Caries-Associated Biosynthetic Gene Clusters in Streptococcus mutans.

Early childhood caries (ECC) is a chronic disease affecting the oral health of children globally. This disease is multifactorial, but a primary factor is cariogenic microorganisms such as Streptococcus mutans. Biosynthetic gene clusters (BGCs) encode small molecules with diverse biological activities that influence the development of many microbial diseases, including caries. The purpose of this study was to identify BGCs in S. mutans from a high-caries risk study population using whole-genome sequencing and assess their association with ECC. Forty representative S. mutans isolates were selected for genome sequencing from a large-scale epidemiological study of oral microbiology and dental caries in children from a localized Alabama population. A total of 252 BGCs were identified using the antiSMASH BGC-mining tool. Three types of BGCs identified herein-butyrolactone-like, ladderane-like, and butyrolactone-ladderane-like hybrid (BL-BGC)-have not been reported in S. mutans. These 3 BGCs were cross-referenced against public transcriptomics data, and were found to be highly expressed in caries subjects. Furthermore, based on a polymerase chain reaction screening for core BL genes, 93% of children with BL-BGC had ECC. The role of BL-BGC was further investigated by examining cariogenic traits and strain fitness in a deletion mutant using in vitro biofilm models. Deletion of the BL-BGC significantly increased biofilm pH as compared to the parent strain, while other virulence and fitness properties remained unchanged. Intriguingly, BL-BGC containing strains produced more acid, a key cariogenic feature, and less biofilm than the model cariogenic strain S. mutans UA159, suggesting the importance of this BL-BGC in S. mutans-mediated cariogenesity. The structure of any BL-BGC derived metabolites, their functions, and mechanistic connection with acid production remain to be elucidated. Nevertheless, this study is the first to report the clinical significance of a BL-BGC in S. mutans. This study also highlights pangenomic diversity, which is likely to affect phenotype and virulence.

Comparative genotyping of Streptococcus mutans by repetitive extragenic palindromic polymerase chain reaction and multilocus sequence typing.

The genetic diversity of Streptococcus mutans has been extensively studied using a variety of genotyping methods. Repetitive extragenic palindromic-polymerase chain reaction (rep-PCR) is a genotyping approach used for screening large numbers of bacterial isolates. This two-part study used multilocus sequence typing (MLST) analysis to evaluate genotypes previously identified as unique using rep-PCR. In part one, an isolate was selected from each of the 22 S. mutans rep-PCR genotype groups representing 8000 clinical isolates. For part two, four additional isolates were selected from the six most commonly occurring genotype groups (GG) for further analysis. Real-time PCR was performed using eight housekeeping S. mutans gene loci and the amplicons were sequenced. Sequence data analysis was performed using CLC DNA Workbench and alleles were compared with the PubMLST database for Oral Streptococcus using the Nakano scheme. Concatenated sequences were evaluated with MEGA using a minimum evolution method with bootstrap. All 22 rep-PCR genotypes were unique by MLST analysis. Within rep-PCR GGs, MLST matched rep-PCR in three groups demonstrating clonality; three groups exhibited more diversity with MLST. The discovery of three clonal groups is unique to this study and suggests that S. mutans genotypes are shared between unrelated subjects. Furthermore, MLST defined 19 new alleles and 26 new sequence types that have been confirmed and registered with PubMLST. Methods for processing were streamlined and a process for using MLST with rep-PCR is suggested. In conclusion, MLST verified that rep-PCR is a reliable and cost-effective method for screening large numbers of S. mutans strains for epidemiological study.

Genetic diversity of plaque mutans streptococci with rep-PCR.

Mutans streptococci (MS) are key organisms associated with the etiology of dental caries. Using probabilities that were tested by oversampling, we designed this study to determine the minimal number of MS isolates from an individual required to evaluate diversity of genotypes. MS isolates were genotyped by repetitive extragenic palindromic-polymerase chain-reaction (rep-PCR). Analysis of 20 isolates from individuals resulted in a mean of 1.6 and 2.4 genotypes in children (N = 12) and adults (N = 10), respectively. In a follow-up study, reducing the number of isolates to 7-10 resulted in a theoretical probability of up to 78% for detecting up to 4 genotypes. A mean of 1.5 genotypes was found in 35 children and 10 adults. These findings provide evidence for the design of studies of MS genotyping that can serve as a model for the analysis of genotypes within individuals.

Variability of two plaque sampling methods in quantitation of Streptococcus mutans.

The aim of this study was to determine the reproducibility of individual versus pooled plaque sampling of permanent first molars (PFM) to quantitate Streptococcus mutans (SM)/total streptococci (TS). Ten individual and pooled plaque samples were collected from 35 subjects, randomly assigned to individual-first or pooled-first group. Plaque samples were processed and quantified for SM and TS. SM/TS ratio was used to determine the reproducibility within two group samples. Mean percentage of SM/TS in both methods were not significantly different. However, within subject detection of SM was found to be significantly more sensitive for individual sampling method. Despite the lack of a difference between both methods for SM/TS quantitation, the difference in SM detection suggests that individual sampling is more sensitive.

Repetitive extragenic palindromic PCR for study of Streptococcus mutans diversity and transmission in human populations.

Pulsed-field gel electrophoresis (PFGE) is considered the "gold standard" for molecular epidemiological study. Repetitive extragenic palindromic PCR (rep-PCR) is less time-consuming and more suitable for analyzing large numbers of bacterial strains in human populations. PFGE and rep-PCR provide comparable genotyping results for investigating Streptococcus mutans diversity and transmission.

Immune response in humans to a nasal boost with Streptococcus mutans antigens.

We previously reported that a Streptococcus mutans enriched-glucosytransferase (E-GTF) preparation induces an immune response following intranasal, but not tonsillar, immunization of humans. In this study, we determined whether intranasal immunization of these subjects 2 years later resulted in augmented immune responses compared to those seen in control subjects. Subjects previously immunized via the intranasal (IN, n = 7) or tonsillar (IT, n = 7) route and control (n = 12) subjects were immunized via the intranasal route with E-GTF. Nasal wash, saliva, and serum were collected before immunization and then weekly for 3 months after immunization. Significant (P < 0.05) mucosal and serum immunoglobulin A (IgA) anti-E-GTF responses were observed in all three groups. Nasal and serum IgA anti-E-GTF responses were significantly higher (P < 0.05) in the IN group. The salivary responses in the three groups were, in general, similar. These results indicate that intranasal immunization primes the immune system for a localized secondary response to S. mutans antigens.

Comparative analysis of the antibacterial effects of combined mouthrinses on Streptococcus mutans.

BACKGROUND/AIMS: Chlorhexidine has been proposed as a potent chemotherapeutic agent against oral bacteria. However, there are some inconsistent results regarding the usefulness of chlorhexidine mouthrinse as an antimicrobial for Streptococcus mutans. The purpose of this study was to investigate the effectiveness of combining oral rinses to reduce S. mutans levels in human saliva. METHODS: Sixteen healthy adult subjects were randomly assigned to one of four rinse groups using a 4-cell crossover design. The groups rinsed twice a day for 7 days with one of the following: 0.12% chlorhexidine (PerioGard), 1.5% hydrogen peroxide (Peroxyl), a combined chlorhexidine+hydrogen peroxide, or water (control). Every 5 weeks, each group initiated a different rinse. Saline wash samples were collected on days 7 and 21 for assessment of S. mutans and total streptococci. RESULTS: No significant differences were seen in S. mutans levels among the groups; however, the levels of total streptococci on day 7 samples were significantly lower in the chlorhexidine and chlorhexidine+hydrogen peroxide groups than in the hydrogen peroxide and control groups. There was no additional decrease seen in S. mutans or total streptococci levels in the group receiving chlorhexidine+hydrogen peroxide compared to chlorhexidine alone. CONCLUSIONS: Sample variation was high throughout the study, with a significant trend toward lower counts as the study progressed. Adding hydrogen peroxide to the chlorhexidine mouthrinse did not result in a further decrease in S. mutans levels.

Intranasal immunization of humans with Streptococcus mutans antigens.

To evaluate the effectiveness of a low dose of soluble or liposomal (L) glucosyltransferase-enriched preparation (E-GTF) in inducing mucosal immune responses after intranasal immunization, 12 adults were immunized on days 0 and 7 by the IN route with 62.5 microg of soluble E-GTF or L-E-GTF. An increase in the mean salivary IgA anti-E-GTF response (P < 0.03) was seen in the L-E-GTF but not the soluble E-GTF group. A significant increase (P < 0.05) in the mean specific IgA antibody activity was also seen in nasal wash from both groups. Although the nasal wash responses were higher in the L-E-GTF than in the soluble E-GTF group, they were not significantly different. The soluble E-GTF immunized group showed a higher serum IgG response than the L-E-GTF immunized group on day 90 (P < 0.05). These results indicate that as little as 62.5 microg of E-GTF, when given by the intranasal route, induced an IgA response in secretions.

Effect of age on immunoglobulin A subclass distribution in human parotid saliva.

Two subclasses of immunoglobulin A (IgA) antibodies are produced in humans, IgA1 and IgA2, IgA2 being more resistant to digestion by bacterial proteases than IgA1. The amount of IgA in saliva has been shown to vary with age; however, little is known about the correlation between IgA subclass distribution in saliva and age. The purpose of this study was to determine whether differences exist in the levels and ratio of IgA subclasses in parotid saliva of children and adults. Parotid saliva was obtained from healthy children (age range 6-12 years, n = 14) and adults (age range 22-51 years, n = 20) using Schaefer cups. Samples were analyzed for levels of total IgA, IgA1, and IgA2 by ELISA. IgA and IgA1 levels were significantly higher in adults than in children. However, no differences were seen in the ratio of IgA1 and IgA2 in the two groups of subjects. These findings indicate that levels of IgA increase with age, whereas the IgA subclass ratio is established early in life.

Humans immunized with Streptococcus mutans antigens by mucosal routes.

Strategies aimed at the prevention of Streptococcus mutans infection and dental caries include mucosal immunization, which results in salivary anti-S. mutans responses. The purpose of this study was to evaluate the effectiveness of nasal vs. tonsillar immunization with S. mutans antigens in inducing salivary immune responses. Twenty-one adult subjects were immunized twice, within a seven-day interval, with a glucosyltransferase-enriched preparation (E-GTF) administered by nasal or tonsillar topical spray. Parotid saliva, nasal wash, and serum were collected prior to and at one- to two-week intervals for 3 months following immunization and were assayed by ELISA for anti-E-GTF activity. Results were analyzed by means of the mixed-models procedure with p < 0.05 level of significance. Significantly higher anti-E-GTF responses were detected in saliva and nasal wash samples from the group immunized by the nasal compared with the tonsillar route, indicating that nasal immunization was more effective in inducing mucosal responses in adults.

Utilization of dental sealants by Alabama Medicaid children: barriers in meeting the year 2010 objectives.

PURPOSE: As a national objective, 50% of US children are expected to have dental sealants on at least one permanent molar by the age of 14 years. The present study was conducted to estimate the prevalence of dental sealants among Alabama Medicaid children and to evaluate the characteristics of the sealant users and non-users so the potential barriers in meeting the year 2000/2010 sealant objectives can be identified. METHODS: Alabama Medicaid 1990-1997 claims for children (N = 3,683,842) were analyzed using basic descriptive statistics, likelihood ratio, Chi-Square and t-tests, and ANOVA. Logistic regression analysis was used in identifying the predictors of dental sealant utilization. RESULTS: Nearly 22 percent of children had at least one sealant claim by 12 to 14 years of age (white = 28.3%; Black = 19.8%; OR = 1.6, 95% CI = 1.4-1.8; female = 23.6%; male = 19.7%; OR = 1.3, 95% CI = 1.1-1.4). 5- to 9-year-olds were more likely to have sealants compared to 11- to 14-year-olds (OR = 4.1, 95% CI = 2.7-6.1). The availability of a Medicaid accepting dentist within the county of residence was a significant predictor (OR = 1.5, 95% CI = 1.1-2.2). Less than 2% of the annual amount claimed for total dental services in Alabama was related to sealants and the providers were reimbursed only for 50% to 70% of the amount claimed for sealant procedures. CONCLUSIONS: Racial and gender disparities in obtaining care, non-availability of a Medicaid-participating dentist within the county, and lower payment/claim ratio may make the national sealant objective difficult to achieve in Alabama.

Human salivary immunoglobulin and antigen-specific antibody activity after tonsillectomy.

The importance of the lymphoid tissue collectively known as Waldeyer's ring, which includes the palatine, lingual and nasopharyngeal tonsils, in the induction and contribution of specific antibody responses in human saliva is not clear. The purpose of this study was to determine whether salivary immunoglobulin A (IgA) levels differ in quantity and quality between subjects who have had a tonsillectomy and age, sex and race-matched controls. Parotid saliva, whole saliva, and blood serum samples were collected from 25 volunteer children who had undergone tonsillectomy (T-) within 6-14 months of sampling and from 25 age, sex and race-matched controls. The levels of total IgA (and subclasses) in saliva, and of antigen-specific salivary IgA and serum IgA and IgG antibodies to 4-9 relevant antigens were analyzed by ELISA. No significant difference was observed in the mean total IgA and IgA subclass levels in parotid and whole saliva, although the mean levels for children with a T- were slightly lower. Children with a T- had significantly higher parotid salivary IgA and IgA1 specific/total activity than controls. The total and specific whole saliva IgA and the specific serum IgA or IgG activities were not significantly different from controls. These results indicate an association between the removal of tonsils and increased levels of specific IgA activity in parotid saliva within the first year after a T-.

A vaccine against dental caries: an overview.

Dental caries continues to be a costly and prevalent oral disease. Research efforts towards developing a well tolerated and effective vaccine against dental caries were initiated following the demonstration of a specific bacterial aetiology for this disease. The cariogenic mutans streptococci are the principal bacteria causing this disease. Specific immune defence against these bacteria is provided mainly by secretory immunoglobulin (Ig) A antibodies present in saliva, which are generated by the common mucosal immune system. Progress in the development of a vaccine against dental caries has increased due to both advancements in molecular biology and our understanding of the mucosal immune system and mucosal vaccines. Advancements in molecular biology have facilitated the cloning and functional characterisation of virulence factors of the mutans streptococci, including the cell-surface fibrillar proteins, which mediate adherence to the tooth surface, and the glucosyltransferase enzymes, which synthesise adhesive glucans and allow microbial accumulation on the teeth. Current strategies for immunisation against dental caries are using these virulence factors as key antigens and incorporating them into novel mucosal vaccine systems and delivering them with or without adjuvants to mucosal IgA inductive sites. The most popular routes of mucosal immunisation are via the oral or nasal route. The mucosal immune system is functional in newborn infants, who develop salivary IgA antibodies as they become colonised by oral micro-organisms. Mucosal immunisation strategies result in the induction of salivary IgA antibody responses and pose fewer problems than parenteral injection of antigen. Therefore, mucosal immunisation of infants prior to the appearance of their first teeth may be a well tolerated and effective way to induce immunity against the colonisation of teeth by mutans streptococci and protection against subsequent dental caries. The purpose of this article is to provide an overview of the recent progress on the development of a vaccine against infection by Streptococcus mutans for the prevention of dental caries, with emphasis on the mucosal immune system and vaccine design.

Adjuvant activity of monophosphoryl lipid A for nasal and oral immunization with soluble or liposome-associated antigen.

The effectiveness of monophosphoryl lipid A (MPL) as a mucosal adjuvant was investigated following oral or intranasal (i.n.) administration of an aqueous adjuvant formulation of MPL (MPL-AF) added to soluble antigen or liposomal antigen or incorporated into liposomal antigen membranes. Groups of BALB/c female mice were immunized with 50 to 100 microg of free or liposomal Streptococcus mutans crude glucosyltransferase (C-GTF) with or without MPL-AF added to the vaccine or incorporated into the liposomal membrane. Plasma, saliva, vaginal wash, and fecal extract samples were collected biweekly following immunization and assessed for antigen-specific antibody activity by enzyme-linked immunosorbent assay (ELISA). Mice immunized by the i.n. route had higher levels of salivary, plasma, and vaginal immunoglobulin A (IgA) anti-C-GTF responses and higher levels of plasma IgG anti-C-GTF than the orally immunized groups. A second administration of the vaccine 14 weeks after the initial immunization resulted in an anamnestic response to C-GTF resulting in 10- and 100-fold increases in saliva and plasma IgA and plasma IgG, respectively (in the i.n. immunized groups). Mice receiving a second i.n. immunization with liposomal antigen and MPL-AF had higher salivary IgA anti-C-GTF responses than mice immunized with antigen plus MPL-AF or liposomal antigen (P < 0.05). Plasma IgG anti-C-GTF activity was highest in mice immunized by the i.n. route with antigen formulations containing MPL-AF (P < 0.05). These results demonstrate the effectiveness of MPL-AF as an adjuvant for potentiating mucosal and systemic immune responses to liposomal C-GTF following i.n. immunization.

Anticipatory guidance in infant oral health: rationale and recommendations.

If appropriate measures are applied early enough, it may be possible to totally prevent oral disease. The American Academy of Pediatric Dentistry recommends that infants be scheduled for an initial oral evaluation within six months of the eruption of the first primary tooth but by no later than 12 months of age. The rationale for this recommendation is provided, although the recommendation itself is not universally accepted. Specific recommendations include elimination of bottles in bed, early use of soft-bristled toothbrushes (with parental supervision) and limitation of high-carbohydrate food intake after teeth have been brushed.

A controlled clinical study of the effect of nasal immunization with a Streptococcus mutans antigen alone or incorporated into liposomes on induction of immune responses.

Recent attention to mucosal immunization strategies has been focused on the nasal route for vaccine delivery. This study was designed to determine the effectiveness of a liposome-protein vaccine compared to that of a protein-only vaccine in inducing immune responses in humans. Healthy subjects were randomly assigned to two groups and immunized intranasally with a crude antigen preparation rich in glucosyltransferase (C-GTF) from Streptococcus mutans, alone or in liposomes. Parotid saliva, nasal wash, and serum were collected prior to and at weekly intervals following immunization and were analyzed for anti-C-GTF activity by enzyme-linked immunosorbent assay. The levels of immunoglobulin A (IgA) anti-C-GTF activity in the nasal wash from both groups after immunization increased to a mean peak of fivefold over the baseline level on day 28. Salivary IgA anti-C-GTF responses were induced to a lesser extent. IgG and IgA anti-C-GTF responses in serum were detected on day 14. The IgA responses were predominantly of the IgA1 subclass. These results show that C-GTF vaccines were more effective in inducing a local secretory IgA antibody response than a salivary or serum response when they were given intranasally. The IgA1 anti-C-GTF response in nasal wash samples for liposomal antigen versus antigen only was the only response which was significantly different (P < 0.04). This suggests that the form of the antigen affects the magnitude of the local mucosal response but not that of a disseminated response. These results provide evidence for the effective use of a nasal protein vaccine in humans for the induction of mucosal and systemic responses.

Secretory immunity in defense against cariogenic mutans streptococci.

Specific immune defense against cariogenic mutans streptococci is provided largely by salivary secretory IgA antibodies, which are generated by the common mucosal immune system. This system is functional in newborn infants, who develop salivary IgA antibodies as they become colonized by oral microorganisms. The mechanisms of action of salivary IgA antibodies include interference with sucrose-independent and sucrose- dependent attachment of mutans streptococci to tooth surfaces, as well as possible inhibition of metabolic activities. The goal of protecting infants against colonization by mutans streptococci might be accomplished by applying new strategies of mucosal immunization that would induce salivary IgA antibodies without the complications of parenteral immunization. Strategies of mucosal immunization against mutans streptococci currently under development include the use of surface adhesins and glucosyltransferase as key antigens, which are being incorporated into novel mucosal vaccine delivery systems and adjuvants. The oral application of preformed, genetically engineered antibodies to mutans streptococcal antigens also offers new prospects for passive immunization against dental caries.

Physicians' views on pediatric preventive dental care.

Physicians who provide primary care for children are considered to be in a unique position to provide dental preventive care to their patients. No literature relates the amount of preventive oral health education that physicians receive during training. The purpose of this study was to assess the knowledge, attitudes, and beliefs of pediatricians and family physicians toward preventive dental care in children. A questionnaire was mailed to 398 pediatricians and 632 family physicians licensed to practice in the state of Alabama. The response rate after one mailing and a reminder was 46%. Physician's knowledge about many aspects of preventive dental care was good, but areas of great concern were identified. Overall, most respondents received 2 hr or less of preventive dental education during medical and specialty training. Pediatricians were better informed than family physicians in the areas of general dental knowledge and prevention counseling related to oral health (P < 0.05).

An alternative restorative method for regional odontodysplasia: case report.

A 5-year-old Caucasian male presented with early loss of multiple deciduous teeth. All the characteristics were consistent with the diagnosis of regional odontodysplasia (ROD). Significant initial findings included premature loss of multiple primary mandibular teeth and some malformed permanent teeth. The affected teeth showed hypoplastic enamel and dentin, short roots, and wide pulp chambers, and were localized in the mandibular dentition. Treatment objectives for this patient were to provide improved esthetics, restored chewing function, and space maintenance by the construction of a temporary prosthetic restoration. However, with limited tooth support and an unusual occlusal pattern, it is difficult to obtain satisfactory retention and esthetics with traditional prosthetic techniques. In this article we introduce an alternative method for fabricating a custom removable denture and discuss the prognosis of the malformed permanent dentition and further treatment plan.

Passive immunization against dental plaque formation in humans: effect of a mouth rinse containing egg yolk antibodies (IgY) specific to Streptococcus mutans.

Passive immunization involving the delivery of antibodies specific to pathogens of infectious diseases to the host has been an attractive approach to establish protective immunity against a variety of microbial pathogens, including Streptococcus mutans, which is the principal etiologic agent of dental caries in humans. The overall purpose of the present study was to determine the effectiveness of a mouth rinse containing antibodies to S. mutans in preventing the establishment of this bacterium in dental plaque of humans. The antibodies were derived from egg yolks obtained from hens immunized with whole cells of S. mutans grown in sucrose-containing medium. The immunoglobulin derived from the yolks (IgY) of immunized hens was characterized in vitro and in vivo in human volunteers. Cross-reactivity tests showed that immune IgY reacted with every serotype, except serotype b, which had lost its GTase activity, when the bacteria were cultured in sucrose-containing medium. Immune IgY inhibited S. mutans adherence to saliva-coated hydroxyapatite discs by 59.2%, while control IgY caused an inhibition of only 8.2%. In the short-term (4-hour) test using a mouth rinse containing 10% sucrose, immune IgY decreased the ratio of the percentage of S. mutans per total streptococci in saliva. In the long-term (7-day) test using a mouth rinse without sucrose, the ratio in saliva was not significantly reduced in the volunteers using the immune IgY due to the large standard deviation. However, comparing the ratios of the percentage of S. mutans per total streptococci in plaque of individual subjects, there was a tendency for a reduction of the ratios in the volunteers receiving the mouth rinse containing immune IgY. These results support the effectiveness of IgY with specificity to S. mutans grown in the presence of sucrose as an efficient method to control the colonization of mutans streptococci in the oral cavity of humans.