Human strains of mutans streptococci show different cariogenic potential in the hamster model

The cariogenic potential of fresh humans strains of Streptococcus mutans and Streptococcus sobrinus was examined in hamsters maintained on a high sucrose diet. The strains 1B 16 (S. mutans) and B13 (S. sobrinus) isolated 20-25 yrs ago served as positive controls. Three series of experiments were run. Some strains were tested once and some strains were tested in all 3 experiments. The animals infected with S. sobrinus strains generally showed lower caries scores than those infected with S. mutans strains. Among the groups infected with the different strains of S. mutans the caries scores varied. In one of the experiments the caries score of the animals infected with one of the Icelandic S. mutans strains was significantly higher than that of the positive control group and the group infected with one of the fresh Swedish isolates. Thus, different strains of mutans streptococci where shown to induce varying caries activity in the hamster model.     

The earlier the colonization by mutans streptococci, the higher the caries prevalence at 4 years of age

Seventy-eight 4-year-old children, examined earlier for the presence of mutans streptococci at 4-month intervals from 15 months of age, were recalled for recording of dental caries and salivary sampling. The saliva samples were analysed for the presence of mutans streptococci, including the species Streptococcus mutans and Streptococcus sobrinus and lactobacilli. The results showed that the earlier the mutans streptococci had been detected in the children, the higher the caries experience. Eighty-nine percent of the children colonized at 2 years of age had experienced caries and had a mean dfs of 5.0 compared with 25% of the non-colonized 4-year-olds with a mean dfs of 0.3. S. mutans was the predominant species. S. sobrinus was usually found in combination with S. mutans , except in 2 children where S. sobrinus was the only species detected. More children with multiple species had higher numbers of total mutans streptococci and a tendency to higher caries prevalence than children with only S. mutans.     

Lactobacillus-mediated interference of mutans streptococci in caries-free vs. caries-active subjects

In order to assess whether naturally occurring oral lactobacilli have probiotic properties, lactobacilli were isolated from saliva and plaque from children and adolescents, with or without caries lesions. The interference capacities of these lactobacilli were investigated against a panel of 13 clinical isolates and reference strains of Streptococcus mutans and Streptococcus sobrinus, as well as against the subject's autologous mutans streptococci, using the agar-overlay technique. Lactobacillus-mediated inhibition differed significantly between the three subject groups (no caries, arrested caries, or active caries), demonstrating increased inhibition in subjects without present or previous caries experience compared to subjects with arrested caries or subjects presenting with frank lesions. Lactobacilli from subjects lacking S. mutans inhibited the growth of the test panel of mutans streptococci significantly better than lactobacilli from subjects who were colonized. Furthermore, subjects without caries experience harbored lactobacilli that more effectively repressed the growth of their autologous mutans streptococci. Twenty-three Lactobacillus spp. completely inhibited the growth of all mutans streptococci tested. Species with maximum interference capacity against mutans streptococci included Lactobacillus paracasei, Lactobacillus plantarum, and Lactobacillus rhamnosus. Naturally occurring oral lactobacilli significantly inhibited the growth of both test strains of mutans streptococci and the subject's autologous mutans streptococci in vitro, and this effect was more pronounced in caries-free subjects.     

Inhibition of cultivable bacteria by chlorhexidine treatment of dentin lesions treated with the ART technique

The aim of this study was to examine the changes in the cultivable microflora of carious dentin before and after atraumatic restorative treatment (ART) and investigate the inhibitory effect of chlorhexidine-gluconate-based cavity disinfectant in the microflora. Using a split mouth design, 35 primary molar pairs with class II carious lesions in 35 patients (mean age 7.31+/-0.47 years) were selected. The total viable counts (TVC), Streptococcus mutans and lactobacilli were first measured in the center of the infected demineralized lesion and then from the hard dentine after caries removal by the ART technique. Chlorhexidine-gluconate (2%)-based cavity disinfectant was applied to one of the molar pairs and the other molar received no disinfectant treatment. Thereafter, all of the teeth were restored with glass ionomer cement (GIC). Cavities were reassessed after 6 months and again dentine samples were microbiologically investigated. Removal of carious dentine by ART significantly reduced TVC, S. mutans and lactobacilli. After 6 months, application of chlorhexidine exhibited a greater significant reduction in TVC (p=0.013), and a significant reduction in S. mutans compared to the nondisinfected group (p<0.001). A significant reduction in lactobacilli counts was observed in both groups after 6 months, but the difference between the disinfected and nondisinfected groups was not significant (p=0.056). ART was found to be effective in reducing the cultivable microflora and chlorhexidine-gluconate-based cavity disinfectant might serve as a suitable additional agent in inhibiting the residual bacteria in the dentine.     

Streptococcus mutans genotypes isolated from root and coronal caries

The aim of the present study was to evaluate the colonization profile and clonal distribution of Streptococcus mutans isolated from oral cavities that presented coronal and root caries lesions. The isolation and biochemical identification of mutans streptococci were carried out by using saliva samples, dental plaque, and tissue from the caries lesions. In order to confirm their molecular identity, S. mutans and Streptococcus sobrinus were submitted to the PCR method, using specific primers for portions of the glucosyltransferase genes (gtfB and gtfI, respectively). The AP-PCR method was used to detect the genetic polymorphism of S. mutans strains. Among the isolated and identified species, S. mutans showed a significantly greater frequency of isolation (59.2%) than the other mutans streptococci. Each of the subjects harbored two to ten genotypes of S. mutans, randomly distributed in different sites. S. mutans genotypes showed no evidence of variability in colonizing noncarious and carious surfaces within the same individual, nor evidence of etiologic differences between coronal and root caries. This study showed that no particular genotype of S. mutans is uniquely associated with the initiation and progression of caries, and that root and coronal caries can emerge in the presence of a broad spectrum of S. mutans clones.     

Mutans streptococci and lactobacilli in carious dentine before and after Atraumatic Restorative Treatment

OBJECTIVES: This study was performed to examine the cultivable flora in dentine after manual excavation of caries lesions using the Atraumatic Restorative Treatment (ART) technique and to examine for associations between the microbiological results and cavity size, dentine colour and consistency. METHODS: Dentine samples from 40 caries lesions were collected before and after treatment and cultured for total viable counts (TVC), mutans streptococci (MS) and lactobacilli. RESULTS: The bacterial bioburden between the two samples showed a significant reduction in the frequency and proportions of TVC and MS but not lactobacilli. CONCLUSION: Cavity preparation produced a clinical change in dentine colour and consistency from dark shades and soft dentine at enamel dentine junction to light shades and hard dentine at the cavity floor. The results show that cavity size, dentine colour and consistency are not absolute indicators of the microbiological bioburden in an ART cavity.     

Mutans streptococci, lactobacilli and caries prevalence in 11-and 12-year-old Icelandic children

Since the caries prevalence has not declined in Iceland as it has in other European countries, it was of interest to study the presence of cariogenic bacteria in Icelandic children. The prevalence of salivary mutans streptococci and lactobacilli was examined in 217 11- and 12-yr-old Icelandic children and was related to their caries prevalence. In 2% of the children mutans streptococci were not found whereas 31% and 35% of the 11-yr-olds and 12-yr-olds respectively carried more than 10(6) CFU per ml saliva. Lactobacilli were not detected in 8% of the children whereas 28% of the 11-yr-olds and 23% of the 12-yr-olds had more than 10(5) CFU per ml saliva. The mean caries prevalence (DFS), initial caries included, for 11-yr-olds was 21.6 and for 12-yr-olds 28.8. Both mutans streptococci and lactobacilli were significantly correlated to each other as well as to caries prevalence. An increased number of these microorganisms, especially the mutans streptococci, were associated with an increased DFS. Children with high salivary counts of mutans streptococci and lactobacilli showed four times higher DFS than children with low numbers. The magnitude of salivary cariogenic bacteria as well as caries prevalence was found to resemble the situation in Sweden 10 yr ago. S. mutans (serotype c/e/f) was carried by all mutans streptococci positive children. S. sobrinus (serotype d/g) was found in 60 children (30.2%). Children with both S. mutans and S. sobrinus had significantly higher salivary counts of mutans streptococci and lactobacilli as well as caries prevalence than children with only S. mutans.     

小沟聚合物探针实时检测变形链球菌和远缘链球菌

目的　寻求一种快速检测变形链球菌(S.mutans)和远缘链球菌(S.sobrinus)的方法,研究变形链球菌群在儿童猛性龋患者口中的分布。方法　设计并合成针对S.mutans和S.sobrinus葡糖基转移酶基因(gtf)的特异性引物和小沟聚合物探针(MGB探针),对9株变形链球菌群参考菌株直接提取DNA及扩增纯培养后提取DNA分别进行检测,比较二者检测结果的异同。采集92例猛性龋儿童菌斑样本,用MGB探针进行实时检测。结果　采用MGB 探针可以特异性地鉴别S.mutans和S.sobrinus,直接检测和扩增纯培养后的定性检测结果完全一致,前者荧光出现的时间略迟。92例猛性龋患儿中S.mutans检出率为96.7%,S.sobrinus检出率为32.6%,所有检出S.sobrinus的菌斑样本均可检出S.mutans。结论　采用特异性MGB探针方法可以对菌斑中S.mutans和S.sobrinus进行实时检测, 提高检测效率。     

Identification of mutans streptococci by restriction fragment length polymorphism analysis of polymerase chain reaction-amplified 16S ribosomal RNA genes

Mutans streptococci are frequently isolated from dental plaque and carious lesions. These bacteria have been identified by conventional methods such as biochemical and serologic tests followed by the isolation of colonies on the mitis-salivarius agar, which are sometimes inconsistent. Recently, species-specific polymerase chain reaction (PCR) has been reported to rapidly identify Streptococcus mutans and Streptococcus sobrinus. However, in the case of identification and classification into several species, e.g. within the group of mutans streptococci consisting of seven species, the identification using species-specific PCR seems somewhat inefficient because of need for the development and preparation of specific primers for each species. Therefore, in this study we developed a simple method using restriction fragment length polymorphism analysis of PCR-amplified 16S ribosomal RNA genes (16S rRNA genes PCR-RFLP) for the identification of seven different species included in the group of mutans streptococci. We amplified 16S rRNA gene sequences from genomic DNA samples by PCR using universal primers and digested the PCR products with the restriction endonucleases, HpaII and HaeIII. HpaII produced six RFLP patterns for eight reference strains, since the patterns for S. sobrinus, Streptococcus downei and Streptococcus ferus were similar. RFLP patterns produced with HaeIII could separate these three species. Furthermore, the RFLP patterns predicted from the 16S rRNA gene sequences in the GenBank database agreed with the actual RFLP patterns produced in the present study. The 16S rRNA sequence comparisons can be used to identify oral mutans streptococci; however, the identification by sequencing is sometimes difficult in large-scale studies and for small laboratories. Therefore, 16S rRNA genes PCR-RFLP, using HpaII and HaeIII, could be an alternative method for the identification of mutans streptococci, and may be applicable for large-scale studies on the cariogenicity of mutans streptococci.     

High prevalence of mutans streptococci in a population with extremely low prevalence of dental caries

The quantitative distribution of the mutans streptococci was investigated in populations with very low caries prevalence. Three hundred and seven school children from rural Sudan with an estimated age of 12 years were examined for dental status and salivary mutans streptococci. Forty-two isolates from the salivary cultures were determined to species level by studying the serogroup and biotype. Denial caries in permanent teeth was diagnosed in 12% of the children and the mean DMFT was 0.17 (range 0-6 DMFT). Caries was confined to fissures. Mutans streptococci were found in 96% of the children and high counts were obtained in 45%. Of 42 isolated strains, 37 belonged to Streptococcus mutans , 5 to Streptococcus sobrinus and none to Streptococcus rattus. The observations show that the mutans streptococci can be widespread in human populations with extremely low prevalence of dental caries and consequently that these bacteria may be readily propagated in human populations without association to a caries-promoting life style.     

Differentiation of mutans streptococci by intact cell matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

It is difficult to distinguish mutans streptococci on the species level, and even more so on the subspecies level. Intact cell matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) (ICM) was applied to reference strains of five of the species of the mutans group (Streptococcus criceti, Streptococcus downei, Streptococcus mutans, Streptococcus ratti, Streptococcus sobrinus), nonmutans streptococci (Streptococcus oralis, Streptococcus mitis, Streptococcus salivarius, and Streptococcus sanguinis), and 177 mutans streptococci isolated from saliva of 10 children. From the analysis of the reference strains, readily distinguishable ICM mass spectra were obtained for the different species. Based on multivariate statistical analysis, a correct and unambiguous assignment was made of the spectra of 159 isolated mutans streptococci to S. mutans and 16 isolates to S. sobrinus. Two isolates were sorted out and were identified by sequencing of their 16S rRNA genes as Streptococcus anginosus. In addition, ICM indicated a misclassification for some reference strains (AHT, V 100 and E 49) and re-classified AHT and E 49 as S. ratti and V 100 as S. sobrinus. This was confirmed by 16S rDNA sequencing. Based on a statistical similarity analysis of the spectra of reference strains and a quantitative assessment of the reproducibility of ICM, the isolates identified as either S. mutans or S. sobrinus were phenotyped on the subspecies level. In the population of the clinical isolates, 14 unambiguously different S. mutans and three different S. sobrinus phenotypes were detected. ICM proved to be a powerful tool for a differentiation of mutans streptococci down to the subspecies level.     

Immunoglobulin A reaction to oral streptococci in saliva of subjects with different combinations of caries and levels of mutans streptococci

The aim of this study, performed in Bangkok, was to study whether a particular salivary immunoglobulin A (IgA) antibody profile against mutans streptococci could be related to the absence or presence of caries. A group of 12-year-old individuals representing various combinations of mutans streptococci levels and caries experience was selected. Whole saliva stimulated by paraffin-chewing was collected, and the children were investigated for decayed, missing and filled surfaces (DMFS) and teeth (DMFT), following WHO criteria and methods, at baseline and after 2 years. The total amount of salivary IgA was determined by an immunobead enzyme-linked immunosorbent assay, and SDS-PAGE and Western blot analysis was performed using sonicated antigens of Streptococcus mutans and Streptococcus sobrinus strains and, as a control, a Streptococcus parasanguis strain. The results showed that Thai children with low caries prevalence had more distinct immunoblot bands to antigens from mutans streptococci than did the high-caries children. A similar picture was not seen for S. parasanguis. On the whole, the Thai children also showed fewer bands than usual Swedish saliva samples from comparable age groups. The complexity of the relationship between dental caries and IgA in saliva is highlighted.     

Automutanolysin disrupts clinical isolates of cariogenic streptococci in biofilms and planktonic cells

Introduction:  Dental caries remains one of the most common chronic infectious diseases throughout the world. The formation of dental plaque is one of the caries risk factors. As a consequence, the removal of plaque may reduce the incidence of caries development. We identified an autolysin produced by Streptococcus mutans named auto-mutanolysin (Aml). Aml selectively lyses S. mutans and Streptococcus sobrinus . The specificity towards these cariogenic bacteria suggests that Aml may be used to prevent dental caries. Here, with the aim towards therapeutic application, we investigated the lytic activity of Aml against clinical isolates of S. mutans and S. sobrinus using planktonic cells and biofilms.
Methods:  Planktonic cell suspensions and biofilms of clinically isolated streptococci were treated with Aml in the absence or the presence of Triton X-100. The lytic activity of Aml was monitored as the change in turbidity. The disruption of biofilms was evaluated by detecting the released DNA by polymerase chain reaction and observing the alteration of optical density of treated biofilms.
Results:  Triton X-100 enhances the lytic ability of Aml. Using planktonic cells, Aml had various lysis levels against clinical strains. Repeated Aml treatment showed disruption of the biofilm using the representative clinical strains.
Conclusion:  Our study demonstrates that Aml has an ability to lyse planktonic and biofilm cells of clinically isolated mutans streptococci in the presence of Triton X-100. These results suggest the possibility of using Aml as an alternative or additional approach for caries prevention.     

Effect of a bacteriocin-producing strain of Streptococcus sobrinus on infection and establishment of Streptococcus mutans on tooth surfaces in rats

The effect of bacteriocin produced by Streptococcus sobrinus MT6223 on infection and establishment of Streptococcus mutans MT6222 was studied in specific pathogen-free rats. These strains were isolated from a carious lesion of a single subject. S. mutans MT6222 was found to be susceptible to the growth inhibitory action of S. sobrinus MT6223. When simultaneously inoculated into the oral cavity of rats, even a small inoculum (10(5) CFU) of S. sobrinus MT6223 completely inhibited colonization of S. mutans MT6222 on the tooth surface. Also, S. sobrinus MT6223 eliminated S. mutans MT6222 when MT6223 (10(8) CFU) was inoculated 2 days after the inoculation of 10(8) CFU cells of MT6222. Similar results were obtained in dental plaque samples from the tooth surface and the fissures of the upper molars at the end of the experiment. However, when S. sobrinus MT6223 (10(8) CFU) was inoculated 2 weeks after the inoculation of S. mutans MT6222 (10(8) CFU), MT6223 coexisted with MT6222. However, the plaque samples showed that MT6223 inhibited the establishment of MT6222 on smooth surfaces, but not in fissures. In addition, MT6223 protected against subsequent infection with MT6222. However, a nonbacteriocinogenic mutant of S. sobrinus MT6223 did not inhibit the infection and establishment of S. mutans MT6222.     

猛性龋儿童变链菌分离株耐酸性实验研究

目的 :确定猛性龋儿童变链菌和远缘链球菌临床株的耐酸性。方法 :采用紫外分光光度计比较猛性龋、非猛性龋、无龋儿童变链菌 (各 6株 )和远缘链球菌 (猛性龋儿童 6株 ,非猛性龋和无龋儿童各 3株 )临床株在体外不同初始 pH条件下的生长情况。 结果 :初始 pH 4.5～ 5 .5条件下 ,各组变链菌生长抑制程度均明显大于远缘链球菌(P <0 .0 5 )。初始pH 4.5条件下 ,猛性龋儿童远缘链球菌分离株耐酸性明显强于非猛性龋和无龋儿童分离菌株(P <0 .0 5 )。结论 :远缘链球菌的耐酸性强于变链菌 ;猛性龋儿童远缘链球菌分离株耐酸性强     

猛性龋儿童变链菌分离株的初始粘附能力

目的 :探讨猛性龋儿童变链菌和远缘链球菌临床分离株的初始粘附能力。方法 :采用唾液包被羟磷灰石 (SHA)及同位素标记方法 ,检测猛性龋、非猛性龋、无龋儿童变链菌 (各 6株 )和远缘链球菌 (猛性龋儿童6株 ,非猛性龋和无龋儿童各 3株 )临床株对SHA的粘附情况。结果 :各组变链菌分离株之间及各组远缘链球菌分离株之间对SHA的粘附率无显著差异 ;在无蔗糖条件下 ,各组远缘链球菌分离株对SHA的粘附百分率均低于各组变链菌 ,差异有显著性 (P <0 .0 5 )。结论 :变链菌的初始粘附能力强于远缘链球菌 ;猛性龋儿童变链菌和远缘链球菌临床株对SHA的初始粘附能力与非猛性龋及无龋儿童无差别。     

Prevalence of mutans streptococci and lactobacilli in elderly Swedish individuals

The number of Streptococcus mutans, Streptococcus sobrinus, and lactobacilli in stimulated whole saliva was examined in 149 elderly persons. More than 10(6) CFU/ml of mutans streptococci were found in 57% of the subjects and 85% had more than 10(5) lactobacilli per ml of saliva. There was no difference in the prevalence of mutans streptococci between edentulous and dentate subjects whereas significantly more lactobacilli were detected in denture wearers. S. mutans was most frequent and was found in 72% of the subjects. S. sobrinus, which was carried by 44% of the persons, was found proportionally in more subjects with dentures than in dentate individuals. In most persons with S. sobrinus, S. mutans was also present but in 8% of the subjects only S. sobrinus was detected. The subjects with both S. mutans and S. sobrinus had higher salivary levels of mutans streptococci than individuals with only S. mutans or S. sobrinus.     

Dental caries in congenitally athymic rats

The importance of the immune response in dental infection was evaluated in heterozygous (rnu/+) normal and homozygous (rnu/rnu) congenitally athymic "nude" Rowett rats. Animals of both types were infected, or immunized and infected, with mutans streptococci (Streptococcus sobrinus strain 6715). The mean numbers of S. sobrinus cells recovered from the nude rats were higher than those from comparable (immune/nonimmune) normal rats in 10 of 12 possible comparisons. Also, S. sobrinus constituted a greater percentage of the total streptococci in the nude rats compared with normal animals (6 of 6 possible comparisons). Antibody to S. sobrinus whole cells or to S. sobrinus glucosyltransferase from nude rats in serum or in saliva was significantly lower (or absent) than that of comparable normal rats. This was seen after infection, but was most pronounced after immunization (and infection). Dental caries was also significantly elevated in the congenitally athymic animals. Immunologic deficiency of congenitally athymic rats can lead to a greater infection level with mutans streptococci and increased dental caries.     

Site-specific oral colonization of mutans streptococci detected by arbitrarily primed PCR fingerprinting

The clonal diversity and colonization pattern of mutans streptococci within an individual were studied by using a total of 50 tooth site samples obtained from seven 3- to 7-year-old children. Five children contributed saliva samples. From the 7 subjects, 90, 74, 81, 53, 83, 103, and 114 isolates (a total of 598) representing Streptococcus mutans and Streptococcus sobrinus were included for arbitrarily primed polymerase chain reaction (AP-PCR) fingerprinting. The children harbored one to four different AP-PCR types of mutans streptococci. In 3 of the 7 children, the distribution of AP-PCR types of S. mutans differed depending on tooth site (chi-square test for heterogeneity, p<0.001), suggesting that mutans streptococcal clones may selectively colonize specific hard-tissue sites.     

Clinical and microbiological performance of resin-modified glass-ionomer liners after incomplete dentine caries removal

The aims of this study were to evaluate clinically and microbiologically the effects of two resin-modified glass-ionomer cements (RMGICs) used as liners after incomplete dentine caries removal and to identify Streptococcus mutans and Streptococcus sobrinus strains isolated from dentine samples, before and after indirect pulp treatment. Twenty-seven primary molars with deep carious lesions, but without signs and symptoms of irreversible pulpitis, were submitted to indirect pulp treatment. Treatment consisted of incomplete excavation of the carious dentine, application of one of the RMGICs (Vitrebond or Fuji Lining LC) or calcium hydroxide cement (Dycal), and sealing for 3 months. Clinical evaluation (consistency, color, and wetness of dentine) and carious dentine collects were performed before temporary sealing and after the experimental period. Microbiological samples were cultivated in specific media for subsequent counting of mutans streptococci (MS) and lactobacilli (LB). MS colonies were selected for identification of S. mutans and S. sobrinus by polymerase chain reaction. After 3 months, the remaining dentine was hard and dry, and there was a significant decrease in the number of MS and LB, in all groups, although complete elimination was not achieved in 33% and 26% of the teeth for MS and LB, respectively. From 243 MS colonies selected, 216 (88.9%) were identified as S. mutans and only 2 (0.8%) as S. sobrinus. The use of resin-modified glass-ionomer liners after incomplete caries removal, as well as a calcium hydroxide cement, promoted significant reduction of the viable residual cariogenic bacteria in addition to favorable clinical changes in the remaining carious dentine.