Effect of Radiotherapy on Cariogenic Organism Streptococcus sobrinus in Saliva in Head and Neck Cancer: A Clinical Study.

AIM: Aim of the study was to assess salivary Streptococcus sobrinus in head and neck cancer using quantitative polymerase chain reaction (PCR). MATERIALS AND METHODS: Unstimulated saliva samples were collected from head and neck cancer patient preradiotherapy. Unstimulated saliva samples were collected from oral and laryngeal cancer patients after 6 weeks of radiotherapy (dose 60 Gy). The subjects were explained not to consume solids or liquids or carry out any dental hygiene activity 1 hour prior to saliva collection. Accumulated unstimulated saliva was collected in cylindrical tube through funnel. The collected saliva was then transferred to Eppendorf tube containing Tris-ethylenediamine-tetraacetic acid (EDTA) (TE) buffer and was transported to lab for real-time PCR analysis. RESULTS: Streptococcus sobrinus significantly increased post-radiotherapy as compared with preradiotherapy in head and neck cancer patients. CONCLUSION: Within the limitation of this study, we conclude that amount of S. sobrinus increases postradiotherapy in head and neck cancer patients. CLINICAL SIGNIFICANCE: As radiation therapy has harmful effects on hard and soft tissues of oral cavity, dentists should provide motivation for oral health care to the patients.

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.

Streptococcus mutans and Streptococcus sobrinus are able to adhere and invade human gingival fibroblast cell line.

Streptococcus mutans and Streptococcus sobrinus, the principal etiologic agents of caries decay of teeth, are generally acquired in oral cavity at the moment of tooth eruption. However, as S. mutans has been detected in oral cavity of predentate children, the eruption of teeth seems not to be a necessary prerequisite, suggesting that this species may be not confined to dental plaque. Here, we evaluate the ability of S. mutans and S. sobrinus in planktonic and biofilm lifestyle to adhere, invade and survive within human gingival fibroblast (HGF-1) cells. Planktonic and biofilm streptococci adhered and invaded host cells to different extents, showing higher efficiencies of biofilm than planktonic counterparts. Moreover, planktonic and biofilm streptococci showed the same percentage of survival within host cells. Transmission electron and confocal microscopy observations confirmed intracellular localization of planktonic and biofilm bacteria. The adhesion, invasion and survival abilities within human oral cells may be considered S. mutans and S. sobrinus virulence mechanisms to colonize and persist in the oral cavity in the absence of tooth surface.

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.

Frequency and distribution of Mutans Streptococci in dental plaque from caries-free and caries-affected Venezuelan children.

Mutans Streptococci, in particular S. mutans and S. sobrinus, are generally considered to be the prime etiological bacteria of human dental caries. The aim of this study was to determine the frequency of mutans streptococci in dental plaque in three groups of caries-free and caries-affected Venezuelan children aged 2-6, 7-12, 13-19 years, and their possible association with dental caries. The frequency of mutans streptococci was determined in samples of pooled dental plaque collected from all detectable sources of 30 (62.5%) caries-affected and 18 (37.5%) caries-free children. The samples were collected from all available tooth sites using a Hollenbak probe and immediately suspended in Ringer's solution, serially diluted and cultured in Mitis Salivarius (MS) agar for total streptococci determination and Mitis Salivarius Bacitracin (MSB) for isolation of mutans streptococci. The bacterial identification procedure was done using the API Rapid Strep System. The criteria used to determine dmft and DMFT was established by Klein and Palmer (1941). Mean dmft and DMFT were 6.4 +/- 3.2 and 4.4 +/- 2.9, respectively. Ten (33%) out of 30 caries-affected children harbored mutans streptococci. The species most frequently found were S. mutans (20%), S. sobrinus (10%) and S. rattus (3.3%). Meanwhile, in the caries-free group only 6 out of 18 children (33%) harbored mutans streptococci, specifically S. mutans and S. sobrinus, both at 17%, with no significant difference between the two groups. These results indicate that the percentage of children that harbored mutans streptococci was similar in both groups, suggesting that other acidogenic species may be responsible for caries development.

The frequency and severity of dental caries, and counts of cariogenic bacteria in rheumatoid arthritis patients.

BACKGROUND: It has been reported that patients with rheumatoid arthritis (RA) are more likely to exhibit periodontitis than patients without RA. However, the frequency and severity of dental caries in patients with RA is still unknown. OBJECTIVES: The aim of the study was to investigate whether higher counts of cariogenic bacteria are present in RA patients in contrast to healthy subjects, and to ascertain whether the frequency and severity of dental caries are increased in RA patients. MATERIAL AND METHODS: The study involved 160 adults: an RA group (n = 80) and a control group matched by age and gender (n = 80). The participants' dental status scores were determined based on the following indices: the Decayed, Missing and Filled Teeth (DMFT) index, the Filled and Sound Teeth (FS-T) index, Treatment Needs Index (TNI), Care Index (CI), and Integrative Dental Caries Index (IDCI). DNA copies of Streptococcus mutans (S. mutans) and Streptococcus sobrinus (S. sobrinus) were quantified using realtime polymerase chain reaction (PCR). RESULTS: The IDCI showed that the RA group was more affected, mainly presenting moderate to severe dental caries. The RA group also had higher global DMFT scores than the control group and scored higher on the decayed component of the DMFT index. The TNI and CI indicated that RA patients required more dental attention and appropriate treatment. The Streptococcus mutans count was significantly higher in the RA group. CONCLUSIONS: A complete basic oral examination, along with oral health instruction including adequate oral and dental hygiene, is crucial to prevent dental caries and associated complications in RA patients, since they appear to be more vulnerable than the non-RA population.

Five-year longitudinal study of dental caries risk associated with Streptococcus mutans and Streptococcus sobrinus in individuals with intellectual disabilities.

Streptococcus mutans (S. mutans) and Streptococcus sobrinus (S. sobrinus) are important etiologic agents in human dental caries. Using quantitative real-time polymerase chain reaction assays for the presence of those strains, we examined 145 outpatients with intellectual disability (ID), calculated the proportion of each of these strains to total bacteria, and compared dental caries incidence over 5 years. Plaque samples were collected from all erupted tooth sites, and dental examinations were performed annually to determine numbers of decayed, missing, and filled teeth (DMFT score; World Health Organization caries diagnostic criteria). Elevated DMFT scores were calculated as ∆DMFT, and sites of newly affected caries (∆SNAC) were identified. Sixty-six patients had both strains. The proportion of S. mutans to total bacteria was moderately correlated with DMFT in year 2, ∆DMFT in years 2 and 5, and ∆SNAC in years 2 and 5 (correlation coefficient = 0.470, P < 0.001), while the proportion of S. sobrinus to total bacteria was moderately correlated with DMFT in years 2 and 5, ∆DMFT in years 1, 2, and 5, and ∆SNAC in years 2 and 5 (correlation coefficient = 0.695, P < 0.001). Individuals with ID who harbored both bacterial strains had a higher risk of dental caries and a significantly higher proportion of S. sobrinus to total bacteria.

Zinc-Containing Hydroxyapatite Enhances Cold-Light-Activated Tooth Bleaching Treatment In Vitro.

Cold-light bleaching treatment has grown to be a popular tooth whitening procedure in recent years, but its side effect of dental enamel demineralization is a widespread problem. The aim of this study was to synthesize zinc-substituted hydroxyapatite as an effective biomaterial to inhibit demineralization or increase remineralization. We synthesized zinc-substituted hydroxyapatite containing different zinc concentrations and analysed the product using X-ray diffraction (XRD), Fourier transform infrared (FTIR) spectroscopy, and energy dispersive spectrometer (EDS). The biological assessment of Zn-HA was conducted by CCK-8 assay and bacterial inhibition tests. pH cycling was performed to estimate the effect of Zn-HA on the enamel surface after cold-light bleaching treatment. The XRD, FTIR, and EDS results illustrated that zinc ions and hydroxyapatite combined in two forms: (1) Zn2+ absorbed on the surface of HA crystal and (2) Zn2+ incorporated into the lattice of HA. The results indicated that 2% Zn-HA, 4% Zn-HA, and 8% Zn-HA effectively inhibited the growth of bacteria yet showed poor biocompatibility, whereas 1% Zn-HA positively affected osteoblast proliferation. The XRD and scanning electron microscopy (SEM) results showed that the use of Zn-HA in pH cycling is obviously beneficial for enamel remineralization. Zinc-substituted hydroxyapatite could be a promising biomaterial for use in cold-light bleaching to prevent enamel demineralization.

Tansmission, diversity and virulence factors of Sreptococcus mutans genotypes.

Dental caries is an infectious and transmissible disease, in which many genetic, environmental and behavioral risk factors interact. The mutans streptococci (MS), mainly Streptococcus mutans and Streptococcus sobrinus are the microorganisms most strongly associated with this disease. The main virulence factors associated with MS cariogenicity include adhesion, acidogenicity and acid tolerance. These properties work together to modify the physico-chemical properties of the biofilm, resulting in ecological changes in the form of increased proportions of S. mutans and other acidogenic and aciduric species. In addition, reports of higher numbers of S. mutans genotypes with increased virulence in caries-active subjects suggest the importance of microenvironmental factors in increasing the risk of caries. This review focuses on the transmission and establishment of different genotypes of S. mutans and the role they play in the development of dental caries.

Dental caries: an infectious and transmissible disease.

By definition, dental caries is an infectious and transmissible disease because it is caused by bacteria colonizing the tooth surfaces. Unlike most infectious diseases affecting humans, caries is the result of an imbalance of the indigenous oral biota rather than a nonindigenous, exogenous pathogen. The introduction of refined sugar into modern society's diet has tipped the balance from health to disease. New insight into the natural history of the leading cariogenic bacteria, the mutans streptococci, may contribute ways to control or prevent this infectious disease. Here, we use the host-parasite model as a platform for viewing the pathogenicity of the caries process in contrast to other infectious diseases.

Cariogenic virulence characteristics of mutans streptococci isolated from caries-active and caries-free adults.

The objective of the study was to compare aciduricity (ability, to live in acid), acidogenicity (ability to produce acid), and intracellular polysaccharide production of mutans streptococci (MS) strains isolated from caries-active (CA, with one or more cavitated lesions) and caries-free (CF, with no clinically observable new caries in the last five years) adults. Forty-three MS strains from 17 of 17 CA adults, and 14 strains from eight of 12 CF adults were investigated. MS isolates' growth, survival, and pH reduction in pH 3.5-7.0 broths were evaluated to compare their acidogenicity and aciduricity. Extracellular water-soluble polysaccharide (WSP) and water-insoluble polysaccharide (WISP) was extracted from MS culture in BHI broth with 5 percent sucrose and assessed by a colorimetric anthrone-sulfuric acid microassay. No significant differences in mean aciduricity were found between CA and CF MS isolates (P>0.05, t test). However, significantly more CA subjects (29 percent) were colonized by MS strains with aciduricity above the average than CF subjects (13 percent, Fisher's exact test, P<0.05). Furthermore, CA MS strains produced significantly more acid at pH<5 (Mann-Whitney, P<0.05) and significantly more CA subjects were colonized with more acidogenic MS at pH<4.5 (Fisher's exact test, P<0.01). Similarly, CA MS isolates produced significantly more WISP than CF (Mann-Whitney test, P<0.01) while no statistical difference was found in WSP between the two groups. More CA subjects were colonized by multiple strains with aciduricity, acidogenicity, and polysaccharide synthesis ability above average. The study indicated that differences in acidogenicity, aciduricity, and polysaccharide synthesis in strains of MS may partially contribute to increased caries activity.

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.

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.

In vitro demineralization by strains of Actinomyces viscosus and Streptococcus sobrinus of sound and demineralized root surfaces.

Root sections were inoculated with one of two strains of Actinomyces viscosus or a strain of Streptococcus sobrinus and then incubated for 9 days in Trypticase soy broth (TSB) containing 0.25% glucose or TSB supplemented with 0.25% sucrose (TSB+S). Lesion progression was measured from microradiographs. One section from each group was examined with a transmission electron microscope. Lesion progression was associated with a fall in the pH of the medium. However, lesion progression was not correlated with the mean 48-hour pH of the medium. In both TSB and TSB + S, lesion progression with A. viscosus was significantly greater than in the S. sobrinus group. However, the mean 48-hour pH value in TSB + S was lower than that in the S. sobrinus group. Plaque formation in TSB was, subjectively, least in the S. sobrinus group. Examination of transmission electron micrographs revealed bacteria penetrating the surfaces of the sections and extending into the lesion in the A. viscosus groups but not in the S. sobrinus group. This in vitro bacterial plaque model of root caries may be suitable for investigations of the bacterial etiology of root-surface caries and the virulence factors associated with pathogenicity.

Characterization of glucosyltransferaseB, GtfC, and GtfD in solution and on the surface of hydroxyapatite.

GlucosyltransferaseB, GtfC, and GtfD were purified by hydroxyapatite column chromatography, followed by ultrafiltration from the culture supernatant fluids of three Streptococcus milleri constructs (gift from Dr. H.K. Kuramitsu) which harbored individual gtf genes of Streptococcus mutans GS5. GtfB, GtfC, and GtfD were enzymatically active both in solution and in an experimental pellicle (HA-CWS-Gtf) formed by adsorbing Gtf onto the surface of clarified human whole saliva (CWS)-coated hydroxyapatite (HA). The Km values for sucrose for all three enzymes were lower when the enzyme was adsorbed to a surface, compared with when it was in solution. In solution phase assays, and in the absence of primer dextran, glucan production was enhanced 75% when both GtfB and GtfD were present in the reaction mixture, compared with the sum of the individual enzyme activities (p < 0.005). This enhancement did not occur when GtfC was additionally present, or when the GtfB+GtfD enzyme pair was adsorbed onto HA-CWS. In additional experiments, glucan formed by GtfB or GtfC, but not by GtfD, on a HA-CWS-Gtf surface increased adherence of Streptococcus mutans GS5 and Streptococcus sobrinus 6715 by seven- to nine-fold compared with adherence when no glucan was present on the pellicle surface (p < 0.001). Further, treatment of the HA-CWS-GtfB-glucan or HA-CWS-GtfC-glucan pellicle with alpha-1,6 dextranase significantly reduced adherence of both streptococcal strains (p < 0.001). These results show that GtfB, GtfC, and GtfD are enzymatically active in an adsorbed state and that the nature of their product glucan can influence the adherence of cariogenic oral streptococci to an experimental pellicle.

Influence of rampant caries in dams on caries activity in their offspring.

Available evidence indicates that infants, for the most part, acquire their oral flora, in particular Streptococcus mutans, from their mothers. The purpose of this study was to explore the effect of rampant caries in rat dams on subsequent caries activity in their offspring. Rampant caries was induced by surgical desalivation of four dams, and subsequent infection with Streptococcus sobrinus 6715. Two intact dams served as controls. Desalivated dams, having rampant caries, reared litters that experienced significantly higher caries scores (P = 0.0001) on smooth and sulcal surfaces than litters from intact dams. Fluoride given to the dams did not affect caries scores of their pups. The results suggest that the magnitude and virulence of the maternal reservoir of S. sobrinus is a critical factor in the primary infection and caries activity in the offspring. Results support the concept that caries activity in mothers should be controlled in any program designed to prevent caries in infants and children.

Effects of phenytoin on dental caries and plaque in Streptococcus sobrinus-infected rats.

Effects of phenytoin (PHT) administration on dental caries in rats infected with Streptococcus sobrinus 6715 were investigated. Twenty-day-old specific pathogen-free Fischer male rats were infected with S. sobrinus 6715 and fed diet 2000 containing 56% sucrose with or without PHT for 52 days. Antibody responses against anti-S. sobrinus in serum and saliva failed to show a statistical difference between PHT-treated and nontreated rats. These results indicate that PHT treatment increased plaque deposition and dental caries in the rats infected with S. sobrinus 6715 and fed diet 2000 containing PHT (1-2 mg/g), as compared with those similarly infected and fed diet without PHT (P < 0.001).

[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.

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.

Genotypic diversity and virulence traits of streptococcus sobrinus isolated from caries-free children and children suffering severe early childhood caries.

OBJECTIVE: To evaluate the genotypic diversity and some virulence traits of Streptococcus sobrinus (S. sobrinus) isolated from caries-free children and children suffering severe early childhood caries (SECC). METHODS: S. sobrinus isolated from stimulated whole saliva samples of 91 caries-free children and 87 SECC children were subcultured, identified by polymerase chain reaction and genotyped by arbitrarily primed polymerase chain reaction. Polysaccharide synthesis ability, acidogenicity, aciduricity and the adherence ability of these S. sobrinus isolates were measured. RESULTS: The frequency of S. sobrinus detection was 18.39% (16/87) in SECC children, which was significantly higher than that (3.30%, 3/91) in caries-free children. One to three different genotypes of S. sobrinus were detected in each SECC child. Only one genotype was colonised in each caries-free child. In SECC children, the production of water-insoluble glucan (WIG) was positively correlated with the ability of S. sobrinus adhering to a glass surface. CONCLUSION: The presence of S. sobrinus could be a risk factor for high caries activity in severe early childhood caries. The multi-genotypes could be related to different caries suceptibility. Water-insoluble glucan plays an important role in the adherence and accumulation of S. sobrinus on tooth surfaces.