Nested PCR for detection of mutans streptococci in dental plaque

AIMS: Mutans streptococci such as Streptococcus mutans and Streptococcus sobrinus have been implicated in human dental caries. In an attempt to develop a rapid and sensitive method for detecting Strep. mutans and Strep. sobrinus in dental plaque, a nested PCR amplification based on the 16S rRNA gene was employed. METHODS AND RESULTS: A universal set of PCR primers for bacterial 16S rRNA gene was introduced for the first PCR, and then two sets of primers specific for the 16S rRNA gene sequences of either Strep. mutans or Strep. sobrinus were used for the second PCR. Eighteen plaque samples were analyzed, and a nested PCR was shown to be more sensitive for detecting Strep. mutans and Strep. sobrinus than direct PCR. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: The 16S rRNA gene-based nested PCR method is a rapid and sensitive method for the detection of mutans streptococci, and may also be suitable for carrying out large-scale studies on the cariogenicity of mutans streptococci.     

Effect of mutacin administration on Streptococcus mutans-induced dental caries in rats

A bacteriocin from serotype c Streptococcus mutans strain C3603 was examined for its inhibitory effect on experimental dental caries in rats infected with S. mutans MT8148R (serotype c). Significant reduction in the incidence of dental caries was found only when bacteriocin was incorporated both in the drinking water and in the diet at a high concentration. However, caries reduction was not as great as expected and the addition of bacteriocin to drinking water alone had no effect on the recovery of S. mutans, plaque deposition or caries incidence. The bacteriocin activity must have been reduced in the oral cavity of rats, and the reasons were examined. Bacteriocin-resistant mutants were not detected and the bacteriocin was not inactivated by saliva. Whereas the bacteriocin did not kill the S. mutans cells grown in a sucrose-containing medium, it completely killed the cells grown in a sucrose-free medium.     

Complete Genome Sequence of Streptococcus mutans GS-5, a Serotype c Strain

Streptococcus mutans, a principal causative agent of dental caries, is considered to be the most cariogenic among all oral streptococci. Of the four S. mutans serotypes (c, e, f, and k), serotype c strains predominate in the oral cavity. Here, we present the complete genome sequence of S. mutans GS-5, a serotype c strain originally isolated from human carious lesions, which is extensively used as a laboratory strain worldwide.     

Quantitative detection of Streptococcus mutans in the dental plaque of Japanese preschool children by real-time PCR

AIMS: To detect quantitatively the total bacteria and Streptococcus mutans in dental plaque by real-time PCR with prbac, Sm and GTF-B primers, and to compare their presence with the prevalence of dental caries in Japanese preschool children. METHODS AND RESULTS: Human dental plaque samples were collected from the labial surfaces of the upper primary central incisors of 107 children. The dental status was recorded as dft by WHO caries diagnostic criteria. Positive dt and dft scores by the Sm or GTF-B primer were significantly higher than negative scores (P < 0.01). The proportions of Strep. mutans to the total bacteria from sound, and sound and/or filled upper primary incisors were significantly lower than those from decayed or filled, and decayed incisors, respectively (P < 0.01). CONCLUSIONS: The ratios of Strep. mutans to total bacteria in plaque detected by real-time PCR with Sm and GTF-B primers were closely associated with the prevalence of dental caries in Japanese preschool children. SIGNIFICANCE AND IMPACT OF THE STUDY: These assays may be useful for the assessment of an individual's risk of dental caries.     

Effect of Nutrients upon Streptococcus mutans BHT and Streptococcus mitior LPA-1 Growing in Pure or Mixed Culture on Human Teeth in an Artificial Mouth

Streptococcus mutans and Streptococcus mitior were both capable of colonizing the surfaces of human teeth in an artificial mouth. Although fermentable carbohydrate was not essential for colonization, highest numbers were recovered after 5 d if 5–0°_***0 (w/v) sucrose had been available intermittently. When grown together in mixed culture the interaction of the two species was affected profoundly by the available nutrients. In the presence of synthetic saliva alone, Strep. mitior was strongly antagonistic to Strep. mutans. When a nutrient broth containing sucrose was also provided intermittently there was slight inhibition of Strep. mutans accompanied by an increase in the proportion of Strep. mitior in the plaque, although the former was the dominant organism under these conditions. When 0–5% (w/v) glucose replaced the sucrose, mutual antagonism occurred and fewer organisms were recovered than if only synthetic saliva had been available. One reason why a high-sucrose diet encourages colonization by Strep. mutans may be that insoluble extracellular polysaccharide confers a competitive advantage upon it in the face of antagonistic agents such as the hydrogen peroxide produced by Strep. mitior.     

Effect of inoculation sequence and nutrients upon Streptococcus mutans BHT and Streptococcus mitior LPA-1 growing on human teeth in an artificial mouth

Human teeth in an artificial mouth were inoculated with Streptococcus mutans BHT, Streptococcus mitior LPA-1, or sequentially with both organisms. Incubation was continued for 90 h. Mixed populations were largest when a nutrient supplement containing 5.0% (w/v) sucrose was supplied. Fewer organisms were recovered from experiments with synthetic saliva only, or when a supplement containing 0.05% (w/v) glucose was available. The inoculation sequence determined the total viable count and a larger population resulted when Strep. mutans was the initial colonizer (P less than 0.01). Strep. mutans was always able to become established even when super-infected on to a 24 h plaque of Strep. mitior. The final proportion of Strep mutans was lower when it was the superinfecting organism and the sucrose (P less than 0.01) or glucose (P less than 0.05) nutrient supplement was provided. This work confirms the importance of inoculation sequence and presence of sugars in plaque accumulation and demonstrates the fundamental role of microbial interactions in this process.     

The Effects of Nisin on the Microbial Flora of the Dental Plaque of Monkeys (Macaca fascicularis)

Nisin was incorporated in a soft diet and fed to eight monkeys for five months. At the end of this period dental plaque was removed from one site in the mouths of these animals and from a similar site in eight control animals. Twelve different selective media were used for the cultivation of the bacteria in the plaque. Streptococci, Haemophilus spp. and Gram negative anaerobes were the organisms most frequently isolated. There was some evidence that streptococci constituted a lesser proportion of the flora in the nisin-fed animals.     

Effect of inoculation sequence and nutrients upon Streptococcus mutans BHT and Streptococcus mitior LPA-1 growing on human teeth in an artificial mouth

Human teeth in an artificial mouth were inoculated with Streptococcus mutans BHT, Streptococcus mitior LPA-1, or sequentially with both organisms. Incubation was continued for 90 h. Mixed populations were largest when a nutrient supplement containing 5–0% (w/v) sucrose was supplied. Fewer organisms were recovered from experiments with synthetic saliva only, or when a supplement containing 0–05% (w/v) glucose was available. The inoculation sequence determined the total viable count and a larger population resulted when Strep, mutans was the initial colonizer ( P < 0–01). Strep, mutans was always able to become established even when super-infected on to a 24 h plaque of Strep, mitior. The final proportion of Strep mutans was lower when it was the superinfecting organism and the sucrose ( P < 0–01) or glucose ( P < 0–05) nutrient supplement was provided. This work confirms the importance of inoculation sequence and presence of sugars in plaque accumulation and demonstrates the fundamental role of microbial interactions in this process.     

复方茶多酚含漱液对正畸儿童牙面菌斑中致龋菌的影响

目的观察复方茶多酚含漱液对正畸儿童牙面菌斑中细菌总数和变形链球菌数的影响,以及牙菌斑内原位pH的改变。方法选择42例戴用固定矫治器的正畸儿童,随机分为2组,试验组用复方茶多酚含漱液漱口,对照组用蒸馏水漱口。分别于戴用矫治器前,戴入后1月采集上下颌牙唇颊面菌斑,测定菌斑中细菌总数及变形链球菌数,同时测定牙菌斑原位pH。结果对照组戴用后1月,细菌总数及变形链球菌数较戴用前明显增加(P0.01),牙菌斑原位pH较戴用前降低(P0.01)。试验组与对照组戴用后1个月相比,试验组细菌总数及变形链球菌数明显少于对照组(P0.01),牙菌斑原位pH高于对照组(P0.01)。结论戴用固定矫治器后,牙面菌斑内细菌总数及变形链球菌数较戴用前增加,牙菌斑原位pH较戴用前降低,应用茶多酚含漱液可明显抑制正畸儿童口腔内变形链球菌数,减少龋坏发生。     

Differences in microbiological composition of saliva and dental plaque in subjects with different drinking habits

Several foods have been shown to contain natural components (especially polyphenols) which display anti-adhesive properties against Streptococcus mutans, the aetiological agent responsible for dental crown caries, as well as inhibition of glucosyltransferases, which are the S. mutans enzymes involved in the synthesis of an adherent, water-insoluble glucan from sucrose. Other studies have demonstrated an in vitro action on oral plaque biofilm formation and desorption. This study evaluated whether the activity displayed in vitro by food compounds could affect the microbiological composition of saliva and dental plaque of subjects with a diet rich in these foods, comparing the results with those obtained from subjects with a different diet. The foods considered were: coffee, barley coffee, tea and wine. A total of 93 subjects were recruited into the study. Six samples of both plaque and saliva were collected from each subject at roughly one-monthly intervals. Total bacteria, total streptococci, S. mutans and lactobacilli counts were determined by culture in both saliva and dental plaque. The highest bacterial titres were recorded for the control population, while each drinking habit subgroup showed counts roughly one log lower than the controls. These differences in bacterial counts proved statistically significant (P<0.05). As far as dental plaque was concerned, while total counts did not significantly vary per mg of plaque in the subjects belonging to the different drinking habit subgroups, a significant decrease (P<0.05) was observed in those subjects drinking coffee, tea, barley coffee and wine when mutans streptococci and lactobacilli were evaluated. In several cases a more than one log decrease was observed. Plaque indices were also determined, and a significant (P<0.05) reduction in values was recorded in the subjects belonging the specific drinking habit subgroups compared to the control group. This study indicates that there is a correlation between consumption of specific foods and oral health in terms of reduced plaque deposition and lower counts of odontopathogens.     

The serotype-specific glucose side chain of rhamnose–glucose polysaccharides is essential for adsorption of bacteriophage M102 to Streptococcus mutans

Bacteriophage M102 is a virulent siphophage that propagates in some serotype c Streptococcus mutans strains, but not in S. mutans of serotype e, f or k. The serotype of S. mutans is determined by the glucose side chain of rhamnose–glucose polysaccharide (RGP). Because the first step in the bacteriophage infection process is adsorption of the phage, it was investigated whether the serotype specificity of phage M102 was determined by adsorption. M102 adsorbed to all tested serotype c strains, but not to strains of different serotypes. Streptococcus mutans serotype c mutants defective in the synthesis of the glucose side chain of RGP failed to adsorb phage M102. These results suggest that the glucose side chain of RGP acts as a receptor for phage M102.     

Immunological relationships between glucosyltransferases synthesizing insoluble glucan from Streptococcus cricetus, Streptococcus sobrinus and Streptococcus downei.

The Mr values and isoelectric points of glucosyltransferases synthesizing insoluble glucan (GTF-Is) were determined, and the immunological relationships between them studied. The GTF-I enzymes were from Streptococcus cricetus (mutans group serotype a), Streptococcus sobrinus (mutans group serotypes d and g) and Streptococcus downei (mutans group serotype h). By double immunodiffusion tests, the GTF-I enzymes from the three species possessed a common antigenic determinant; in addition, the GTF-I enzymes of serotypes d, g and h shared a further determinant. The S. sobrinus serotypes d and g GTF-I enzymes were immunologically identical. The GTF-I enzymes of S. sobrinus serotypes d and g, and of S. downei, had an Mr of 161,000 and isoelectric points of 4.8-4.9, while S. cricetus GTF-I had a lower Mr (150,000) and a higher isoelectric point (5.2). This suggests that the S. cricetus GTF-I enzyme may lack a sequence of amino acids which include the determinant shared by S. sobrinus and S. downei GTF-I enzymes. Antibodies specific to the determinant shared by all four serotypes inhibited the homologous and heterologous enzymes by 94-100%.     

Immunological properties of the primer-independent glucosyltransferase of Streptococcus mutans serotypes d and g

Streptococcus mutans serotype g secretes at least three kinds of glucosyltransferase with different enzymological and immunological properties. One of them is a primer-independent enzyme and seems to be the source of primer for the others, both of which are primer-dependent enzymes. Recently, we purified the primer-independent enzyme, the third glucosyltransferase in this group from S. mutans strain AHT-k serotype g. In the present study, we examined the specificity of the antiserum against the primer-independent glucosyltransferase using extracellular culture-conditioned fluids of many strains of the various serotypes of S. mutans. The antiserum cross-reacted with the extracellular culture fluids from strains of serotypes d and a, in addition to serotype g, but not with those of other serotypes, indicating that the primer-independent glucosyltransferase is secreted by the S. sobrinus and S. cricetus, but not by S. mutans and S. rattus. The antiserum did not completely inhibit the activity of the enzyme, even at more than twofold antibody excess, determined by indirect precipitation with immobilized staphylococcal protein A.     

Intrafamilial distribution of mutans streptococci in Japanese families and possibility of father-to-child transmission

The purpose of this study was to investigate the intrafamilial distribution of mutans streptococci in Japanese families using chromosomal DNA fingerprinting with three endonucleases; EcoRI, HindIII and HaeIII. The analysis of 1,908 isolates cultured from the dental plaque of 76 subjects from 20 families (20 married couples and 36 of their children) resulted in the identification of 144 genotypes containing 114 strains of Streptococcus mutans (serotype c, 66.7%; e, 12.5%) and 30 strains of S. sobrinus (d, 13.2%; g, 7.6%). A mean of 1.89 genotypes (from one to four) was harbored in individual subjects, and a mean of 4.10 genotypes from two to seven was harbored in individual families. Among the 70 genotypes found in the children, 36 (51.4%) were in agreement with their mothers and 22 (31.4%) were in agreement with their fathers. The other genotypes (18.6%) did not correspond with the parents. Homologous strains between parents were found in only two couples. This result showed that fathers or others as well as mothers can be sources of transmission. Further, the serotype d, e and g strains showed significantly higher probabilities of transmission than serotype c.     

Purification and properties of extracellular mutacin, a bacteriocin from Streptococcus sobrinus.

Mutacin MT6223, a cell-free bacteriocin produced by Streptococcus sobrinus MT6223, was purified by ammonium sulphate precipitation, chromatofocusing with PBE 94 and column chromatography on SP Sephadex C-25. The specific activity of the purified mutacin was increased 1950-fold with a recovery of 9.7%. The molecular mass of the purified mutacin preparation was estimated to be 6.5 kDa. The mutacin activity was stable from pH 2-7, and was resistant to treatment at 100 degrees C for 20 min. It was inactivated by papain or ficin digestion, and was partially inhibited by alpha-chymotrypsin. The mutacin was found to be active against strains of serotypes c, e and f of Streptococcus mutans and the addition of purified mutacin MT6223 to growing cells of S. mutans MT8148 resulted in a rapid inhibition of incorporation of [3H]thymidine, [3H]uracil or L-[3H]glutamic acid into DNA, RNA or protein, respectively. Specific pathogen-free Fischer rats fed diet 2000 and infected with S. mutans MT8148R showed significantly fewer caries and lower plaque scores when mutacin was administered through drinking water. The present study demonstrates that mutacin MT6223 inhibited the growth of mutans streptococci. Thus, mutacin MT6223 may be a candidate for use in dental caries prevention.     

Inhibition by yeast killer toxin-like antibodies of oral Streptococci adhesion to tooth surfaces in an ex vivo model

BACKGROUND: Monoclonal (KTmAb) and recombinant (KTscFv) anti-idiotypic antibodies, representing the internal image of a yeast killer toxin, proved to be microbicidal in vitro against important eukaryotic and prokaryotic pathogens such as Candida albicans, Pneumocystis carinii, Mycobacterium tuberculosis, Staphylococcus aureus, S. haemolyticus, Enterococcus faecalis, E. faecium, and Streptococcus pneumoniae, including multidrug-resistant strains. KTmAb and KTscFv exerted a strong therapeutic effect in well-established animal models of candidiasis and pneumocystosis. Streptococcus mutans is the most important etiologic agent of dental caries that might result from the metabolic end products of dental plaque. Effective strategies to reduce the disease potential of dental plaque have considered the possibility of using antibiotics or antibodies against oral streptococci in general and S. mutans in particular. In this study, the activity of KTmAb and KTscFv against S. mutans and the inhibition and reduction by KTmAb of dental colonization by S. mutans and other oral streptococci in an ex vivo model of human teeth were investigated. MATERIALS AND METHODS: KTscFv and KTmAb were used in a conventional colony forming unit (CFU) assay against a serotype C strain of S. mutans, and other oral streptococci (S. intermedius, S. mitis, S. oralis, S. salivarius). An ex vivo model of human teeth submerged in saliva was used to establish KTmAb potential of inhibiting or reducing the adhesion to dental surfaces by S. mutans and other oral streptococci. RESULTS: KTmAb and KTscFv kill in vitro S. mutans and other oral streptococci. KTmAb inhibit colonization of dental surfaces by S. mutans and oral streptococci in the ex vivo model. CONCLUSIONS: Killer antibodies with antibiotic activity or their engineered derivatives may have a potential in the prevention of dental caries in vivo.     

Inter-serotype comparison of polysaccharides produced by extracellular enzymes from Streptococcus mutans

The biochemical and morphological characteristics of polysaccharides synthesized from sucrose by extracellular enzymes from D-glucose-grown Streptococcus mutans representing serotypes a-g were compared. The polysaccharides synthesized by the enzymes from serotypes a, d, and g formed visible aggregates and firmly adhered to glass surfaces, whereas those formed by the enzymes from serotypes b, c, e, and f floated homogeneously and were poorly adherent. The enzymes of serotypes a, d, and g produced large amounts of water-insoluble polysaccharides (IPs, D-glucans), and those of serotypes b, c, e, and f water-soluble polysaccharides (SPs, D-glucans and D- fructans ). As compared with the IPs of serotypes b, c, e, and f, the IPs of serotypes a, d, and g (a) contained a higher proportion of (1----3)-alpha-D-glucosidic linkages and alpha-D-(1----3,6) branch linkages; (b) showed higher susceptibility to (1----3)-alpha-D-glucanase (serotype a excepted) and lower (1----6)-alpha-D-glucanase sensitivity; (c) contained larger amounts of high-molecular-weight fractions; (d) showed higher intrinsic viscosities (serotype b excepted); and (e) had lower S. mutans cell-agglutination activities. On electron-microscope observation, the IPs of all serotypes showed two fibrillar components; a double-stranded fibril, with short, fluffy protrusions extending out of its periphery, and a fine, single-stranded fibril. Thus, the serotypes could be divided into two major groups: a, d, and g; and b, c, e, and f. No similar grouping of serotypes was indicated by the chemical and morphological properties of SPs.     

氟对正畸儿童牙面菌斑变形链球菌的影响

目的:观察和探讨2%氟化钠对正畸患者牙面菌斑内细菌总数及变形链球菌数的影响.方法:选择34例正畸儿童,分为两组,试验组涂布2%氟化钠;对照组不做处理.分别于戴用矫治器前,戴入后1月采集上颌牙唇颊面菌斑,测定菌斑中细菌总数及变形链球菌数.结果:对照组戴用后1月,细菌总数及变形链球菌数较戴用前明显增加(P＜0.01).对照组与试验组戴用后1月相比,试验组细菌总数及变形链球菌数明显少于对照组(P＜0.05).结论:戴用固定矫治器后,牙面菌斑内细菌总数及变形链球菌数较戴用前增加,应用2%氟化钠可明显抑制正畸患者口腔内变形链球菌数,减少龋坏发生.     

Sucrose-dependent cell adherence and cariogenicity of serotype c Streptococcus mutans

Four strains of serotype c Streptococcus mutans differing in glucosyltransferase (GTase) and fructosyltransferase (FTase) activities were examined. These strains had been made resistant to streptomycin. FTase activity of an S. mutans clinical variant, MT6801R, which forms large mucoid colonies on sucrose-containing agar, was considerably higher than that of a typical serotype c strain, MT8148R, which forms small, rough colonies on the same agar. Two mutants, NG14 and NG7183, were induced from strain MT6801R by N-methyl-N'-nitro-N-nitrosoguanidine, and were found to be streptomycin-resistant. GTase and FTase activities of mutant NG14 were similar to those of the typical serotype c strain, while in mutant NG7183 the two enzyme activities were very low. Growing cells of these strains (except NG7183) adhered firmly to a glass surface in sucrose broth. Resting cells of all strains attached in small numbers to saliva-coated hydroxyapatite in the absence of sucrose. On the other hand, the presence of sucrose markedly enhanced the attachment of cells of strains MT8148R, MT6801R and NG14, but not NG7183. Cell-surface hydrophobicity and acid production of all strains were similar. Both strain MT8148R and NG14 colonized tooth surfaces and produced significant dental caries in specific-pathogen-free rats. Strain MT6801R had lower colonization ability and cariogenicity when compared with strains MT8148R and NG14. Furthermore, mutant NG7183 was able to colonize the tooth surfaces in small numbers, but failed to cause dental caries. These results indicate that sucrose-dependent cell adherence mediated by de novo glucan synthesis is necessary for the accumulation of serotype c S. mutans cells on the tooth surface and the induction of dental caries.     

Application of carbon source utilization patterns to measure the metabolic similarity of complex dental plaque biofilm microcosms

Biolog technology was applied to measure the metabolic similarity of plaque biofilm microcosms, which model the complex properties of dental plaque in vivo. The choice of Biolog plate, incubation time, and incubation conditions strongly influenced utilization profiles. For plaque biofilm microcosms, Biolog GP2 plates incubated anaerobically in an H2-free atmosphere gave the clearest profile. To test the application of the Biolog GP2 assay, plaque microcosms were developed under different nutrient conditions in which the frequency of sucrose application was varied. Cluster analysis of Biolog GP2 data from 10 microcosm biofilms correlated with sucrose frequency. Aciduric bacteria (Streptococcus mutans plus lactobacilli) predominated in the plaques receiving high-frequency sucrose applications. Agreement between the Biolog GP2 groupings with nutrient and compositional changes suggests that Biolog analysis is a valuable technique for analyzing the metabolic similarity of dental plaque biofilm microcosms and other high-nutrient or predominantly anaerobic ecosystems.