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A randomized clinical trial studied the effects of early ad-ministration of Bifidobacterium animalis subsp. lactis BB-12 (BB-12) on oral colonization of (1) mutans streptococci (MS), and (2) BB-12. In this double-blind, placebo-controlled study, infants (n = 106) received probiotic bacteria (BB-12 group), xylitol (X group), or sorbitol (S group). Test tablets were administered twice a day (from the age of 1-2 months) with a novel slow-release pacifier or a spoon (daily dose of BB-12 1010 CFU, polyol 200-600 mg). Samples were collected from mucosa/teeth at the age of 8 months and 2 years for BB- 12 determination (qPCR) and plate culturing of MS (MSB, TYCSB), lactobacilli (Rogosa) and yeasts (Sabouraud). The MS levels of the mothers were determined (Dentocult SM Strip Mutans). The baseline characteristics of the three groups were similar. Mean duration of tablet delivery was 14.9 ± 6.7 months. In all groups, >90% of the mothers showed high MS counts (log CFU ≧5). MS colonization percentages of the children at the age of 2 years were rather low (BB-12 group: 6%; X group: 31%; S group: 10%; p < 0.05). The levels of lactobacilli and yeasts did not differ between the groups. BB-12 cell counts barely exceeding the detection limit were found in three of the oral samples of the 8-month-old children; however, the 2-year samples did not contain BB-12. The early administration of BB-12 did not result in permanent oral colonization of this probiotic or significantly affect MS colonization in the children.

The aims of this study were to evaluate clinically and microbiologically the effects of two resin-modified glass-ionomer cements (RMGICs) used as liners after incomplete dentine caries removal and to identify Streptococcus mutans and Streptococcus sobrinus strains isolated from dentine samples, before and after indirect pulp treatment. Twenty-seven primary molars with deep carious lesions, but without signs and symptoms of irreversible pulpitis, were submitted to indirect pulp treatment. Treatment consisted of incomplete excavation of the carious dentine, application of one of the RMGICs (Vitrebond or Fuji Lining LC) or calcium hydroxide cement (Dycal), and sealing for 3 months. Clinical evaluation (consistency, color, and wetness of dentine) and carious dentine collects were performed before temporary sealing and after the experimental period. Microbiological samples were cultivated in specific media for subsequent counting of mutans streptococci (MS) and lactobacilli (LB). MS colonies were selected for identification of S. mutans and S. sobrinus by polymerase chain reaction. After 3 months, the remaining dentine was hard and dry, and there was a significant decrease in the number of MS and LB, in all groups, although complete elimination was not achieved in 33% and 26% of the teeth for MS and LB, respectively. From 243 MS colonies selected, 216 (88.9%) were identified as S. mutans and only 2 (0.8%) as S. sobrinus . The use of resin-modified glass-ionomer liners after incomplete caries removal, as well as a calcium hydroxide cement, promoted significant reduction of the viable residual cariogenic bacteria in addition to favorable clinical changes in the remaining carious dentine.

We report a clinical trial of the effects of test tablets containing bovine lactoferrin and lactoperoxidase on oral malodor and salivary bacteria. Fifteen subjects with volatile sulfur compounds (VSCs) in mouth air above the olfactory threshold (H 2 S >1.5 or CH 3 SH >0.5 ng/10 ml) as detected by gas chromatography were enrolled in the trial. Either a test or a placebo tablet was ingested twice at 1-h intervals in two crossover phases. Mouth air was monitored for VSC levels at the baseline before ingestion of a tablet, 10 min after the first ingestion, 1 h (just before the second ingestion), and 2 h after the first ingestion. Whole saliva was analyzed at the baseline and at 2 h for bacterial numbers. At 10 min, the level of CH 3 SH was significantly lower in the test group (median [interquartile range] = 0.28 [0.00–0.68] ng/10 ml) compared to that in the placebo group (0.73 [0.47–1.00] ng/10 ml; P  = 0.011). The median concentration of CH 3 SH in the test group was below the olfactory threshold after 10 min until 2 h, whereas the level in the placebo group was above the threshold during the experimental period. No difference in the numbers of salivary bacteria was detected by culturing or quantitative PCR, but terminal restriction fragment length polymorphism detected one fragment with a significantly lower copy number at 2 h in the test group (mean ± standard error, 4.89 ± 0.11 log 10 copies/10 µl) compared to that in the placebo group (5.38 ± 0.15 log 10 copies/10 µl; P  = 0.033). These results indicate a suppressive effect of the test composition on oral malodor and suggest an influence on oral bacteria.

Dental caries in pre-school children has significant public health and health disparity implications. To determine microbial risk markers for this infection, this study aimed to compare the microbiota of children with early childhood caries with that of caries-free children. Plaque samples from incisors, molars, and the tongue from 195 children attending pediatricians’ offices were assayed by 74 DNA probes and by PCR to Streptococcus mutans. Caries-associated factors included visible plaque, child age, race, and snacking habits. Species were detected more frequently from tooth than tongue samples. Lactobacillus gasseri (p < 0.01), Lactobacillus fermentum, Lactobacillus vaginalis, and S. mutans with Streptococcus sobrinus (all p < 0.05) were positively associated with caries. By multifactorial analysis, the probiotic Lactobacillus acidophilus was negatively associated with caries. Prevotella nigrescens was the only species (p < 0.05) significantly associated with caries by the ‘false discovery’ rate. Analysis of the data suggests that selected Lactobacillus species, in addition to mutans streptococci, are risk markers for early childhood caries.

Aim This was to examine the occurrence of S. mutans , S. sobrinus and C. albicans in dental plaque and saliva from caries-free and caries-active Greek children. Methods Saliva and dental plaque samples from 46 caries-free and 51 caries-active 3-to-13-year-old children were examined using selective media for the three microbes. Identification of isolated mutans streptococci ( S. mutans and S. sobrinus ) was performed with biochemical test and specific DNA probes. The salivary levels of mutans streptococci were additionally determined by a chair-side test (Dentocult ® SM strips). Results The isolation frequencies of S. mutans , S. sobrinus and C. albicans were 66, 11 and 18 %, respectively. Caries-active children harboured more frequently and at significantly higher numbers the specific microbes than caries-free children. A similar pattern was observed with the Dentocult ® SM strip scores. No correlation was found between the presence of these microbes and the age or gender of the children. Conclusions Caries experience was statistically significantly related to the presence of all three microbes under study, both in dental plaque and saliva.

The aim of this study was to examine the colonization of Streptococcus mutans and Streptococcus sobrinus in supra-gingival plaque samples and to determine their correlation with the prevalence of early childhood caries (ECC) in Thai children. A total of 344 Thai children, ages 3 and 5 years, were invited to participate in this study. Caries status of the children was examined. Supra-gingival plaque samples were collected. Quantitative real-time PCR was performed to evaluate DNA levels of S. mutans and S. sobrinus. Eighty-five percent of the children were colonized by S. mutans and 50.9% of them were colonized by S. sobrinus. The prevalence of ECC was 43.8% and 56.2% among 3- and 5-year-old children, respectively, and was significantly associated with the presence of S. mutans and S. sobrinus. The severity of ECC was significantly correlated with increased DNA levels of the two bacteria. Children who were positive for S. mutans and S. sobrinus (Sm+/Sb+) were 8 times or 44 times more likely to experience ECC than children who were Sm-/Sb + or were Sm-/Sb-. The study evidence further suggest that children colonized by both S. mutans and S. sobrinus are at the higher risk for ECC. Molecular-based qPCR can be used to detect and quantify S. mutans and S. sobrinus colonization for epidemiological and clinical studies for ECC risk assessment.

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.

The aim of the study was to identify the number and distribution of genotypes of Streptococcus mutans (S. mutans) and Streptococcus sobrinus (S. sobrinus) isolated from caries-free and caries-active subjects. Non-stimulated saliva, buccal smooth surface of the right upper teeth, fissures of sound occlusal surface, and carious surface were sampled from 7 caries-free and 7 caries-active (DMFT ≥ 6) students aged 22-24 years. S. mutans and S. sobrinus were isolated using Chelex-100 and primarily identified by colony morphology and biochemical characteristics. The isolates of S. mutans were genotyped using arbitrarily primed polymerase chain reaction. A total of 516 isolates of S. mutans were genotyped from 47 sites in 14 students, and 44 different genotypes were determined. All of the caries-free individuals harbored S. mutans but not S. sobrinus, although individuals 3 and 7 had no S. mutans in their saliva. The CFU value of S. mutans on carious surfaces was the highest, and values in saliva, fissures, and occlusal surfaces were higher in caries-active individuals than in caries-free individuals. We detected 28 genotypes of S. mutans in caries-free individuals, each of who carried more than 3 genotypes. However, we found only 16 genotypes of S. mutans in caries-active individuals, each of who carried no more than 3 genotypes. More genotypes are harbored in the saliva, fissures, and smooth surfaces of caries-free individuals than of caries-active individuals. The proportion of samples positive for S. mutans and S. sobrinus was significantly higher in caries-active individuals than in caries-free individuals, and the presence of these species is a risk factor for high DMFT in dental caries. Isolates of S. mutans exist that have apparent genetic diversity. The genotypes of isolates might relate to differences in caries susceptibility.

Background Streptococcus mutans and Streptococcus sobrinus are associated with the development of dental caries in humans. However, previous diagnostic systems are unsuitable for monitoring viable cell numbers in oral specimens. Assessing the relationship between the numbers of viable and dead bacterial cells and oral status is important for understanding oral infectious diseases. Propidium monoazide (PMA) has been reported to penetrate dead cells following membrane damage and to cross-link DNA, thereby inhibiting DNA amplification. In the present study, we established an assay for selective analysis of two viable human cariogenic pathogens, S. mutans and S. sobrinus , using PMA combined with real-time PCR (PMA-qPCR). Results We designed species-specific primer sets for S. mutans and S. sobrinus , generated standard curves for measuring cell numbers, and evaluated the dynamic range of the assay. To determine the effectiveness of the assay, PMA was added to viable and autoclave-killed cell mixtures. PMA treatment effectively prevented DNA amplification from dead cells. No amplification of DNA from dead cells was observed in these organisms. In addition, we applied this assay to analyze viable cell numbers in oral specimens. A significant correlation was found between the number of viable S. mutans cells in saliva and that in plaque among caries-free patients, whereas no correlation was observed between saliva and carious dentin. The total and viable cell numbers in caries-positive saliva were significantly higher than those in caries-free saliva. Finally, we analyzed the usefulness of this assay for in vitro oral biofilm analysis. We applied PMA-qPCR for monitoring viable S. mutans cell numbers in vitro in planktonic cells and oral biofilm treated with hydrogen peroxide (H 2 O 2 ). In planktonic cells, the number of viable cells decreased significantly with increasing H 2 O 2 concentration, whereas only a small decrease was observed in biofilm cell numbers. Conclusions PMA-qPCR is potentially useful for quantifying viable cariogenic pathogens in oral specimens and is applicable to oral biofilm experiments. This assay will help to elucidate the relationship between the number of viable cells in oral specimens and the oral status.

Aim: We aimed to assess caries experience and microbiota in systemically healthy children with black stain (BS) and non-discoloured plaque. Methods: Forty-six children with BS and 47 counterparts with non-discoloured plaque aged 7.9 ± 1.3 years were clinically examined. Dental caries was scored using WHO criteria. Samples of BS and non-discoloured dental plaque were collected from tooth surfaces. The DNA of the samples was extracted and real-time PCR was performed to determine the total number of bacteria and the species Streptococcus mutans, S. sobrinus, Lactobacillus sp., Actinomyces naeslundii, Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia and Fusobacterium nucleatum. Results: Children with BS had lower DMFT (p = 0.013), lower DT values (p = 0.005) and a tendency to lower caries prevalence (p = 0.061) than children with non-discoloured plaque. Plaque samples of the BS group contained higher numbers of A. naeslundii (p = 0.005) and lower numbers of F. nucleatum (p = 0.001) and Lactobacillus sp. (p = 0.001) compared to the non-discoloured plaque samples of the control group. Comparing the children with BS and non-discoloured plaque, higher counts for A. naeslundii (p = 0.013) were observed in caries-free children with BS while in caries-affected children with BS, lower counts of F. nucleatum (p = 0.007) were found. Counts of Lactobacillus sp. were higher in non-discoloured plaque samples than in BS of caries-free and caries-affected children. Conclusion: Results suggest that the different microbial composition of BS might be associated with the lower caries experience in affected subjects. The role of black-pigmented bacteria associated with periodontitis needs further studies.