Inhibitory effects of citrus lemon oil and limonene on Streptococcus sobrinus - Induced dental caries in rats.

OBJECTIVE: This study aimed to examine and compare the anti-caries effects of citrus lemon oil (CLO) and limonene in rats. METHODS: The minimal inhibitory concentrations of CLO and limonene were measured using the disk diffusion method. The rats were infected with Streptococcus sobrinus and assigned into four groups: (1) Chlorhexidine, (2) CLO, (3) limonene, and (4) distilled water (H2O). The total cultivable microbiota and Streptococcus sobrinus in the mouth of the rats were counted, and the caries lesions were measured by Keyes' scoring and DIAGNOdent examination. RESULTS: The minimal inhibitory concentrations of CLO and limonene against Streptococcus sobrinus were 4.50 and 21.00 mg/mL, respectively. The chlorhexidine group had the lowest total microbiota counts (p < 0.05), while there were no significant differences among the CLO, limonene and H2O groups (p > 0.05). The proliferation of Streptococcus sobrinus was remarkably inhibited by chlorhexidine, limonene and CLO (p < 0.05). The Keyes' scoring and DIAGNOdent results indicated that the caries lesions were reduced in the CLO and limonene groups compared to that of the vehicle control group (p < 0.05). There was no significant difference between CLO and limonene (p > 0.05). CONCLUSION: Limonene and CLO have similar anti-caries abilities in a bacteriostatic manner in vivo.

Rapid and sensitive PCR-dipstick DNA chromatography for multiplex analysis of the oral microbiota.

A complex of species has been associated with dental caries under the ecological hypothesis. This study aimed to develop a rapid, sensitive PCR-dipstick DNA chromatography assay that could be read by eye for multiplex and semiquantitative analysis of plaque bacteria. Parallel oligonucleotides were immobilized on a dipstick strip for multiplex analysis of target DNA sequences of the caries-associated bacteria, Streptococcus mutans, Streptococcus sobrinus, Scardovia wiggsiae, Actinomyces species, and Veillonella parvula. Streptavidin-coated blue-colored latex microspheres were to generate signal. Target DNA amplicons with an oligonucleotide-tagged terminus and a biotinylated terminus were coupled with latex beads through a streptavidin-biotin interaction and then hybridized with complementary oligonucleotides on the strip. The accumulation of captured latex beads on the test and control lines produced blue bands, enabling visual detection with the naked eye. The PCR-dipstick DNA chromatography detected quantities as low as 100 pg of DNA amplicons and demonstrated 10- to 1000-fold higher sensitivity than PCR-agarose gel electrophoresis, depending on the target bacterial species. Semiquantification of bacteria was performed by obtaining a series of chromatograms using serial 10-fold dilution of PCR-amplified DNA extracted from dental plaque samples. The assay time was less than 3 h. The semiquantification procedure revealed the relative amounts of each test species in dental plaque samples, indicating that this disposable device has great potential in analysis of microbial composition in the oral cavity and intestinal tract, as well as in point-of-care diagnosis of microbiota-associated diseases.

Effects of Withania somnifera on the growth and virulence properties of Streptococcus mutans and Streptococcus sobrinus at sub-MIC levels.

The aim of this study was to evaluate the effect of the methanol extract of Withania somnifera (MEW) on the growth and virulence properties of Streptococcus mutans and Streptococcus sobrinus at sub-minimum inhibitory concentration (MIC) levels and to identify the main components of MEW. First, antibacterial activity of MEW against oral bacteria was determined using a micro-dilution method. Then, the effect of MEW on the growth of S. mutans and S. sobrinus was investigated at sub-MIC levels. To test the effect of MEW on the virulence properties of S. mutans and S. sobrinus, assays for acid production, acid tolerance, and biofilm formation were performed at sub-MIC levels. A GC-MS analysis for the main components of MEW was also carried out. MEW showed a broad antibacterial range against oral bacteria (MIC: 0.125-2 mg/mL). At sub-MIC levels, MEW dose-dependently increased doubling times of S. mutans and S. sobrinus up to 258% and 400%, respectively. Furthermore, MEW inhibited acid production, acid tolerance, and biofilm formation of S. mutans and S. sobrinus at sub-MIC levels. The GC-MS analysis revealed the presence of mono- and disaccharides, sugar alcohols, and organic acids as main components. These data suggest that MEW might be useful for restraining physiological activities of cariogenic bacteria.

A high molecular mass cranberry constituent reduces mutans streptococci level in saliva and inhibits in vitro adhesion to hydroxyapatite.

Previous investigations showed that a high molecular mass, non-dialyzable material (NDM) from cranberries inhibits the adhesion of a number of bacterial species and prevents the co-aggregation of many oral bacterial pairs. In the present study we determined the effect of mouthwash supplemented with NDM on oral hygiene. Following 6 weeks of daily usage of cranberry-containing mouthwash by an experimental group (n = 29), we found that salivary mutans streptococci count as well as the total bacterial count were reduced significantly (ANOVA, P < 0.01) compared with those of the control (n = 30) using placebo mouthwash. No change in the plaque and gingival indices was observed. In vitro, the cranberry constituent inhibited the adhesion of Streptococcus sobrinus to saliva-coated hydroxyapatite. The data suggest that the ability to reduce mutans streptococci counts in vivo is due to the anti-adhesion activity of the cranberry constituent.

[Anti-caries vaccines]. / Vaccini anticarie.

Even though the reduction of caries-incidence in developed countries, its increasing has been observed nowadays. The use of a vaccine was object of many researches, going under modifications and evaluations during years. Wallace and McCollum showed the chance to induce experimental cavities, while Clarke and McIntosh were the first underlining the roll of S. mutans and Lactobacilli as efforts of the pathology. Williams was the first working with humans and Zinner and Fitzgerald continued. So since Bowen the research tried to build a vaccine made of single bacterial molecules with antigenic power. We can count about a large number of targets, like: the Ag I/II, the glucosyltransferase enzyme (GTF), the glucan-binding-protein (GBP), the destranase, the fruttosyltransferase and the glucans. Among the substances used to obtain a vaccine cacao revealed its capacity against bacteria able to develop cavities, thanks to its cariostatic and anti-glucosyltransferase activity due to polyphenols, that we can find in green tea too. It's also interesting a technique that gives passive antibodies like cow's milk, but in particular the one of a monoclonal antibody made with biotechnology of plants: the Guy's 13. It does not show substantial differences in comparison with the human Ig and it's able to prevent the installation of micro-organism and to reduce cavities in adult patients already infected. For the setting-up of a vaccine, however, only studies, comparison and research will be able to show precise instruments of defence.

Powdered milk micellar casein prevents oral colonization by Streptococcus sobrinus and dental caries in rats: a basis for the caries-protective effect of dairy products.

Three animal studies were performed to investigate the influence of the macromolecular structure of milk casein on caries incidence and the possible ecological changes of the oral microbiota by such casein fractions. Towards this end, rats were infected with mixed bacterial suspensions of Streptococcus sobrinus OMZ 176 and Actinomyces viscosus Ny1. Various milk protein fractions were incorporated into carefully balanced powdered cariogenic diets to constitute the sole major protein component. Diets containing micellar casein had a pronounced and highly significant effect on almost all clinical and microbiological parameters examined. Both the formation of advanced dentinal fissure (B) and smooth surface (E) caries lesions was inhibited by diets containing micellar casein; this caries-inhibiting effect appeared to be due mainly to modifications within the plaque microbiota. The proportion of S. sobrinus in the oral cavity of rats was reduced (73-80%) by micellar casein-containing preparations, whereas the A. viscosus population was increased. Both these microbiological parameters were always negatively correlated. This appears to be the first example of a food component other than dietary sugars, selectively modifying the composition of the dental plaque microbiota of rats in such a way as to reduce its pathogenic potential. It also demonstrates the importance of establishing a molecular basis for the role of food components, which prove to be beneficial to oral health.

Comparison of the cariostatic effects between regimens to administer oolong tea polyphenols in SPF rats.

The cariostatic effect of oolong tea polyphenols administered according to several regimens was examined in specific pathogen-free (SPF) Sprague-Dawley rats given both a diet containing 20% sucrose and infected with S. sobrinus 6715. The crude preparation (OTE) of oolong tea polyphenols showed the most prominent effect on caries reduction in SPF rats when OTE was administered in the drinking water beginning 1 day prior to the inoculation of S. sobrinus 6715, when compared with chromatographically isolated polyphenol fractions (OTF1 and OTF6) of OTE. Reduction in caries development was found even when OTE was given 1 day after inoculation of the organism. OTE was shown to significantly inhibit dental caries in rats at the concentrations of either more than 5 microg/ml in drinking water or more than 10 microg/g in diet. OTF1 and OTF6 also showed significant inhibition of caries induction, with the minimum inhibitory concentration of OTF6 being 50 microg/ml in drinking water and the minimum inhibitory concentration of OTF1 being 100 microg/g in diet. These results indicate that cariostatic activity of OTE was effective even after the establishment of S. sobrinus in the oral cavity and was more effective in drinking water than in diet. Furthermore, OTE may contain some anticaries substances that affect the virulence of S. sobrinus other than glucosyltransferases.

Peroxidase-catalyzed generation of catechin oligomers that inhibit glucosyltransferase from Streptococcus sobrinus.

Oolong tea extract (OTE) and the purified polymeric polyphenols from OTE have been found to inhibit glucosyltransferase (GTase) of mutans streptococci. In view of the partial fermentation characteristic of oolong tea, we describe here an in vitro model reaction system to produce partially fermented products of D-(+)-catechin or green tea extract (GTE) using horseradish peroxidase. A dimeric catechin molecule was identified as dehydro-dicatechin A by instrumental analyses. The molecular size of some oligomeric catechins was estimated by the elution profile with HPLC. These catechin oligomers markedly inhibited GTase from Streptococcus sobrinus 6715. As the degree of polymerization of catechin or GTE increased, GTase was inhibited more effectively. These results suggest that polymeric polyphenols found in OTE are synthesized by partial fermentation due to oxidases/peroxidases present in tea leaves.