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。     

高龋儿童口腔中变形链球菌基因型与传播来源的AP-PCR分析

龋病是儿童最常见的口腔疾病，S．mutans是主要致龋菌。了解高龋儿童口腔中的S．mutans基因型分布及其传播来源和方式对儿童龋病的预防具有积极的意义。目前的研究认为，儿童口腔中的S．mutans主要是通过母子垂直传播等途径获得，所获得的S．mutans存在明显的基因多态性，不同基因型的S．mutans菌株可能在致龋过程中发挥了不同的作用，但是，S．mutans的基因型与致龋性之间的关系尚存在争议。     

不同龋状况母子间口腔微生物群落结构分析

目的：应用PCR-DGGE（变性梯度凝胶电泳）技术,分析20对不同龋状况母子口腔唾液中微生物群落组成的多样性。方法：分别采集20名不同龋状况儿童及母亲的唾液样本,提取细菌总DNA,进行16SrDNA的PCR扩增及DGGE,克隆测序分析。结果：母子口腔唾液中分别检出42、48个菌种,其共同菌种为37个,归属于16个菌属,同时,检出新菌种Megasphaeraspp.,且在无龋儿童及高龋母亲唾液中分别检出Abiotrophiaspp.和Olsenellaspp.两个特异菌属。无龋组母子唾液中嗜血菌属的相对数量显著高于高龋组（P〈0.001）;无龋儿童携带的放线菌属相对数量也多于高龋组（P〈0.001）;而链球菌属,奈瑟菌属低于高龋组（P〈0.001）,而高龋组母亲口腔微生物中相对数量最多的为乳杆菌属,其次为放线菌属,奈瑟菌属,与无龋组比较差异显著（P〈0.001）。结论：母子口腔唾液菌群组成具有多样性,且随龋状况不同而有所差异。     

高龋者口腔中c血清型变形链球菌的基因组指纹分析

目的：确定高龋者口腔中c血清型变形链球菌的基因型分布。方法：从20例高龋者口腔中分离出c血清型变形链球菌，采用chelex法提取细菌染色体DNA，通过AP—PCR进行临床分离株的基因组指纹分析。结果：共分离获得了87株c血清型变形链球菌，不同个体所携带的变形链球菌临床分离株的基因型不同，同一个体可携带不同基因型的c血清型变形链球菌，26．7％的高龋者携带1种基因型，60．0％携带2种基因型，13．3％携带3种基因型。结论：大多数高龋者口腔中定植有2种或2种以上基因型c血清型变形链球菌。     

不同龋敏感儿童口腔变异链球菌不同基因型临床分离株产酸力的实验研究

目的比较不同龋敏感儿童口腔变异链球菌不同基因型临床分离株对蔗糖进行酵解产生有机酸的能力。方法从不同龋敏感的3~5岁学龄前儿童口腔变异链球菌中选取66株变异链球菌临床分离株,用气相色谱法测定高龋、中龋和无龋儿童口腔变异链球菌不同基因型临床分离株产生各有机酸的量。结果不同龋敏感儿童不同基因型变异链球菌临床分离株产生有机酸的量的差异有统计学意义（P<0.05）,同一龋敏感儿童变异链球菌携带不同基因型菌株产酸量的差异也有统计学意义（P<0.05）,携带基因型数目越多的菌株其产酸量越多。变异链球菌临床分离株产生乳酸、乙酸、甲酸的量的差异有统计学意义（P<0.05）,乳酸多于乙酸和甲酸。结论不同龋敏感儿童口腔变异链球菌不同基因型菌株产酸力不同,携带基因型数目越多的菌株其产酸力越强。     

维吾尔族高龋儿童口腔中白色念珠菌基因分型研究

目的：研究乌鲁木齐市维吾尔族儿童口腔白色念珠菌的分布及高龋儿童口腔白色念珠菌基因分型。方法：利用科玛嘉念珠菌显色培养基对144名维吾尔族3～5岁儿童进行口腔白色念珠菌分离培养及鉴定,进行 PCR25SrDNA 基因分型。结果：35个儿童（24．3％）的口腔中检出白色念球菌。无龋、有龋、高龋儿童口腔白色念珠菌检出率分别为3．8％、50．0％和35．7％（χ2＝22．246,P ＝0．000）;25例高龋儿童的 PCR25rDNA 分3型,以 A 型为主（72．0％）。结论：白色念珠菌可能是维吾尔族儿童龋病的危险因素,白色念珠菌 PCR25SrDNA 基因的 A 型者可能更易致龋。     

变形链球菌F-ATP酶亚基基因uncA遗传多态性的研究

目的研究变形链球菌临床分离株耐酸因子F—ATP酶亚基α的结构基因uncA的遗传多态性，并探讨基因多态性与细菌耐酸力及龋病发生的关系。方法分别从高龋、无龋个体中分离变形链球菌34和30株，其中包括18株高耐酸株、20株低耐酸株。从细菌组DNA扩增uncA，行限制性内切酶长度多态性分析（RFLP）及核酸测序比较。结果不同限制性内切酶RFLP产生不同的基因型，测序证实了导致多态出现的基因变异；内切酶Hph Ⅰ产生的A、B基因型在不同患龋个体分离菌株的分布不同（P〈0．05），A型uncA在高龋分离株的检出率高于无龋分离株；内切酶MboⅡ产生的C、D基因型在不同耐酸力菌株中的分布不同（P〈0．05），C型uncA在高耐酸力菌株的检出率高于低耐酸力菌株。结论变形链球菌F—ATP酶的α亚基基因uncA具有明显遗传多态性，酸性环境下生存力强的菌株可能出现基因的适应性变异，不同基因型uncA分布与菌株的耐酸力及致龋力相关。     

表兄链球菌的检测与儿童猛性龋的关系

目的：探讨表兄链球菌(Streptococcus sobrinus,S. sobrinus)与儿童猛性龋的关系。方法：根据前期郑州市区猛性龋调查结果,随机抽样选择3～5岁儿童66例,其中猛性龋、普通高龋及无龋组各22例。采用TYCSB培养基作变形链球菌(Streptococcus mutans, S. mutans )及S. sobrinus初步筛选,结合生理生化鉴定,并采用聚合酶链反应作最终鉴定。采用 SPSS10.0软件包对实验组与对照组S.mutans和S.sobrinus的检出率进行χ²检验,组间均数作t检验。结果： S. sobrinus在各组儿童牙菌斑中均不能脱离S. mutans而单独检出,猛性龋组S. mutans检出率高于高龋组,差异无显著性(P>0.05),而2组儿童S. sobrinus检出率的差异有显著性(P<0.05);猛性龋组与无龋组S. mutans检出率差异显著(P<0.05),S. sobrinus检出率差异显著(P< 0.01)。同时检出S. mutans和S. sobrinus的样本,其猛性龋的发生率及龋失补牙数、龋失补牙面数和平滑面龋均数与只能检出S. mutans及2种细菌均不能检出的样本的差异有显著性(P< 0.01)。结论：S. mutans与S. sobrinus是儿童猛性龋的主要致病菌,S. sobrinus与儿童猛性龋的发生有关。S. sobrinus对儿童猛性龋的发生、发展具有协同作用。     

有龋和无龋儿童四种唾液蛋白成分的比较

目的比较3～4岁有龋儿童和无龋儿童非刺激性和刺激性唾液中IgA、乳酸脱氢酶、溶菌酶和碱性磷酸酶含量的差异。方法分别采集98例有龋儿童和94名无龋儿童非刺激性和刺激性唾液，测定上述各成分含量。结果非刺激性和刺激性唾液中IgA、乳酸脱氢酶和溶菌酶含量有龋组均显著高于无龋组（P〈0．01），非刺激性唾液中碱性磷酸酶含量有龋组显著高于无龋组（P〈0．05）；但刺激性唾液中碱性磷酸酶含量两组间差异无统计学意义（P〉0．05）。结论非刺激性和刺激性唾液中IgA、乳酸脱氢酶和溶菌酶升高可能与婴幼儿龋的发生、发展有关。     

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。     

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...     

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 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.     

Colonization of Streptococcus mutans and Streptococcus sobrinus genotypes and caries development in children to mothers harboring both species

The major route of early acquisition of mutans streptococci in humans is a vertical transmission from mother to child. The purpose of this longitudinal study was to examine the acquisition, distribution and persistence of Streptococcus mutans and Streptococcus sobrinus in children whose mothers harbored both species and to study the caries incidence in relation to colonization of these bacteria. Fifteen mother-child pairs were followed during the child's first 7 years. Stimulated salivary samples were taken from the mothers and the children. Plaque samples were also collected from the teeth and the tongue of the children. The samples were analyzed by cultivating techniques together with genomic fingerprinting and hybridizing. The caries experience was evaluated on the sampling occasions and retrospectively using the records of caries registrations from the community clinics. During the 7-year period 10 of the 15 children acquired mutans streptococci. Only 4 of them were colonized by both S. mutans and S. sobrinus despite the fact that their mothers harbored both species. In 2 of the children S. sobrinus was found later than S. mutans. A total of 26 genotypes were found in the children and 9 of them were identical to their mothers. New genotypes and a gain-loss pattern were noted especially in the children but also in their mothers. The groups of teeth first positive for the two species were the deciduous molars. The caries experience was low during the study period with 8 children showing no caries.     

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.     

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     

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.     

变形链球菌葡糖基转移酶遗传多态性与龋病发生关系的研究

目的:探讨变形链球菌葡糖基转移酶遗传多态性与龋病发生的关系。方法:菌株分别选自本实验室前期工作中分离鉴定的合成水不溶性多糖能力较强和较弱的血清c型变形链球菌临床分离株，提取全菌DNA，经PCR扩增gtfBC(961 - 5574 by)后，分别采用限制性内切酶Hinf工、Mb。工和Taq工进行限制性片段长度多态性分析。结果: 不同龋敏感人群变形链球菌血清。型临床分离株经Hinf工酶切后限制性片段长度多态性分析酶谱出现了差异，有 1.9 kb长片段的菌株在高龋组中的比例高于无龋组(P < 0.05)。结论:萝基因型的不同是导致菌株合成水不溶性多糖能力差异的原因之一。     

奶瓶龋儿童口腔变链菌的研究

目的：研究儿童口腔内的变链菌基因型与奶瓶龋易感性的关系。方法：取20名奶瓶龋及20名无龋儿童牙菌斑及唾液接种于TYCSB培养基计数并筛选MS,采用AP－PCR法鉴定变链菌基因型。结果：奶瓶龋组儿童唾液及菌斑变链菌计数高于无龋组;并且有两种以上基因型变链菌的比例也高于对照组（14／20vs 5/20)。结论：患有奶瓶龋的儿童不仅变链菌计数高,而且多数被两个以上基因型的MS感染。