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Immunoglobulin A antibody activity to mutans streptococci in parotid, submandibular and whole saliva.

Antigen extracts of 9 strains of Streptococcus mutans, Streptococcus sobrinus, Streptococcus salivarius and Streptococcus mitis were separated in acrylamide gels by electrophoresis. Strains of the same genotype of mutans streptococci were found to have virtually identical sodium dodecyl sulfate-polyacrylamide gel electrophoresis patterns but differed clearly from other genotypes of mutans or with the reference strains S. salivarius and S. mitis. Immunoglobulin A (IgA) antibody activity to antigens of S. mutans, S. sobrinus and S. mitis in the parotid, submandibular and whole saliva of 12 people was detected by Western blotting. IgA antibodies reacted with several of the antigens, and the reaction pattern was unique for each of the individuals. To certain antigens, all the salivas blotted bands if both clear and questionable bands were counted. Significantly more blotted bands were revealed with antigens from S. mutans than S. sobrinus. The results of the 3 different salivas of the same individual had a high degree of resemblance but in a few individuals, more blotted bands were obtained with parotid saliva to S. mutans and S. sobrinus antigens.

Adult↗

Differential toxic effects of lactate and acetate on the metabolism of Streptococcus mutans and Streptococcus sanguis.

Experiments were conducted with Streptococcus mutans NCTC 10449 and Streptococcus sanguis ATCC 10556 to determine whether the acid end-products, lactate and acetate, were involved in the regulation of cellular growth and metabolism. The growth rate and culture biomass of both organisms was inhibited by the addition of lactate and acetate at concentrations as high as 200 mM to the cultures, although the final pH values of the lactate and acetate cultures were similar. In addition, the metabolic conversion of glucose to lactate was decreased by external lactate but stimulated by acetate. In spite of this, calculation of the yield of cell biomass per mole of ATP (YATP) showed that the yield of both organisms actually increased in the presence of added lactate, but decreased with acetate. This indicates that the two acids interacted with the cells of the organisms by different mechanisms. For both organisms, the final external undissociated lactic acid was relatively constant at concentrations between 0 and 200 mM added lactate, 24.9-32.5 mM for S. mutans and 8.0-11.5 mM for S. sanguis. On the other hand, the final concentration of undissociated acetic acid in the S. mutans cultures increased from 2.9 to 83.7 mM as the medium acetate concentration increased, and from 1.0 to 36.0 mM with the S. sanguis cultures. Counterflow experiments provided evidence for a lactate carrier in both S. mutans and S. sanguis, but an acetate carrier in these organisms could not be demonstrated. [14C]-lactate and [14C]-acetate were taken up into de-energized, chemostatgrown cells of S. mutans and S. sanguis in response to an artificially generated pH gradient but not by an imposed electrical gradient. Thus, under these conditions lactate uptake occurred via a symport process with only one proton. Growth of both organisms in the presence of increasing concentrations of acetate resulted in a small reduction (27%) in the transmembrane pH gradient (delta pH) as measured by the permeant acid, [14C]-salicylate. However, the uptake of [14C]-acetate for the estimation of delta pH revealed significant inhibition of the acetate concentration gradient in the presence external acetate, indicating that the cells expelled the acetate anion. The results indicate that, unlike acetate uptake, lactate transport by S. mutans and S. sanguis was strictly regulated via the lactate carrier in order to prevent excessive dissipation of the pH gradient. Clearly, the formation of acetate by oral streptococci is more problematic for cellular homeostasis than the formation of lactate.

Acetic Acid↗

Ribosomal RNA (rrn) operons in Streptococcus mutans and nucleotide sequence of tRNA(Pro) gene associated with rrnB.

Using a cloned 0.5-kb probe containing an internal fragment of 23S ribosomal RNA from the rrnB operon of Streptococcus mutans, we analyzed various endonuclease digests of the chromosomal DNA isolated from human-derived strains of mutans streptococci. Thus far, the examined S. mutans strains exhibited five ribosomal operons. Here, we describe a ribotyping technique for S. mutans based on restriction and Southern blot analyses with the biotin-labeled homologous probe and chemiluminescence detection. We cloned and sequenced a unique gene encoding tRNA(Pro) downstream from 23S rRNA gene at the 3' end of the operon. Primers designed to the 3' end of the rrnB operon PCR-amplified a 2.3-kb DNA fragment in all tested strains. Restriction fragment length polymorphism analysis of the amplicon revealed a diversity of the single locus among S. mutans isolates, thus establishing a potential use of the technique for the molecular epidemiology of mutans streptococci.

Base Sequence↗

Arbitrarily primed polymerase chain reaction fingerprinting for the genotypic identification of mutans streptococci from humans.

Determining whether two strains of bacteria are unique, identical or clonally related depends upon comparisons of phenotypic and/or genotypic traits. Individual isolates can then be grouped according to differences or similarities among those traits. One method of genotyping strains of bacteria is commonly referred to as chromosomal DNA fingerprinting. Previously, we generated chromosomal DNA fingerprints of mutans streptococci to study the transmission of this organism within families. Here, we developed and evaluated an arbitrarily primed polymerase chain reaction (AP-PCR) method for the genotypic characterization of mutans streptococci. Results were compared to those derived from the more conventional chromosomal DNA fingerprinting method. First, we showed that randomly selected clinical isolates displayed a unique banding profile by both methods; the mean similarity indices between DNA fragment patterns were 0.69 for chromosomal DNA fingerprinting and 0.74 for AP-PCR. This indicated that AP-PCR demonstrated less diversity than chromosomal DNA fingerprinting. Subsequently, we tested the agreement between chromosomal DNA fingerprinting and AP-PCR in determining genotypic similarities among 21 mutans streptococci strains obtained from 10 mother-child pairs, and 5 mutans streptococci strains from 5 fathers. The Kappa value for agreement was 0.88. We conclude that AP-PCR, which generates patterns of 8 to 12 amplicons, is capable of distinguishing strains of mutans streptococci among non-related individuals. Moreover, AP-PCR can discern both homogeneity and heterogeneity of mutans streptococci genotypes among mother and child pairs. Overall, we found that AP-PCR gave results comparable to those of chromosomal DNA fingerprinting.

Chromosomes, Bacterial↗

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.

Adult↗

DnaK expression in response to heat shock of Streptococcus mutans.

The oral pathogen, Streptococcus mutans, persistently colonizes human hosts and initiates oral disease despite extreme variations in environmental conditions. To begin to investigate the role of the stress protein, DnaK (Hsp70), in environmental stress responses by S. mutans, pulse-chase experiments were initially used to establish that a functional heat shock response existed in this organism. A C-terminal fragment of the S. mutans dnaK gene was cloned and engineered to be expressed with a histidine tag. Using the recombinant DnaK protein that had been purified by nickel affinity chromatography, an antibody specific for the S. mutans DnaK protein was generated to analyse DnaK expression under homeostatic and heat shock conditions. Western blot analysis indicated that the anti-recombinant DnaK antibody specifically recognized a protein (molecular mass approx. 68 kDa) which was induced in response to thermal stress. Elucidating the role of DnaK in responses by S. mutans to various environmental stressors will provide a better understanding of how DnaK is involved in survival of extreme environments and the contribution of the DnaK protein to the virulence of S. mutans.

Amino Acid Sequence↗

Construction and characterization of a recombinant ureolytic Streptococcus mutans and its use to demonstrate the relationship of urease activity to pH modulating capacity.

To begin to understand the contribution of oral microbial ureolysis to the inhibition of dental caries, we sought to construct a recombinant, ureolytic mutans streptococcus and correlate the ureolytic capacity of plaque bacteria with pH moderating ability. Streptococcus mutans GS-5 was transformed with a plasmid containing the urease genes from Streptococcus salivarius 57.I. The recombinant strain, S. mutans AC04, stably maintained the urease genes. High levels of urease activity were detected, with a maximum specific activity of 0.9 mumol of urea hydrolyzed/min/mg cell dry weight when the growth medium was supplemented with 50 microM exogenous NiCl2. Harboring the recombinant plasmid, or growth in NiCl2, did not markedly affect the glycolytic capacity of S. mutans. In vitro pH drop analysis of S. mutans AC04, metabolizing glucose and physiologically relevant concentrations of urea simultaneously, demonstrated that increasing the urease activity of plaque bacteria resulted in a corresponding reduction in the depth and the duration of the glycolytic pH fall. The results demonstrate the feasibility of engineering urease producing S. mutans and suggest that enhancing the ureolytic capacity of dental plaque, particularly cariogenic plaque, may help to offset the progression of the caries process.

Hydrogen-Ion Concentration↗

Caries prevalence, salivary Streptococcus mutans and dietary scores in 13-year-old Swedish schoolchildren.

388 13-yr-old schoolchildren from Karlstad, Sweden, participated. Dental caries experience was expressed as number of decayed (D) and filled (F) tooth surfaces (S), and registered on bite-wing radiographs and obtained from records. Salivary Streptococcus mutans counts were determined by a spatula method. A dietary score was calculated for each individual based on an interview concerning the intake frequency of 23 sugar-containing products. Significantly lower DFS values were found in the group with no detectable S. mutans compared to three of the four groups with salivary S. mutans (P less than 0.01). No differences were found between the DFS values of individuals with high, moderate or low dietary scores. There was no statistically significant association between dietary scores and levels of S. mutans. Among the children with no detectable S. mutans, there were higher DFS-values with increasing intake frequency. No relationship between initial caries lesions, S. mutans and diet was found.

Adolescent↗

Dental caries and mutans streptococci in selected groups of urban and native Indian schoolchildren in Mexico.

Two groups of Mexican children aged 12-14 yr were examined for dental caries and salivary mutans streptococci counts. One hundred lived in Mexico City and 100 belonged to a native Mazahua Indian population. The prevalence of caries, diagnosed according to WHO, was 90% in Mexico City and 82% in the Indian community. Mean DMFT was 5.98 and 3.57 respectively. Saliva samples were analyzed for mutans streptococci by the "Strip mutans" method. Further identification of the biotype/serotype was done. Mutans streptococci were found in 95% of the urban children and 75% of the Indians. There was a statistically significant difference in the prevalence of mutans streptococci between the city and the rural samples. Streptococcus mutans was more frequently found than Streptococcus sobrinus.

Adolescent↗

Streptococcus mutans in plaque and saliva and the development of caries.

Plaque samples from 10 different tooth surfaces of 10 schoolchildren with varied caries experience (DFS 10-33) were collected five times during 2.5 years. The samples were examined with an immunofluorescent technique for identification and enumeration of Streptococcus mutans serotypes c/e/f and d/g. At each sampling occasion the children were scored for caries. A stimulated saliva sample was also collected and the number of S. mutans per ml saliva was determined. The saliva level of S. mutans was shown to reflect the prevalence and proportion of this microorganism on the selected surfaces. Five surfaces carried S. mutans at each sampling. Four of these surfaces showed progressive cries S. mutans infection was also found to precede the development of incipient caries on four surfaces. Eighty percent of the surfaces that stayed sound were only transiently carriers of S. mutans in mainly very low numbers. Serotype c/e/f dominated in prevalence and proportion on the surfaces with a history of caries during the study.

Adolescent↗

Streptococcus mutans establishment and dental caries experience in children from 2 to 4 years old.

39 children were studied longitudinally at the age of 2, 3, and 4 yr for the colonization of S. mutans in plaque and saliva and for caries experience. S. mutans was found in 38% of the children, and the predominant serotype group was c/e/f. A total of 16 children got caries before the age of 4. Children who harbored S. mutans in their plaque at the age of 2, appeared to be the most caries-active individuals. Their caries index values (number of decayed, missed and filled surfaces, dmfs = 10.6 +/- 5.3) at the age of 4 differed significantly from the values of children who harbored S. mutans later (dmfs = 3.4 +/- 1.8, P less than 0.005) or remained free from S. mutans infection (dmfs = 0.3 +/- 1.1, P less than 0.0003). It was thus concluded that the early establishment of S. mutans in the plaque of primary incisors indicated early and extensive caries attack in young primary dentition.

Child, Preschool↗

Crevicular IgG antibodies and recovery of Streptococcus mutans implanted by mouthrinsing.

After challenge with a streptomycin-resistant strain of Streptococcus mutans (S. mutans), a tendency to higher recovery of S. mutans was found in gingival crevicular fluid (GCF) from surfaces with a low IgG antibody activity against S. mutans than in GCF from surfaces with a high antibody activity. This suggests that antibodies in GCF may interfere with the establishment of S. mutans on gingival tooth surfaces. In GCF collected from some sites, considerably higher IgG antibody activity was observed than in homologous serum, indicating that part of the IgG response to S. mutans was locally derived.

Adult↗

Deletion of gtfC of Streptococcus mutans has no influence on the composition of a mixed-species in vitro biofilm model of supragingival plaque.

Glucosyltransferases from Streptococcus mutans are thought to play an important role in bacterial adherence to the tooth surface. The goal of the present study was to determine the effect of the deletion of the gtfC gene, which encodes a glucosyltransferase that catalyses primarily the formation of insoluble glucan (mutan), on colonization of S. mutans in a mixed-species biofilm model of supragingival plaque. A gtfC deletion mutant of S. mutans UA159 grew poorly in biofilms on a polystyrene surface in Todd-Hewitt medium containing sucrose, but biofilm formation in the semi-defined fluid universal medium (FUM) was not affected. The S. mutans gtfC mutant colonized with the same efficiency as the wild-type strain when grown together with five other species in a mixed-species biofilm on hydroxyapatite in a mixture of FUM and saliva with pulses of sucrose and showed the same ability to demineralize enamel in vitro. Colonization of mutant and wild-type strains was also equal in an association experiment in specific-pathogen-free rats. However, the gtfC mutant gave rise to more dentinal fissure lesions and smooth surface caries than the wild-type strain; this could be caused by a change in diffusion properties as a result of to the lack of mutan.

Animals↗

Targeted killing of Streptococcus mutans by a pheromone-guided "smart" antimicrobial peptide.

Within the repertoire of antibiotics available to a prescribing clinician, the majority affect a broad range of microorganisms, including the normal flora. The ecological disruption resulting from antibiotic treatment frequently results in secondary infections or other negative clinical consequences. To address this problem, our laboratory has recently developed a new class of pathogen-selective molecules, called specifically (or selectively) targeted antimicrobial peptides (STAMPs), based on the fusion of a species-specific targeting peptide domain with a wide-spectrum antimicrobial peptide domain. In the current study, we focused on achieving targeted killing of Streptococcus mutans, a cavity-causing bacterium that resides in a multispecies microbial community (dental plaque). In particular, we explored the possibility of utilizing a pheromone produced by S. mutans, namely, the competence stimulating peptide (CSP), as a STAMP targeting domain to mediate S. mutans-specific delivery of an antimicrobial peptide domain. We discovered that STAMPs constructed with peptides derived from CSP were potent against S. mutans grown in liquid or biofilm states but did not affect other oral streptococci tested. Further studies showed that an 8-amino-acid region within the CSP sequence is sufficient for targeted delivery of the antimicrobial peptide domain to S. mutans. The STAMPs presented here are capable of eliminating S. mutans from multispecies biofilms without affecting closely related noncariogenic oral streptococci, indicating the potential of these molecules to be developed into "probiotic" antibiotics which could selectively eliminate pathogens while preserving the protective benefits of a healthy normal flora.

Anti-Bacterial Agents↗

Genes involved in bacitracin resistance in Streptococcus mutans.

Streptococcus mutans is resistant to bacitracin, which is a peptide antibiotic produced by certain species of Bacillus. The purpose of this study was to clarify the bacitracin resistance mechanism of S. mutans. We cloned and sequenced two S. mutans loci that are involved in bacitracin resistance. The rgp locus, which is located downstream from rmlD, contains six rgp genes (rgpA to rgpF) that are involved in rhamnose-glucose polysaccharide (RGP) synthesis in S. mutans. The inactivation of RGP synthesis in S. mutans resulted in an approximately fivefold-higher sensitivity to bacitracin relative to that observed for the wild-type strain Xc. The second bacitracin resistance locus comprised four mbr genes (mbrA, mbrB, mbrC, and mbrD) and was located immediately downstream from gtfC, which encodes the water-insoluble glucan-synthesizing enzyme. Although the bacitracin sensitivities of mutants that had defects in flanking genes were similar to that of the parental strain Xc, mutants that were defective in mbrA, mbrB, mbrC, or mbrD were about 100 to 120 times more sensitive to bacitracin than strain Xc. In addition, a mutant that was defective in all of the mbrABCD genes and rgpA was more sensitive to bacitracin than either the RGP or Mbr mutants. We conclude that RGP synthesis is related to bacitracin resistance in S. mutans and that the mbr genes modulate resistance to bacitracin via an unknown mechanism that is independent of RGP synthesis.

Anti-Bacterial Agents↗

Functional genomics approach to identifying genes required for biofilm development by Streptococcus mutans.

Streptococcus mutans, the primary etiological agent of human dental caries, is an obligate biofilm-forming bacterium. The goals of this study were to identify the gene(s) required for biofilm formation by this organism and to elucidate the role(s) that some of the known global regulators of gene expression play in controlling biofilm formation. In S. mutans UA159, the brpA gene (for biofilm regulatory protein) was found to encode a novel protein of 406 amino acid residues. A strain carrying an insertionally inactivated copy of brpA formed longer chains than did the parental strain, aggregated in liquid culture, and was unable to form biofilms as shown by an in vitro biofilm assay. A putative homologue of the enzyme responsible for synthesis of autoinducer II (AI-2) of the bacterial quorum-sensing system was also identified in S. mutans UA159, but insertional inactivation of the gene (luxS(Sm)) did not alter colony or cell morphology or diminish the capacity of S. mutans to form biofilms. We also examined the role of the homologue of the Bacillus subtilis catabolite control protein CcpA in S. mutans in biofilm formation, and the results showed that loss of CcpA resulted in about a 60% decrease in the ability to form biofilms on an abiotic surface. From these data, we conclude that CcpA and BrpA may regulate genes that are required for stable biofilm formation by S. mutans.

Amino Acid Sequence↗

Intranasal immunization against dental caries with a Streptococcus mutans-enriched fimbrial preparation.

Streptococcus mutans has been identified as the major etiological agent of human dental caries. The first step in the initiation of infection by this pathogenic bacterium is its attachment (i.e., through bacterial surface proteins such as glucosyltransferases, P1, glucan-binding proteins, and fimbriae) to a suitable receptor. It is hypothesized that a mucosal vaccine against a combination of S. mutans surface proteins would protect against dental caries by inducing specific salivary immunoglobulin A (IgA) antibodies which may reduce bacterial pathogenesis and adhesion to the tooth surface by affecting several adhesins simultaneously. Conventional Sprague-Dawley rats, infected with S. mutans at 18 to 20 days of age, were intranasally immunized with a mixture of S. mutans surface proteins, enriched for fimbriae and conjugated with cholera toxin B subunit (CTB) plus free cholera toxin (CT) at 13, 15, 22, 29, and 36 days of age (group A). Control rats were either not immunized (group B) or immunized with adjuvant alone (CTB and CT [group C]). At the termination of the study (when rats were 46 days of age), immunized animals (group A) had significantly (P < 0.05) higher salivary IgA and serum IgG antibody responses to the mixture of surface proteins and to whole bacterial cells than did the other two groups (B and C). No significant differences were found in the average numbers of recovered S. mutans cells among groups. However, statistically fewer smooth-surface enamel lesions (buccal and lingual) were detected in the immunized group than in the two other groups. Therefore, a mixture of S. mutans surface proteins, enriched with fimbria components, appears to be a promising immunogen candidate for a mucosal vaccine against dental caries.

Administration, Intranasal↗

Stability of the resident microflora and the bacteriocinogeny of Streptococcus mutans as factors affecting its establishment in specific pathogen-free rats.

The outcome of the experimental implantation of Streptococcus mutans strains in humans and animals is unpredictable, and neither success nor failure can be explained. It seems logical to assume that, apart from dietary and host factors, the characteristics of the S. mutans strain involved and those of the resident plaque microflora are important in colonization. For example, previous work in this laboratory suggested that bacteriocin production accounts at least in part for the establishment of an invading bacterium in a microbial ecosystem. In the present study, a complex specific pathogen-free Ny plaque ecosystem was obtained by the inoculation of specific pathogen-free rats with Actinomyces viscosus Ny1 and S. sanguis Ny101, and the establishment of S. mutans in such rats was then examined. It was found that bacteriocinogenic (bac(+)) strains generally colonized in much higher proportions than non-bacteriocinogenic (bac(-)) strains. Moreover, the longer the delay in introducing S. mutans, the poorer was its establishment. Shortly after inoculation of strains Ny1 and Ny101, there is probably a transient state in which microbial equilibrium has not been reached, but later the specific pathogen-free Ny system attains a stable climax community which more strongly resists invaders. The ability of a number of S. mutans strains to establish in such a climax community was then examined, and it was found that bac(+) strains generally established at a higher level than did bac(-) strains. In summary, it was concluded that, although the bac(+) state is an important property in the successful invasion of a plaque by S. mutans, the stability of the resident microflora is also an important factor.

Actinomyces↗