Can insoluble polysaccharide concentration in dental plaque, sugar exposure and cariogenic microorganisms predict early childhood caries? A follow-up study.

BACKGROUND: Insoluble polysaccharide (IP) has been associated with caries prevalence in young children. However, the power of IP to predict ECC needs to be demonstrated. AIMS: To assess the relationships between early childhood caries (ECC) and extracellular insoluble polysaccharides (IP) in dental plaque, sugar exposure and cariogenic microorganisms. DESIGN: Visible plaque on maxillary incisors was recorded, followed by caries diagnosis in 65 preschoolers (3-4 years) at baseline and after 1 year. Plaque was collected for mutans streptococci (MS), total microorganism (TM) and lactobacilli (LB) enumerations in selective media, as well as for IP analysis, which was later assessed by colorimetry. Sugar/sucrose exposure was assessed by a diet chart. RESULTS: Positive correlations were found among the prevalence of caries and MS, TM, LB, solid sucrose and visible dental plaque. Additionally, children with IP concentrations in dental plaque higher than 2.36 µg/mg (odds ratio-OR=6.8), with visible plaque on maxillary incisors (OR=4.3), harbouring LB (OR=13) and exposed to solid sugar more than twice/day (OR=5) showed higher risk of developing caries (p<0.05). CONCLUSION: Extracellular insoluble polysaccharides, solid sugar/sucrose, visible dental plaque and cariogenic microorganisms could predict caries development, partially explaining the ECC pattern.

Antimicrobial peptides and nitric oxide production by neutrophils from periodontitis subjects

Neutrophils play an important role in periodontitis by producing nitric oxide (NO) and antimicrobial peptides, molecules with microbicidal activity via oxygen-dependent and -independent mechanisms, respectively. It is unknown whether variation in the production of antimicrobial peptides such as LL-37, human neutrophil peptides (HNP) 1-3, and NO by neutrophils influences the pathogenesis of periodontal diseases. We compared the production of these peptides and NO by lipopolysaccharide (LPS)-stimulated neutrophils isolated from healthy subjects and from patients with periodontitis. Peripheral blood neutrophils were cultured with or without Aggregatibacter actinomycetemcomitans-LPS (Aa-LPS), Porphyromonas gingivalis-LPS (Pg-LPS) and Escherichia coli-LPS (Ec-LPS). qRT-PCR was used to determine quantities of HNP 1-3 and LL-37 mRNA in neutrophils. Amounts of HNP 1-3 and LL-37 proteins in the cell culture supernatants were also determined by ELISA. In addition, NO levels in neutrophil culture supernatants were quantitated by the Griess reaction. Neutrophils from periodontitis patients cultured with Aa-LPS, Pg-LPS and Ec-LPS expressed higher HNP 1-3 mRNA than neutrophils from healthy subjects. LL-37 mRNA expression was higher in neutrophils from patients stimulated with Aa-LPS. Neutrophils from periodontitis patients produced significantly higher LL-37 protein levels than neutrophils from healthy subjects when stimulated with Pg-LPS and Ec-LPS, but no difference was observed in HNP 1-3 production. Neutrophils from periodontitis patients cultured or not with Pg-LPS and Ec-LPS produced significantly lower NO levels than neutrophils from healthy subjects. The significant differences in the production of LL-37 and NO between neutrophils from healthy and periodontitis subjects indicate that production of these molecules might influence individual susceptibility to important periodontal pathogens.

Antimicrobial peptides and nitric oxide production by neutrophils from periodontitis subjects.

Neutrophils play an important role in periodontitis by producing nitric oxide (NO) and antimicrobial peptides, molecules with microbicidal activity via oxygen-dependent and -independent mechanisms, respectively. It is unknown whether variation in the production of antimicrobial peptides such as LL-37, human neutrophil peptides (HNP) 1-3, and NO by neutrophils influences the pathogenesis of periodontal diseases. We compared the production of these peptides and NO by lipopolysaccharide (LPS)-stimulated neutrophils isolated from healthy subjects and from patients with periodontitis. Peripheral blood neutrophils were cultured with or without Aggregatibacter actinomycetemcomitans-LPS (Aa-LPS), Porphyromonas gingivalis-LPS (Pg-LPS) and Escherichia coli-LPS (Ec-LPS). qRT-PCR was used to determine quantities of HNP 1-3 and LL-37 mRNA in neutrophils. Amounts of HNP 1-3 and LL-37 proteins in the cell culture supernatants were also determined by ELISA. In addition, NO levels in neutrophil culture supernatants were quantitated by the Griess reaction. Neutrophils from periodontitis patients cultured with Aa-LPS, Pg-LPS and Ec-LPS expressed higher HNP 1-3 mRNA than neutrophils from healthy subjects. LL-37 mRNA expression was higher in neutrophils from patients stimulated with Aa-LPS. Neutrophils from periodontitis patients produced significantly higher LL-37 protein levels than neutrophils from healthy subjects when stimulated with Pg-LPS and Ec-LPS, but no difference was observed in HNP 1-3 production. Neutrophils from periodontitis patients cultured or not with Pg-LPS and Ec-LPS produced significantly lower NO levels than neutrophils from healthy subjects. The significant differences in the production of LL-37 and NO between neutrophils from healthy and periodontitis subjects indicate that production of these molecules might influence individual susceptibility to important periodontal pathogens.

Influence of VicRK and CovR on the interactions of Streptococcus mutans with phagocytes.

OBJECTIVE: Streptococcus mutans are members of the oral microbiota that are implicated in dental caries and infective endocarditis. To adapt to environmental stresses encountered during host colonization, these bacteria employ two-component regulatory systems, which modulate global changes in gene expression. These include the systems VicRK and CovR. In this study, we investigate the influence of VicRK and CovR in S. mutans interactions with mononuclear and polymorphonuclear (PMN) phagocytes. METHODS: Patterns of S. mutans uptake by murine macrophages were determined in strains, which differ in the production of proteins regulated by VicRK and CovR. Bacterial uptake by murine macrophages and by PMN in human blood was analyzed in vicK and covR knockout mutants obtained in strains UA159 and LT11. RESULTS: Inactivation of covR did not affect uptake by macrophages, while vicK inactivation transiently reduced uptake only in LT11 (P < 0.05). In the two strains, inactivation of vicK and covR impaired uptake by PMN for a period of 1 h or more (P < 0.01-0.05). Mutant complementation with vicK or covR restored the PMN uptake phenotypes. CONCLUSION: This study indicates that VicRK and CovR regulate functions that influence bacterial susceptibility to phagocytosis, suggesting a novel role for these systems in the virulence of S. mutans.

Immunological and microbiologic changes during caries development in young children.

We explored the association between caries development, colonization with caries-associated microflora, and immunity as children begin the transition to mixed dentition. Forty children received dental examinations at 3-4 years of age, repeated a year later. Children were grouped into caries-free (n = 23; CF) and caries-active (n = 17; CA ≥3 new lesions on follow-up). Salivary IgA and IgA antibody to Streptococcus mutans virulence epitopes were measured by Luminex assay. Mutans streptococci (MS), lactobacilli and total microorganisms were enumerated on selective media from plaque samples. There was no significant difference in baseline levels of MS or lactobacilli between CF and CA groups. However, both MS and lactobacilli levels were higher at follow-up in the CA group. Furthermore, children with detectable lactobacilli at baseline had significantly higher caries risk. Salivary IgA concentrations increased significantly in both groups during the study. Both CF and CA groups also displayed significant increases in salivary IgA antibody levels to glucosyltransferase, glucan-binding protein (Gbp) and antigen I/II salivary binding region. CF antibody levels to seven peptides associated with domains of biological importance increased at follow-up, in contrast to increases to only three peptides in CA saliva samples. Multivariate modeling showed that a lower baseline level of salivary IgA anti-GbpB was associated with higher caries risk. These data indicate that MS and lactobacilli are associated with caries in this population, that the secretory immune system is undergoing significant maturation during this period, and that the breadth of mucosal IgA response to epitopes of S. mutans virulence components may influence the degree to which these cariogenic microorganisms can cause disease.

Transcriptional analysis of gtfB, gtfC, and gbpB and their putative response regulators in several isolates of Streptococcus mutans.

BACKGROUND: Streptococcus mutans, a major dental caries pathogen, expresses several virulence genes that mediate its growth, accumulation on tooth surfaces, and acid-mediated tooth demineralization. GtfB and GtfC catalyze the extracellular synthesis of water-insoluble glucan matrix from sucrose, and are essential for accumulation of bacteria in the dental biofilm. GbpB, an essential protein of S. mutans, might also mediate cell-surface interaction with glucan. AIM/METHODS: In this study, we determined the transcription levels of gtfB, gtfC, and gbpB, and several putative transcriptional response regulators (rr) at different phases of planktonic growth in 11 S. mutans strains. RESULTS: Activities of gtfB and gtfC were growth-phase dependent and assumed divergent patterns in several strains during specific phases of growth, while gbpB activities appeared to be under modest influence of the growth phase. Transcription patterns of the rr vicR, covR, comE, ciaR, and rr1 were growth-phase dependent and some of these genes were expressed in a highly coordinated way. Each rr, except comE, was expressed by all the strains. Patterns of virulence and regulatory genes were, however, strain-specific. CONCLUSIONS: The findings suggest that mechanisms controlling virulence gene expression are variable among genotypes, providing the notion that the genetic diversity of S. mutans may have important implications for understanding mechanisms that regulate the expression of virulence genes in this species.

Mutans streptococcal infection induces salivary antibody to virulence proteins and associated functional domains.

The interplay between mucosal immune responses to natural exposure to mutans streptococci and the incorporation and accumulation of these cariogenic microorganisms in oral biofilms is unclear. An initial approach to explore this question would be to assess the native secretory immunity emerging as a consequence of Streptococcus mutans infection. To this end, we analyzed salivary immunoglobulin A (IgA) antibody to mutans streptococcal glucosyltransferase (Gtf) and glucan binding protein B (GbpB) and to domains associated with enzyme function and major histocompatibility complex (MHC) class II binding in two experiments. Salivas were collected from approximately 45-day-old Sprague-Dawley rats, which were then infected with S. mutans SJ32. Infection was verified and allowed to continue for 2 to 2.5 months. Salivas were again collected following the infection period. Pre- and postinfection salivas were then analyzed for IgA antibody activity using peptide- or protein-coated microsphere Luminex technology. S. mutans infection induced significant levels of salivary IgA antibody to Gtf (P < 0.002) and GbpB (P < 0.001) in both experiments, although the levels were usually far lower than the levels achieved when mucosal immunization is used. Significantly (P < 0.035 to P < 0.001) elevated levels of postinfection salivary IgA antibody to 6/10 Gtf peptides associated with either enzyme function or MHC binding were detected. The postinfection levels of antibody to two GbpB peptides in the N-terminal region of the six GbpB peptides assayed were also elevated (P < 0.031 and P < 0.001). Interestingly, the patterns of the rodent response to GbpB peptides were similar to the patterns seen in salivas from young children during their initial exposure to S. mutans. Thus, the presence of a detectable postinfection salivary IgA response to mutans streptococcal virulence-associated components, coupled with the correspondence between rat and human mucosal immune responsiveness to naturally presented Gtf and GbpB epitopes, suggests that the rat may be a useful model for defining mucosal responses that could be expected in humans. Under controlled infection conditions, such a model could prove to be helpful for unraveling relationships between the host response and oral biofilm development.

Secretory immunity following mutans streptococcal infection or immunization.

Salivary IgA antibody responses to mutans streptococci can be observed in early childhood, sometimes even before permanent colonization of the oral biofilm occurs. Many of these early immune responses are directed to components thought to be essential for establishment and emergence of mutans streptococci in the oral biofilm. Initial responses are likely to be modulated by antigen dose, by immunological maturity, and by previous encounters with similar antigenic epitopes in the pioneer commensal flora. Our understanding of these modulating factors is modest and is an opportunity for continued investigation. Under controlled conditions of infection, experimental vaccine approaches have repeatedly shown that infection and disease can be modified in the presence of elevated levels of antibody in the oral cavity. Protection can be observed regardless of antibody isotype or method used to actively or passively provide the immune reagent. Limited clinical trials have supported the utility of both of these approaches in humans. Refinements in antigen formulation, delivery vehicles, enhancing agents and routes of application, coupled with approaches that are timed to intercept most vulnerable periods of infection of primary and permanent dentition may well provide the healthcare practitioner with an additional tool to maintain oral health.

Determination of mutacin activity and detection of mutA genes in Streptococcus mutans genotypes from caries-free and caries-active children.

Relationships between genetic diversity, mutacin production and sensitivity to mutacins in Streptococcus mutans were evaluated in 19 clinical isolates from caries-free and caries-active children. Mutacin production was tested against 30 indicator strains; results showed significant variations in the inhibitory spectra of the clinical isolates. There was no association between the inhibitory spectrum of the infecting strain and the caries experience or the level of mutans streptococci infection of the host. Homology to the mutA gene coding for mutacin II was detected in one clinical isolate; none of the clinical isolates showed homology to the mutA genes coding for mutacins I or III. Genotyping by random amplified polymorphic DNA (RAPD) reactions grouped the isolates into three clusters, but no correlation was found between any of the clusters and mutacin activity, caries experience or level of mutans streptococci in the host.

Mutans streptococci oral colonization in 12-30-month-old Brazilian children over a one-year follow-up period.

OBJECTIVES: The infection levels of mutans streptococci were investigated during a one-year follow-up in children aged 12 to 30 months attending school nurseries where a sucrose-rich diet was provided. METHODS: Oral levels of mutans streptococci obtained from 101 children at baseline and after a one-year follow-up were compared by age, number of teeth, feeding habits, and presence of visible plaque at baseline. Baseline predictors and changes in mutans streptococci levels during the study were compared to caries incidence after one year. RESULTS: Fluctuations in mutans streptococci levels during the follow-up period were not related to feeding habits or presence of visible plaque. Mutans streptococci levels increased after one year among children aged 12 to 24 months, while a significantly higher proportion of those aged 25-30 months showed a decrease in mutans streptococci levels during the study. Multiple logistic regression analysis suggested that high levels of mutans streptococci (> or = 100 cfu) at baseline were associated with a higher caries increment, while reduction in mutans streptococci was negatively associated with caries incidence. CONCLUSIONS: Our data suggest that despite early mutans streptococci infection and high exposure to sucrose, mutans streptococci may achieve relatively stable levels after 2 years of age. Heavy colonization by mutans streptococci in an early age was related to an extremely high caries incidence during childhood, while decreasing levels of mutans streptococci can be associated to the decrease in caries activity.

Cloning of the Streptococcus mutans gene encoding glucan binding protein B and analysis of genetic diversity and protein production in clinical isolates.

Streptococcus mutans, the primary etiological agent of dental caries, produces several activities that promote its accumulation within the dental biofilm. These include glucosyltransferases, their glucan products, and proteins that bind glucan. At least three glucan binding proteins have been identified, and GbpB, the protein characterized in this study, appears to be novel. The gbpB gene was cloned and the predicted protein sequence contained several unusual features and shared extensive homology with a putative peptidoglycan hydrolase from group B streptococcus. Examination of gbpB genes from clinical isolates of S. mutans revealed that DNA polymorphisms, and hence amino acid changes, were limited to the central region of the gene, suggesting functional conservation within the amino and carboxy termini of the protein. The GbpB produced by clinical isolates and laboratory strains showed various distributions between cells and culture medium, and amounts of protein produced by individual strains correlated positively with their ability to grow as biofilms in an in vitro assay.

Genotypic diversity of mutans streptococci in Brazilian nursery children suggests horizontal transmission.

Streptococcus mutans strains were isolated from cohorts of Brazilian nursery school children and genotyped by arbitrarily primed PCR and restriction fragment length polymorphism analysis. Of 24 children with two to five S. mutans isolates, 29% carried two or more genotypes. The presence of matching genotypes of S. mutans among children attending one nursery suggests horizontal transmission.

Relation of oral yeast infection in Brazilian infants and use of a pacifier.

The frequency of oral yeast ingestion and its relationship with sucking and feeding habits was described in children from one to 18 months of age. Yeasts were detected in 58.3 percent of children and the most prevalent species were Candida parapsilosis and Candida albicans. The use of a pacifier was positively associated with the frequency of yeast infection and with the levels of these microorganisms in the mouth. No relationship was detected between the prevalence of yeast and breast-feeding or bottle-feeding habits. The results suggest that use of a pacifier is an important local factor in the colonization and proliferation of yeast in the oral cavity.

Water-insoluble glucan synthesis by mutans streptococcal strains correlates with caries incidence in 12- to 30-month-old children.

Early mutans streptococci (MS) infection has been associated with higher caries activity in childhood. Since colonization with MS does not always lead to caries activity, additional factors may be involved in MS cariogenicity. For example, MS may differ in virulence traits such as the potential to synthesize glucan polymers from sucrose. In the present study, we tested the hypothesis that caries activity can be associated with variations in virulence factor expression of MS-infecting strains. At baseline, levels of MS obtained by the tongue-blade sampling method, and the presence of visible plaque on upper incisors, were measured in 101 12- to 30-month-old children. Dental caries lesions were diagnosed at baseline and after one year. Caries incidence data were then used to select ten caries-free and nine caries-active children from whom a total of 20 MS fresh isolates was studied. Water-insoluble glucan (WIG) synthesis, final pH, and sucrose-dependent adherence on glass surfaces were measured in these MS isolates. Concentrated culture supernatants were separated in duplicate SDS-PAGE gels, which were then either stained for protein or incubated with 5% sucrose. The intensities of the WIG bands developed in the 5% sucrose PAGE gels and the corresponding protein-stained GTF bands were measured by scanning densitometry. High MS levels (> or = 100 CFU) were associated with high caries incidence (p < 0.01). The presence of visible plaque did not correlate with caries incidence. The intensities of WIG bands were positively correlated with caries incidence (p < 0.05) and with the ability of MS to adhere to glass surfaces (p < 0.05). Analysis of our data suggests that the ability to synthesize WIG is an important virulence factor in initial caries development by increasing MS adherence and accumulation in the plaque of young children.

Association between caries prevalence and clinical, microbiological and dietary variables in 1.0 to 2.5-year-old Brazilian children.

The association between caries prevalence and clinical (presence of visible plaque in the labial surfaces of maxillary incisors), microbiological (salivary levels of mutans streptococci) and dietary variables was evaluated in 142 1.0- to 2.5-year-old children attending public day-care nurseries in the city of Piracicaba - São Paulo. A significant difference in caries prevalence was observed between those children with and without visible plaque (chi2 = 12.08, p < 0.001). The mean ds (decayed surfaces) was significantly higher in children with visible plaque on the maxillary incisors than in children without it (p < 0.001). Mutans streptococci were detected in 114 (80.3%) of the children. A significantly higher caries prevalence was observed in children with high levels of mutans streptococci compared to children with low levels (chi2 = 28.67, p < 0.001). The mean ds was significantly higher in children with levels of mutans streptococci greater than 50 CFU when compared to children with 0 CFU or 1-50 CFU of mutans streptococci (p < 0.05). Children who were either never breast-fed or only until 3 months exhibited a significantly higher caries prevalence than those breast-fed for a longer time (chi2 = 4.11, p < 0.05). A significantly higher caries prevalence was also observed between children that used bottle containing milk with sucrose and cereal than children using bottle with milk with or without sucrose (chi2 = 6.24, p < 0.05). Children who started to eat salty meals at or after 7 months of age showed a significant higher caries prevalence than children who started earlier (chi2 = 10.30, p < 0.01). These data support the evidence of an association between caries prevalence in young children and mutans streptococci levels, clinical and dietary factors.

Anomalies of tooth form and number in the permanent dentition: report of two cases.

Two cases of bilateral double teeth involving the permanent maxillary central incisors are described. The difficulties in establishing the precise diagnosis are considered. The treatment plans also are discussed. The etiology and nomenclature of these dental formations and number of anomalies are reviewed.