全托儿童口腔变形链球菌水平传播的初步研究

目的　通过对幼儿园全托儿童之间口腔变形链球菌水平传播的研究,为儿童龋病预防提供新的思路。方 法　选择24名3~4岁全托儿童为研究对象,其中无龋组和有龋组各12名。采用无菌牙签采集牙面菌斑,厌氧培 养48 h,根据变形链球菌的典型形态挑取单个菌落进行次代纯培养,经形态学和微量生化鉴定后将分离的菌株行 AP-PCR扩增,对来自不同个体基因型相似的菌株再进行DNA指纹分析。结果　24名受检儿童中,变形链球菌检出 率为66·7%,无龋组和有龋组检出率分别为58·3%和75·0%。经AP-PCR扩增,24名儿童口腔中检测出29个不同 基因型的变形链球菌,其中携带2个以上基因型儿童占45·8%。经DNA指纹分析,有2种基因型在11名全托儿童 口腔中有重复检出。结论　变形链球菌在全托儿童口腔中可能存在水平传播。     

变形链球菌在日托儿童口腔中水平传播的初探

目的 通过对日托儿童口腔中变形链球菌（S.mutans）水平传播的研究，为儿童龋的早期预防提供新思路。方法 选择32名3～4岁日托儿童，根据是否患龋分为有龋组和无龋组。用无菌牙签采集牙面菌斑，MSB琼脂培养，微量生化反应鉴定分离S.mutans菌落，纯培养后提取基因组DNA，用AP-PCR检测其基因多态性，对来自不同个体的具有相似AP-PCR扩增图谱的菌株再进行DNA指纹分析。结果 32名受检儿童中，变形链球菌的检出率为78.1%；有龋组和无龋组的变形链球菌检出率分别为100%和69.6%。在检出变形链球菌的25名儿童中，携带有2种及以上基因型的儿童占88%；有龋组为88.9%，无龋组为87.5%，OR值为1.143，二者之间的差异无统计学意义。DNA指纹分析表明，有2种基因型变形链球菌分别在2名儿童口腔中重复检出。结论 变形链球菌在日托儿童中可能存在水平传播，但其基因型检出数目与龋的发生关系不明显。     

儿童口腔中变形链球菌传播方式的研究

目的通过对婴儿院儿童及其母亲之间、全托儿童之间口腔中变形链球菌（简称变链菌）传播方式的研究,为儿童龋病预防提供新的思路.方法选取四川省实验婴儿院3～4岁的20名非全托儿童及其母亲和24名全托儿童作为研究对象,无菌牙签收集其菌斑样本,MSB培养基中培养48 h,每种表型菌落挑取一个代表株在TPY平板上次代纯培养,经形态学和微量生化鉴定后对分离的变链菌株行AP-PCR扩增,对不同个体出现相似基因型的变链菌株进行分析.结果44名儿童中,65.9%的儿童口腔内检出变链菌,20对母子中有10对均检出变链菌,44名儿童及20名母亲口腔中共分离出98株变链菌;均有变链菌检出的10对母子口腔中分辨出32个不同的基因型,其中7对母子有相似基因型出现;24名全托儿童口腔中共分辨出29个不同的基因型,有2种基因型在13名全托儿童口腔中有重复检出.结论变链菌在婴儿院儿童中存在水平传播及垂直传播两种传播方式.     

Horizontal transmission of Streptococcus mutans in schoolchildren

Objetive: The aim of this study was to analyze possible horizontal transmission patterns of S. mutans among 6-7-yr-old schoolchildren from the same class, identifying genotypes and their diversity and relationship with caries disease status. Study Design: Caries indexes and saliva mutans streptococci and lactobacilli counts were recorded in 42 schoolchildren. Mutans streptococci colonies were identified by means of biochemical tests and all S. mutans strains were genotyped by arbitrarily primed polymerase chain reaction. A child was considered free of S. mutans when it could not be isolated in 3 samples at 1-week intervals. Results: S. mutans was isolated in 30 schoolchildren: 20 having one genotype and 10 two genotypes. Higher mutans streptococci and caries index values were found in those with two genotypes. Five genotypes were isolated in more than 1 schoolchild and one of these was isolated in 3 schoolchildren. Our results suggest that horizontal transmission may take place. Conclusion: Schoolchildren aged 6-7 yrs may be the source of mutual transmission of S. mutans. Key words:Streptococcus mutans, Horizontal transmission, AP-PCR, genotyping     

Transmission of Streptococcus mutans in a group of Turkish families

BACKGROUND/AIMS: To investigate the transmission of Streptococcus mutans in a group of Turkish families using AP-polymerase chain reaction (PCR) detection. METHODS: Eight mothers who had high S. mutans levels in unstimulated saliva and 8 children aged between 2 and 3 years participated in the study. Plaque samples from each child were collected with the tips of sterile toothpicks for S. mutans counts. Although not part of the original study design, S. mutans samples were also obtained from the unstimulated saliva of the three fathers who shared the same households. Three typical isolates of S. mutans were isolated from TYCSB agar of each subject and identified by sugar fermentation tests. S. mutans ATCC 10449 was used as the reference strain. AP-PCR was conducted with OPA-05 primer. RESULTS: All of the mothers and fathers shared the similar genotypes within their children. The fathers also harbored similar genotypes to their spouses. CONCLUSION: The mothers or the fathers could be the source for the transmission of S. mutans to their children.     

AP-PCR基因指纹用于变形链球菌传播株与非传播株的筛选

目的　应用AP-PCR基因指纹筛选变形链球菌(mutans streptococci,MS)的传播株(transmitted strains)与非传播株(nontransmitted strains),探讨影响MS传播的因素。方法　选取20对口腔中已定居有MS的儿童(3~4岁) 与他们的母亲,取牙面菌斑样本涂于轻唾-杆菌肽培养基。每人随机挑取45株分离株,提取染色体DNA,AP-PCR 基因指纹检测。结果　①从200个分离株中共分辨出45个不同的基因型,其中10位(50%)母亲和15位(75%)儿童分别带有1种类型,另5位(25%)儿童带2种类型,另10位(50%)母亲带有2种或2种以上类型(2位母亲带有5 种型),表明人群口腔中定居的MS存在基因多态性;②比较母亲与其子女MS基因型的相似性发现,20对母子中 16对(80%)有相似基因型出现,提示MS在此人群中的高传播现象;③对有传播现象的16位母亲的S.mutans进行传播株与非传播株的筛选发现,10(50%)位母亲口腔中传播株与非传播株共存,表明并非所有基因型的S.mutans 都能传播。结论　①AP-PCR基因指纹能清晰地分辨出S.mutans的传播株和非传播株;②在S.mutans的母婴传播过程中某一型菌株的优先传播是普遍存在的,进一步探讨造成这种现象的原因是很有必要的。     

Genotypic profiles by AP-PCR of streptococcus mutans in caries-active and caries-free preschoolers

Streptococcus mutans, an acidogenic and aciduric microorganism that colonizes the oral cavity is recognized as the main causal agent of dental caries. Epidemiological studies have shown a strong correlation between the number of S. mutans in the oral cavity and prevalence and incidence of caries. At present, different genotypic and phenotypic methods are known to determine the profiles of settling and epidemiological distribution of S. mutans. The aim of this study was to investigate the profiles of S. mutans isolated from children with and without dental caries by using the AP-PCR (arbitrarily primed polymerase chain reaction) and api-Zym methods. In the AP-PCR method, random DNA segments of the target bacterium are amplified with single primers of arbitrary sequence. The api-Zym system (bioMirieux, Marcy-létoile, France) is a phenotypic micro-method that allows simultaneous detection of 19 enzymatic activities from bacterial inoculum. A transversal observational study was conducted, which finally included 1203- to 5-year-old children (75 with and 45 without dental caries), who attended a preschool institution in Bogota (Colombia). S. mutans was isolated from 15 of the 45 children without dental caries (33.3%) and from 31 of the 75 children with caries (41.33%). In the 46 children, 69 S. mutans isolates were identified: 24 isolates in the 15 children without dental caries and 45 isolates in 31 children with dental caries. With api-Zym system, 36 different phenotypes were detected: 22 in the caries group and 15 in the caries-free group. The phenotype XX was present in both groups. With the AP-PCR method, 27 different fingerprinting profiles were identified: 22 for the caries group and 9 of the healthy group; the two groups of patients shared four of these genomic profiles. In conclusion, the information shows a great diversity in S. mutans genotypes and phenotypes in the population studied.     

母子口腔中变形链球菌基因型的对照研究

目的：分析高龋、无龋儿童及其母亲口腔中变形链球菌（S．mutans）菌株的基因型，探讨S．mutans基因型与致龋活性的关系。方法：试剂盒法提取细菌染色体DNA，对20名儿童（高龋组10名，无龋组10名）及其母亲的共800株S．mutans临床分离株进行AP—PCR基因型分析。结果：高龋儿童携带的S．mutans基因型数目多于无龋儿童（P〈0．05），高龋儿童的母亲携带的S．mutans基因型数目显著多于无龋儿童的母亲（P〈0．01），高龋儿童母亲的DMFT值显著高于无龋儿童母亲（P〈0．01），高龋组和无龋组的共有基因型数目无统计学差异（P〉0．05）。结论：高龋个体比无龋个体携带更多的S．mutans基因型，个体携带的S．mutnm基因型数目与其致龋活性有关。     

随意引物PCR法鉴别口腔变形链球菌的初步研究

目的 :探讨口腔变形链球菌族细菌中各个种和血清型的关系。方法 :采用随意引物PCR法对变形链球菌 (血清型c ,d ,f)及与其同源性相近的血链球菌标准菌株进行PCR扩增 ,建立口腔变形链球菌基因图谱。结果 :变形链球菌与血链球菌基因图谱差异较大 ;变形链球菌各个种基因图谱有差异 ;属于同一个种的变形链球菌不同血清型的菌株基因图谱虽相似但有差异。结论 :运用随意引物PCR法可鉴别不同种或同一种中血清型不同的变形链球菌。提示 :随意引物PCR法是一种鉴别口腔变形链球菌比较简便、准确的方法。     

Genotyping of Streptococcus mutans by using arbitrarily primed polymerase chain reaction in children with Down Syndrome

OBJECTIVE: The aim of this study was to compare the caries prevalence between Down Syndrome (DS) and non-DS children and to investigate the difference between the genotypes of Streptococcus mutans (S. mutans) colonized in both DS and non-DS groups. DESIGN: Sixty children with DS and 64 non-DS children aged between 7 and 12 years old were included to this study. All erupted teeth were evaluated according to the criteria recommended by the World Health Organization. Unstimulated saliva samples were carried out from the children and cultivated on S. mutans selective Tryptone-yeast-cystine (TYC) agar with 0.2 U/ml bacitracin and 15% sucrose. Molecular typing of S. mutans strains was performed by using arbitrarily primed polymerase chain reaction (AP-PCR) with OPA-05 primer. All data were analysed by using SPSS (SPSS Inc., Chicago, IL, USA) 11.0 software program for windows. RESULTS: The caries index scores were found significantly lower in DS individuals than the non-DS group (p < 0.05). The salivary S. mutans levels between DS and non-DS groups did not show significant difference (p > 0.05). The difference between dental caries and salivary S. mutans levels also was not statistically significant (p > 0.05). According to the results of the AP-PCR typing, all profiles of S. mutans which colonized in DS group were different from the control group. The relationship between these different profiles and dental caries prevalence was statistically significant (p < 0.05). CONCLUSION: The profiles of S. mutans colonized in DS group might be a reason of low caries prevalence.     

Mutacin production in Streptococcus mutans genotypes isolated from caries-affected and caries-free individuals

Relationships between genetic diversity and mutacin production in Streptococcus mutans were evaluated in 319 clinical isolates from eight caries-affected and eight caries-free individuals. The isolates were submitted to mutacin typing and AP-PCR (arbitrarily primed polymerase chain reaction) assay. The mutacin production was detected for 12 Streptococcus sp. indicator strains. Results showed significant variations in the mutacin production profiles and the inhibitory spectra of both groups. A possible association was seen between mutacin activity and the distinct patterns of Streptococcus sp. colonization in the two groups. Genotyping by AP-PCR using the primers OPA-02 and OPA-13 revealed 101 distinct genotypes against 48 phenotypes identified by mutacin typing. No correlation was observed between the inhibitory spectra of mutacin and genotypic similarities based on AP-PCR analyses. According to our results, strains of the same S. mutans genotype showed different mutacin profiles, suggesting a high degree of interstrain diversity. In conclusion, mutacin production seems to be of clinical importance in the colonization of S. mutans and is highly diversified in the S. mutans species.     

Genotypes of Streptococcus mutans in saliva versus dental plaque

OBJECTIVE: The aim of this study was to investigate whether genotypes of Streptococcus mutans strain can be detected as effectively in saliva samples as in plaque samples from buccal surfaces and occlusal surface fissures of permanent first molars. DESIGN: The study included 20 school children aged 6-7 years who were positive for mutans streptococci. Samples of stimulated saliva and of dental plaque on buccal surfaces and occlusal surface fissures of permanent first molars were collected. Samples were cultivated in MSB agar. Up to nine isolates compatible with mutans streptococci were obtained and identified by means of biochemical tests. All isolates identified as S. mutans were genotyped by arbitrarily primed polymerase chain reaction. RESULTS: A total of 28 genotypes of S. mutans were isolated: 23 in saliva samples, 23 in buccal surface plaque samples, and 16 in plaque samples from occlusal surface fissures. CONCLUSIONS: Although, saliva sampling did not reveal all genotypes isolated, it was equally as effective as plaque sampling from the buccal surfaces of permanent first molars, and more effective than plaque sampling from fissures on their occlusal surfaces.     

Genotypic and phenotypic analysis of Streptococcus mutans from different oral cavity sites of caries-free and caries-active children

INTRODUCTION: Streptococcus mutans exhibits extensive genotypic diversity, but the role of this variation is poorly understood. This study aimed to determine the number and distribution of genotypes of S. mutans isolated from caries-active and caries-free children and to evaluate some of their phenotypic traits. METHODS: Stimulated saliva, tongue surface and biofilms over sound and carious teeth surfaces were sampled from 10 caries-free and 11 caries-active children aged 5-8 years. A total of 339 isolates of S. mutans were genotyped by arbitrarily primed polymerase chain reaction using OPA2 primer. One isolate from each genotype was tested for its acid susceptibility and its ability to form a biofilm. RESULTS: Fifty-one distinct genotypes were determined, one to three genotypes in each oral sample. A single genotype was detected in seven children, whereas the remaining 14 children exhibited two to seven genotypes. There were no significant differences in the number of genotypes detected in caries-free and caries-active children. No correlation was observed between the number of genotypes and the mutans streptococci salivary levels. Five of the six high biofilm-forming genotypes were obtained from caries-active children, although the differences in biofilm formation between isolates from caries-free and caries-active children were not statistically significant. Genotypes with low susceptibility to acid challenge were statistically more frequent among isolates from caries-active children than among those from caries-free children. CONCLUSION: The present data suggested that there were differences in the distribution of genotypes of S. mutans according to the oral site and that S. mutans populations differ in their acid susceptibility and ability to form biofilms, factors allowing their colonization of sucrose-rich environments.     

新疆维吾尔族儿童乳牙龋与变形链球菌基因型相关性的初步研究

目的 探讨新疆维吾尔族不同龋敏感儿童口腔中变形链球菌幕冈多态性与乳才龋发生的关系,以期为新疆维吾尔族儿童乳牙龋危险性预测和防治提供依据.方法 选取新疆乌鲁小齐市3～5岁维吾尔族高龋(龋失补牙数≥5)和无龋(龋失补牙数=0)儿童各17名为研究对象,收集唾液样本,轻唾一杆菌肽琼脂培养基分离培养变形链球菌.典型菌落行革兰染色...     

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.     

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.     

重度龋儿童远缘链球菌基因多态性研究

目的 采用随机引物聚合酶链反应(arbitrarily primed-polymerase chain reaction,AP-PCR)法初步探讨远缘链球菌在重度低龄儿童龋(severe early childhood caries,S-ECC)儿童与无龋儿童口腔中基因型分布的情况,分析其与婴幼儿龋发生之间的关系.方法 选取北京市海淀区和西城区14所幼儿园178名42～54个月龄儿童,S-ECC(患龋牙数≥5)组87例,无龋组91人.嚼蜡法采集刺激性唾液进行分离培养,典型菌落行革兰染色、生化鉴定并保种,提取基因组DNA,PCR鉴定变形链球菌和远缘链球菌,AP-PCR法对远缘链球菌临床分离株行基因型分析.结果 S-ECC组远缘链球菌检出率18%(16/87),显著高于无龋组的3%(3/91),差异有统计学意义(P＜0.01).53株远缘链球菌临床分离株共检出22种基因型,S-ECC组个体基因型为1～3种,无龋组均为1种;此外,S-ECC组3名个体间存在相同基因型的菌株.S-ECC组基因型数与龋失补牙数间存在相关性(r=0.50,P＜0.05).结论 S-ECC儿童远缘链球菌检出率明显高于无龋儿童,且菌株间存在基因多态性,个体携带基因型的种类数与其致龋性间存在相关性;远缘链球菌无关个体间存在相同基因型的菌株.     

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.     

儿童猛性龋病原菌母子传播研究

目的 :探讨幼儿猛性龋病原菌母子传播途径。方法 :随机选择可获得变形链球菌群 (MS)纯培养的 2～ 5岁猛性龋、非猛性龋及无龋儿童母子各 10对 ,进行DNA指纹实验 ,检测儿童牙菌斑和母亲唾液中MS菌株的遗传相似性 ,并检查母亲唾液MS水平。结果 :猛性龋儿童MS菌株基因型与母亲的一致性为 70 % ,与非猛性龋和无龋儿童(均为 6 0 % )无显著差异 ;但猛性龋儿童MS基因型数目显著多于非猛性龋和无龋儿童 (P <0 0 5 )。猛性龋儿童母亲唾液变链菌水平与非猛性龋和无龋儿童母亲相似 ,但唾液远缘链球菌水平及DMFT均数显著高于无龋儿童母亲。结论 :母亲是儿童MS的主要来源 ;幼儿猛性龋与母亲的DMFT计数及唾液远缘链球菌水平成正相关 ,母子传播对幼儿猛性龋的发生具有一定作用。     

Mutans streptococci strains prevalence before and after cavity preparation during Atraumatic Restorative Treatment

Critics argue that all carious dentine is not removed from the hand-prepared cavity during the Atraumatic Restorative Treatment (ART) procedure, and that the caries process is soon resumed. The aim of this study was to determine the effectiveness of ART in removing carious tissue, by investigating the numbers of mutans streptococci and lactobacilli, with emphasis on the prevalence of Streptococcus mutans and Streptococcus sobrinus strains before, and after ART treatment of dental caries. Two microbiology samples were collected. The first sample was removed from the centre of the carious lesion at the enamel-dentine junction, and the second was collected from the centre of the hard cavity wall above the pulp, after the soft infected dentine had been manually removed. A total of 71 mutans streptococci isolates from 31 children and 40 carious teeth were subcultured, biochemically characterised and genotyped by the arbitrarily primed polymerase chain reaction (AP-PCR). Results showed a significant decrease in TVC (P<0.0001), mutans streptococci (P < 0.0001) and lactobacilli (P = 0.0002) after cavity preparation. AP-PCR identified S. mutans strains that were undetectable during biotyping, and divided clinical isolates into two main clusters. In all, 63% (45/71) of isolates from the carious lesions comprised S. mutans strains. After cavity preparation, this was reduced to 35% (25/71), of which 30% (21/71) were S. mutans and the remaining 6% (4/71) S. sobrinus strains. The number of mutans streptococci strains was below detectable levels in 19 of the prepared cavities. The significant decrease in bacteria after manual cavity preparation demonstrates the reliability of a standardized ART technique, yet the presence of S. mutans strains shows that the effectiveness of the ART procedure can vary during treatment and between dental practitioners.