Metagenomics of the modern and historical human oral microbiome with phylogenetic studies on Streptococcus mutans and Streptococcus sobrinus.

We have recently developed bioinformatic tools to accurately assign metagenomic sequence reads to microbial taxa: SPARSE for probabilistic, taxonomic classification of sequence reads; EToKi for assembling and polishing genomes from short-read sequences; and GrapeTree, a graphic visualizer of genetic distances between large numbers of genomes. Together, these methods support comparative analyses of genomes from ancient skeletons and modern humans. Here, we illustrate these capabilities with 784 samples from historical dental calculus, modern saliva and modern dental plaque. The analyses revealed 1591 microbial species within the oral microbiome. We anticipated that the oral complexes of Socransky et al., which were defined in 1998, would predominate among taxa whose frequencies differed by source. However, although some species discriminated between sources, we could not confirm the existence of the complexes. The results also illustrate further functionality of our pipelines with two species that are associated with dental caries, Streptococcus mutans and Streptococcus sobrinus. They were rare in historical dental calculus but common in modern plaque, and even more common in saliva. Reconstructed draft genomes of these two species from metagenomic samples in which they were abundant were combined with modern public genomes to provide a detailed overview of their core genomic diversity. This article is part of the theme issue 'Insights into health and disease from ancient biomolecules'.

Quantitative analysis of changes in salivary mutans streptococci after orthodontic treatment.

INTRODUCTION: The purpose of this study was to analyze the initial changes in salivary mutans streptococci levels after orthodontic treatment with fixed appliances. METHODS: Our subjects consisted of 58 adults. Whole saliva and simplified oral hygiene index values were obtained at 4 time points: at debonding (T1), 1 week after debonding (T2), 5 weeks after debonding (T3), and 13 weeks after debonding (T4). Repeated measures analysis of variance was used to determine the time-related differences in salivary bacterial levels and the simplified oral hygiene index values among the 4 time points after quantifying the salivary levels of Streptococcus mutans, Streptococcus sobrinus, and total bacteria with real-time polymerase chain reaction. RESULTS: Simplified oral hygiene index values and total bacteria significantly decreased, but salivary mutans streptococci levels significantly increased after orthodontic treatment. The amounts of total bacteria in saliva significantly decreased at T3 (T1, T2 > T3, T4), and the simplified oral hygiene index values decreased at T2 (T1 > T2, T3, T4). However, salivary S mutans and S sobrinus significantly increased at T3 and T4, respectively (T1, T2 < T3 < T4). Furthermore, the proportion of mutans streptococci to total bacteria significantly increased at T4 (T1, T2, T3 < T4). CONCLUSIONS: This study suggests that careful hygienic procedures are needed to reduce the risk for dental caries after orthodontic treatment, despite overall improved oral hygiene status.

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.

Genotypic diversity of Streptococcus mutans and Streptococcus sobrinus in 3-4-year-old children with severe caries or without caries.

BACKGROUND. The genotypic diversity of both Streptococcus mutans and Streptococcus sobrinus in children with different caries experience remains unclear. AIM. To investigate the genotypic diversity of S. mutans and S. sobrinus in children with severe early childhood caries (SECC) and in caries-free (CF) children. METHODS. Stimulated saliva of 87 SECC and 91 CF children aged 3-4 years was collected and submitted to cultivation, and MS colonies were enumerated. The genomic fingerprint analysis of S. mutans and S. sobrinus was carried out using AP-PCR. RESULTS. One to five genotypes of S. mutans were colonized in an oral cavity of SECC and CF children; 85.5% SECC children and 57.9% CF children harboured more than one genotype of S. mutans. One to three genotypes of S. sobrinus were detected from each SECC child; 31.25% SECC children harboured more than one genotype of S. sobrinus. And one genotype was colonized in each CF child. S. mutans isolates from different individuals displayed distinctive DNA fingerprints. CONCLUSIONS. DNA fingerprints of S. mutans and S. sobrinus isolates from 3- to 4-year-old children displayed genetic polymorphism, and S. mutans has greater genetic diversity than S. sobrinus. SECC children harboured more genotypes of S. mutans and S. sobrinus than CF children.

Dental caries experience in relation to salivary findings and molecular identification of S. mutans and S. sobrinus in subjects with Down syndrome.

This study investigated the association between clinical and salivary or molecular parameters in Down syndrome subjects. Sixty individuals (1- to 48-year old) were clinically examined using DMFT/DMFS. Stimulated saliva was collected; salivary flow was calculated (mL/min), buffering capacity was measured using a standard pH tape. In addition, 25 µL of saliva was diluted using 10-fold-dilution method and then placed on Mitis-Salivarius-Bacitracin agar to count colony forming units (CFU/mL) of mutans streptococci. Polymerase chain reaction analysis identified species. Caries indexes were 0.65-13.5 (DMFT) and 0.65-26.0 (DMFS) according to groups. Ninety-four percent of subjects had low flow rate (0.7-1.0 mL/min) and 44% had low buffering capacity (pH < 4). Besides, 60% had more than 1 × 10(6) CFU/mL, 60% had S. mutans, and 41.4% had S. sobrinus. Caries indexes did not significantly correlate with flow rate, buffering capacity, CFU/mL by Pearson's correlation (p > 0.05), and showed no significant association with prevalence of species by Chi-square (p > 0.05). There is no association between clinical picture and salivary or molecular parameters in Down syndrome subjects.

Morphological differentiation between S. mutans and S. sobrinus on modified SB-20 culture medium.

Due to the major role of Streptococcus mutans and Streptococcus sobrinus in the etiology of dental caries, it is important to use culture media that allow for differentiating these bacterial species. The aim of this study was to evaluate the suitability of a modified SB-20 culture medium (SB-20M) for the isolation and morphological differentiation of S. mutans and S. sobrinus, compared to biochemical identification (biotyping). Saliva samples were collected using the spatula method from 145 children, seeded on plates containing the SB-20M, in which sucrose was replaced by coarse granular cane sugar, and incubated in microaerophilia at 37°C during 72 h. Identification of the microorganisms was performed under stereomicroscopy based on colony morphology of 4904 colonies. The morphological identification was examined by biochemical tests of 94 randomly selected colonies with the macroscopic characteristic of S. mutans and S. sobrinus using sugar fermentation, resistance to bacitracin and production of hydrogen peroxide. There was no statistically significant difference (p>0.05) between morphological identification in the SB-20M medium and biochemical identification (biotyping). Biotyping confirmed that S. mutans and S. sobrinus colonies were correctly characterized in the SB-20M in 95.8% and 95.5% of the cases, respectively. Of the mutans streptococci detected in the children 98% were S. mutans and 2% S. sobrinus. The SB-20M medium is reliable for detection and direct morphological identification of S. mutans and S. sobrinus.

Human milk compounds inhibiting adhesion of mutans streptococci to host ligand-coated hydroxyapatite in vitro.

Acquisition of mutans streptococci at an early age is a risk factor for later caries development. Following our recent finding that human milk may inhibit adhesion of Streptococcus mutans the aim of the present study was to identify compounds in human milk preventing adhesion of mutans streptococci to saliva- or gp340-coated hydroxyapatite (s-HA and gp340-HA) using an in vitro model system. Superdex 200 fractions of human milk and purified proteins were screened for binding inhibition of the S. mutans strain Ingbritt. Avid inhibition was seen to both s-HA and gp340-HA for caseins, lactoferrin, IgA and IgG, and moderate inhibition for alpha-lactalbumin and bile salt-stimulated lipase, whereas albumin and lysozyme had no effect. The inhibitory epitope in beta-casein was delineated to its C-terminal LLNQELLNPTHQIYPVTQPLAPVHNPISV stretch by use of synthetic peptides. Similarly, a peptide (SCKFDEYFSQSCA) corresponding to the human lactoferrin stretch that is highly homologous to the previously shown inhibitory stretch of bovine lactoferrin was found to inhibit S. mutans Ingbritt binding. Inhibition by human milk, IgA, and the inhibitory beta-casein peptide was universal among 4 strains of S. mutans (Ingbritt, NG8, LT11, JBP) and 2 strains of S. sobrinus (6715 and OMZ176). IgG inhibited 4, alpha-lactalbumin 3 and lactoferrin 2 of these 6 strains. It was also confirmed that none of the milk components coated on HA mediated S. mutans Ingbritt adhesion, which was consistent with the finding that no milk protein was recognized on Western blots by gp340/DMBT1 monoclonal antibodies.

Isolation and characterization of the mutans streptococci from the dental plaques in Koreans.

Mutans streptococci have been implicated as cariogenic bacteria in dental caries because they can produce high levels of dental caries-causing lactic acid and extracellular polysaccharide. The aim of this study was to isolate and characterize the mutans streptococci from the dental plaque obtained from Koreans. The dental plaque samples were collected from the anterior and molar teeth of both jaws in 155 subjects (aged 2 to 33.2 years, average age 13.7+/-4.7 years). The samples were diluted by 100-fold in 1x PBS and plated on mitis-salivarius bacitracin (MSB) agar plates. The mutans streptococci grown on MSB plates were screened by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) targeting dextranase gene (dex). The mutans streptococci were identified at the species level using a 16S rDNA sequencing comparison method. The biochemical tests were carried out to biotype the mutans streptococci. Ninety-five strains of the mutans streptococci out of 358 colonies, which were derived from 141 subjects, were isolated. Of them, 77 strains and 18 strains were Streptococcus mutans and Streptococcus sobrinus, respectively. The biotyping data showed that 62, 1, 20, 10, and 2 strains were biotypes I, II, IV, V and variant, respectively. Of the two strains of variant biotype, one strains was similar to biotype IV except that it was positive to the arginine hydrolysis test. We considered this one strain a new biotype, and classified it as biotype VII. In conclusion, S. mutans and its biotype I was most frequently isolated in Korean dental plaque. The mutans streptococci strains isolated in this study might be useful for the study of the pathogenesis and the prevention of dental caries.

Quantitative determination of adhesion patterns of cariogenic streptococci to various orthodontic adhesives.

OBJECTIVE: To investigate the adhesion of various cariogenic streptococci to orthodontic adhesives. MATERIALS AND METHODS: Five light-cure orthodontic adhesives (one fluoride-releasing composite, three non-fluoride-releasing composites, and one resin-modified glass ionomer cement) were used. The adhesive type, bacterial strain, incubation time, and saliva coating were studied. Thirty specimens of each adhesive were incubated with unstimulated whole saliva or phosphate-buffered saline for 2 hours. Binding assays were then performed by incubating tritium-labeled streptococci with the adhesives for 3 or 6 hours. RESULTS: The results showed a characteristic adhesion pattern according to the type of bacterial strains used. Streptococcus mutans LM7 showed the highest amount of adhesion, whereas S sobrinus B13 showed the lowest amount of adhesion. The cariogenic streptococci adhered to the glass ionomer significantly more than to the composites, whereas there was no significant difference in the adhesion amount among the four composites. The extended incubation time significantly increased bacterial adhesion. However, saliva coating did not significantly alter adhesion patterns of cariogenic streptococci. CONCLUSIONS: This study suggests that cariogenic streptococci can adhere diversely according to adhesive type and that the adhesion of the cariogenic streptococci is not influenced by its fluoride-releasing properties.

Effect of mixed mutans streptococci colonization on caries development.

OBJECTIVE: To evaluate the clinical importance of mixed mutans streptococci colonization in predicting caries in preschool children. METHODS: Caries prevalence was examined twice, with a 6-month interval, in 410 preschool children aged 3-4 years at baseline. A commercial strip method was used to evaluate the mutans streptococci score in plaque collected from eight selected interdental spaces and in saliva. Mutans streptococci typing polymerase chain reaction (PCR) assays (Streptococcus sobrinus and Streptococcus mutans, including serotypes c, e, and f) were performed using colonies on the strips as template. RESULTS: Twenty variables were examined in a univariate analysis to predict caries development: questionnaire variables, results of clinical examination, mutans streptococci scores, and PCR detection of S. sobrinus and S. mutans (including serotypes c, e, and f). Sixteen variables showed statistically significant associations (P < 0.04) in the univariate analysis. However, when entered into a logistic regression, only five variables remained significant (P < 0.05): caries experience at baseline; mixed colonization of S. sobrinus and S. mutans including S. mutans serotypes; high plaque mutans streptococci score; habitual use of sweet drinks; and nonuse of fluoride toothpaste. CONCLUSION: 'Mixed mutans streptococci colonization' is a novel measure correlated with caries development in their primary dentition.

Differentiation of mutans streptococci by intact cell matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

It is difficult to distinguish mutans streptococci on the species level, and even more so on the subspecies level. Intact cell matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) (ICM) was applied to reference strains of five of the species of the mutans group (Streptococcus criceti, Streptococcus downei, Streptococcus mutans, Streptococcus ratti, Streptococcus sobrinus), nonmutans streptococci (Streptococcus oralis, Streptococcus mitis, Streptococcus salivarius, and Streptococcus sanguinis), and 177 mutans streptococci isolated from saliva of 10 children. From the analysis of the reference strains, readily distinguishable ICM mass spectra were obtained for the different species. Based on multivariate statistical analysis, a correct and unambiguous assignment was made of the spectra of 159 isolated mutans streptococci to S. mutans and 16 isolates to S. sobrinus. Two isolates were sorted out and were identified by sequencing of their 16S rRNA genes as Streptococcus anginosus. In addition, ICM indicated a misclassification for some reference strains (AHT, V 100 and E 49) and re-classified AHT and E 49 as S. ratti and V 100 as S. sobrinus. This was confirmed by 16S rDNA sequencing. Based on a statistical similarity analysis of the spectra of reference strains and a quantitative assessment of the reproducibility of ICM, the isolates identified as either S. mutans or S. sobrinus were phenotyped on the subspecies level. In the population of the clinical isolates, 14 unambiguously different S. mutans and three different S. sobrinus phenotypes were detected. ICM proved to be a powerful tool for a differentiation of mutans streptococci down to the subspecies level.

Arbitrarily primed polymerase chain reaction for fingerprinting the genotype identification of mutans streptococci in children with Down syndrome.

This study investigated the possible intrafamilial similarity of mutans streptococcal strains in some families with a child with Down syndrome using chromosomal DNA fingerprinting. The isolates were genotyped using arbitrarily primed polymerase chain reaction with the OPA 02 and OPA 03 primers. The results showed that five children with Down syndrome harbored mutans streptococci genotypes different from those of their mothers. A matching of genotypes was observed within the control pair (mother/child without Down syndrome). After six months, new samples were collected from all participants. Analysis showed that samples from children with Down syndrome were colonized by a new strain of Streptococcus mutans that did not match the previously collected one. The results suggest the S. mutans indigenous bacteria change more than once in children with Down syndrome.

Longitudinal study of transmission, diversity, and stability of Streptococcus mutans and Streptococcus sobrinus genotypes in Brazilian nursery children.

The aim of this study was to perform a follow-up evaluation of the Streptococcus mutans and Streptococcus sobrinus colonization profile of children's oral cavities, which included the pattern of vertical transmission from mother to child, genotypic diversity, and stability of the strains. The subjects were 16 mother-child pairs, who were monitored for 20 months. Samples of saliva, tongue dorsum, alveolar ridge mucosa, and dental plaque from the children were collected bimonthly. Saliva samples from the mothers were also collected. After isolation and identification, the arbitrarily primed PCR method was performed for the genotypic characterization of S. mutans (968 isolates) and S. sobrinus (111 isolates). At the time the strains were acquired, the children harbored one to four distinct genotypes of S. mutans and only one genotype of S. sobrinus. Although S. mutans prevalence and genotypic diversity were greater than those of S. sobrinus, the presence of matching genotypes of S. mutans and S. sobrinus was similar (in 81.25 and 83.33% of mother-child pairs, respectively), suggesting vertical transmission for both species. This longitudinal study showed an increase in genotypic diversity of S. mutans in the oral cavity during the follow-up period: most of the initially acquired genotypes persisted, normally those genotypes transmitted by the mother, and some were lost during follow-up; new strains were also acquired. In conclusion, S. mutans and S. sobrinus genotypes acquired from maternal or alternative sources may show effective persistence in the oral cavity and/or transitory detection in the children's mouths, reflecting the continuous development of oral microbiota in children.

Time of initial acquisition of mutans streptococci by human infants.

The aim of this study was to detect and monitor the acquisition of mutans streptococci (MS) in healthy Brazilian children. Samples of 4 different sites (saliva, tongue dorsum, dental ridges, and dental plaque, if teeth were present) were collected from 33 edentulous nursery school infants (5.9+/-1.5 month-old), using sterilized swabs, bi-monthly for 24 months. Saliva samples from the mothers were collected only once. After inoculation, and incubation typical morphotype colonies, were isolated and submitted to amplification by the technique of polymerase chain reaction (PCR) for identification. The PCR method identified 1667 strains as MS In 29 of the children's samples, the first positive culture for MS occurred at 15.3+/-4.6 months. At the end of the follow-up period, 77% of the children were classified as colonized and in 33% MS was found as a transient microorganism. A positive correlation was found between the time of MS acquisition by the infant and the number of erupted teeth (p<0.0001), and the time of emergence of the first tooth (p=0.0048). After 24 months, there were no dental caries, and 77% of children remained caries-free. These results indicate that MS colonization in this sample of low-income pre-school children may begin earlier than suggested by some investigations.

A method for mapping the distribution pattern of cariogenic streptococci within dental plaque in vivo.

This study was carried out to develop a method for mapping the distribution of cariogenic oral streptococci, Streptococcus mutans and Streptococcus sobrinus, from the outermost to the innermost plaque. Ten consenting subjects were asked to form plaque by abstaining from tooth brushing over 3 days within in situ plaque-generating devices, which were placed on the upper molars. The plaque formed in the devices was separated into 8-10 layered fractions (100 microm thick). Genomic DNA was extracted from each plaque fraction by a commercial DNA purification kit and used for the amplification of the 16S ribosomal RNA gene sequences by polymerase chain reaction (PCR) with universal primers. The products were then amplified by PCR with S. mutans- or S. sobrinus-specific nested primers. The final products were separated on agarose gels, stained and photographed to confirm the existence of S. mutans and S. sobrinus. The results showed that S. mutans was detected in the plaque obtained from all of the 10 subjects and S. sobrinus in the plaque of 7 subjects. However, the distribution patterns of fractions positive for S. mutans and S. sobrinus varied among the subjects, with a tendency for frequent detection of both species in the outer to middle layers of dental plaque. There were no plaque fractions in which only S. sobrinus was found. This method could be useful to map the distribution of cariogenic microorganisms and to estimate the bacterial ecology for oral biofilm.

Streptococcus mutans genotypes isolated from root and coronal caries.

The aim of the present study was to evaluate the colonization profile and clonal distribution of Streptococcus mutans isolated from oral cavities that presented coronal and root caries lesions. The isolation and biochemical identification of mutans streptococci were carried out by using saliva samples, dental plaque, and tissue from the caries lesions. In order to confirm their molecular identity, S. mutans and Streptococcus sobrinus were submitted to the PCR method, using specific primers for portions of the glucosyltransferase genes (gtfB and gtfI, respectively). The AP-PCR method was used to detect the genetic polymorphism of S. mutans strains. Among the isolated and identified species, S. mutans showed a significantly greater frequency of isolation (59.2%) than the other mutans streptococci. Each of the subjects harbored two to ten genotypes of S. mutans, randomly distributed in different sites. S. mutans genotypes showed no evidence of variability in colonizing noncarious and carious surfaces within the same individual, nor evidence of etiologic differences between coronal and root caries. This study showed that no particular genotype of S. mutans is uniquely associated with the initiation and progression of caries, and that root and coronal caries can emerge in the presence of a broad spectrum of S. mutans clones.

Colonization of Streptococcus mutans and Streptococcus sobrinus genotypes and caries development in children to mothers harboring both species.

The major route of early acquisition of mutans streptococci in humans is a vertical transmission from mother to child. The purpose of this longitudinal study was to examine the acquisition, distribution and persistence of Streptococcus mutans and Streptococcus sobrinus in children whose mothers harbored both species and to study the caries incidence in relation to colonization of these bacteria. Fifteen mother-child pairs were followed during the child's first 7 years. Stimulated salivary samples were taken from the mothers and the children. Plaque samples were also collected from the teeth and the tongue of the children. The samples were analyzed by cultivating techniques together with genomic fingerprinting and hybridizing. The caries experience was evaluated on the sampling occasions and retrospectively using the records of caries registrations from the community clinics. During the 7-year period 10 of the 15 children acquired mutans streptococci. Only 4 of them were colonized by both S. mutans and S. sobrinus despite the fact that their mothers harbored both species. In 2 of the children S. sobrinus was found later than S. mutans. A total of 26 genotypes were found in the children and 9 of them were identical to their mothers. New genotypes and a gain-loss pattern were noted especially in the children but also in their mothers. The groups of teeth first positive for the two species were the deciduous molars. The caries experience was low during the study period with 8 children showing no caries.

Longitudinal study of intrafamilial mutans streptococci ribotypes.

To explore the intrafamilial homology and longitudinal stability of colonization by early acquired mutans streptococci, genomic fingerprinting was performed on 254 strains (192 Streptococcus mutans and 62 Streptococcus sobrinus strains) collected from 16 families (16 mother-child pairs, seven fathers and four siblings). Genomic DNA was digested by the restriction endonuclease HindIII, followed by gel electrophoresis, Southern blotting, and hybridization with a digoxigenin-labeled 16S rDNA probe, and hybrid detection by enhanced chemiluminescence. One to five ribotypes were identified per person, and between two and nine (median five) within each family. Fourteen of the 16 mother-child pairs showed homology for at least one ribotype (range 1-4). Six of the seven father-child pairs had one ribotype in common. Ten of the 13 longitudinally examined children showed persistence of at least one ribotype over a period of up to 16 yr. The results support the notion of intrafamilial transfer of mutans streptococci, and suggest that colonization of early acquired strains persists into young adulthood.

Identification of mutans streptococci by restriction fragment length polymorphism analysis of polymerase chain reaction-amplified 16S ribosomal RNA genes.

Mutans streptococci are frequently isolated from dental plaque and carious lesions. These bacteria have been identified by conventional methods such as biochemical and serologic tests followed by the isolation of colonies on the mitis-salivarius agar, which are sometimes inconsistent. Recently, species-specific polymerase chain reaction (PCR) has been reported to rapidly identify Streptococcus mutans and Streptococcus sobrinus. However, in the case of identification and classification into several species, e.g. within the group of mutans streptococci consisting of seven species, the identification using species-specific PCR seems somewhat inefficient because of need for the development and preparation of specific primers for each species. Therefore, in this study we developed a simple method using restriction fragment length polymorphism analysis of PCR-amplified 16S ribosomal RNA genes (16S rRNA genes PCR-RFLP) for the identification of seven different species included in the group of mutans streptococci. We amplified 16S rRNA gene sequences from genomic DNA samples by PCR using universal primers and digested the PCR products with the restriction endonucleases, HpaII and HaeIII. HpaII produced six RFLP patterns for eight reference strains, since the patterns for S. sobrinus, Streptococcus downei and Streptococcus ferus were similar. RFLP patterns produced with HaeIII could separate these three species. Furthermore, the RFLP patterns predicted from the 16S rRNA gene sequences in the GenBank database agreed with the actual RFLP patterns produced in the present study. The 16S rRNA sequence comparisons can be used to identify oral mutans streptococci; however, the identification by sequencing is sometimes difficult in large-scale studies and for small laboratories. Therefore, 16S rRNA genes PCR-RFLP, using HpaII and HaeIII, could be an alternative method for the identification of mutans streptococci, and may be applicable for large-scale studies on the cariogenicity of mutans streptococci.

Detection of Streptococcus mutans and Streptococcus sobrinus on the permanent first molars of the Mosuo people in China.

Streptococcus mutans and Streptococcus sobrinus are associated with the development of dental caries. The purpose of this study was to differentiate these bacteria by morphology, biochemical characteristics and PCR, and to compare their occurrence with the prevalence of dental caries in the Mosuo people. Plaque samples were collected from the permanent first molar in 126 Mosuo people (83 females, 43 males, aged 25-55 years, average age 36.1 +/- 7.73). Dental status was recorded as DMFT by WHO caries diagnostic criteria. Males had a significantly lower prevalence of caries and DMFT than females: 11.4 vs. 86.9% and 1.65 vs. 6.95, respectively (p<0.001). Morphological and biochemical tests gave unreliable results. The prevalence of S. mutans and S. sobrinus was 75.4 and 57.1%, respectively. 26.5% of females and 53.5% of males were positive for S. mutans alone, 18.1% of females and 16.3% of males were positive for S. sobrinus alone, while 50.6% of females and 18.6% of males were positive for both S. mutans and S. sobrinus and 4.8% of females and 11.6% of males were negative for both species. The DMFT scores of subjects positive for both S. mutans and S. sobrinus were significantly higher than of those positive for either S. mutans or S. sobrinus alone. These results indicate that subjects harboring both S. mutans and S. sobrinus have a significantly higher prevalence of dental caries than those with S. mutans or S. sobrinus alone.