using Dentocult MS and salivary buffering capacity did not predict dental caries. Thus, detection of salivary LB level using Dentocult LB may be a useful tool for detecting approximal and secondary caries.     

Effect of frequency of sucrose exposure on dental biofilm composition and enamel demineralization in the presence of fluoride

It has been suggested that enamel would resist higher frequencies of sucrose exposure if fluoride from water or dentifrice is being used. However, the effect of increasing frequencies of sugar on dental biofilm composition is not well known. Ten volunteers living in a fluoridated area wore palatal appliances bearing human enamel slabs during 14 days. The slabs were exposed to 20% sucrose solution 0 (control), 2, 4, 6, 8 or 10 times/day and the volunteers used fluoride dentifrice 3 times/day. Enamel demineralization was significantly greater than control for sucrose frequencies higher than 6 times/day. However, biofilm mass, total microbiota, total streptococci, lactobacilli counts and insoluble extracellular polysaccharide concentration increased, while Ca, P(i) and F concentration in whole biofilm decreased significantly, with frequencies of sucrose exposure lower than 6 times/day. The findings confirm that fluoride can reduce enamel demineralization if sucrose consumption is not higher than 6 times/day, but changes in the biochemical and microbiological composition of the biofilm are observed with lower frequencies of sucrose use.     

伊犁黑蜂蜂胶对口腔主要致龋细菌生物膜作用的实验研究

目的 研究伊犁黑蜂蜂胶对口腔常见致龋细菌生物膜的影响,探讨其防龋效果及可能的防龋机制。方法 通过结晶紫染色法测定黑蜂蜂胶对口腔常见致龋菌（变异链球菌、表兄链球菌、血链球菌、黏性放线菌、内氏放线菌）的最小生物膜清除浓度（MBEC）;培养测试细菌24 h单菌生物膜,加入MBEC及以下的3个浓度配置成初始pH值为7.0的含药培养基,厌氧培养24 h后测pH值,并计算pH变化值以检测不同浓度黑蜂蜂胶对测试菌单菌生物膜产酸能力的影响。蒽酮法测定MBEC及以下的3个浓度的黑蜂蜂胶对变异链球菌24 h单菌生物膜产生水不溶性胞外多糖的影响。结果 黑蜂蜂胶对变异链球菌、表兄链球菌、血链球菌、黏性放线菌、内氏放线菌的MBEC分别是6.25、1.56、3.13、0.78、0.78 mg•mL-1;黑蜂蜂胶可使各测试菌单菌生物膜ΔpH降低,蜂胶各组与对照组相比差异均有统计学意义（P<0.05）;在MBEC浓度时,蜂胶可使变异链球菌合成水不溶性胞外多糖的能力降低。结论 伊犁黑蜂蜂胶具有一定的防龋效果,其可能的防龋机制是通过有效清除口腔主要致龋细菌单菌生物膜,抑制测试菌株产酸、合成水不溶性胞外多糖的能力起作用的。     

Extracellular matrix influence in Streptococcus mutans gene expression in a cariogenic biofilm

Caries etiology is biofilm–diet‐dependent. Biofilms are highly dynamic and structured microbial communities enmeshed in a three‐dimensional extracellular matrix. The study evaluated the expression dynamics of Streptococcus mutans genes associated with exopolysaccharides (EPS) (gtfBCD, gbpB, dexA), lipoteichoic acids (LTA) (dltABCD, SMU_775c) and extracellular DNA (eDNA) (lytST, lrgAB, ccpA) during matrix development within a mixed‐species biofilm of S. mutans, Actinomyces naeslundii and Streptococcus gordonii. Mixed‐species biofilms using S. mutans strains UA159 or ΔgtfB formed on saliva‐coated hydroxyapatite discs were submitted to a nutritional challenge (providing an abundance of sucrose and starch). Biofilms were removed at eight developmental stages for gene expression analysis by quantitative polymerase chain reaction. The pH of spent culture media remained acidic throughout the experimental periods, being lower after sucrose and starch exposure. All genes were expressed at all biofilm developmental phases. EPS‐ and LTA‐associated genes had a similar expression profile for both biofilms, presenting lower levels of expression at 67, 91 and 115 hours and a peak of expression at 55 hours, but having distinct expression magnitudes, with lower values for ΔgtfB (eg, fold‐difference of ~382 for gtfC and ~16 for dltB at 43 hours). The eDNA‐associated genes presented different dynamics of expression between both strains. In UA159 biofilms lrgA and lrgB genes were highly expressed at 29 hours (which were ~13 and ~5.4 times vs ΔgtfB, respectively), whereas in ΔgtfB biofilms an inverse relationship between lytS and lrgA and lrgB expression was detected. Therefore, the deletion of gtfB influences dynamics and magnitude of expression of genes associated with matrix main components.