Comparative Analysis of Streptococcus Mutans and Streptococcus Sobrinus from Dental Plaque Samples of Nigerian Pre-school Children with and Without Caries.

BACKGROUND: Early childhood caries (ECC), despite being a preventable disease, remains prevalent in sub-Saharan Africa. This study compared the occurrence of Streptococcus mutans and Streptococcus sobrinus in dental plaque from caries free and caries active Nigerian preschool children. METHODS: Structured questionnaire were administered to eighty (80) mothers to inquire about the sociodemographic characteristics of their children. Diagnosis of dental caries was made according to the diagnostic criteria of the World Health Organization (WHO). Supragingival plaque sample collected from caries free children and children with dental caries (cavitated and non-cavitated lesions) using wooden toothpicks were cultured anaerobically. Samples were cultured in anaerobiosis and isolates obtained were identified based on molecular characterization performed by polymerase chain reaction (PCR) analysis. Statistical package for the social sciences (SPSS 25.0) was used for analysis. RESULTS: Streptococcus mutans was detected in 26(65.0%) children with early childhood caries, and S. sobrinus in 10(25.0%) children with early childhood caries. The presence of S. mutans and S. sobrinus occurring together was associated with a higher deft score. Occurrence of S. mutans did not show significant difference between children with ECC and caries free children (p=0.099), however, the proportion of children with ECC positive for S. sobrinus was significantly higher than that of caries free children (p= 0.003). CONCLUSIONS: S. mutans and S. sobrinus had a similar frequency of occurrence in children with ECC and caries free children. The occurrence of S. mutans alongside S. sobrinus in ECC is related to its severity.

CONTEXTE: La carie de la petite enfance (CPE), bien qu'étant une maladie évitable, reste prévalente en Afrique sub-saharienne. Cette étude a comparé la présence de Streptococcus mutans et Streptococcus sobrinus dans la plaque dentaire d'enfants nigérians d'âge préscolaire avec et sans caries. MÉTHODES: Des questionnaires structurés ont été administrés à quatrevingts (80) mères pour connaître les caractéristiques sociodémographiques de leurs enfants. Le diagnostic de la carie dentaire a été établi selon les critères de diagnostic de l'Organisation mondiale de la santé (OMS). Des échantillons de plaque supragingivale prélevés sur des enfants exempts de caries et des enfants atteints de caries dentaires (lésions cavitaires et non cavitaires) à l'aide de cure-dents en bois ont été mis en culture en anaérobiose. Les échantillons ont été mis en culture en anaérobiose. Les isolats obtenus ont été identifiés sur la base d'une caractérisation moléculaire effectuée par une analyse de réaction en chaîne par polymérase (PCR). Le progiciel statistique pour les sciences sociales (SPSS 25.0) a été utilisé pour l'analyse. RÉSULTATS: Streptococcus mutans a été détecté chez 26(65,0%) enfants atteints de caries de la petite enfance, et S. sobrinus chez 10(25,0%) enfants atteints de caries de la petite enfance. La présence simultanée de S. mutans et de S. sobrinus était associée à un score de déformation plus élevé. La présence de S. mutans n'a pas montré de différence significative entre les enfants atteints de CEC et les enfants indemnes de caries (p=0,099), cependant, la proportion d'enfants atteints de CEC positifs pour S. sobrinus était significativement plus élevée que celle des enfants indemnes de caries (p= 0,003). CONCLUSIONS: S. mutans et S. sobrinus ont une fréquence d'apparition similaire chez les enfants atteints de CEC et les enfants indemnes de caries. La présence de S. mutans et de S. sobrinus dans l'ECC est liée à sa gravité. MOTS CLÉS: Streptococcus mutans, Streptococcus sobrinus, caries de lapetite enfance, plaque supragingivale.

Antimicrobial effects of the ginsenoside Rh2 on monospecies and multispecies cariogenic biofilms.

AIMS: To investigate the effects of the ginsenoside Rh2 on monospecies and multispecies cariogenic biofilms and explore the mechanism of the antibiofilm effect of Rh2 in vitro. METHODS AND RESULTS: Streptococcus mutans, Streptococcus sobrinus and Streptococcus sanguinis were chosen to form the monospecies or multispecies biofilms. Crystal violet staining and laser scanning confocal microscopy were used to observe the effect of Rh2 on biofilms in vitro. Cytotoxicity was examined by the Cell Counting Kit-8. The effects of Rh2 on bacterial membranes were observed via transmission electron microscopy (TEM). The isobaric tags for relative and absolute quantification (iTRAQ) method were used to profile the common differentially expressed proteins. Gene expression was analysed by reverse transcription quantitative polymerase chain reaction. In general, the treatment of cariogenic biofilms with Rh2 significantly decreased biomass accumulation by inhibiting bacterial growth and extracellular polysaccharide synthesis without any cytotoxic effects. TEM imaging showed that Rh2 could disrupt the cell membranes of these bacteria. The iTRAQ results indicated that the levels of mannose-specific IIC/D and acetaldehyde/alcohol dehydrogenase were substantially down-regulated, while the mRNA expression of the corresponding genes were significantly changed. CONCLUSIONS: Our data revealed a potential application for Rh2 in the protection against dental caries via the inhibition of cariogenic biofilms. SIGNIFICANCE AND IMPACT OF THE STUDY: This study describes the first application of a ginsenoside against multispecies cariogenic biofilms. Rh2 may serve as an alternative agent to prevent dental caries by effectively modulating the pathogenic potentials of oral biofilms.

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.

Frequency of dental caries in active and inactive systemic lupus erythematous patients: salivary and bacterial factors.

OBJECTIVE: The objective of this study was to determine dental caries frequency and to analyze salivary and bacterial factors associated with active and inactive systemic lupus erythematous (SLE) patients. Also, a proposal to identify dental caries by a surface, teeth, and the patient was developed. MATERIAL AND METHODS: A cross-sectional, blinded study that included 60 SLE patients divided into two groups of 30 subjects each, according to the Activity Index for Diagnosis of Systemic Lupus Erythematous (SLEDAI). The decayed, missing, and filled teeth (DMFT) index and Integrative Dental Caries Index (IDCI) were used for analyzing dental caries. The saliva variables recorded were: flow, pH, and buffer capacity. The DNA copies of Streptococcus mutans and Streptococcus sobrinus were estimated by real-time PCR. RESULTS: The caries frequency was 85% for SLE subjects (73.3% for inactive systemic lupus erythematous (ISLE) and 100% for active systemic lupus erythematous (ASLE)); DMFT for the SLE group was 12.6 ± 5.7 and the IDCI was (9.8 ± 5.9). The ASLE group showed a salivary flow of 0.65 compared with 0.97 ml/1 min from the ISLE group; all variables mentioned above showed a statistical difference (p < 0.05). The salivary pH was 4.6 (6.06 for ISLE and 3.9 for ASLE). The DNA copies of S. mutans and S. sobrinus were high; all variables mentioned above show a significant statistical difference (p < 0.05) between groups. CONCLUSION: SLE patients had high DMFT and IDCI scores that were associated with a decrease in salivary flow, pH, and buffer capacity. There were high counts of S. sobrinus and S. mutans species, and IDCI is a useful tool to provide more detail about dental caries in epidemiological studies.

Penetration of Streptococcus sobrinus and Streptococcus sanguinis into dental enamel.

The aim of this pilot study was to assess the difference in virulence of acidogenic and aciduric oral streptococci in an in vitro caries model using their penetration depths into dental enamel. 30 caries-free extracted molars from 11- to 16-year-olds were cleaned ultrasonically for 1 min with de-ionized water and, after air-drying, embedded in epoxy resin. After 8-h of setting at room temperature, the specimens were ground on the buccal side with SiC-paper 1200 (particle size 13-16 µm). Enamel was removed in circular areas sized 3 mm in diameter; the mean depth of removed enamel was 230 ± 60 µm. 15 specimens each were incubated anaerobically under standardized conditions with 24 h-cultures of Streptococcus sanguinis 9S or Streptococcus sobrinus OMZ 176 in Balmelli broth at 37 ± 2 °C; the pH-values of the broths were measured at the beginning and end of each incubation cycle. After 2, 4, 6, 8, and 10 weeks 3 teeth each were fixed in 2.5% glutaraldehyde in cacodylate buffer for 24 h, washed 3× and dehydrated 30-60min by sequential washes through a series of 30-100% graded ethanol. The teeth were cut in half longitudinally; afterward, two slits were made to obtain fracture surfaces in the infected area. After critical-point-drying the fragments were gold-sputtered and viewed in a scanning electron microscope at magnifications of ×20-20,000. After 10 weeks of incubation, penetration of S. sanguinis of 11.13 ± 24.04 µm below the break edges into the enamel was observed. The invasion of S. sobrinus reached depths of 87.53 ± 76.34 µm. The difference was statistically significant (paired t test: p = 0.033). The experimental penetration depths emphasize the importance of S. sanguinis versus S. sobrinus in the context of the extended ecological plaque hypothesis.

AP-PCR detection of Streptococcus mutans and Streptococcus sobrinus in caries-free and caries-active subjects.

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.

Streptococcus sobrinus as a Predominant Oral Bacteria Related to the Occurrence of Dental Caries in Polish Children at 12 Years Old.

Dental caries is listed by the WHO as one of the major non-communicable diseases that need to be prevented and treated. The aim of the study was to evaluate the prevalence and severity of caries expressed as the Decayed, Missing and Filled Permanent Teeth (DMFT) index in 12-year-old Polish children and to verify bacterial species related to the occurrence of dental caries. Quantitative real-time PCR analysis of DNA isolated from saliva samples was performed to detect 8 cariogenic and periopathogenic bacterial strains. A total of 118 Polish children were enrolled in the study. They had low mean DMFT scores of 1.58 ± 1.98. The prevalence of dental caries in the children tested was low (53.4%), with a tendency to decrease compared to previous oral surveys. Bacterial abundance of other species in the dental caries and caries-free groups did not differ; however, periopathogenic Prevotella pallens, Fusobacterium nucleatum along with cariogenic Streptococcus mutans and Lactobacillus fermentum were significantly strongly correlated in the caries-active subjects. The prevalence of S. sobrinus was significantly higher in children with dental caries (p = 0.023) and correlated with higher DMFT. It may temporarily play an important role in the initiation of the cariogenic process or in its enhancement due to an ecological imbalance in dental microbiota.

Streptococcus mutans and Streptococcus sobrinus contributions in dental caries in Iranian and Afghan children: A report from serotype distribution and novel STs.

OBJECTIVE: This study evaluated the frequency and the effects of S. mutans and S. sobrinus on Decayed, Missing, and Filled Teeth (DMFT) scores in Iranian and Afghan populations. Serotyping of S. mutans isolates and multi-locus sequence typing (MLST) were the secondary goals. DESIGN: This study was performed on 360 saliva and plaque samples from people from age groups of 4-7 and 15-17 years with Iranian and Afghan nationality who were residents of Tehran province. The DMFT index of the study population was determined, and S. mutans and S. sobrinus were identified using species-specific primers. Following the collagen-binding protein of S. mutans (cnm) gene identification, serotypes were determined, and genotyping was performed on eight selected isolates by assessing eight loci in the existing MLST scheme. RESULTS: Of 360 samples, 300 were recruited as population study. Of these, 204 (51%) harbored S. mutans alone. In 42 specimens (10.5%), both specious were detected, and 54 (13.5%) were free of both. The frequencies of c, f, e, and k serotypes were 47.5%, 17.9%, 13.8%, and 8.1%, respectively. The frequency of serotype f was significantly higher in four-year-old Iranian children. MLST showed eight different sequence types (STs), which were confirmed as novel singleton sequence types. CONCLUSIONS: The high frequency of serotypes k and f as systemic serotypes with the cnm gene among the Iranian population suggests the need for more worldwide studies on serotype distribution. Since very few studies have reported the epidemiological status of mutans streptococci (MS), the molecular properties of the isolates are unknown. Thus, the STs reported in this study should be considered as emerging strains.

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.

A Novel Competence Pathway in the Oral Pathogen Streptococcus sobrinus.

Streptococcus sobrinus is an etiologic cause of dental caries (tooth decay) in humans. Our knowledge of S. sobrinus is scant despite the organism's important role in oral health. It is widely believed that S. sobrinus lacks the natural competence pathways that are used by other streptococci to regulate growth, virulence, and quorum sensing. The lack of natural competence has also prevented genetic manipulation of S. sobrinus, limiting our knowledge of its pathogenicity. We discovered that most strains of S. sobrinus contain a new class of the ComRS competence system. Although S. sobrinus is typically placed among the mutans group streptococci, the S. sobrinus ComRS system is most similar to the competence pathways in the salivarius group. Unlike all other ComRS systems, the S. sobrinus pathway contains 2 copies of the transcriptional regulator ComR and has a peptide pheromone (XIP) that lacks any aromatic amino acids. Synthetic XIP enables transformation of S. sobrinus with plasmid or linear DNA, and we leverage this newfound genetic tractability to confirm that only 1 of the ComR homologs is required for induced competence while the other appears to suppress competence. Exogenous XIP increases the expression of bacteriocin gene clusters and produces an antimicrobial response that inhibits growth of S. mutans. We also identified 2 strains of S. sobrinus that appear to be "cheaters" by either not responding to or not producing XIP. We show how a recombination event in the nonresponsive strain could restore function of the ComRS pathway but delete the gene encoding XIP. Thus, the S. sobrinus ComRS pathway provides new tools for studying this pathogen and offers a lens into the evolution of ecological cheaters.

Caries experience and quantification of Streptococcus mutans and Streptococcus sobrinus in saliva of Sudanese schoolchildren.

Streptococcus mutans and Streptococcus sobrinus are among the most commonly isolated bacterial species implicated as etiological agents of dental caries. Details of the composition of the oral microflora related to dental caries should aid in assessing the prevalence and risk of disease at an individual level. The aim of the present study was to determine the presence and relative amounts of S. mutans and S. sobrinus in the saliva samples obtained from schoolchildren in Khartoum State, the Sudan, and to study the association of the amounts of S. mutans and S. sobrinus with caries experience, socioeconomic status and sugar-sweetened snacks in this population. 140 samples, 30 of which were from individuals with caries experience, were examined using quantitative real-time polymerase chain reaction (qRT-PCR) with specific oligonucleotide primers. The mean ratio of fold differences of S. mutans to S. sobrinus was 0.77 (SD 5.4) and 2.29 (SD 6.0) for samples obtained from caries-free and caries-active individuals, respectively. This suggested that the proportion of S. sobrinus was higher than S.mutans in the caries-active group when compared to the caries-free group. An association was found between children with caries-active lesions and the frequent consumption of sticky desserts and higher socioeconomic status. S. sobrinus seems to be associated with caries experience in the studied population. A proposal of caries screening programs designed to test for S. sobrinus in this population may be developed.

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'.

Culture-based PCR analysis of plaque samples of Japanese school children to assess the presence of six common cariogenic bacteria and its association with caries risk.

The aim of the study was to assess the presence of six common cariogenic bacteria from Cariostat-inoculated plaque samples of Japanese elementary school children through PCR analysis and check its associations with caries risk testing the validity of Cariostat as a caries risk assessment tool. This epidemiological school-based study investigated plaque samples of 399 Japanese elementary school children. Assessed using the Cariostat, 48.2% of the children had high caries risk. DNA detection of Streptococcus mutans, Streptococcus sobrinus, Streptococcus salivarius, Lactobacillus casei, Lactobacillus plantarum, Lactobacillus fermentum and both S. mutans and S. sobrinus was seen in 65.2%, 24.1%, 69.7%, 17.5%, 7.8%, 19.3%, and 17.3% of the participants, respectively. Except for S. salivarius, the presence of all other investigated bacteria resulted in a statistically significant increase among the proportion of subjects with high caries risk. Caries risk assessed using Cariostat was significantly influenced by the presence of cariogenic bacteria. Being a selective medium for cariogenic bacteria, the Cariostat can be a useful and direct source of cariogenic bacterial DNA for PCR analysis while effectively assessing caries risk.

Genes responsible for dextran-dependent aggregation of Streptococcus sobrinus strain 6715.

INTRODUCTION: Streptococcus sobrinus exhibits more significant dextran-dependent aggregation mediated by glucan-binding proteins than Streptococcus mutans. We have identified four glucan-binding protein C gene (gbpC) homologues designated as gbpC1, gbpC2, dblA and dblB in S. sobrinus in contrast to the single gene gbpC in S. mutans. We attempted to determine which gene is most responsible for the dextran-dependent aggregation of S. sobrinus. METHODS: We introduced mutation with a chemical mutagen, 1-methyl-3-nitro-1-nitrosoguanidine, into S. sobrinus strain 6715 and analysed the four gbpC homologous gene sequences in the parental strain 6715 and an obtained aggregation-negative mutant NUM-Ssg99. We also examined the localization of proteins encoded by these genes in the mutant NUM-Ssg99. RESULTS: The nucleotide sequences of the gbpC1, gbpC2 and dblA genes in NUM-Ssg99 were 100% identical to the homologous genes in parental strain 6715. In contrast, a truncated mutation was detected in the dblB gene and the mutant protein devoid of the LPXTG motif was confirmed by Western blot analysis to be released into the extracellular milieu. CONCLUSION: We conclude that the dblB gene among the four GbpC homologous protein genes is most responsible for aggregation in strain 6715.

Use of checkerboard DNA-DNA hybridization to evaluate the internal contamination of dental implants and comparison of bacterial leakage with cast or pre-machined abutments.

AIMS: To evaluate the checkerboard DNA-DNA hybridization method for detection and quantitation of bacteria from the internal parts of dental implants and to compare bacterial leakage from implants connected either to cast or to pre-machined abutments. MATERIALS AND METHODS: Nine plastic abutments cast in a Ni-Cr alloy and nine pre-machined Co-Cr alloy abutments with plastic sleeves cast in Ni-Cr were connected to Branemark-compatible implants. A group of nine implants was used as control. The implants were inoculated with 3 microl of a solution containing 10(8) cells/ml of Streptococcus sobrinus. Bacterial samples were immediately collected from the control implants while assemblies were completely immersed in 5 ml of sterile Tripty Soy Broth (TSB) medium. After 14 days of anaerobic incubation, occurrence of leakage at the implant-abutment interface was evaluated by assessing contamination of the TSB medium. Internal contamination of the implants was evaluated with the checkerboard DNA-DNA hybridization method. RESULTS: DNA-DNA hybridization was sensitive enough to detect and quantify the microorganism from the internal parts of the implants. No differences in leakage and in internal contamination were found between cast and pre-machined abutments. Bacterial scores in the control group were significantly higher than in the other groups (P<0.05). CONCLUSION: Bacterial leakage through the implant-abutment interface does not significantly differ when cast or pre-machined abutments are used. The checkerboard DNA-DNA hybridization technique is suitable for the evaluation of the internal contamination of dental implants although further studies are necessary to validate the use of computational methods for the improvement of the test accuracy.

Development of species-specific primers for detection of Streptococcus mutans in mixed bacterial samples.

Streptococcus mutans is the major microbial pathogen associated with dental caries in children. The objectives of this study were to design and evaluate species-specific primers for the identification of S. mutans. Validation of the best primer set, Sm479F/R, was performed using seven S. mutans reference strains, 48 ATCC non-S. mutans strains, 92 S. mutans clinical isolates, DNA samples of S. mutans-Streptococcus sobrinus or S. mutans-Streptococcus sanguinis, and mixed bacterial DNA of saliva samples from 33 18-month-old children. All of the S. mutans samples tested positive, and no PCR products were amplified from members of the other streptococci or nonstreptococci strains examined. The lowest detection level for PCR was 10(-2) ng of S. mutans DNA (c. 4.6 x 10(3) copies) in the test samples. The results of this study suggest that the Sm479F/R primer pair is highly specific and sensitive for identification of S. mutans in either purified or mixed DNA samples.

[Microbiologic and molecular diagnostic of cariogenic bacteria in pregnant women from the Araucania Region of Chile]. / Diagnóstico microbiológico y molecular de bacterias cariogénicas en mujeres embarazadas de la Región de La Araucanía, Chile.

Dental caries is a transmissible infectious disease in which Streptococcus mutans is a principal protagonist. Although it is widely believed that pregnancy is harmful to teeth, the effect of pregnancy on the development of caries is not clear. Considering this situation, the aim of the present study was to evaluate the levels of infection and to differentiate bacterial species with cariogenic potential in pregnant women from the Araucania region in Chile, by bacteriological and molecular analysis. In this work, we evaluated 51 pregnant women aged 15 to 40 years. The results show that 100% of women are infected by mutans streptococci Group, and 70.6% exhibited high levels of infection (> 500.000 cfu/mL). The molecular analysis shows that Streptococcus mutans and Streptococcus sobrinus frequencies were 92.1% and 1.9%, respectively. In conclusion, our data suggest that pregnant women are a high risk group for caries development.

Improved method for rapid and accurate isolation and identification of Streptococcus mutans and Streptococcus sobrinus from human plaque samples.

Mutans streptococci (MS), specifically Streptococcus mutans (SM) and Streptococcus sobrinus (SS), are bacterial species frequently targeted for investigation due to their role in the etiology of dental caries. Differentiation of S. mutans and S. sobrinus is an essential part of exploring the role of these organisms in disease progression and the impact of the presence of either/both on a subject's caries experience. Of vital importance to the study of these organisms is an identification protocol that allows us to distinguish between the two species in an easy, accurate, and timely manner. While conducting a 5-year birth cohort study in a Northern Plains American Indian tribe, the need for a more rapid procedure for isolating and identifying high volumes of MS was recognized. We report here on the development of an accurate and rapid method for MS identification. Accuracy, ease of use, and material and time requirements for morphological differentiation on selective agar, biochemical tests, and various combinations of PCR primers were compared. The final protocol included preliminary identification based on colony morphology followed by PCR confirmation of species identification using primers targeting regions of the glucosyltransferase (gtf) genes of SM and SS. This method of isolation and identification was found to be highly accurate, more rapid than the previous methodology used, and easily learned. It resulted in more efficient use of both time and material resources.

Inhibition of streptococcal biofilm by hydrogen water.

OBJECTIVES: The accumulation of oral bacterial biofilm is the main etiological factor of oral diseases. Recently, electrolyzed hydrogen-rich water (H-water) has been shown to act as an effective antioxidant by reducing oxidative stress. In addition to this general health benefit, H-water has antibacterial activity for disease-associated oral bacteria. However, little is known about the effect of H-water on oral bacterial biofilm. The objective of this study was to confirm the effect of H-water on streptococcal biofilm formation. METHODS: In vitro streptococcal biofilm was quantified using crystal violet staining after culture on a polystyrene plate. The effect of H-water on the expression of genes involved in insoluble glucan synthesis and glucan binding, which are critical steps for oral biofilm formation, was evaluated in MS. In addition, we compared the number of salivary streptococci after oral rinse with H-water and that with control tap water. Salivary streptococci were quantified by counting viable colonies on Mitis Salivarius agar-bacitracin. RESULTS: Our data showed that H-water caused a significant decrease in in vitro streptococcal biofilm formation. The expression level of the mRNA of glucosyltransferases (gtfB, gtfc, and gtfI) and glucan-binding proteins (gbpC, dblB) were decreased remarkably in MS after H-water exposure for 60s. Furthermore, oral rinse with H-water for 1 week led to significantly fewer salivary streptococci than did that with control tap water. CONCLUSIONS: Our data suggest that oral rinse with H-water would be helpful in treating dental biofilm-dependent diseases with ease and efficiency.

Expression of the gtfI gene from Streptococcus sobrinus in Streptococcus anginosus using integration-mediated transformation system.

We have constructed a Streptococcus anginosus transformant expressing the gtfI gene from Streptococcus sobrinus, using a previously developed integration-mediated transformation system to introduce foreign genes onto the oral streptococcal chromosome, and attempted to evaluate the gene expression. In this system, one cloning plasmid and three pACYC184 derivatives, anchor, heterodimer, and integration plasmids were used for the construction of a series of integrants via homologous recombination. A portion of S. sobrinus gtfI gene devoid of approximately 1 kb of the 5'-region derived from pMD39 was cloned into the integration plasmid and introduced onto the S. anginosus chromosome. Next, the polymerase chain reaction product corresponding to 2.0 kb of the 5'-region of the gtfI gene from S. sobrinus chromosome was further cloned into the cloning plasmid, and the intact gtfI gene was reconstructed following integration. The final S. anginosus integrant successfully secreted the enzymatically active gtfI gene products and extracellular enzyme was characterized. This enzyme produced water-insoluble glucans and glucan-forming activity was stimulated by the addition of dextranT10. When this integrant was grown in Todd-Hewitt broth supplemented with sucrose, the integrant adhered to the glass surface in vitro and this integrant exhibited the different colony morphology on Mitis-Salivarius agar plates compared to S. sobrinus and S. anginosus. These observations strongly suggest that the construction of S. anginosus integrant expressing S. sobrinus gtfI gene using this transformation system may be an effective means of analysis of cariogenic biofilm formation.