Correlation between Streptococcus mutans levels in dental plaque and saliva of children.

PURPOSE: This study was designed to compare the levels of Streptococcus mutans (S. mutans) in saliva with those in occlusal plaque on posterior teeth at different stages of dentition, and to explore the correlation with caries experience to determine the most suitable source of S. mutans for research. METHODS: Samples of saliva and occlusal plaque were collected from 83 patients (aged 3-17 years) over three months. S. mutans levels were determined by culture-based selective plating, morphological identification, and S.mutans-specific monoclonal antibody labeling. RESULTS: The mean age of the participants was 8.8 (±3.7) years, and 74.7% of them were Hispanic. Mean caries experience for children with primary, mixed, and permanent dentition was 5.2 (±4.7), 4.0 (±3.3), and 0.8 (±1.3), respectively. Children with primary and mixed dentition had a higher caries experience than children with permanent dentition (P < 0.01), despite having similar S. mutans levels and total bacteria. A positive correlation was observed between S. mutans levels in plaque and those in saliva, but not between S. mutans levels and caries experience. It was noteworthy that plaque samples harbored higher S. mutans levels (>105 CFU/mL) than saliva samples. CONCLUSION: Both plaque and saliva samples are useful sources for S. mutans isolation. S. mutans levels from both sources were not significantly correlated with caries experience, but occlusal plaque had greater sensitivity for quantification of high S. mutans levels.

Tooth-Specific Streptococcus mutans Distribution and Associated Microbiome.

Dental caries is multifactorial and polymicrobial in nature and remains one of the most common oral diseases. While caries research has focused on Streptococcus mutans as the main etiological pathogen, its impact at the tooth level is not fully understood. In this cross-sectional study, the levels and distribution of S. mutans in the posterior teeth at different dentition stages were investigated along with the corresponding tooth-specific microbiome. Occlusal plaque samples of 87 individual posterior teeth were collected from thirty children in three dentition stages (primary, mixed, and permanent). The S. mutans levels in the occlusal plaque of individual posterior teeth were quantified with qPCR, and those with preferential colonization were selected for tooth-specific microbiome analysis using 16S rRNA sequencing. Results: Quantification of S. mutans levels in the occlusal plaque confirmed the preferential colonization on the first primary and permanent molars. These teeth were selected for further tooth-specific microbiome sequencing, as they also displayed high caries experience. There were significant differences in the relative abundance of the four most abundant genera: Neisseria, Streptococcus, Rothia, and Veillonella. Furthermore, the tooth-level caries experience was correlated with a reduction in the microbiome diversity. Analyzing the different tooth-associated microbial communities, distinct tooth-specific core microbiomes were identified. Conclusions: Our findings suggest that caries susceptibility at the tooth level, depending on tooth type and dentition stage, is influenced by individual species as well as plaque community.

Oral Microbiome: Streptococcus mutans/Caries Concordant-Discordant Children.

Dental caries remains the most common chronic disease in children, and the respective etiology is not fully understood. Though Streptococcus mutans is an important factor in the initiation and progression of caries, its presence is not always associated with the disease. The existence of caries discordant populations, in which S. mutans counts do not correlate with caries experience, poses a challenging problem. This study explored the possible correlation of S. mutans and other microorganism levels on caries-associated ecology of caries-concordant and discordant populations. A total of forty-seven children were analyzed in this study and stratified into four clinical groups based on their S. mutans levels in saliva (HS/LS: High/low S. mutans) and caries experience. Streptococcus mutans levels were determined by culture-based selective plating. The salivary microbiome of caries concordant and discordant populations was investigated by 16S rRNA gene sequencing and downstream bioinformatics analysis. The salivary microbial communities significantly clustered based on S. mutans levels and independent of their caries experience. In addition to S. mutans levels, significant differences in the abundance of other species were observed between HS and LS groups. Interestingly, disease-associated species such as Veillonella dispar, Streptococcus spp., and Prevotella spp. were significantly increased in HS groups and may contribute, in combination with S. mutans, to the caries progression. Furthermore, health-associated species exhibited higher abundance in the LS groups, such as Veillonella rogosae, Haemophilus sp., and Alloprevotella spp. but their possible contribution to the caries process remains to be elucidated. This study provides evidence that S. mutans may play a role in shaping the salivary microbial community. Our results highlight that future caries research should consider additional species as health/disease microbial markers in conjunction with S. mutans to improve diagnosis and caries management of the caries-discordant population.

The impact of fixed orthodontic appliances and clear aligners on the oral microbiome and the association with clinical parameters: A longitudinal comparative study.

INTRODUCTION: Orthodontic treatment interferes with oral hygiene and promotes plaque retention, which leads to gingival inflammation and enamel demineralization. Although removable clear aligners (CAs) are designed to improve oral hygiene compared with fixed appliances (FAs), comprehensive studies comparing their respective effects on the oral microbiome are limited. This longitudinal study investigated the microbial changes during orthodontic treatment with FA and CA in correlation with clinical parameters. METHODS: Clinical parameters and supragingival plaque were collected from 12 study participants for the FA or CA treatment groups at baseline and at least twice at the 1, 3, 6, and 12-month follow-up appointments. The plaque was also harvested from the aligner tray for the CA group. Microbiome composition was determined via 16S rRNA gene sequencing, compared between groups, and correlated with clinical parameters. RESULTS: Plaque (PI) and gingival indexes (GI) increased significantly in the FA but not the CA group. Beta but not alpha diversities of the microbial communities were distinct between the 2 treatment groups, even though genus-level differences were not significant except for Leptotrichia. The CA tray harbors a unique plaque community. Elevated PI and GI in the FA group correlated with a higher abundance of disease-related genera. CONCLUSIONS: Orthodontic treatments trigger appliance-related plaque community shifts from baseline, and the CA tray environment attracts distinct microbial communities. In comparison with FA, the use of CA resulted in better oral health index outcomes, which is reflected by the corresponding PI and GI-associated oral microbial communities.

Oral colonization of Candida albicans and Streptococcus mutans in children with or without fixed orthodontic appliances: A pilot study.

BACKGROUND/PURPOSE: Adolescents undergoing fixed orthodontic therapy have an increased risk of oral diseases due to additional plaque accumulation sites. However, the effect of fixed orthodontics appliances (FOAs) on the colonization of Candida albicans (Ca) and Streptococcus mutans (Sm), two synergistic oral pathogens, is largely unknown and was, therefore, the primary objective of this pilot investigation. MATERIAL AND METHODS: Sixteen children aged 10-15 years were enrolled, nine in the FOA and seven in the control groups. Saliva and occlusal plaque were collected, and the Ca and Sm levels were quantified with a quantitative real-time polymerase chain reaction (qPCR) assay. RESULTS: A trend of higher Ca levels was observed in the saliva and occlusal plaque of the FOA group, while the control group contained higher levels of Sm. Furthermore, for Sm levels, a positive correlation between saliva and occlusal plaque was shown in both the FOA and control groups; in contrast, Ca levels were negatively correlated between these samples only in the FOA group. Between Ca and Sm, a positive correlation was observed in saliva and occlusal plaque in the control group; however, this relationship was disrupted in the FOA group. CONCLUSION: Our preliminary study demonstrated that the presence of FOAs disturbs the colonization of Ca and Sm within the oral cavity. This perturbation might increase orthodontic patients' risk for Ca- and Sm-related diseases.

Pilot study on selective antimicrobial effect of a halitosis mouthrinse: monospecies and saliva-derived microbiome in an in vitro model system.

BACKGROUND: Halitosis refers to malodor emanating from the oral cavity. Several mouthrinses with halitosis-reduction exist on the market, but their effect on the oral microbiome is largely unknown. In this study, we used an efficient in vitro model system to investigate a test mouthrinse's impact on the oral microbiome. METHODS: Single halitosis-associated species and other common oral microorganism cultures were exposed to the test mouthrinse over time, and their viability was determined by culture-based selective plating. Next, the saliva-derived microbiome from healthy and halitosis-associated individuals was cultured in the presence of the test mouthrinse over time using the previously developed in vitro model system. The microbiome composition was assessed with 16S rRNA gene sequencing and downstream bioinformatics analyses. RESULTS: The test mouthrinse displayed antimicrobial activity against known anaerobic bacterial species producing halitosis-related compounds such as Fusobacterium nucleatum, F. periodonticum, and Prevotella intermedia but not against other common oral microorganisms. In the multispecies, saliva-derived cultures, mouthrinse exposure decreased the relative abundance of the Fusobacterium and Prevotella genera while not affecting overall diversity. CONCLUSIONS: The test mouthrinse had promising anti-halitosis characteristics at the microbiome level, as demonstrated by the reduction in the relative abundance of halitosis-associated taxa while maintaining microbial diversity.

Omics and interspecies interaction.

Interspecies interactions are key determinants in biofilm behavior, ecology, and architecture. The cellular responses of microorganisms to each other at transcriptional, proteomic, and metabolomic levels ultimately determine the characteristics of biofilm and the corresponding implications for health and disease. Advances in omics technologies have revolutionized our understanding of microbial community composition and their activities as a whole. Large-scale analyses of the complex interaction between the many microbial species residing within a biofilm, however, are currently still hampered by technical and bioinformatics challenges. Thus, studies of interspecies interactions have largely focused on the transcriptional and proteomic changes that occur during the contact of a few prominent species, such as Porphyromonas gingivalis, Streptococcus mutans, Candida albicans, and a few others, with selected partner species. Expansion of available tools is necessary to grow the revealing, albeit limited, insight these studies have provided into a profound understanding of the nature of individual microbial responses to the presence of others. This will allow us to answer important questions including: Which intermicrobial interactions orchestrate the myriad of cooperative, synergistic, antagonistic, manipulative, and other types of relationships and activities in the complex biofilm environment, and what are the implications for oral health and disease?

Deep metagenomics examines the oral microbiome during dental caries, revealing novel taxa and co-occurrences with host molecules.

Dental caries, the most common chronic infectious disease worldwide, has a complex etiology involving the interplay of microbial and host factors that are not completely understood. In this study, the oral microbiome and 38 host cytokines and chemokines were analyzed across 23 children with caries and 24 children with healthy dentition. De novo assembly of metagenomic sequencing obtained 527 metagenome-assembled genomes (MAGs), representing 150 bacterial species. Forty-two of these species had no genomes in public repositories, thereby representing novel taxa. These new genomes greatly expanded the known pangenomes of many oral clades, including the enigmatic Saccharibacteria clades G3 and G6, which had distinct functional repertoires compared to other oral Saccharibacteria. Saccharibacteria are understood to be obligate epibionts, which are dependent on host bacteria. These data suggest that the various Saccharibacteria clades may rely on their hosts for highly distinct metabolic requirements, which would have significant evolutionary and ecological implications. Across the study group, Rothia, Neisseria, and Haemophilus spp. were associated with good dental health, whereas Prevotella spp., Streptococcus mutans, and Human herpesvirus 4 (Epstein-Barr virus [EBV]) were more prevalent in children with caries. Finally, 10 of the host immunological markers were significantly elevated in the caries group, and co-occurrence analysis provided an atlas of potential relationships between microbes and host immunological molecules. Overall, this study illustrated the oral microbiome at an unprecedented resolution and contributed several leads for further study that will increase the understanding of caries pathogenesis and guide therapeutic development.

Effectiveness of the GumChucks flossing system compared to string floss for interdental plaque removal in children: a randomized clinical trial.

Flossing, an important oral hygiene skill, is technique-sensitive and challenging for children with developing manual dexterity. GumChucks is a novel flossing device designed to assist children with proper flossing technique. The aim of this study was to assess the efficacy of the GumChucks flossing device compared to string floss (SF). We conducted a randomized trial with 40 children aged 4-15 years at the UCLA Children's Dental Center from January- April 2017. Participants were randomly assigned to either GumChucks or SF. Interdental plaque score (IPS) and gingival index (GI) were recorded at baseline and 4-week post-usage. Flossing speed and interdental plaque reduction were also determined immediately after first use. In addition, questionnaires were completed by children, parents and dentists. Overall, children flossed significantly faster (p < 0.001) and achieved greater IPS reduction after first use (47.0% vs. 26.8%) with GumChucks compared to SF. After 4-week post-usage, children ages 10-15 in the GumChucks group demonstrated significantly greater improvement in GI and IPS from baseline (p < 0.01) and greater efficacy in interdental plaque removal compared to the SF group (p < 0.01). Children ages 4-9 flossed more effectively (p < 0.01) with GumChucks after first use, but no significant IPS and GI improvement after 4-week post-usage. Children preferred GumChucks (92.5%) over SF, with a similar positive attitude reported by parents and dentists. GumChucks is an effective alternative interdental plaque removal aid that allows children to floss with greater speed and efficacy, with recommended parental supervision for children under age 10.

Lollipop containing Glycyrrhiza uralensis extract reduces Streptococcus mutans colonization and maintains oral microbial diversity in Chinese preschool children.

The anticariogenic activity of the extract of Glycyrrhiza uralensis (licorice) has been well documented. We recently developed an herbal lollipop containing licorice extracts with Glycyrrhizol A, the compound displaying strong antimicrobial activity against Streptococcus mutans. Preliminary testing showed that the herbal lollipop reduced salivary S. mutans counts in vivo. In this study, we aimed to further test the efficacy of this herbal lollipop for reducing salivary S. mutans levels, and investigate its impact on salivary microbiome. Using a well-established in vitro oral microbiome model, we showed that licorice extract displays targeted killing against S. mutans without affecting the biodiversity of the community. In vivo study corroborated in vitro findings, showing for high caries-risk children aged 3-6 with salivary S. mutans levels >5x105 cells/ml, daily use of 2 licorice-containing lollipops for 3 weeks significantly reduced salivary S. mutans levels compared to the control group. Salivary microbiome analysis showed either no change or even increase in phylogenetic diversity of the oral community following herbal lollipop usage. Although further study with longer term observation is needed, these results suggest that use of licorice extract-containing lollipops can be as a simple and effective way to reduce the risk of dental caries in children.

Identification of the Bacterial Biosynthetic Gene Clusters of the Oral Microbiome Illuminates the Unexplored Social Language of Bacteria during Health and Disease.

Small molecules are the primary communication media of the microbial world. Recent bioinformatic studies, exploring the biosynthetic gene clusters (BGCs) which produce many small molecules, have highlighted the incredible biochemical potential of the signaling molecules encoded by the human microbiome. Thus far, most research efforts have focused on understanding the social language of the gut microbiome, leaving crucial signaling molecules produced by oral bacteria and their connection to health versus disease in need of investigation. In this study, a total of 4,915 BGCs were identified across 461 genomes representing a broad taxonomic diversity of oral bacteria. Sequence similarity networking provided a putative product class for more than 100 unclassified novel BGCs. The newly identified BGCs were cross-referenced against 254 metagenomes and metatranscriptomes derived from individuals either with good oral health or with dental caries or periodontitis. This analysis revealed 2,473 BGCs, which were differentially represented across the oral microbiomes associated with health versus disease. Coabundance network analysis identified numerous inverse correlations between BGCs and specific oral taxa. These correlations were present in healthy individuals but greatly reduced in individuals with dental caries, which may suggest a defect in colonization resistance. Finally, corroborating mass spectrometry identified several compounds with homology to products of the predicted BGC classes. Together, these findings greatly expand the number of known biosynthetic pathways present in the oral microbiome and provide an atlas for experimental characterization of these abundant, yet poorly understood, molecules and socio-chemical relationships, which impact the development of caries and periodontitis, two of the world's most common chronic diseases.IMPORTANCE The healthy oral microbiome is symbiotic with the human host, importantly providing colonization resistance against potential pathogens. Dental caries and periodontitis are two of the world's most common and costly chronic infectious diseases and are caused by a localized dysbiosis of the oral microbiome. Bacterially produced small molecules, often encoded by BGCs, are the primary communication media of bacterial communities and play a crucial, yet largely unknown, role in the transition from health to dysbiosis. This study provides a comprehensive mapping of the BGC repertoire of the human oral microbiome and identifies major differences in health compared to disease. Furthermore, BGC representation and expression is linked to the abundance of particular oral bacterial taxa in health versus dental caries and periodontitis. Overall, this study provides a significant insight into the chemical communication network of the healthy oral microbiome and how it devolves in the case of two prominent diseases.

Group A Streptococcus emm3 strains induce early macrophage cell death.

Group A Streptococcus (GAS) infections present high morbidity and mortality rates and consequently remain a significant health problem. The emm3 isolates induce more severe pathologies than all others. In this study, we tested, on a collection of invasive and non-invasive emm3 clinical isolates, whether in that genotype the invasive status of the strains affects the innate immune response. We show that phagocytosis is dependent on the invasiveness of the isolates. Interestingly, all emm3 isolates compromise macrophage integrity, already noticeable 1 h after infection. Inflammatory modulators (IL-6, TNF-α and IFN-ß) are nevertheless detected during at least 6 h post-infection. This is a likely consequence of the macrophages not being all infected. The efficient and rapid induction of macrophage death could explain the virulence of the emm3 strains.

AMA1-deficient Toxoplasma gondii parasites transiently colonize mice and trigger an innate immune response that leads to long-lasting protective immunity.

The apical membrane antigen 1 (AMA1) protein was believed to be essential for the perpetuation of two Apicomplexa parasite genera, Plasmodium and Toxoplasma, until we genetically engineered viable parasites lacking AMA1. The reduction in invasiveness of the Toxoplasma gondii RH-AMA1 knockout (RH-AMA1(KO)) tachyzoite population, in vitro, raised key questions about the outcome associated with these tachyzoites once inoculated in the peritoneal cavity of mice. In this study, we used AMNIS technology to simultaneously quantify and image the parasitic process driven by AMA1(KO) tachyzoites. We report their ability to colonize and multiply in mesothelial cells and in both resident and recruited leukocytes. While the RH-AMA1(KO) population amplification is rapidly lethal in immunocompromised mice, it is controlled in immunocompetent hosts, where immune cells in combination sense parasites and secrete proinflammatory cytokines. This innate response further leads to a long-lasting status immunoprotective against a secondary challenge by high inocula of the homologous type I or a distinct type II T. gondii genotypes. While AMA1 is definitively not an essential protein for tachyzoite entry and multiplication in host cells, it clearly assists the expansion of parasite population in vivo.

The innate immune response elicited by Group A Streptococcus is highly variable among clinical isolates and correlates with the emm type.

Group A Streptococcus (GAS) infections remain a significant health care problem due to high morbidity and mortality associated with GAS diseases, along with their increasing worldwide prevalence. Macrophages play a key role in the control and clearance of GAS infections. Moreover, pro-inflammatory cytokines production and GAS persistence and invasion are related. In this study we investigated the correlation between the GAS clinical isolates genotypes, their known clinical history, and their ability to modulate innate immune response. We constituted a collection of 40 independent GAS isolates representative of the emm types currently prevalent in France and responsible for invasive (57.5%) and non-invasive (42.5%) clinical manifestations. We tested phagocytosis and survival in mouse bone marrow-derived macrophages and quantified the pro-inflammatory mediators (IL-6, TNF-α) and type I interferon (INF-ß) production. Invasive emm89 isolates were more phagocytosed than their non-invasive counterparts, and emm89 isolates more than the other isolates. Regarding the survival, differences were observed depending on the isolate emm type, but not between invasive and non-invasive isolates within the same emm type. The level of inflammatory mediators produced was also emm type-dependent and mostly invasiveness status independent. Isolates of the emm1 type were able to induce the highest levels of both pro-inflammatory cytokines, whereas emm89 isolates induced the earliest production of IFN-ß. Finally, even within emm types, there was a variability of the innate immune responses induced, but survival and inflammatory mediator production were not linked.

Molecular epidemiology of sil locus in clinical Streptococcus pyogenes strains.

Streptococcus pyogenes (group A Streptococcus [GAS]) causes a wide variety of diseases, ranging from mild noninvasive to severe invasive infections. Mutations in regulatory components have been implicated in the switch from colonization to invasive phenotypes. The inactivation of the sil locus, composed of six genes encoding a quorum-sensing complex, gives rise to a highly invasive strain. However, studies conducted on limited collections of GAS strains suggested that sil prevalence is around 15%; furthermore, whereas a correlation between the presence of sil and the genetic background was suggested, no link between the presence of a functional sil locus and the invasive status was assessed. We established a collection of 637 nonredundant strains covering all emm genotypes present in France and of known clinical history; 68%, 22%, and 10% were from invasive infections, noninvasive infections, and asymptomatic carriage, respectively. Among the 637 strains, 206 were sil positive. The prevalence of the sil locus varied according to the emm genotype, being present in >85% of the emm4, emm18, emm32, emm60, emm87, and emm90 strains and absent from all emm1, emm28, and emm89 strains. A random selection based on 2009 French epidemiological data indicated that 16% of GAS strains are sil positive. Moreover, due to mutations leading to truncated proteins, only 9% of GAS strains harbor a predicted functional sil system. No correlation was observed between the presence or absence of a functional sil locus and the strain invasiveness status.

Extracellular nucleotide catabolism by the Group B Streptococcus ectonucleotidase NudP increases bacterial survival in blood.

Streptococcus agalactiae (Group B Streptococcus) is a commensal of the human intestine and vagina of adult women but is the leading cause of invasive infection in neonates. This Gram-positive bacterium displays a set of virulence-associated surface proteins involved in the interaction with the host, such as adhesion to host cells, invasion of tissues, or subversion of the immune system. In this study, we characterized a cell wall-localized protein as an ecto-5'-nucleoside diphosphate phosphohydrolase (NudP) involved in the degradation of extracellular nucleotides which are central mediators of the immune response. Biochemical characterization of recombinant NudP revealed a Mn(2+)-dependent ecto-5'-nucleotidase activity on ribo- and deoxyribonucleoside 5'-mono- and 5'-diphosphates with a substrate specificity different from that of known orthologous enzymes. Deletion of the gene coding the housekeeping enzyme sortase A led to the release of NudP into the culture supernatant, confirming that this enzyme is anchored to the cell wall by its non-canonical LPXTN motif. The NudP ecto-5'-nucleotidase activity is reminiscent of the reactions performed by the mammalian ectonucleotidases CD39 and CD73 involved in regulating the extracellular level of ATP and adenosine. We further demonstrated that the absence of NudP activity decreases bacterial survival in mouse blood, a process dependent on extracellular adenosine. In vivo assays in animal models of infection showed that NudP activity is critical for virulence. These results demonstrate that Group B Streptococcus expresses a specific ecto-5'-nucleotidase necessary for its pathogenicity and highlight the diversity of reactions performed by this enzyme family. These results suggest that bacterial pathogens have developed specialized strategies to subvert the mammalian immune response controlled by the extracellular nucleotide signaling pathways.

Leukocyte populations and cytokine expression in the mammary gland in a mouse model of Streptococcus agalactiae mastitis.

Streptococcus agalactiae is a contagious, mastitis-causing pathogen that is highly adapted to survive in the bovine mammary gland. This study used a BALB/c mouse model of Streptococcus agalactiae mastitis to evaluate leukocyte populations in regional lymph nodes and cytokine expression in the mammary gland involved in the immune response against Streptococcus agalactiae. It was found that the bacteria replicated efficiently in the mammary gland, peaking after 24 h and increasing by 100-fold. Dissemination of bacteria to systemic organs was observed 6 h after infection. At the same time, a massive infiltration of polymorphonuclear cells and an increase in the inflammatory cytokines interleukin (IL)-1beta, IL-6 and tumour necrosis factor-alpha were detected in mammary glands, indicating an early inflammatory response. A decrease in the levels of inflammatory cytokines in mammary glands was observed 72 h after infection, accompanied by an increase in the levels of IL-12 and IL-10, which were related to a gradual decrease in bacterial load. An increase in the number of macrophages and B220(+) lymphocytes and similar increases in both CD4(+) and CD8(+) T cells in regional lymph nodes were observed, being most pronounced 5 days after infection. Moreover, increased levels of anti-Streptococcus agalactiae antibodies in the mammary gland were observed 10 days after infection. Overall, these data suggest that the host exhibits both innate and acquired immune responses in response to Streptococcus agalactiae mastitis.

Oral therapeutic vaccination with Streptococcus sobrinus recombinant enolase confers protection against dental caries in rats.

Dental caries is among the more prevalent chronic human infections for which an effective human vaccine has not yet been achieved. Enolase from Streptococcus sobrinus has been identified as an immunomodulatory protein. In the present study, we used S. sobrinus recombinant enolase (rEnolase) as a target antigen and assessed its therapeutic effect in a rat model of dental caries. Wistar rats that were fed a cariogenic solid diet on day 18 after birth were orally infected with S. sobrinus on day 19 after birth and for 5 consecutive days thereafter. Five days after infection and, again, 3 weeks later, rEnolase plus alum adjuvant was delivered into the oral cavity of the rats. A sham-immunized group of rats was contemporarily treated with adjuvant alone. In the rEnolase-immunized rats, increased levels of salivary IgA and IgG antibodies specific for this recombinant protein were detected. A significant decrease in sulcal, proximal enamel, and dentin caries scores was observed in these animals, compared with sham-immunized control animals. No detectable histopathologic alterations were observed in all immunized animals. Furthermore, the antibodies produced against bacterial enolase did not react with human enolase. Overall, these results indicate that rEnolase could be a promising and safe candidate for testing in trials of vaccines against dental caries in humans.

Identification of immunoreactive extracellular proteins of Streptococcus agalactiae in bovine mastitis.

Streptococcus agalactiae is a common pathogen that causes bovine mastitis. The aims of this study were to evaluate the antibody response against S. agalactiae extracellular proteins in the whey and serum of naturally infected bovines and to identify possible immunodominant extracellular antigens. IgG1 antibodies against S. agalactiae extracellular proteins were elevated in the whey and serum of naturally infected bovines. In the whey, the levels of IgG1 specific for S. agalactiae extracellular proteins were similar in infected and noninfected milk quarters from the same cow, and the production of antibodies specific for S. agalactiae extracellular proteins was induced only by infection with this bacterium. The immunoreactivity of extracellular proteins with bovine whey was clearly different in infected versus control animals. Group B protective surface protein and 5'-nucleotidase family protein were 2 major immunoreactive proteins that were detected only in the whey of infected cows, suggesting that these proteins may be important in the pathogenesis of S. agalactiae-induced mastitis. This information could be used to diagnose S. agalactiae infection. In addition, these antigens may be useful as carrier proteins for serotype-specific polysaccharides in conjugate vaccines.

Identification of NAD+ synthetase from Streptococcus sobrinus as a B-cell-stimulatory protein.

Streptococcus sobrinus, one agent of dental caries, secretes a protein that induces lymphocyte polyclonal activation of the host as a mechanism of immune evasion. We have isolated from culture supernatants of this bacterium a protein with murine B-cell-stimulatory properties and subsequently cloned the relevant gene. It contains an open reading frame of 825 bp encoding a polypeptide with 275 amino acid residues and a molecular mass of 30 kDa. The protein displays high sequence homology with NAD(+) synthetases from several organisms, including a conserved fingerprint sequence (SGGXD) characteristic of ATP pyrophosphatases. The polypeptide was expressed in Escherichia coli as a hexahistidine-tagged protein and purified in an enzymatically active form. The recombinant NAD(+) synthetase stimulates murine B cells after in vitro treatment of spleen cell cultures, as demonstrated by its ability to induce up-regulation of the expression of CD69, an early marker of lymphocyte activation. Stimulation with the recombinant NAD(+) synthetase was also observed with other B-cell markers, such as CD19(+), B220(+), and CD21(+). Cell proliferation follows the activation induced by the recombinant NAD(+) synthetase.