变形链球菌、远缘链球菌耐氟菌株与亲代菌株基因组REA的比较分析

目的：利用限制性核酸内切酶(REA)技术，对变形链球菌、远缘链球菌及耐氟菌株的基因组进行比较，判定变形链球菌、远缘链球菌耐氟突变后，遗传型是否发生改变。方法：采用REA技术，经多次实验自行确立REA反应体系，对变形链球菌、远缘链球菌及耐氟菌株的基因组进行比较。结果：变形链球菌、远缘链球菌耐氟菌株的基因组已发生改变。结论：变形链球菌、远缘链球菌耐氟突变后，已具有自身的遗传特性。     

变形链球菌耐氟菌株、远缘链球菌耐氟菌株与亲代菌株基因组AP-PCR的比较

目的：利用随机引物聚合酶链反应(AP-PCR)技术，对变形链球菌、远缘链球菌和耐氟菌株的基因组进行比较，判定变形链球菌耐氟突变、远缘链球菌耐氟突变后，遗传型是否发生改变方法：采用AP-PCR技术，经多次实验自行确立AP-PCR反应条件，对变形链球菌、远缘链球菌及耐氟闲株的基因组进行比较结果：变形链球菌耐氟菌株、远缘链球蒲耐氟菌株的基因组已发生改变。结论：变形链球菌耐氟突变、远缘链球菌耐氟突变后，已具有自身的遗传性。     

变形链球菌和远缘链球菌耐氟菌株粘附能力的体外研究

目的：探讨变形链球菌和远缘链球菌耐氟突变后,其粘附能力的改变。方法用同位素标记方法测定变形链球菌,远缘链球菌亲代及耐氟菌株粘附至唾液包被羟基磷灰石（ＳＨＡ）表面的粘附量及粘附百分率,并比较两者的粘附能力。结果变形链球菌、远缘链球菌亲人菌株与耐菌株间粘附百分率无明显差异（Ｐ〉０．０５）;远缘链球菌与变形链球菌相比,其粘附百分率偏低,差异有显著性（Ｐ〈０．０１）结论变形链球菌与远缘链球菌耐氟菌株粘附至     

变形链球菌和远缘链球菌耐氟菌株DNA G+C含量测定

目的　探讨变形链球菌和远缘链球菌耐氟菌株基因型是否发生改变。方法　采用高效液相色谱法测定变形链球菌和远缘链球菌及相应耐氟菌株DNA碱基含量,即DNAG＋C摩尔百分比(mol%)。结果　四株耐氟菌株DNAG＋Cmol%为54.45,44.86,54.41,55.39;相应亲代菌株G+Cmol%为35.8,37.4,43.35和46.62。两者明显不同,差值大于5％。结论　变形链球菌和远缘链球菌的耐氟菌株已发生基因型改变,且可能包括多个基因的突变。     

洗必泰对变形链球菌、远缘链球菌耐氟菌株与亲代菌株生长抑制的测定

目的：通过洗必泰对变形链球菌、远缘链球菌耐氟菌株与亲代菌株生长抑制的测定，探讨一种新的防龋途径。方法：将醋酸洗必泰液稀释成不同的浓度，分别加入变形链球菌、远缘链球菌及耐氟菌株菌悬液，测得最小抑菌浓度（MIC）、最小杀菌浓度（MBC）。结果：洗必泰对变形链球菌、远缘链球菌及耐氟菌株均有抑菌、杀菌作用，4种细菌最小抑菌浓度（MIC）均值为338μg/L，最小杀菌浓度（MBC）均值为375μg/L。结论：洗必泰可有效抑制变形链球菌、远缘链球菌及耐氟菌株生长。     

变形链球菌耐氟菌株脱矿能力的体外研究

目的：探讨变形链球菌耐氟突变后，其致釉质片脱矿能力的改变。方法：采用原子吸收分光光度计，测定不同氟浓度条件下变形链球菌耐氟菌株培养物上清液中钙离子浓度，并与亲代菌株进行比较。结果：耐氟菌株脱矿量在无氟化物存在时，小于亲代菌株；而当０．５ｍｍｏｌ／Ｌ氟离子存在情况下，其脱矿是大于亲代菌株。差异具显著性。结论：耐氟菌株的产生将增加变形链球菌的致龋力。     

远缘链球菌6715及其耐氟菌株适应性耐酸能力的比较研究

目的：探讨远缘链球菌6715（Streptococcus sobrinus 6715，以下简称S．6715）及其耐氟菌株的适应性耐酸能力。方法：在含有不同氟浓度的TSA上逐步诱导S．6715产生耐氟突变株（以下简称S．6715-FR）。通过测定在各种酸性pH值培养基中预酸化2h后，能否使S．6715及其耐氟突变株抵抗致死性pH杀伤而存活及存活率，来测定其适应性耐酸能力的有无及大小。结果：S．6715属强适应性耐酸能力类菌株，最大生存率9．98％出现在pH5．5预酸化组，其耐氟突变株S．6715-FR亦具有强适应性耐酸能力，最大生存率10．55％出现在pn值5．0预酸化组。结论：在亚致死性pH值中生长能引发远缘链球菌6715及其耐氟菌株的某些生理变化，产生适应性耐酸能力，抵抗酸性环境的杀伤。     

变形链球菌耐氟菌株中耐酸相关基因ffh突变的检测

目的检测变形链球菌耐氟菌株中耐酸相关基因ffh是否存在突变。方法体外诱导变形链球菌耐氟菌株,碱裂解法提取细菌基因组,根据GenBank上公布的变形链球菌耐酸相关基因ffh的序列,设计一对引物进行PCR,利用PGEM-TEasy载体进行T-A克隆,构建重组质粒,酶切电泳,并测序鉴定。结果变形链球菌耐氟菌株中耐酸相关基因ffh发生了突变。结论变形链球菌耐氟菌株中耐酸相关基因ffh发生了突变,并可能与其耐酸性增强有关。     

套式PCR检测唾液中变形链球菌及远缘链球菌

目的 探索一种快速、简便地从人类唾液中同时检测变形链球菌和远缘链球菌的方法。方法 分别以变形链球菌gtfI和远缘链球菌gtfB基因设计两组成套引物，首先用套式PCR（二次PCR）检测变形链球菌和远缘链球菌标准株和临床株，然后用套式PCR直接从唾液中检测这两种细菌。结果 变链菌（血清型c,e,f)的标准株及临床株第1次PCR扩增产物为517bp，第2次扩增产物为468bp；远缘链球菌（血清型d,g)及道勒链球菌（血清型h)的标准株及临床株第1次PCR扩增产物为712bp，第2次扩增产物为663bp;其他异种菌均不能扩增出产物，因此该PCR检测具有高度的特异性。细菌纯培养物及唾液PCR检测的敏感性分别是：第1次PCR为10^5CFU，第2次PCR为10^3CFU。结论 套式PCR能快速在人类唾液中同时检测变形链球菌和远缘链球菌。该检测方法有望运用于临床检测，对揭示两种细菌与龋病发生关系的研究具有一定价值。     

变形链球菌耐氟菌株对葡萄糖摄取的体外研究

目的　探讨变形链球菌发生耐氟突变后摄取葡萄糖能力的变化。方法　采用逐步法诱导变形链球菌产生耐氟菌株 ,用葡萄糖氧化酶法测定不同PH值 ,有氟和无氟条件下变形链球菌耐氟菌株培养物上清液中葡萄糖减少量 ,并与亲代菌株进行比较。结果　在氟化物存在时 ,耐氟菌株糖摄取量大于亲代菌株 ;耐氟菌株和亲代株在有无氟化物条件下糖摄取量不同 ,差异有显著性 (P <0 .0 0 1)。结论　变形链球菌耐氟菌株具有致龋性 ;其致龋力大于亲代菌株。     

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。     

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。     

Susceptibility of Streptococcus mutans and Streptococcus sobrinus to cell wall inhibitors and development of a novel selective medium for S. sobrinus

Representative strains of Streptococcus mutans and Streptococcus sobrinus showed differences in susceptibility to members of the monobactam group of beta-lactam antibiotics: S. sobrinus was less sensitive than S. mutans. The minimum inhibitory concentrations of aztreonam (AZT) and carumonam, both of which belong to this group, were 2,000 microg/ml for S. sobrinus and 125 microg/ml for S. mutans. Further addition of fosfomycin, bacitracin and sodium chloride to Mitis Salivarius agar (MS) supplemented with AZT resulted in growth inhibition of S. mutans and oral streptococci other than S. sobrinus, and was therefore used as a selective medium for S. sobrinus (MS-SOB medium). The average growth recovery of laboratory and clinically isolated strains of S. sobrinus on MS-SOB medium was 74.1% compared to that on MS medium. Seventy-eight percent of clinical samples in which S. sobrinus was detected yielded pure growth of S. sobrinus on MS-SOB medium.     

变异链球菌耐氟菌株的研究进展

氟化物作为防龋制剂,其广泛应用可能导致变异链球菌耐氟菌株的产生。为了解变异链球菌耐氟菌株致龋毒力的变化,学者们对其超微结构、黏附力、耐酸性、产酸性及脱矿能力等致龋相关特性进行了研究,并对耐氟菌株的致龋相关基因和其他分子生物学改变予以深入的研究和探讨。本文就此作一综述。     

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。     

PCR同时检测变形链球菌和远缘链球菌

目的:建立一种快速、简便地同时检测变形链球菌和远缘链球菌的方法。方法:以变形链球菌Dextranase基因设计引物,用PCR检测变形链球菌群细菌。结果:变形链球菌(血清型c、e、f)的PCR扩增产物为720 bp;远缘链球菌(血清型d、g)的PCR扩增产物为460 bp;道勒链球菌(血清型h)910 bp;仓鼠链球菌(血清型a),鼠链球菌(血清型b)均为1 270 bp。其它异种菌均不能扩增出产物,因此该PCR检测具有高度的特异性。从细菌纯培养物中PCR检测的敏感性为105菌落形成单位(colony-form ing un its,CFU)。结论:PCR能同时检测变形链球菌和远缘链球菌。该检测方法具有较高的敏感性和特异性,有望运用于临床检测。     

Streptococcus mutans and Streptococcus sobrinus detection by Polymerase Chain Reaction and their relation to dental caries in 12 and 15 year-old schoolchildren in Valencia (Spain)

A cross-sectional study was carried out to determine the prevalence of Streptococcus mutans and Streptococcus sobrinus and the association of the two in a random sample (n=614) of the child population of the region of Valencia (Spain). Saliva samples were analyzed by the quantitative polymerase chain reaction (PCR) method to study the relation of these bacteria to caries prevalence and the DMFT index. The prevalence of S. mutans was 35.4% at age 12 and 22.9% at age 15, that of S. sobrinus 18.9% and 8.4% and that of the S. mutans-S. sobrinus association 18.2% and 6.8% respectively. At both 12 and 15 years of age, the caries prevalence rates were lower in the Streptococcus-free group of children (37.6% and 48.5% respectively) and higher in the S.mutans-only group (67.3% and 74.0%). At the age of 12, the DMFT index was significantly higher in the mutans-only carriers (2.1) than in the Streptococcus-free and S. mutans-S. sobrinus association groups (both 0.9). At the age of 15, the DMFT index was significantly higher in the S. mutans-S. sobrinus association (3.71) and mutans-only (3.1) carrier groups than in the Streptococcus-free group (1.4). Determination of S. mutans and S. sobrinus by real-time quantitative PCR can provide valuable information for caries risk assessment in epidemiological studies. Key words:Streptococcus mutans, Streptococcus sobrinus, polymerase chain reaction, dental caries, cross-sectional studies.     

Interactions between and within Streptococcus mutans and Streptococcus sobrinus isolated from humans harboring both species

Abstract— The prevalence of Streptococcus mutans and Streptococcus sobrinus was examined in plaque samples from small discrete areas of the buccal tooth surfaces of seven subjects. Strains of S. mutans and S. sobrinus were isolated and tested for bacteriocin-mediated interactions between and within the two species, using the stab inoculation technique. S. mutans and S. sobrinus did not colonize each tooth surface uniformly and, in plaque from small discrete sites, S. mutans and S. sobrinus were either undetected or present in different interspecies proportions. Within the same subject, there were no bacteriocin-mediated interactions between strains of the same mutans species and no difference in bacteriocin activity was found between the strains of S. mutans and S. sobrinus from different sites. When bacteriocin interactions were tested between isolated strains from all seven subjects a somewhat higher inhibition ability was found for producer strains isolated from plaque compared with those isolated from saliva. S. mutans appeared to be more bacteriocinogenic than S. sobrinus . Replacing the glucose in the medium with sucrose enhanced the bacteriocin activity of S. mutans towards other S. mutans strains but reduced the inhibitory interaction towards strains of S. sobrinus .     

儿童猛性龋变链菌分离株的产酸性

目的：确定儿童猛性龋变链菌和远缘链球菌临床分离株的产酸性。方法：采用酸度计和自动气相色谱仪比较儿童猛性龋、非猛性龋、无龋变链菌（各6株）和远缘链球菌（儿童猛性龋6株,非猛性龋和无龋各3株）的临床株降低环境pH值的能力（ΔpH）和乳酸产量,并以此推测其致龋力。结果：远缘链球菌ΔpH及乳酸产量均高于变链菌,特别是在低pH水平下,二者差异更为显著（P＜0．05）。儿童猛性龋远缘链球菌分离株ΔpH和乳酸产量显著大于非猛性龋和无龋分离菌株（P＜0．05）。结论：远缘链球菌的产酸能力强于变链菌;儿童猛性龋远缘链球菌分离株较非猛性龋和无龋儿童分离菌株产酸性更强。