Comprehensive analysis of bacteriocins in Streptococcus mutans.

Streptococcus mutans produces bacteriocins that show antibacterial activity against several bacteria. However, comprehensive analysis of these bacteriocins has not been well done. In this study, we isolated 125 S. mutans strains from volunteers and determined their whole genome sequence. Based on the genome analysis, the distribution of each bacteriocin gene (mutacins I-IV, K8 and Smb) was investigated. We found 17, 5, and 2 strains showing 100% matches with mutacin I, mutacin II and mutacin III, respectively. Five mutacin III-positive strains had 2 mismatches compared to mature mutacin III. In 67 mutacin IV-positive strains, 38 strains showed 100% match with mutacin IV, while 29 strains showed some variations. In 23 mutacin K8- and 32 mutacin Smb-positive strains, all except one mutacin K8-positive strain showed 100% match with the mature peptides. Among 125 strains, 84 (65.1%), 26 (20.2%), and 5 (3.9%) strains were positive for one, two and three bacteriocin genes, respectively. Then, the antibacterial activity against oral streptococci and other oral bacterial species was investigated by using bacteriocin gene single-positive strains. Each bacteriocin gene-positive strain showed a different pattern of antibacterial activity. These results speculate that individual S. mutans strains may affect the bacterial composition of dental plaques.

Bacterial diversity and functional analysis of severe early childhood caries and recurrence in India.

Dental caries is the most prevalent oral disease affecting nearly 70% of children in India and elsewhere. Micro-ecological niche based acidification due to dysbiosis in oral microbiome are crucial for caries onset and progression. Here we report the tooth bacteriome diversity compared in Indian children with caries free (CF), severe early childhood caries (SC) and recurrent caries (RC). High quality V3-V4 amplicon sequencing revealed that SC exhibited high bacterial diversity with unique combination and interrelationship. Gracillibacteria_GN02 and TM7 were unique in CF and SC respectively, while Bacteroidetes, Fusobacteria were significantly high in RC. Interestingly, we found Streptococcus oralis subsp. tigurinus clade 071 in all groups with significant abundance in SC and RC. Positive correlation between low and high abundant bacteria as well as with TCS, PTS and ABC transporters were seen from co-occurrence network analysis. This could lead to persistence of SC niche resulting in RC. Comparative in vitro assessment of biofilm formation showed that the standard culture of S. oralis and its phylogenetically similar clinical isolates showed profound biofilm formation and augmented the growth and enhanced biofilm formation in S. mutans in both dual and multispecies cultures.

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'.

A Core Genome Multilocus Sequence Typing Scheme for Streptococcus mutans.

Streptococcus mutans is one of the primary pathogens responsible for the development of dental caries. Recent whole-genome sequencing (WGS)-based core genome multilocus sequence typing (cgMLST) approaches have been employed in epidemiological studies of specific human pathogens. However, this approach has not been reported in studies of S. mutans Here, we therefore developed a cgMLST scheme for S. mutans We surveyed 199 available S. mutans genomes as a means of identifying cgMLST targets, developing a scheme that incorporated 594 targets from the S. mutans UA159 reference genome. Sixty-eight sequence types (STs) were identified in this cgMLST scheme (cgSTs) in 80 S. mutans isolates from 40 children that were sequenced in this study, compared to 35 STs identified by multilocus sequence typing (MLST). Fifty-six cgSTs (82.35%) were associated with a single isolate based on our cgMLST scheme, which is significantly higher than in the MLST scheme (11.43%). In addition, 58.06% of all MLST profiles with ≥2 isolates were further differentiated by our cgMLST scheme. Topological analyses of the maximum likelihood phylogenetic trees revealed that our cgMLST scheme was more reliable than the MLST scheme. A minimum spanning tree of 145 S. mutans isolates from 10 countries developed based upon the cgMLST scheme highlighted the diverse population structure of S. mutans This cgMLST scheme thus offers a new molecular typing method suitable for evaluating the epidemiological distribution of this pathogen and has the potential to serve as a benchmark for future global studies of the epidemiological nature of dental caries.IMPORTANCEStreptococcus mutans is regarded as a major pathogen responsible for the onset of dental caries. S. mutans can transmit among people, especially within families. In this study, we established a new epidemiological approach to S. mutans classification. This approach can effectively differentiate among closely related isolates and offers superior reliability relative to that of the traditional MLST molecular typing method. As such, it has the potential to better support effective public health strategies centered around this bacterium that are aimed at preventing and treating dental caries.

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.

Temporal development of the oral microbiome and prediction of early childhood caries.

Human microbiomes are predicted to assemble in a reproducible and ordered manner yet there is limited knowledge on the development of the complex bacterial communities that constitute the oral microbiome. The oral microbiome plays major roles in many oral diseases including early childhood caries (ECC), which afflicts up to 70% of children in some countries. Saliva contains oral bacteria that are indicative of the whole oral microbiome and may have the ability to reflect the dysbiosis in supragingival plaque communities that initiates the clinical manifestations of ECC. The aim of this study was to determine the assembly of the oral microbiome during the first four years of life and compare it with the clinical development of ECC. The oral microbiomes of 134 children enrolled in a birth cohort study were determined at six ages between two months and four years-of-age and their mother's oral microbiome was determined at a single time point. We identified and quantified 356 operational taxonomic units (OTUs) of bacteria in saliva by sequencing the V4 region of the bacterial 16S RNA genes. Bacterial alpha diversity increased from a mean of 31 OTUs in the saliva of infants at 1.9 months-of-age to 84 OTUs at 39 months-of-age. The oral microbiome showed a distinct shift in composition as the children matured. The microbiome data were compared with the clinical development of ECC in the cohort at 39, 48, and 60 months-of-age as determined by ICDAS-II assessment. Streptococcus mutans was the most discriminatory oral bacterial species between health and current disease, with an increased abundance in disease. Overall our study demonstrates an ordered temporal development of the oral microbiome, describes a limited core oral microbiome and indicates that saliva testing of infants may help predict ECC risk.

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.

[Isolation of cariogenic Streptococcus mutans and the inhibitory effect of egg yolk antibody on caries].

Objective: To isolate the cariogenic Streptococcus mutans (Sm) strains and study the therapeutical effect of egg yolk antibody (IgY) of the Sm on dental caries development. Methods: Sm strains were isolated from the children's dental plaque samples. Morphological, biochemical and molecular biological methods were applied to identify the serotype, acid producing and adhesion abilities of isolated Sm strains. After inactivation one of the Sm strains was used as antigen to immune laying hens to collect and extract the specific anti-Sm IgY. The rats were infected with Sm (serotype e). After 16 weeks of infection, all the rats were found developing dental caries. The rats were then randomly divided into two groups. The rats in experimental group were supplied with diet containing anti-Sm IgY while the rats in control group with normal IgY. All rats were sacrificed after another 8 weeks' observation. The degree of caries for each rat was assessed using Keyes' method. Results: We isolated 7 Sm strains from the children's dental plaque samples in the present study. The numbers of serotype c, e, f, k were 3, 2, 0 and 2, respectively. All strains showed similar morphological and biochemical characters as standard UA159 Sm strain, and possessed strong capabilities of acid production and adherence. Interestingly, even the same serotypec strains, such as No.3 and No.7 strains, demonstrated significant difference on acid producing and adherence capabilities. After 16 weeks infection with serotype e strain, the rats' mandibular teeth were apparently decayed, and treatment with specific anti-Sm IgY obviously attenuated the development of caries in the experiment group rats (16.4±2.0) compared with that in the control group rats (30.2±9.3) (P<0.05) determined by Keyes' method. Conclusions: Seven cariogenic Sm strains of different serotypes were isolated, which possesses similar morphology and biochemical characters. Although belonging to the same serotype strains they always show significant difference in acid-producing and adherencec apabilities. Further experiment provides evidences that the serotype e strain could obviously induce caries independently, and employment of specific anti-Sm IgY as passive immunotherapy additive might effectively inhibit the further development of dental caries.

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.

Contribution of the Collagen-Binding Proteins of Streptococcus mutans to Bacterial Colonization of Inflamed Dental Pulp.

Streptococcus mutans is a major pathogen of dental caries. Collagen-binding proteins (CBPs) (approximately 120 kDa), termed Cnm and Cbm, are regarded as important cell surface antigens related to the adherence of S. mutans to collagenous tissue. Furthermore, CBP-positive S. mutans strains are associated with various systemic diseases involving bacteremia, such as infective endocarditis. Endodontic infection is considered to be an important cause of bacteremia, but little is known regarding the presence of S. mutans in dental pulp tissue. In the present study, the distribution and virulence of S. mutans in dental pulp tissues were investigated by focusing on CBPs. Adhesion and invasion properties of various S. mutans strains were analyzed using human dental pulp fibroblasts (HDPFs). CBP-positive strains had a significantly higher rate of adhesion to HDPFs compared with CBP-defective isogenic mutant strains (P<0.001). In addition, CBP-positive strains induced HDPF proliferation, which is a possible mechanism related to development of hyperplastic pulpitis. The distribution of S. mutans strains isolated from infected root canal specimens was then analyzed by PCR. We found that approximately 50% of the root canal specimens were positive for S. mutans. Approximately 20% of these strains were Cnm-positive, while no Cbm-positive strains were isolated. The Cnm-positive strains isolated from the specimens showed adhesion to HDPFs. Our results suggest that CBP-positive S. mutans strains exhibit high colonization in dental pulp. This could be a possible virulence factor for various systemic diseases.

Influência da contaminação cavitária na adaptação marginal das restaurações de resina composta / Influence of cavity contamination on marginal adaptation of composite resin restorations

Objetivo: avaliar a influencia da contaminação cavitária na adaptação marginal das restauraçõesde resina composta e a associação da lisozima ao sistema adesivo. Materiais e Métodos:Foram selecionados 40 terceiros molares permanentes e confeccionados preparos cavitários classeV com pontas diamantadas cilíndricas 1090 (4 mm de largura, 6 mm de comprimento e 1,5 mmde profundidade). Todos os dentes foram esterilizados e divididos aleatoriamente em 4 grupos(n=10): G1: sistema adesivo (SA) e restauração com resina composta (RC); G2: cavidadescontaminadas com 10 μl de Streptococcus mutans (0,5 de Mc Farland), SA e RC; G3:sistema adesivo associado com lisozima (ADL) e RC e G4: cavidades contaminadas com 10 μlde Streptococcus Mutans (0,5 de Mc Farland), ADL e RC. A lisozima foi adicionada ao SA com ummicrobrush embebido no SA e na lisozima em pó. Os dentes foram impermeabilizados e imersosem azul de metileno por 4 horas a 37ºC. Os dentes foram seccionados e fotografados. Resultados:A avaliação da microinfiltração foi feita por dois avaliadores utilizando o Programa Tpsdig. Paraavaliar a calibração entre os examinadores, foi utilizado o teste de Correlação de Pearson. As médiasdas porcentagens de fendas e bolhas foram submetidas ao teste de Kruskal Wallis com 5% designificância. Conclusão: a contaminação cavitária com Streptococcus mutans acarretou na formaçãode fendas entre a resina composta e as margens cavitárias. A lisozima associada ao sistemaadesivo não influenciou na formação de fendas e bolhas nas restaurações de resina composta.

Objective: evaluate the influence of cavity contamination on marginal adaptation of compositerestorations and the association of lysozyme to the adhesive system. Materials and Methods:Were selected 40 permanent third molars and made cavity preparations class V with cylindricaldiamond burs 1090 (4 mm wide, 6 mm long and 2 mm deep). All teeth were sterilized and randomlydivided into 4 groups (n = 10): G1: adhesive system (SA) and restoration with compositeresin (CR); G2: cavity contaminated with 10 μl of Streptococcus mutans (0.5 McFarland), SA andRC; G3: adhesive system associated with lysozyme (ADL) and RC and G4: cavity contaminated with10 μl of Streptococcus mutans (0.5 Mc Farland), ADL and RC. Lysozyme was added to the SA witha microbrush embedded in the SA and powdered lysozyme. The teeth were sealed and immersedin a methylene blue for 4 hours at 37 ° C. The teeth were sectioned and photographed. Results:The evaluation of microleakage was made by two reviewers using the Tpsdig Program. To assessthe calibration of examiners, we used the Pearson correlation test. The mean percentages of cracksand bubbles were submitted to Kruskal Wallis test at 5% significance level. Conclusion: the cavityStreptococcus mutans contamination resulted in the formation of cracks between the compositeresin and the cavity margins. Lysozyme associated with the adhesive system did not influence theformation of cracks and bubbles in the composite restorations.

Avaliação dos métodos de higienização sobre S. mutans dos protetores bucais / Evaluation of cleaning methods on custom-made mouthguards of S. mutans

Objetivo: avaliar a eficácia de três diferentes métodos de higienização sobre cepas de S.mutans dos protetores bucais feitos sob medida: dentifrício fluoretado, detergente neutro eclorexidina. Materiais e Métodos: foram confeccionados 30 protetores bucais feitos sob medida com placas de silicone de 3 mm que foram contaminados em meio de sobrevivência BHI (BrainHeart Infusion) suplementado com 1% de glicose e sacarose e 0,5% de extrato de levedura e incubados em jarra de anaerobiose a 37°C em estufa bacteriológica por 15 dias. O meio foi renovado a cada 24 horas. Após a contaminação, foi feita a coleta dos protetores bucais, esfregaço e plaqueamento na superfície do meio de cultura mitis salivarius com bacitracina. Os protetores foram higienizados com escova dental, com três diferentes meios de higienização:dentifrício fluoretado, detergente neutro e clorexidina e, após a higienização foram submetidos a uma nova coleta, esfregaço e plaqueamento. Após incubação, foi feita a contagem do total de bactérias viáveis. Os resultados obtidos foram submetidos à análise estatística e ao teste não paramétrico de Wilcoxon. Resultados: houve redução microbiana significativa da contagem deS. mutans utilizando clorexidina e detergente (p<0.05). Não houve diferença significante na contagem antes e depois utilizando o dentifrício (p>0.05). Conclusão: a clorexidina e o detergente podem ser utilizados para desinfecção dos protetores bucais.

Objective: The effect of three different cleaning methods on custom-made mouth guards’strains of S. mutans: fluoridated tooth paste, neutral detergent and chlorhexidine. Materials and Methods: it were made 30 mouthguards custom-made with 3mm silicone slabs that have been subjected to a microbiological processing in supplemented BHI (Brain Heart Infusion). They were in cubated in anaerobic jar at 37 ° bacteriological green house for 15 days, changing the medium every 24 hours. After the contamination, samples were drawn and it was made the collection, smear and plating on surface of the culture medium mitis salivarius with bacitracin and then they were cleaned with tooth brush with three different modes of cleaning: chlorhexidine, neutral detergent and fluoridated tooth paste. After incubation, the count was made of total viable bacteria. The results were statistically analyzed by the Wilcoxon non-parametric test. Results: there wasa significant reduction in microbial counts before and after using chlorhexidine and detergent (p<0.05). There wasn’t significant difference in the count before and after using the tooth paste(p>0.05). Conclusion: chlorhexidine and detergent can be used for disinfection of mouth guards

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.

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.

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.

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.

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.