The aims of this study were to identify hydrogen sulfide (H 2 S)-producing bacteria among tongue biofilm microflora and to investigate the relationship between bacterial flora and H 2 S levels in mouth air. Oral malodour levels in 10 subjects (age 21-56 years) were assessed by gas chromatography, and Breathtron and organoleptic scores. Based on these assessments, subjects were divided into two groups: an odour group and a no/low odour group. Tongue coatings were sampled and spread onto Fastidious Anaerobe Agar plates containing 0 . 05 % cysteine, 0 . 12 % glutathione and 0 . 02 % lead acetate, and were then incubated anaerobically at 37 8C for 2 weeks. Bacteria forming black or grey colonies were selected as H 2 S-producing phenotypes. The numbers of total bacteria (P , 0 . 005) and H 2 S-producing bacteria (P , 0 . 05) in the odour group were significantly larger than those in the no/low odour group. Bacteria forming black or grey colonies (126 isolates from the odour group; 242 isolates from the no/low odour group) were subcultured, confirmed as producing H 2 S and identified according to 16S rRNA gene sequencing. Species of Veillonella (38 . 1 % in odour group; 46 . 3 % in no/low odour group), Actinomyces (25 . 4 %; 17 . 7 %) and Prevotella (10 . 3 %; 7 . 8 %) were the predominant H 2 S-producing bacteria in both the odour and no/low odour groups. These results suggest that an increase in the number of H 2 S-producing bacteria in the tongue biofilm is responsible for oral malodour, although the bacterial composition of tongue biofilm was similar between the two groups.

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.

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.

Bifidobacterium is frequently detected in early childhood caries and white spot lesions, indicating that it is a novel caries-associated bacterium. Bifidobacterium is known to possess a unique metabolic pathway, the “bifid shunt,” which might give it cariogenic potential by increasing its acid production. Thus, we evaluated the acid-producing activity of Bifidobacterium and its sensitivity to fluoride, a caries preventive reagent. Bifidobacterium longum , Bifidobacterium dentium , and Streptococcus mutans were used. Acid-producing activity was measured using a pH-stat in the absence and presence of fluoride under anaerobic conditions. Furthermore, metabolomic analysis was performed to elucidate the mechanism underlying the inhibitory effects of fluoride. The acid production of Bifidobacterium at pH 5.5 was as high as that seen at pH 7.0, indicating that Bifidobacterium has high cariogenic potential, although it produced less acid than S. mutans . In addition, Bifidobacterium produced acid in the absence of extracellular carbohydrates, suggesting that it can store intracellular polysaccharides. Bifidobacterium produced more acid from lactose than from glucose. Bifidobacterium mainly produced acetate, whereas S. mutans mainly produced lactate. The 50% inhibitory concentration (IC 50 ) of fluoride for acid production was 6.0–14.2 times higher in Bifidobacterium than in S. mutans . Fluoride inhibited enolase in the glycolysis, resulting in the intracellular accumulation of 3-phosphoenolpyruvate, glucose 6-phosphate, and erythrose 4-phosphate. However, the bifid shunt provides a bypass pathway that can be used to produce acetate, suggesting that Bifidobacterium is able to metabolize carbohydrates in the presence of fluoride. It is suggested that its exclusive acetate production contributes to the pathogenesis of dental caries.

Veillonella species are one of the major anaerobes in the oral cavity and are frequently detected in both caries lesions and healthy oral microbiomes. They possess the ability to utilize lactate and convert nitrate (NO3-) into nitrite (NO2-). Recently, interest in NO2- has increased rapidly because of its beneficial effects on oral and general health; i.e., it inhibits the growth and metabolism of oral pathogenic bacteria, such as Streptococcus mutans, and lowers systemic blood pressure. However, there is only limited information about the biochemical characteristics of NO2- production by Veillonella species. We found that NO3- did not inhibit the growth of Veillonella atypica or Veillonella parvula, and it only inhibited the growth of Streptococcus mutans at a high concentration (100 mM). However, NO2- inhibited the growth of Streptococcus mutans at a low concentration (0.5 mM), while a higher concentration of NO2- (20 mM) was needed to inhibit the growth of Veillonella species. The NO2- production of Veillonella species was increased by environmental factors (lactate, acidic pH, and anaerobic conditions) and growth conditions (the presence of NO3-/NO2-), and was linked to anaerobic lactate metabolism. A stoichiometric evaluation revealed that NO3- is reduced to NO2- by accepting reducing power derived from the oxidization of lactate. These findings suggest that the biochemical characteristics of NO2- production from NO3- and its linkage with lactate metabolism in oral Veillonella species may play a key role in maintaining good oral and general health. IMPORTANCE The prevalence of dental caries is still high around the world. Dental caries is initiated when the teeth are exposed to acid, such as lactic acid, produced via carbohydrate metabolism by acidogenic microorganisms. Veillonella species, one of the major oral microorganisms, are considered to be beneficial bacteria due to their ability to convert lactic acid to weaker acids and to produce NO2- from NO3-, which is thought to be good for both oral and general health. Therefore, it is clear that there is a need to elucidate the biochemical characteristics of NO2- production in Veillonella species. The significance of our research is that we have found that lactate metabolism is linked to NO2- production in Veillonella species in the environment found in the oral cavity. This study suggests that Veillonella species are potential candidates for maintaining oral and general health.

Indigenous oral bacteria in the tongue coating such as Veillonella have been identified as the main producers of hydrogen sulfide (H 2 S), one of the major components of oral malodor. However, there is little information on the physiological properties of H 2 S production by oral Veillonella such as metabolic activity and oral environmental factors which may affect H 2 S production. Thus, in the present study, the H 2 S-producing activity of growing cells, resting cells, and cell extracts of oral Veillonella species and the effects of oral environmental factors, including pH and lactate, were investigated. Type strains of Veillonella atypica, Veillonella dispar, and Veillonella parvula were used. These Veillonella species produced H 2 S during growth in the presence of L-cysteine. Resting cells of these bacteria produced H 2 S from L-cysteine, and the cell extracts showed enzymatic activity to convert L-cysteine to H 2 S. H 2 S production by resting cells was higher at pH 6 to 7 and lower at pH 5. The presence of lactate markedly increased H 2 S production by resting cells (4.5-to 23.7-fold), while lactate had no effect on enzymatic activity in cell extracts. In addition to H 2 S, ammonia was produced in cell extracts of all the strains, indicating that H 2 S was produced by the catalysis of cystathionine ␥-lyase (EC 4.4.1.1). Serine was also produced in cell extracts of V. atypica and V. parvula, suggesting the involvement of cystathionine ␤-synthase lyase (EC 4.2.1.22) in these strains. This study indicates that Veillonella produce H 2 S from L-cysteine and that their H 2 S production can be regulated by oral environmental factors, namely, pH and lactate.O ral malodor is due to metabolic products of bacteria in the oral cavity, particularly those living on the dorsum of the tongue (1, 2). Some cases of oral malodor are known to be linked with periodontitis (3, 4), and thus various periodontitis-related bacterial species have been detected in the tongue coating (5, 6). These findings also suggest that the tongue coating plays a role in the reservoir of such bacteria (5). Most of these bacteria have the ability to produce hydrogen sulfide (H 2 S), one of the major components of oral malodor (7,8). In a previous study (9), we focused on oral malodor in patients without oral diseases such as periodontitis or caries and found that the predominant H 2 S-producing bacteria were not periodontitis-related bacteria but were mainly indigenous bacteria of the oral cavity such as Veillonella and Actinomyces. Among these, Veillonella species, including V. atypica, V. dispar, and V. parvula, were dominant (9).Veillonella species are Gram-negative anaerobic micrococci that are frequently detected in the tongue coating (6, 9). These bacteria are asaccharolytic but utilize lactate, pyruvate, and oxaloacetate as energy sources. Although several studies have reported that Veillonella species produce H 2 S (1, 8, 10, 11), the metabolic properties of H 2 S production have not been fully understood. In the tongue coating, environmen...

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 largescale studies on the cariogenicity of mutans streptococci.

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