Divalent cations enhance fluoride binding to Streptococcus mutans and Streptococcus sanguinis cells and subsequently inhibit bacterial acid production.

One preventive effect of topical fluoride application is derived from the fact that fluoride can inhibit bacterial acid production. Furthermore, divalent cations such as Ca(2+) and Mg(2+) increase the binding of fluoride to bacterial cells. These findings suggest that exposure of oral bacteria to fluoride in the presence of divalent cations increases fluoride binding to bacterial cells and subsequently enhances fluoride-induced inhibition of bacterial acid production. This study investigated the effects of fluoride exposure (0-20,000 ppm F) in the presence of Ca(2+) or Mg(2+) prior to glucose challenge on pH fall ability by bacterial sugar fermentation, as well as fluoride binding to bacterial cells by exposure to fluoride, and fluoride release from bacterial cells during bacterial sugar fermentation, using caries-related bacteria, Streptococcus mutans and Streptococcus sanguinis. The pH fall by both streptococci was inhibited by exposure to over 250 ppm F in the presence of Ca(2+) (p < 0.01), whereas in the presence of Mg(2+), the pH fall by S. mutans and S. sanguinis was inhibited after exposure to over 250 and 950 ppm F, respectively (p < 0.05). The amounts of fluoride binding to and released from streptococcal cells increased with the concentration of fluoride the cells were exposed to in the presence of Mg(2+), but were high enough even after 250 ppm F exposure in the presence of Ca(2+). The enhanced inhibition of acid production in the presence of divalent cations is probably due to the improved efficiency of fluoride binding to bacterial cells being improved via these divalent cations.

Transient acid-impairment of growth ability of oral Streptococcus, Actinomyces, and Lactobacillus: a possible ecological determinant in dental plaque.

INTRODUCTION: Dental plaque pH decreases to about 4 through bacterial fermentation of carbohydrates and this low pH is maintained for from several minutes to about an hour. Repeated acidification causes demineralization of the tooth surface, resulting in caries formation. The acidification also influences plaque bacteria. Severe acidification kills bacteria efficiently, while physiological acidification, the condition occurring in plaque, kills bacteria partially and may impair growth ability. We, therefore, investigated the effects of physiological acidification on representative caries-related bacteria. METHODS: Streptococcus mutans, Streptococcus sobrinus, Streptococcus sanguinis, Streptococcus oralis, Lactobacillus paracasei, and Actinomyces naeslundii were used. Effects of physiological acidification at pH 4.0 on cell viability and growth ability, as well as the growth rate of these bacteria at pH 4.0-7.0, were investigated. RESULTS: Mutans streptococci and Lactobacillus grew at pH 4.0 but the growth of S. sanguinis and S. oralis ceased below pH 4.2 and pH 4.2-4.4, respectively. Acidification at pH 4.0 for 1 h killed 43-89%, 45% and 35-76% of S. sanguinis, S. oralis, and Actinomyces, respectively. Furthermore, assessment of bacterial growth curves revealed that the growth ability of the surviving cells of S. sanguinis, S. oralis and Actinomyces was impaired, but it was recovered within 2-5 h after the environmental pH had returned to 7.0. The acidification neither killed nor impaired the growth of mutans streptococci and Lactobacillus. CONCLUSIONS: These results indicate that physiological and transient acidification is not sufficient to kill bacteria, but it causes a temporary acid-impairment of their growth ability, which may function as an ecological determinant for microbial composition in dental plaque.

Effects of alpha-amylase and its inhibitors on acid production from cooked starch by oral streptococci.

This study evaluated acid production from cooked starch by Streptococcus mutans, Streptococcus sobrinus, Streptococcus sanguinis and Streptococcus mitis, and the effects of alpha-amylase inhibitors (maltotriitol and acarbose) and xylitol on acid production. Streptococcal cell suspensions were anaerobically incubated with various carbohydrates that included cooked potato starch in the presence or absence of alpha-amylase. Subsequently, the fall in pH and the acid production rate at pH 7.0 were measured. In addition, the effects of adding alpha-amylase inhibitors and xylitol to the reaction mixture were evaluated. In the absence of alpha-amylase, both the fall in pH and the acid production rate from cooked starch were small. On the other hand, in the presence of alpha-amylase, the pH fell to 3.9-4.4 and the acid production rate was 0.61-0.92 micromol per optical density unit per min. These values were comparable to those for maltose. When using cooked starch, the fall in pH by S. sanguinis and S. mitis was similar to that by S. mutans and S. sobrinus. For all streptococci, alpha-amylase inhibitors caused a decrease in acid production from cooked starch, although xylitol only decreased acid production by S. mutans and S. sobrinus. These results suggest that cooked starch is potentially acidogenic in the presence of alpha-amylase, which occurs in the oral cavity. In terms of the acidogenic potential of cooked starch, S. sanguinis and S. mitis were comparable to S. mutans and S. sobrinus. Alpha-amylase inhibitors and xylitol might moderate this activity.

Metabolomic effects of xylitol and fluoride on plaque biofilm in vivo.

Dental caries is initiated by demineralization of the tooth surface through acid production from sugar by plaque biofilm. Fluoride and xylitol have been used worldwide as caries-preventive reagents, based on in vitro-proven inhibitory mechanisms on bacterial acid production. We attempted to confirm the inhibitory mechanisms of fluoride and xylitol in vivo by performing metabolome analysis on the central carbon metabolism in supragingival plaque using the combination of capillary electrophoresis and a time-of-flight mass spectrometer. Fluoride (225 and 900 ppm F(-)) inhibited lactate production from 10% glucose by 34% and 46%, respectively, along with the increase in 3-phosphoglycerate and the decrease in phosphoenolpyruvate in the EMP pathway in supragingival plaque. These results confirmed that fluoride inhibited bacterial enolase in the EMP pathway and subsequently repressed acid production in vivo. In contrast, 10% xylitol had no effect on acid production and the metabolome profile in supragingival plaque, although xylitol 5-phosphate was produced. These results suggest that xylitol is not an inhibitor of plaque acid production but rather a non-fermentative sugar alcohol. Metabolome analyses of plaque biofilm can be applied for monitoring the efficacy of dietary components and medicines for plaque biofilm, leading to the development of effective plaque control.

Evaluation of pH at the bacteria-dental cement interface.

Physiochemical assessment of the parasite-biomaterial interface is essential in the development of new biomaterials. The purpose of this study was to develop a method to evaluate pH at the bacteria-dental cement interface and to demonstrate physiochemical interaction at the interface. The experimental apparatus with a well (4.0 mm in diameter and 2.0 mm deep) was made of polymethyl methacrylate with dental cement or polymethyl methacrylate (control) at the bottom. Three representative dental cements (glass-ionomer, zinc phosphate, and zinc oxide-eugenol cements) were used. Each specimen was immersed in 2 mM potassium phosphate buffer for 10 min, 24 hrs, 1 wk, or 4 wks. The well was packed with Streptococcus mutans NCTC 10449, and a miniature pH electrode was placed at the interface between bacterial cells and dental cement. The pH was monitored after the addition of 1% glucose, and the fluoride contained in the cells was quantified. Glass-ionomer cement inhibited the bacteria-induced pH fall significantly compared with polymethyl methacrylate (control) at the interface (10 min, 5.16 ± 0.19 vs. 4.50 ± 0.07; 24 hrs, 5.20 ± 0.07 vs. 4.59 ± 0.11; 1 wk, 5.34 ± 0.14 vs. 4.57 ± 0.11; and 4 wks, 4.95 ± 0.27 vs. 4.40 ± 0.14), probably due to the fluoride released from the cement. This method could be useful for the assessment of pH at the parasite-biomaterial interface.

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.

Nested PCR for detection of mutans streptococci in dental plaque.

AIMS: Mutans streptococci such as Streptococcus mutans and Streptococcus sobrinus have been implicated in human dental caries. In an attempt to develop a rapid and sensitive method for detecting Strep. mutans and Strep. sobrinus in dental plaque, a nested PCR amplification based on the 16S rRNA gene was employed. METHODS AND RESULTS: A universal set of PCR primers for bacterial 16S rRNA gene was introduced for the first PCR, and then two sets of primers specific for the 16S rRNA gene sequences of either Strep. mutans or Strep. sobrinus were used for the second PCR. Eighteen plaque samples were analyzed, and a nested PCR was shown to be more sensitive for detecting Strep. mutans and Strep. sobrinus than direct PCR. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: The 16S rRNA gene-based nested PCR method is a rapid and sensitive method for the detection of mutans streptococci, and may also be suitable for carrying out large-scale studies on the cariogenicity of mutans streptococci.

Identification of mutans streptococci by restriction fragment length polymorphism analysis of polymerase chain reaction-amplified 16S ribosomal RNA genes.

Mutans streptococci are frequently isolated from dental plaque and carious lesions. These bacteria have been identified by conventional methods such as biochemical and serologic tests followed by the isolation of colonies on the mitis-salivarius agar, which are sometimes inconsistent. Recently, species-specific polymerase chain reaction (PCR) has been reported to rapidly identify Streptococcus mutans and Streptococcus sobrinus. However, in the case of identification and classification into several species, e.g. within the group of mutans streptococci consisting of seven species, the identification using species-specific PCR seems somewhat inefficient because of need for the development and preparation of specific primers for each species. Therefore, in this study we developed a simple method using restriction fragment length polymorphism analysis of PCR-amplified 16S ribosomal RNA genes (16S rRNA genes PCR-RFLP) for the identification of seven different species included in the group of mutans streptococci. We amplified 16S rRNA gene sequences from genomic DNA samples by PCR using universal primers and digested the PCR products with the restriction endonucleases, HpaII and HaeIII. HpaII produced six RFLP patterns for eight reference strains, since the patterns for S. sobrinus, Streptococcus downei and Streptococcus ferus were similar. RFLP patterns produced with HaeIII could separate these three species. Furthermore, the RFLP patterns predicted from the 16S rRNA gene sequences in the GenBank database agreed with the actual RFLP patterns produced in the present study. The 16S rRNA sequence comparisons can be used to identify oral mutans streptococci; however, the identification by sequencing is sometimes difficult in large-scale studies and for small laboratories. Therefore, 16S rRNA genes PCR-RFLP, using HpaII and HaeIII, could be an alternative method for the identification of mutans streptococci, and may be applicable for large-scale studies on the cariogenicity of mutans streptococci.