Gen Mayanagi

Improvement of periodontal condition by probiotics with Lactobacillus salivarius WB21 : a randomized, double-blind, placebo-controlled study

AIM This randomized clinical study was designed to evaluate the effect of probiotic intervention using lactobacilli on the periodontal condition of volunteers without severe periodontitis. MATERIAL AND METHODS Freeze-dried Lactobacillus salivarius WB21 (WB21)-containing tablets or a placebo were given to volunteers in a double-blind randomized study. A total of 66 volunteers were finally enrolled and randomly assigned to receive tablets containing WB21 (6.7 x 10(8) CFU) with xylitol or xylitol alone (placebo) three times a day for 8 weeks. Periodontal clinical parameters and whole saliva samples were obtained at baseline (BL), 4 weeks, and the end of the interventional period (8 weeks). Salivary lactoferrin (Lf) levels were measured by enzyme-linked immunosorbent assay. Lactobacilli in saliva and plaque samples was detected by semi-quantitative RT-PCR using 16S rRNA primers. RESULTS Periodontal clinical parameters were improved in both groups after an 8-week intervention. Current smokers in the test group showed a significantly greater improvement of plaque index and probing pocket depth from BL when compared with those in the placebo group. Salivary Lf level was also significantly decreased in the test group smokers. CONCLUSION Our results indicate that probiotics could be useful in the improvement/maintenance of oral health in subjects at a high risk of periodontal disease.

Probiotic effects of orally administered Lactobacillus salivarius WB21‐containing tablets on periodontopathic bacteria: a double‐blinded, placebo‐controlled, randomized clinical trial

AIM This study was designed to evaluate whether the oral administration of lactobacilli could change the bacterial population in supra/subgingival plaque. MATERIAL AND METHODS Sixty-six healthy volunteers without severe periodontitis were randomized into two groups to receive lactobacilli or placebo for 8 weeks (8W): the test group (n=34) received 2.01 x 10(9) CFU/day of Lactobacillus salivarius WB21 and xylitol in tablets; the control group (n=32) received placebo with xylitol. Supra/subgingival plaque samples were collected at the baseline and after 4 weeks (4W) and 8W. The bacterial amounts in plaque samples were analysed by quantitative real-time polymerase chain reaction. RESULTS The numerical sum of five selected periodontopathic bacteria in the test group was decreased significantly in subgingival plaque at 4W [odds ratio (OR)=3.13, 95% confidence intervals (CI)=1.28-7.65, p=0.012]. Multivariate analysis showed that significantly higher odds were obtained for the reduction of Tannerella forsythia in subgingival plaque of the test group at both 4W (OR=6.69, 95% CI=2.51-17.9, p<0.001) and 8W (OR=3.67, 95% CI=1.45-9.26, p=0.006). CONCLUSION Oral administration of probiotic lactobacilli reduced the numerical sum of five selected periodontopathic bacteria and could contribute to the beneficial effects on periodontal conditions.

Profiling of subgingival plaque biofilm microflora from periodontally healthy subjects and from subjects with periodontitis using quantitative real-time PCR

BACKGROUND AND OBJECTIVE Qualitative and quantitative changes of the subgingival plaque biofilm microflora in periodontal pockets are thought to be associated with the development and progression of periodontitis. The aims of the present study were to quantify the proportions of nine periodontitis-associated bacterial species and four Streptococcus species in subgingival plaque, and to evaluate their relationship with periodontitis quantitatively. MATERIAL AND METHODS Subgingival plaque samples were obtained from 12 periodontally healthy subjects and from 28 patients with periodontitis. The amounts of total and target bacteria were measured by quantitative real-time PCR using universal and species-specific primers, respectively. RESULTS The proportion of total obligate anaerobes was found to be higher in subjects with periodontitis than in periodontally healthy subjects (p < 0.05). Among obligate anaerobes, Tannerella forsythia (2.04 +/- 5.27%, p < 0.05), Porphyromonas gingivalis (0.54 +/- 1.41%) and Eubacterium saphenum (0.30 +/- 0.96%) were detected at high proportions in subjects with periodontitis, but not in periodontally healthy subjects. By contrast, the proportion of total streptococci was lower in subjects with periodontitis (p < 0.05). Specifically, the proportion of T. forsythia, P. gingivalis or E. saphenum increased (>or= 2.78%) and the proportion of Streptococcus species decreased to virtually undetectable levels, in subjects with periodontitis. CONCLUSION Obligate anaerobes, including T. forthysia, P. gingivalis and E. saphenum, were identified predominantly in microflora from subjects with periodontitis, whereas Streptococcus species were identified predominantly in microflora from periodontally healthy subjects, suggesting a change in the subgingival environment that resulted in conditions more suitable for the survival of obligate anaerobes. The proportion of these obligate anaerobes in the subgingival plaque of subjects with periodontitis appears to be associated with the status of human periodontitis.

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.

Effect of fluoride-releasing restorative materials on bacteria-induced pH fall at the bacteria–material interface: An in vitro model study

OBJECTIVES Inhibition of bacterial acid production by dental restorative materials is one of the strategies for secondary caries prevention. This study aimed to evaluate the effect of fluoride-releasing restorative materials on bacteria-induced pH fall at the bacteria-material interface. METHODS Four fluoride-releasing restorative materials, glass-ionomer cement (GIC), resin-modified glass-ionomer cement (RMGIC), resin composite (RC) and flowable resin composite (FRC) were used. Each specimen was immersed in potassium phosphate buffer at pH 7.0 for 10min and 4 weeks, and in potassium acetate buffer at pH 5.5 for 4 weeks. An experimental apparatus was made of polymethyl methacrylate and had a well with restorative materials or polymethyl methacrylate (control) at the bottom. The well was packed with cells of Streptococcus mutans, and the pH at the interface between cells and materials was monitored using a miniature pH electrode after the addition of 1% glucose for 90min, and the fluoride released into the well was quantified using a fluoride ion electrode. RESULTS The pH of GIC (4.98-5.18), RMGIC (4.77-4.99), RC (4.62-4.75) and FRC (4.54-4.84) at 90min were higher than that of control (4.31-4.49). The fluoride amounts released from GIC were the highest, followed by RMGIC, RC and FRC, irrespective of immersion conditions. Saliva coating on materials had no significant effect. CONCLUSIONS The fluoride-releasing restorative materials inhibited pH fall at the bacteria-material interface. The degree of inhibition of pH fall seemed to correspond to the amount of fluoride detected, suggesting that the inhibition was due to the fluoride released from these materials. CLINICAL SIGNIFICANCE A little amount of fluoride actually released from the fluoride-releasing materials may have caries preventive potential for oral bacteria.

Microbiologically Induced Corrosive Properties of the Titanium Surface

Corrosion of titanium is the major concern when it is used for dental treatment. This study aimed to investigate the mechanism of the microbiologically induced corrosive properties of titanium. An experimental well was made of polymethyl methacrylate with pure titanium at the bottom. Viable or killed cells of Streptococcus mutans were packed into the well, and pH at the bacteria-titanium interface was monitored with and without glucose. Before and after 90-minute incubation, the electrochemical behavior on the titanium surface was measured by means of a potentiostat. The oxygen concentration under bacterial cells was monitored with oxygen-sensitive fluorescent film. The amount of titanium eluted was measured by inductively coupled plasma-mass spectrometry. The corrosion current and passive current under killed cells were low and stable during 90 min, while those under viable cells increased, regardless of the glucose-induced pH fall. The polarization resistance and oxygen concentration under killed cells were high and stable, while those under viable cells decreased. No elution of titanium was detected. Viable bacterial cells may form ‘oxygen concentration cells’ through metabolism-coupled oxygen consumption and subsequently induce corrosive properties of the titanium surface.

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.

Microflora profiling of infected root canal before and after treatment using culture-independent methods

This study aimed to profile the microflora in infected root canals before and after root canal treatment using culture-independent methods. Six infected root canals in single-rooted teeth with periapical lesions from five subjects were included. Quantification of total bacteria was performed by real-time PCR with primers targeting 16S rRNA genes. PCR products with universal 16S rRNA gene primers were cloned and partially sequenced, and bacterial identification at the species level was performed by comparative analysis with the GenBank database. The concentration of extracted DNA before treatment was higher than that after root canal treatment, although the difference was not statistically significant. Sequence analysis revealed that oral bacteria such as Fusobacterium, Streptococcus, Olsenella, and Pseudoramibacter detected in cases before root canal treatment disappeared after treatment. These results suggest that the root canal microflora are distinct before and after root canal treatment, and that treatment changes the microflora in both quantity and quality.

pH Response and Tooth Surface Solubility at the Tooth/Bacteria Interface

Evaluating the physiochemical processes at the tooth surface/bacteria interface is important for elucidating the etiology of dental caries. This study aimed to compare the mineral solubility and protein degradation of coronal enamel (CE) and root dentin (RD), and investigate the involvement of dissolved components in bacteria-induced pH changes using a model of tooth/bacteria interface. An experimental apparatus forming a well was made of polymethyl methacrylate, and a bovine tooth (CE or RD) specimen was fixed at the bottom of the well. A miniature pH electrode was placed on the tooth, and Streptococcus mutans NCTC 10449 cells, grown in 0.5% glucose-containing complex medium, were packed into the well. The pH at the tooth/S. mutans interface was monitored continuously for 120 min after the addition of 0.5% glucose at 37°C. S. mutans cells were recovered from the wells, and the amounts of lactate and calcium were measured using a portable lactate meter and a fluorescent dye, respectively. Proteolytic activity was also evaluated fluorometrically. The pH of the RD/S. mutans interface was significantly higher than that of the CE/S. mutans interface (30 min: 6.37 ± 0.12 vs. 6.18 ± 0.11, 60 min: 6.08 ± 0.14 vs. 5.66 ± 0.27, 90 min: 5.49 ± 0.24 vs. 5.14 ± 0.22, p < 0.05). Greater amounts of calcium were dissolved from RD (3.19 ± 0.74 µg/mL) than from CE (1.84 ± 0.68 µg/mL; p < 0.05), while similar amounts of lactate were produced. Proteolytic activity was not detected at any of the interfaces. These results indicate that RD is more soluble to bacteria-induced acidification than CE. This method can contribute to the evaluation and development of caries-preventive materials.