Synergistic inhibition by combination of fluoride and xylitol on glycolysis by mutans streptococci and its biochemical mechanism

The purpose of this study was to evaluate the combined inhibitory effect of fluoride and xylitol on acid production by mutans streptococci, Streptococcus mutans NCTC10449 and Streptococcus sobrinus 6715, from glucose under strictly anaerobic conditions at fixed pH 5.5 and 7.0. The bacteria were grown in a tryptone-yeast extract broth under strictly anaerobic conditions (N2: 80%; H2: 10%; CO2: 10%). Reaction mixtures for acid production from glucose contained bacterial cells with fluoride (0-6.4 mM) and/or xylitol (60 mM). Acidic end products of glucose fermentation and intracellular glycolytic intermediates were assayed. The combination of fluoride and xylitol inhibited acid production more effectively than fluoride or xylitol alone. In the presence of fluoride and xylitol, the proportion of lactic acid in the total amount of acidic end products decreased, while the proportion of formic and acetic acids increased. Analyses of intracellular glycolytic intermediates revealed that xylitol inhibited the upper part of the glycolytic pathway, while fluoride inhibited the lower part. This study indicates that fluoride and xylitol together have synergistic inhibitory effects on the acid production of mutans streptococci and suggests that xylitol has the potential to enhance inhibitory effects of low concentrations of fluoride.     

Mechanisms of inhibition by fluoride of urease activities of cell suspensions and biofilms of Staphylococcus epidermidis, Streptococcus salivarius, Actinomyces naeslundii and of dental plaque

BACKGROUND/AIMS: Fluoride is known to be a potent inhibitor of bacterial ureases and can also act in the form of hydrofluoric acid as a transmembrane proton conductor to acidify the cytoplasm of intact cells with possible indirect, acid inhibition of urease. Our research objectives were to assess the inhibitory potencies of fluoride for three urease-positive bacteria commonly found in the mouth and to determine the relative importance of direct and indirect inhibition of ureases for overall inhibition of intact cells or biofilms. METHODS: The experimental design involved intact bacteria in suspensions, mono-organism biofilms, cell extracts, and dental plaque. Standard enzymatic assays for ammonia production from urea were used. RESULTS: We found that ureolysis by cells in suspensions or mono-organism biofilms of Staphylococcus epidermidis, Streptococcus salivarius or Actinomyces naeslundii was inhibited by fluoride at plaque levels of 0.1-0.5 mm in a pH-dependent manner. The results of experiments with the organic weak acids indomethacin and capric acid, which do not directly inhibit urease enzyme, indicated that weak-acid effects leading to cytoplasmic acidification are also involved in fluoride inhibition. However, direct fluoride inhibition of urease appeared to be the major mechanism for reduction in ureolytic activity in acid environments. Results of experiments with freshly harvested supragingival dental plaque indicated responses to fluoride similar to those of S. salivarius with pH-dependent fluoride inhibition and both direct and indirect inhibition of urease. CONCLUSION: Fluoride can act to diminish alkali production from urea by oral bacteria through direct and indirect mechanisms.     

Metabolomics of supragingival plaque and oral bacteria

Dental caries is initiated by demineralization of the tooth surface through acid production by sugar metabolism of supragingival plaque microflora. To elucidate the sugar metabolic system, we used CE-MS to perform metabolomics of the central carbon metabolism, the EMP pathway, the pentose-phosphate pathway, and the TCA cycle in supra- gingival plaque and representative oral bacteria, Streptococcus and Actinomyces. Supragingival plaque contained all the targeted metabolites in the central carbon metabolism, except erythrose 4-phosphate in the pentose-phosphate pathway. After glucose rinse, glucose 6-phosphate, fructose 6-phosphate, fructose 1,6-bisphosphate, dihydroxyacetone phosphate, and pyruvate in the EMP pathway and 6-phosphogluconate, ribulose 5-phosphate, and sedoheptulose 7-phosphate in the pentose-phosphate pathway, and acetyl CoA were increased. Meanwhile, 3-phosphoglycerate and phosphoenolpyruvate in the EMP pathway and succinate, fumarate, and malate in the TCA cycle were decreased. These pathways and changes in metabolites observed in supragingival plaque were similar to the integration of metabolite profiles in Streptococcus and Actinomyces.     

Challenge to Metabolomics of Oral Biofilm: —From “What Are They?” to “What Are They Doing?”—

There has been extensive bacteriological research into oral biofilm. Most of these studies examined the bacterial composition of oral biofilm and the bacterial species specific to oral diseases. It is thought that the various metabolites, intracellular metabolic intermediates and end-products produced by metabolic activities of oral biofilm directly relate to oral diseases, such as dental caries and periodontitis. However, the metabolic properties, including functional metabolic pathways and their metabolic regulations, have not been clarified sufficiently because the comprehensive identification and quantification of metabolites, that is, metabolomics (or metabolome analysis) are technically difficult. Recently, a new device consisting of capillary electrophoresis (CE) and a time-of-flight mass spectrometer (TOFMS) has been developed, facilitating the metabolomic investigation of sugar metabolism in oral biofilm. The results clearly showed that oral biofilm metabolomics is possible even with a very small sample, and that almost all metabolites in the central carbon metabolic pathways, the EMP pathway, the pentose-phosphate pathway, and the TCA cycle are present in the oral biofilm in vivo. Metabolomics using CE-TOFMS may offer a new frame of knowledge to answer the question “What are bacteria doing in oral biofilm?” from a metabolic perspective, and possibly provide insight into the relationship between the metabolic properties of oral biofilm and the etiology of oral disease, as well as the influences of medicines, such as fluoride, or a sugar substitute, such as xylitol, on metabolism of the oral biofilm.     

Metabolomic approach to oral biofilm characterization—A future direction of biofilm research

Research approaches to biofilm are stratified by a series of analyses: (1) microbial number and species; (2) microbial proteins, such as enzymes; and (3) microbial activity, such as metabolic activity. On the other hand, the hierarchical structure of biology includes the genome, proteome, and metabolome, in which the metabolome is the final output of biological function. Metabolome analysis is the comprehensive analysis of the metabolome, a new strategy for biological research in the 21st century. The stratified structure of biofilm research corresponds to the biological hierarchy, and the analysis of microbial activity, especially metabolic activity, is comparable to metabolome analysis; however, oral biofilm samples are too small to analyze the metabolome by conventional methods. Recently, a new device involving capillary electrophoresis (CE) and time-of-flight mass spectrometry (MS) has been developed, facilitating metabolomic investigation of the central carbon metabolic pathways (i.e., the EMP pathway, pentose phosphate pathway, and TCA cycle) in oral biofilm. Using CE–MS, we analyzed metabolome profiles of oral biofilm after oral rinsing with glucose in vivo and evaluated the effects of oral rinsing with fluoride and xylitol on the metabolome profiles of oral biofilm. The results were somewhat consistent with previous in vitro data obtained from single bacterial strains, namely, Streptococcus and Actinomyces; however, new information describing the metabolic properties of oral biofilm was also obtained. This metabolomic approach will reveal the functional characteristics of oral biofilm in vivo, potentially providing new insights into the nature of oral biofilm in health and disease.     

The effect of delmopinol and fluoride on acid adaptation and acid production in dental plaque biofilms

Objective

To investigate the effect of delmopinol and fluoride alone or in combination on acid adaptation and acid production in plaque biofilm bacteria in vitro.

Design

The effect of delmopinol and fluoride on acid adaptation was tested by exposing the biofilm bacteria, grown in a mini-flow cell system under static conditions, to pH 5.5 overnight in the presence of 0.16 mM delmopinol, 1 mF NaF or a combination of both. The following day, acid adaptation was evaluated by exposing the cells to an acid challenge for 2 h at a pH known to kill non-adapted cells (pH 2.5). The cells were stained using LIVE/DEAD^® BacLight™ Viability stain and the number of viable (acid tolerant) cells was determined using confocal scanning laser microscopy. Control cells were treated in the same manner but without the exposure to delmopinol or fluoride. How delmopinol and fluoride affected acid production was assessed by measuring the pH-drop after glucose pulsing in the presence of delmopinol and/or different concentrations of fluoride.

Results

Fluoride alone or in combination with delmopinol affected the acid adaptation and significantly reduced the acid tolerance of the plaque biofilm. This effect was more pronounced when the two compounds were combined. Delmopinol alone did not affect acid adaptation. A combination of delmopinol and fluoride also reduced acid production at concentrations where neither of the compounds in isolation had an effect.

Conclusion

Fluoride and delmopinol can work synergistically to affect acid adaptation and acid production in plaque biofilm bacteria.     

Microbiological aspects of some caloric sugar substitutes

Several caloric sugar substitutes are available today, e.g. maltitol, Lycasin, sorbitol, xylitol, palatinit, sorbose, coupling sugar, palatinose, fructose and invert sugar. When evaluating the cariogenicity of these sugar substitutes from a bacteriological point of view, different analytical procedures should be considered. In vitro studies on the capacity of pure oral bacterial strains and dental plaque material to decompose the substitute to acid. Studies on the catabolism of the substitute. Possible adaptation of the oral microflora to metabolize the substitute in vivo as well as in vitro. Studies on any inhibitory effect of the substitute on the microbial activities in dental plaque and on the oral microflora. The capacity of oral bacteria, e.g. Streptococcus mutans, to induce caries in animal experiments should also be considered. Results from such microbiological studies make it possible to classify the caloric sugar substitutes into different groups. The first, most suitable, group seems to consist of xylitol and sorbose. These substitutes are fermented by few oral bacteria and no negative adaptation of the oral flora has been observed. A reducing effect upon dental plaque formation and on acid production from glucose has been reported. A second group consists of maltitol, palatinite, palatinose, sorbitol and Lycasin. These substitutes are fermented by certain groups of organisms within the oral genera Actinomyces, Lactobacillus and Streptococcus. Adaptation of oral bacteria to palatinose and to sorbitol, resulting in a more pronounced acid production, has been observed. No inhibitory effects have been reported. The results from experimental studies suggest that these sugar substitutes have no or little cariogenic action in rodents inoculated with S. mutans.(ABSTRACT TRUNCATED AT 250 WORDS)     

应用新一代高通量测序技术探讨含氟涂料对患龋乳前牙菌斑微生物的影响

目的 应用新一代高通量测序技术,了解含氟涂料处理患龋乳前牙前后菌斑微生物的变化。方法 选取20名乳前牙患龋的学龄前儿童,菌斑生物膜样本采自含氟涂料处理前和处理后3天、7天和30天的乳前牙患龋牙面;抽提全基因组DNA后,用16S rRNA基因V4-V5高变区为引物扩增后,运用MiSeq测序平台测序并分析。结果 4个时间点菌斑生物膜微生物多样性Shannon指数的比较表明,涂氟后3天、后7天比涂氟前的Shannon指数更低（P=0.011;P=0.016）,提示菌斑生物膜的微生物多样性降低;涂氟后30天和涂氟前的Shannon指数持平（P>0.05）,提示涂氟后菌斑生物膜的微生物多样性恢复至涂氟前的状态;PCA分析也进一步表明和Shannon指数一致;4个时间点优势菌属的比较表明涂氟后3天、后7天、后30天和涂氟前优势菌属的种类和相对丰度均没有差异,说明含氟涂料对菌斑生物膜的多样性的抑制作用没有特异性。结论 含氟涂料对患龋乳前牙牙面菌斑微生物在一定时间内有明显的抑制作用,抑制作用无特异性,微生物多样性经过一段时间还能恢复至原有状态。     

Effect of sodium fluoride on oral biofilm microbiota and enamel demineralization

ObjectiveFluoride is widely used as an anti-caries agent, e.g. in toothpastes and mouth rinses. However, the nature of the anti-caries action is not entirely clear. Mechanisms suspected to explain the cariostatic effect include inhibitory effects on acid formation by bacteria, inhibition of extracellular polysaccharide (EPS) production, inhibition of enamel demineralization and enhancement of remineralizaton or combination thereof. The aim of this study was to examine with the supragingival Zurich in vitro biofilm model the effect of fluoride in NaF formulation, on the microbiota and on demineralization.MethodsBiofilms consisting of Actinomyces oris, Candida albicans, Fusobacterium nucleatum, Streptococcus oralis, Veillonella dispar and Streptococcus sobrinus, were grown anaerobically on sintered hydroxyapatite or bovine enamel disks, exposed to 200, 400, and 1400 ppm of NaF, or 0.1% chlorhexidine (positive control). The biofilms were harvested after 64 h and CFUs were assessed for total bacteria. Demineralization of enamel disks was measured by quantitative light-induced fluorescence.ResultsNaF did not affect the bacterial numbers. No enamel mineral loss was observed at 1400 and 400 ppm of fluoride, whereas the pH of the surrounding medium was increased to 5.5 and 5.0, respectively, compared to the untreated control (pH 4.5 and mineral loss ΔF of −32%). At 1400 ppm NaF the biofilm’s EPS volume was also significantly reduced.ConclusionsAdministration of NaF completely prevented demineralization without affecting biofilm composition and growth. This protective effect may be attributed to the observed decrease in acid production or EPS volume, or to a shift in the de/remineralization balance.     

Effect of xylitol on dental plaque in vivo during carbohydrate challenge

Abstract – Xylitol has previously been shown to inhibit acid production in vitro when glucose is used as energy source, and the present studies were carried out to investigate whether this effect was valid in vivo. A solution containing both xylitol and glucose was applied on sucrose—induced 4-day-old plaque in vivo. The xylitol added to the glucose solution inhibited the acid production in the plaque, measured as a drop in pH, compared to using glucose alone. A further reduction in acidogenicity was obtained when xylitol was used as a rinse for 1 hr continuously prior to the glucose challenge.     

Release of mineral ions in dental plaque following acid production

The release of appreciable amounts of calcium, phosphate and fluoride found in whole plaque into the plaque-fluid phase, following bacterial acid production, can potentially reduce the driving force for tooth demineralization. However, limited information is available on this topic, particularly on the release of fluoride. This study sought to determine the change in calcium, phosphate and fluoride concentrations in plaque fluid after sucrose exposure. 48 h overnight-fasted supragingival plaque samples were collected from all tooth surfaces (with the exception of the lower lingual anterior teeth) of one half of an individual mouth, following a 1 min water rinse. Plaque samples were then collected from the other half of the same mouth, following a 292 mM sucrose rinse. Plaque fluid was isolated by centrifugation and analysed for total calcium and phosphate (ion chromatography) and for free fluoride (ion-specific electrode). Samples were collected from seven individuals. Following sucrose exposure, plaque-fluid pH decreased significantly from 6.5+/- 0.3 to 5.4+/-0.2; calcium concentrations (mmol/l) also increased significantly (p < 0.01) from 1.9+/-0.5 to 5.0+/-2.1. Fluoride and phosphate concentrations in plaque fluid, however, did not increase significantly after sucrose exposure: mean concentrations (mmol/l) of fluoride after the water and sucrose rinses were 0.006+/-0.003 and 0.005+/-0.002, respectively, and mean phosphate concentrations (mmol/l) were 11.0+/-2.0 and 12.0+/-3.0, respectively. When results were expressed per wet plaque weight, phosphate concentrations were also found to increase significantly. The same trends were observed when additional plaque samples were treated in vitro with sucrose: fluoride-ion activity did not increase in plaque under in vivo-like conditions.     

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氟是人体可能必需的微量元素,适量摄入可对机体代谢产生积极影响。氟亦可通过降低釉质溶解度和促进再矿化、对微生物产生作用达到预防龋病的目的。氟化物应用可分为全身应用和局部应用。目前在我国,局部用氟已成为广泛使用、安全有效的防龋方法之一。     

Inhibition of acid production from oral bacteria by fluorapatite-derived fluoride

The inhibitory effect of fluorapatite (FAP)-derived fluoride upon resting cell suspensions of Streptococcus mutans incubated at pH 4.5 and 6.5 was studied using lactic acid production from 0.1% sucrose as an indicator of fermentation activity. Cells incubated with FAP produced significantly less lactic acid than did cells incubated with hydroxyapatite (HAP). Addition of HAP to cell suspensions containing FAP reduced this inhibition, suggesting that dissolution of the FAP was necessary for inhibition. Incubation with low concentrations of NaF showed significant inhibition in cell suspensions incubated with as little as 0.45 micrograms/mL F at pH 5.0. These results provide further support to the hypothesis that fluoride levels in plaque and enamel, achievable through use of fluoridated water and/or fluoride dentifrices, may produce appreciable inhibition of glycolysis at the acidic pH levels which are readily achieved in plaque. Thus, bacterial acid production may activate plaque and enamel-bound fluoride, resulting in inhibition of further acid production, and thereby contribute substantially to the other cariostatic mechanisms of fluoride.     

Comparative antimicrobial activity of an essential oil and an amine fluoride/stannous fluoride mouthrinse in vitro

Although laboratory studies are not necessarily predictive of clinical activity; they can help to elucidate mechanisms underlying clinical activity when the latter has been established. In a recent clinical study, an essential oil mouthrinse (Listerine Antiseptic) was shown to be significantly more effective than an amine fluoride/stannous fluoride mouthrinse (Meridol) in inhibiting supragingival plaque formation. This paper reports the results of laboratory studies comparing the antimicrobial effectiveness of these 2 mouthrinses using a kill kinetics assay and a plaque biofilm kill assay. In both assays, the essential oil mouthrinse was considerably more effective than the amine fluoride/stannous fluoride mouthrinse. These findings are consistent with the results of the clinical trial and may help to explain the observed differences in clinical activity.     

Fluoride profiles in dental plaque in vivo formed on fluoride pre-treated human enamel

Using a novel device capable of generating plaque in vivo on a natural enamel substrate, it has been possible to determine fluoride profiles from the saliva-plaque interface towards the enamel surface. Fluoride profiles in dental plaques tended to fall from the saliva-plaque interface towards the enamel. The device also offered the possibility of examining fluoride distributions after pre-treatment of the enamel with fluoride in vitro. Fluoride profiles were determined in plaque generated in vivo on enamel surfaces, which had been previously treated with a 900-ppm fluoride solution. The results showed the previously reported fall from the plaque surface, but in addition, a further rise towards the enamel surface was seen. The data imply that enamel loaded with fluoride can release some of this fluoride back into the plaque and may act as a fluoride reservoir.     

The effect of A Triclosan/copolymer/fluoride dentifrice on plaque formation and gingivitis: a six-month clinical study

A total of 108 adult male and female subjects completed a six-month, double-blind clinical study designed to compare the effect of a dentifrice containing 0.3% triclosan and 2.0% of a copolymer of methoxyethylene and maleic acid (Gantrez copolymer) in a 0.243% sodium fluoride/silica base to a 0.243% sodium fluoride/silica placebo dentifrice on supragingival plaque formation and gingivitis. Subjects were stratified into two balanced groups by their baseline plaque and gingivitis scores. They received an oral prophylaxis and were assigned to use either the triclosan/copolymer dentifrice or the placebo dentifrice for the next six months. After six months, the dentifrice containing triclosan/copolymer provided a 24.93% statistically significant (99% level of confidence) reduction in supragingival plaque, and a 19.72% statistically significant (99% level of confidence) reduction in gingivitis compared to the placebo dentifrice. Twice daily use of a dentifrice containing 0.3% triclosan, 2.0% of a copolymer, and 0.243% sodium fluoride in a silica base, over a six-month period, reduces supragingival plaque formation and gingivitis to a significant degree compared to a sodium fluoride/silica base placebo dentifrice.     

Effect of xylitol on salivary β‐glucosidase in humans

Dental biofilm – in which a diverse set of microorganisms are embedded in a complex polysaccharide matrix that adheres to oral components – is one of the most complex microbial communities in the human body. As biofilm formation is related to oral infections, such as caries and periodontal diseases, strategies for biofilm control are crucial for maintaining oral health. Xylitol, a synthetic sugar used as a sucrose substitute, has been shown to reduce biofilm formation. However, its precise mechanism of action on biofilm reduction has so far not been elucidated. Previous studies demonstrate that bacterial β‐glucosidase action is crucial for biofilm formation. Here, we investigated the correlation between salivary β‐glucosidase activity and dental plaque occurrence. We found a positive correlation between enzymatic activity and the presence of dental biofilm. We observed that xylitol inhibits β‐glucosidase in human saliva. Kinetic studies also confirmed that xylitol acts as a mixed type inhibitor of salivary β‐glucosidase. Based on our data, we suggest that xylitol impairs oral biofilm formation by the inhibition of bacterial β‐glucosidase, which is essential for biofilm formation in the oral cavity.     

The effect of fluoride on Streptococcus sanguis 7863 IGA1 protease production and activity

Fluoride was found to affect the production of the bacterial IgAl protease but to have no effect on IgAl protease activity. The concentrations of fluoride that do affect Streptococcus sanguis growth and IgAl protease production are higher than those normally seen in vivo under normal circumstances. The concentrations of fluoride in dental plaque following use of a fluoride rinse or dentifrice would be sufficient to reduce Strep. sanguis IgAl protease production.     

Evidence for xylitol 5-P production in human dental plaque.

The Turku sugar studies indicated that xylitol may possess a caries-therapeutic effect. More recent data show that xylitol exhibits a bacteriostatic activity on a wide range of bacteria based on uptake and expulsion of xylitol. Intracellular xylitol 5-P appears to be a key substance associated with inhibition of bacterial metabolism by xylitol. This has been shown in studies with pure strains of bacteria, mainly Streptococcus mutans. The aim of the present study was to examine if production of xylitol 5-P occurs in freshly collected dental plaque which is exposed to labeled xylitol. Plaque extracts were analyzed by thin-layer chromatography combined with autoradiography and high performance liquid chromatography. Strong indications were obtained that xylitol 5-P is readily produced by dental plaque. No other significant xylitol metabolites were identified. The bacteriostatic properties of xylitol in plaque are a mechanism which may well account for the caries-therapeutic effect of xylitol.     

Evidence for xylitol 5-P production in human dental plaque

The Turku sugar studies indicated that xylitol may possess a caries-therapeutic effect. More recent data show that xylitol exhibits a bacteriostatic activity on a wide range of bacteria based on uptake and expulsion of xylitol. Intracellular xylitol 5-P appears to be a key substance associated with inhibition of bacterial metabolism by xylitol. This has been shown in studies with pure strains of bacteria, mainly Streptococcus mutans . The aim of the present study was to examine if production of xylitol 5-P occurs in freshly collected dental plaque which is exposed to labeled xylitol. Plaque extracts were analyzed by thin-layer chromatography combined with autoradiography and high performance liquid chromatography. Strong indications were obtained that xylitol 5-P is readily produced by dental plaque. No other significant xylitol metabolites were identified. The bacteriostatic properties of xylitol in plaque are a mechanism which may well account for the caries-therapeutic effect of xylitol.