Molecular and serological typing of Streptococcus mutans strains isolated from young Galician population: relationship with the oral health status.

The aim of this study was to determine the prevalence of Streptococcus mutans and its serotypes in samples from oral cavity of young Galician population and their relationship with the oral health state. The variables generally associated with dental caries, such as salivary flow rate, buffering capacity, eating habits, and lifestyle, were also analysed. No relationship was found between the variables studied and the presence of S. mutans in the oral cavity or the existence of dental caries. Presumptive strains of S. mutans were isolated from saliva samples from 48% of the analysed population. The use of conventional microbiological methods, API 20 Strep system, and species-specific polymerase chain reaction (PCR) allowed to substantiate the identity of the strains as S. mutans. Multiplex PCR protocols, developed in this study for the simultaneous detection of S. mutans and serotypes c, e, and f and for detection of S. mutans and serotype k, also confirmed this result and demonstrated that serotype c was predominant in the studied young Galician population (86%). Serotypes e (8%), k (3%), and f (2%) were also detected. Serotype c was detected in carious and caries-free subjects, while the remaining serotypes were only found in subjects with caries.

Comparison of the salivary and dentinal microbiome of children with severe-early childhood caries to the salivary microbiome of caries-free children.

BACKGROUND: The main objectives of this study were to describe and compare the microbiota of 1) deep dentinal lesions of deciduous teeth of children affected with severe early childhood caries (S-ECC) and 2) the unstimulated saliva of these children and 3) the unstimulated saliva of caries-free children, and to compare microbiota compositional differences and diversity of taxa in these sampled sites. METHODS: Children with S-ECC and without S-ECC were recruited. The saliva of all children with and without S-ECC was sampled along with the deep dentinal microbiota from children affected by S-ECC. The salivary microbiota of children affected by S-ECC (n = 68) was compared to that of caries-free children (n = 70), by Illumina MiSeq sequencing of 16S rRNA amplicons. Finally, the caries microbiota of deep dentinal lesions of those children with S-ECC was investigated. RESULTS: Using two beta diversity metrics (Bray Curtis dissimilarity and UniFrac distance), the caries microbiota was found to be distinct from that of either of the saliva groups (caries-free & caries-active) when bacterial abundance was taken into account. However, when the comparison was made by measuring only presence and absence of bacterial taxa, all three microbiota types separated. While the alpha diversity of the caries microbiota was lowest, the diversity difference between the caries samples and saliva samples was statistically significant (p < 0.001). The major phyla of the caries active dentinal microbiota were Firmicutes (median abundance value 33.5%) and Bacteroidetes (23.2%), with Neisseria (10.3%) being the most abundant genus, followed by Prevotella (10%). The caries-active salivary microbiota was dominated by Proteobacteria (median abundance value 38.2%) and Bacteroidetes (27.8%) with the most abundant genus being Neisseria (16.3%), followed by Porphyromonas (9.5%). Caries microbiota samples were characterized by high relative abundance of Streptococcus mutans, Prevotella spp., Bifidobacterium and Scardovia spp. CONCLUSIONS: Distinct differences between the caries microbiota and saliva microbiota were identified, with separation of both salivary groups (caries-active and caries-free) whereby rare taxa were highlighted. While the caries microbiota was less diverse than the salivary microbiota, the presence of these rare taxa could be the difference between health and disease in these children.

Exploring the Genomic Diversity and Cariogenic Differences of Streptococcus mutans Strains Through Pan-Genome and Comparative Genome Analysis.

Pan-genome refers to the sum of genes that can be found in a given bacterial species, including the core-genome and the dispensable genome. In this study, the genomes from 183 Streptococcus mutans (S. mutans) isolates were analyzed from the pan-genome perspective. This analysis revealed that S. mutans has an "open" pan-genome, implying that there are plenty of new genes to be found as more genomes are sequenced. Additionally, S. mutans has a limited core-genome, which is composed of genes related to vital activities within the bacterium, such as metabolism and hereditary information storage or processing, occupying 35.6 and 26.6% of the core genes, respectively. We estimate the theoretical core-genome size to be about 1083 genes, which are fewer than other Streptococcus species. In addition, core genes suffer larger selection pressures in comparison to those that are less widely distributed. Not surprisingly, the distribution of putative virulence genes in S. mutans strains does not correlate with caries status, indicating that other factors are also responsible for cariogenesis. These results contribute to a more understanding of the evolutionary characteristics and dynamic changes within the genome components of the species. This also helps to form a new theoretical foundation for preventing dental caries. Furthermore, this study sets an example for analyzing large genomic datasets of pathogens from the pan-genome perspective.

Salivary Microbiome Diversity in Caries-Free and Caries-Affected Children.

Dental caries (tooth decay) is an infectious disease. Its etiology is not fully understood from the microbiological perspective. This study characterizes the diversity of microbial flora in the saliva of children with and without dental caries. Children (3-4 years old) with caries (n = 20) and without caries (n = 20) were recruited. Unstimulated saliva (2 mL) was collected from each child and the total microbial genomic DNA was extracted. DNA amplicons of the V3-V4 hypervariable region of the bacterial 16S rRNA gene were generated and subjected to Illumina Miseq sequencing. A total of 17 phyla, 26 classes, 40 orders, 80 families, 151 genera, and 310 bacterial species were represented in the saliva samples. There was no significant difference in the microbiome diversity between caries-affected and caries-free children (p > 0.05). The relative abundance of several species (Rothia dentocariosa, Actinomyces graevenitzii, Veillonella sp. oral taxon 780, Prevotella salivae, and Streptococcus mutans) was higher in the caries-affected group than in the caries-free group (p < 0.05). Fusobacterium periodonticum and Leptotrichia sp. oral clone FP036 were more abundant in caries-free children than in caries-affected children (p < 0.05). The salivary microbiome profiles of caries-free and caries-affected children were similar. Salivary counts of certain bacteria such as R. dentocariosa and F. periodonticum may be useful for screening/assessing children's risk of developing caries.

Variation of expression defects in cell surface 190-kDa protein antigen of Streptococcus mutans.

Streptococcus mutans, which consists of four serotypes, c, e, f, and k, possesses a 190-kDa cell surface protein antigen (PA) for initial tooth adhesion. We used Western blot analysis to determine PA expression in 750 S. mutans isolates from 150 subjects and found a significantly higher prevalence of the isolates with PA expression defects in serotypes f and k compared to serotypes c and e. Moreover, the defect patterns could be classified into three types; no PA expression on whole bacterial cells and in their supernatant samples (Type N1), PA expression mainly seen in supernatant samples (Type N2), and only low expression of PA in the samples of whole bacterial cells (Type W). The underlying reasons for the defects were mutations in the gene encoding PA as well as in the transcriptional processing of this gene for Type N1, defects in the sortase gene for Type N2, and low mRNA expression of PA for Type W. Since cellular hydrophobicity and phagocytosis susceptibility of the PA-defective isolates were significantly lower than those of the normal expression isolates, the potential implication of such defective isolates in systemic diseases involving bacteremia other than dental caries was suggested. Additionally, multilocus sequence typing was utilized to characterize S. mutans clones that represented a proportion of isolates with PA defects of 65-100%. Therefore, we described the molecular basis for variation defects in PA expression of S. mutans. Furthermore, we also emphasized the strong association between PA expression defects and serotypes f and k as well as the clonal relationships among these isolates.

Cloning, characterization and anion inhibition study of a ß-class carbonic anhydrase from the caries producing pathogen Streptococcus mutans.

The oral pathogenic bacterium involved in human dental caries formation Streptococcus mutans, encodes for two carbonic anhydrase (CA, EC 4.2.1.1) one belonging to the α- and the other one to the ß-class. This last enzyme (SmuCA) has been cloned, characterized and investigated for its inhibition profile with a major class of CA inhibitors, the inorganic anions. Here we show that SmuCA has a good catalytic activity for the CO2 hydration reaction, with kcat 4.2×10(5)s(-1) and kcat/Km of 5.8×10(7)M(-1)×s(-1), being inhibited by cyanate, carbonate, stannate, divannadate and diethyldithiocarbamate in the submillimolar range (KIs of 0.30-0.64mM) and more efficiently by sulfamide, sulfamate, phenylboronic acid and phenylarsonic acid (KIs of 15-46µM). The anion inhibition profile of the S. mutans enzyme is very different from other α- and ß-CAs investigated earlier. Identification of effective inhibitors of this new enzyme may lead to pharmacological tools useful for understanding the role of S. mutans CAs in dental caries formation, and eventually the development of pharmacological agents with a new mechanism of antibacterial action.

Potent inhibitory effects of D-tagatose on the acid production and water-insoluble glucan synthesis of Streptococcus mutans GS5 in the presence of sucrose.

We examined and compared the inhibitory effects of D-tagatose on the growth, acid production, and water-insoluble glucan synthesis of GS5, a bacterial strain of Streptococcus mutans, with those of xylitol, D-psicose, L-psicose and L-tagatose. GS5 was cultured for 12h in a medium containing 10% (w/v) of xylitol, D-psicose, L-psicose, D-tagatose or L-tagatose, and the inhibitory effect of GS5 growth was assessed. Each sugar showed different inhibitory effects on GS5. Both D-tagatose and xylitol significantly inhibited the acid production and water-insoluble glucan synthesis of GS5 in the presence of 1% (w/v) sucrose. However, the inhibitory effect of acid production by D-tagatose was significantly stronger than that of xylitol in presence of sucrose.

Human Leucocyte Antigen Profile and Transmission of Mutans Streptococci in Mother-Child Pairs.

PURPOSE: To investigate possible association between the transmission of mutans streptococci and sharing the immune system component Human Leucocyte Antigen (HLA) class II in mother-child pairs. MATERIAL AND METHODS: Plaque samples from 43 mother-child pairs were cultivated and screened for mutans streptococci. In 14 pairs where both mother and child harboured the bacteria, the strains were genotyped by Random Amplified Polymorphic DNA and samples were run on PAGE gels. Analysis of genetic identity between mother and child strains was performed with help of software and Dice similarity index. The distribution of HLA of serogroup DR4 (HLA DR4) was studied in relation to maternal transmission and mutans streptococci colonisation in children. The study hypothesis was that in pairs where both mother and child were HLA DR4 positive, transmission of mutans streptococci was more likely. RESULTS: No correlation between the presence of HLA DR4 in mother and child and maternal transmission of mutans streptococci was established. However, the results showed no linkage between mutans streptococci colonisation and HLA DR 4. Of 15 children with mutans streptococci, 12 were HLA DR4 positive. CONCLUSION: The result suggests that presence of HLA DR4 could be a predisposing factor for colonisation with mutans streptococci in children.

Serotype diversity of Streptococcus mutans and caries activity in children in Argentina.

AIM: The purpose of this study was to analyse the serotype distribution of S. mutans and their association with caries activity in school children from Córdoba, Argentina. MATERIALS AND METHODS: Clinical examination was performed in 133 children. The dmft+DMFT and Significant Caries (SiC) indices were calculated to identify individuals with high caries activity. After DNA extractions of S. mutans strains, serotypes were determined by PCR amplifications. The median caries activity of each serotype group was compared using a non-parametric Kruskall-Wallis test. RESULTS: We obtained S. mutans strains from stimulated saliva of 94 children. The mean dmft+DMFT was 4.14 and the mean SiC index was 8.65. Serotype c was the most frequent (53.2%), followed by e (31.9%), f (8.5%) and k (6.4%). The comparison between the SiC and Non-Sic groups showed significant differences in the frequency of serotypes c and k. The median caries activity was non-significant in the different serotypes. CONCLUSION: The difference between the serotype frequencies detected in Argentina compared to those of other countries could be related with contrasting dietary habits. The results obtained in the present study would increase the knowledge about the epidemiology of dental caries in children from Argentina.

The usefulness of biotyping in the determination of selected pathogenicity determinants in Streptococcus mutans.

BACKGROUND: Streptococcus mutans is known to be a primary etiological factor of dental caries, a widespread and growing disease in Polish children. Recognition of novel features determining the pathogenicity of this pathogen may contribute to understanding the mechanisms of bacterial infections.The goal of the study was to determine the activity of prephenate dehydrogenase (PHD) and to illuminate the role of the enzyme in S. mutans pathogenicity. The strains were biotyped based on STREPTOtest 24 biochemical identification tests and the usefulness of biotyping in the determination of S. mutans pathogenicity determinants was examined. RESULTS: Out of ninety strains isolated from children with deciduous teeth fifty three were classified as S. mutans species. PDH activity was higher (21.69 U/mg on average) in the experimental group compared to the control group (5.74 U/mg on average) (P <0.001). Moreover, it was demonstrated that biotype I, established basing on the biochemical characterization of the strain, was predominant (58.5%) in oral cavity streptococcosis. Its dominance was determined by higher PDH activity compared to biotypes II and III (P = 0.0019). CONCLUSIONS: The usefulness of biotyping in the determination of Streptococcus mutans pathogenicity determinants was demonstrated. The obtained results allow for better differentiation of S. mutans species and thus may contribute to recognition of pathogenic bacteria transmission mechanisms and facilitate treatment.

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.

ClpL is required for folding of CtsR in Streptococcus mutans.

ClpL, a member of the HSP100 family, is widely distributed in Gram-positive bacteria but is absent in Gram-negative bacteria. Although ClpL is involved in various cellular processes, such as the stress tolerance response, long-term survival, virulence, and antibiotic resistance, the detailed molecular mechanisms are largely unclear. Here we report that ClpL acts as a chaperone to properly fold CtsR, a stress response repressor, and prevents it from forming protein aggregates in Streptococcus mutans. In vitro, ClpL was able to successfully refold urea-denatured CtsR but not aggregated proteins. We suggest that ClpL recognizes primarily soluble but denatured substrates and prevents the formation of large protein aggregates. We also found that in vivo, the C-terminal D2-small domain of ClpL is essential for the observed chaperone activity. Since ClpL widely contributes to various cellular functions, we speculate that ClpL chaperone activity is necessary to maintain cellular homeostasis.

[A novel TaqMan® MGB probe for specifically detecting Streptococcus mutans].

OBJECTIVE: To design a new TaqMan® MGB probe for improving the specificity of Streptococcus mutans's detection. METHODS: We extracted six DNA samples from different streptococcal strains for PCR reaction. Conventional nested PCR and TaqMan® MGB real-time PCR were applied independently. The first round of nested PCR was carried out with the bacterial universal primers, while a second PCR was conducted by using primers specific for the 16S rRNA gene of Streptococcus mutans. The TaqMan® MGB probe for Streptococcus mutans was designed from sequence analyses, and the primers were the same as nested PCR. Streptococcus mutans DNA with 2.5 mg/L was sequentially diluted at 5-fold intervals to 0.16 µg/L. Standard DNA samples were used to generate standard curves by TaqMan® MGB real-time PCR. RESULTS: In the nested PCR, the primers specific for Streptococcus mutans also detected Streptococcus gordonii with visible band of 282 bp, giving false-positive results. In the TaqMan® MGB real-time PCR reaction, only Streptococcus mutans was detected. The detection limitation of TaqMan® MGB real-time PCR for Streptococcus mutans 16S rRNA gene was 20 µg/L. CONCLUSION: We designed a new TaqMan® MGB probe, and successfully set up a PCR based method for detecting oral Streptococcus mutans. TaqMan® MGB real-time PCR is a both specific and sensitive bacterial detection method.

Comparison of mutans streptococcal strains of father, mother, and child in indian families using chromosomal DNA fingerprinting.

INTRODUCTION: It is now understood and accepted that there is a direct transmission of mutans streptococci (MS) from the mother to the child. There is also a direct correlation between the levels of MS in the mother and the caries status of the child. Advanced technologies in molecular biology like chromosomal DNA fngerprinting have established beyond doubt that the mother and the child bear similar strains of MS. AIM: A study was designed with the aim of comparing the MS strains between the father, mother and the child in Indian families. MATERIALS AND METHODS: A group of 20 Indian families comprising of the father, mother and child were selected and divided into caries free and caries active groups. Mixed salivary samples were collected from the individuals and were cultured for the growth of Mutans streptococci. The colonies were counted on a colony counter and a comparison was made between the mutans streptococcal counts of the mother and the caries status of the child. Further, the genotypes of the father, mother and the child were isolated and compared using the technique of chromosomal DNA fngerprinting. Following electrophoresis, the band pattern obtained was compared for similarities or differences. The results of the same were tabulated and evaluated statistically. RESULTS: When the colony counts of the mother (in CFU/ml) were compared with the 'dft' status of the child, a positive correlation was seen in group II. Intergroup comparison using the unpaired T test was statistically signifcant. Electrophoretic analysis of the chromosomal DNA on the agarose gels revealed identical band patterns in 13 mother-child pairs, which was statistically signifcant. Three of the father-child pairs showed identical band patterns, which was statistically signifcant. Intergroup comparison using Chi-square test was not statistically signifcant. CONCLUSION: One may conclude that irrespective of the caries status of the child, majority of the mother child pairs share identical strains of MS and hence the mother is the primary source of infection. However, in children with a high dft, the father may also play an important role in the acquisition and transmission of MS.

Prevalence of salivary Streptococcus mutans serotype k in children undergoing congenital heart surgery.

OBJECTIVE: The prevalence of Streptococcus mutans serotype k, which was speculated that might be associated with the development of cardiovascular diseases, has been reported in adult cardiovascular surgery patients. There is no information about presence of serotype k in children with cardiac disease. The aim of this study was to determine the salivary prevalence of S. mutans serotype k in children with congenital heart disease. STUDY DESIGN: Salivary samples of 25 patients undergoing elective surgery for congenital heart defects with cardiopulmonary bypass and an age and gender matched control group of 25 healthy children were enrolled in the study. Species-specific 16SrRNA gene sequences were used for S. mutans and serotype-specific rgpF gene sequences were used for S. mutans serotype k determination in stimulated saliva samples. RESULTS: S. mutans was detected in 19 (76%) of the study and 15 (60%) of the control children. The difference was not shown to be statistically significant. Serotype k was determined from 3 (12%) of the study group, while it was not determined from the samples of the control group. CONCLUSIONS: Our results indicate that those children with congenital heart disease may possess S. mutans serotype k in oral cavity at a higher frequency as similar with the adult cardiac surgery patients.

Complete genome sequence of the serotype k Streptococcus mutans strain LJ23.

Streptococcus mutans is the major pathogen of dental caries and occasionally causes infective endocarditis. Here we report the complete genome sequence of serotype k S. mutans strain LJ23, which was recently isolated from the oral cavity of a Japanese patient.

Complete genome sequence of Streptococcus mutans GS-5, a serotype c strain.

Streptococcus mutans, a principal causative agent of dental caries, is considered to be the most cariogenic among all oral streptococci. Of the four S. mutans serotypes (c, e, f, and k), serotype c strains predominate in the oral cavity. Here, we present the complete genome sequence of S. mutans GS-5, a serotype c strain originally isolated from human carious lesions, which is extensively used as a laboratory strain worldwide.

Subpopulation-specific transcriptome analysis of competence-stimulating-peptide-induced Streptococcus mutans.

Competence-stimulating-peptide (CSP)-mediated competence development in Streptococcus mutans is a transient and biphasic process, since only a subpopulation induces the expression of ComX in the presence of CSP, and the activation of the DNA uptake machinery in this fraction shuts down ~3 to 4 h postinduction. Here, we combine for the first time, to our knowledge, the bacterial flow-cytometric sorting of cells and subpopulation-specific transcriptome analysis of both the competent and noncompetent fraction of CSP-treated S. mutans cells. Sorting was guided by a ComX-green fluorescent protein (ComX-GFP) reporter, and the transcriptome analysis demonstrated the successful combination of both methods, because a strong enrichment of transcripts for comX and its downstream genes was achieved. Three two-component systems were expressed in the competent fraction, and among them was ComDE. Moreover, the recently identified regulator system ComR/S was expressed exclusively in the competent fraction. In contrast, the expression of bacteriocin-related genes was at the same level in all cells. GFP reporter strains for ComE and CipB (mutacin V) confirmed this expression pattern on the single-cell level. Fluorescence microscopy revealed that some ComX-expressing cells committed autolysis in an early stage of competence initiation. In viable ComX-expressing cells, the uptake of DNA could be shown on the single-cell level. This study demonstrates that all cells in the population respond to CSP through the activation of bacteriocin-related genes. Some of these cells start to activate ComX expression but then segregate into two subpopulations, one becoming competent and another one that lyses, resulting in intrapopulation diversity.

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.

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.