伊犁黑蜂蜂胶对口腔主要致龋细菌及生物膜生长的实验研究

目的研究伊犁黑蜂蜂胶对口腔常见致龋细菌及其生物膜生长的影响,观察其防龋效果。方法 (1)通过液体稀释法测定伊犁黑蜂蜂胶对口腔常见致龋菌的最小抑菌浓度(MIC)及最小杀菌浓度(MBC);(2)生物膜形成抑制试验测定伊犁黑蜂蜂胶对口腔常见致龋菌的最小生物膜形成抑制浓度(MBIC50);(3)通过结晶紫染色法测定伊犁黑蜂蜂胶对口腔常见致龋菌的最小生物膜清除浓度(MBEC);结果(1)伊犁黑蜂蜂胶对变形链球菌、远缘链球菌、嗜酸乳杆菌、血链球菌、粘性放线菌和内氏放线菌的最低抑菌浓度分别是0.78、0.39、1.56、0.39、0.20和0.20 mg/mL,最低杀菌浓度分别为1.56、0.78、3.125、0.78、0.39和0.39mg/mL;(2)伊犁黑蜂蜂胶对变形链球菌、远缘链球菌、血链球菌、粘性放线菌和内氏放线菌的MBIC50分别是0.39、0.39、0.39、0.05和0.10mg/mL;(3)伊犁黑蜂蜂胶对变形链球菌、远缘链球菌、血链球菌、粘性放线菌和内氏放线菌的MBEC分别是6.25、1.56、3.1256、0.78和0.78mg/mL。结论伊犁黑蜂蜂胶对口腔主要致龋细菌的生长具有抑制作用,能够抑制主要致龋细菌单菌生物膜的形成,并具有一定清除作用,是具有一定防龋效果的天然药物。     

伊犁黑蜂蜂胶对不同生长状态白假丝酵母菌抑菌作用的体外研究

目的探究伊犁黑蜂蜂胶对白假丝酵母菌游离状态及其生物膜状态的影响。方法采用NCCLS M27-A2酵母微量稀释法结合平板法测定蜂胶对游离状态下白假丝酵母菌的最小抑菌浓度(MIC)和最低杀菌浓度(MBC);玻片法在体外构建白假丝酵母菌生物膜模型,采用MTT法检测蜂胶对白假丝酵母菌生物膜状态下的最低生物膜清除浓度(MBEC)及抑制作用;激光共聚焦显微镜(CLSM)观察不同浓度蜂胶醇提取物对同一时间白假丝酵母菌生物膜的抑菌效果,并进行红蓝荧光染色定量分析。结果伊犁黑蜂蜂胶对浮游白假丝酵母菌的MIC为4mg/mL,MBC为8mg/mL;伊犁黑蜂蜂胶对生物膜状态下白假丝酵母菌的MBEC为16mg/mL,随着药物浓度的增加抑菌性逐渐增大,差异具有统计学意义(P0.05);CLSM观察,伊犁黑蜂蜂胶作用于白假丝酵母菌生物膜后可使生物膜内活菌比例降低,生物膜活性减弱,生物膜内实验菌死亡率随蜂胶浓度增加呈上升趋势,与阴性对照组比较差异有统计学意义(P0.05)。结论伊犁黑蜂蜂胶在体外对不同状态下的白假丝酵母菌均起到了明显的抑制作用。     

没食子鞣质对变形链球菌生长抑制的实验研究

目的研究没食子鞣质对浮游状态和生物膜状态下变形链球菌生长抑制的作用,探讨药物的防龋功效。方法采用纸片扩散法测定不同浓度没食子鞣质溶液对变形链球菌的抑菌圈大小,采用试管稀释法测定没食子鞣质对变形链球菌生长的最低抑菌浓度(MIC)、最低杀菌浓度(MBC)值大小,用半定量可见光测定A值的方法测定生物膜最低抑菌浓度(MBEC)值。结果没食子鞣质对变形链球菌的生长具有明显的抑制作用,经液体稀释法实验测定,没食子对变形链球菌的MIC为4 mg/m L,MBC为16 mg/m L,经半定量可见光测定A值的方法测定没食子鞣质对变形链球菌单菌生物膜生长的MBEC值为8 mg/m L。结论没食子鞣质对变形链球菌有抑制作用。     

汉族与维吾尔族儿童封闭型不同龋敏感中变形链球菌乳酸脱氢酶遗传多态性研究

目的探讨汉族与维吾尔族儿童封闭型不同龋敏感中变形链球菌乳酸脱氢酶活性与ldh基因多态性及龋病发生的关系。方法从前期实验分离的维吾尔族与汉族儿童口腔变形链球菌中筛选出:维吾尔族儿童封闭型口腔变形链球菌高龋组变形链球菌35株[龋失补牙数(dmft)≥5],无龋组变形链球菌37株[龋失补牙数(dmft)=0];27株表现乳酸脱氢酶高活性,24株低活性;汉族儿童口腔中分离的高龋和无龋变形链球菌各35株,高活性和低活性各25株,使用特异引物扩增细菌组DNA,获得结构基因ldh,进行聚合酶链反应-限制性内切酶多态性分析。结果维吾尔族儿童封闭型和汉族儿童MseⅠ酶切ldh-RFLP表现A、B两种基因型;而MnlⅠ、DdeⅠ、NlaⅢ和AluⅠ消化扩增产物未发现多态性。MseⅠ产生的B基因型菌株在维吾尔族儿童封闭型,汉族儿童高龋组的检出均高于无龋组(P0.05),A、B两种基因型在不同酶活性组的分布不同(P0.05),高酶活性组B基因型菌株的比例高于低酶活性组;A、B基因型在维吾尔族儿童封闭型和汉族高龋组、无龋组,高酶活性组和低酶活性组的检出比较差异无统计学意义(P0.05)。结论变形链球菌乳酸脱氢酶基因多态性在维吾尔族与汉族儿童并未表现出差异性,ldh基因多态性与变形链球菌乳酸脱氢酶活性及龋高敏感性相关。ldh基因分布与酶活性及乳牙龋具有正相关性。     

椰子油对变形链球菌抑菌作用的体外研究

目的研究椰子油对变形链球菌的生长抑制作用,通过观察其对生物膜活性、产酸及粘附的影响,探讨其在口腔中防龋的作用。方法采用96孔微量板液体稀释法进行抑菌试验,并测得最低抑菌浓度(MIC)。体外建立变形链球菌生物膜模型,通过激光共聚焦显微镜(CLSM)扫描生物膜,观察不同浓度药物作用24 h后对生物膜活性的影响。其次测定处理后各组培养基上清液的终末pH值。最后通过玻璃棒粘附试验计算出不同浓度药物作用48 h后对生物膜粘附的影响。结果椰子油对变形链球菌的生长有抑制作用,其对变形链球菌的MIC为3.13%。CLSM观察24 h后生物膜内活菌比例逐渐下降,死菌逐渐增多。培养基上清液的终末pH值随椰子油浓度的增大而升高,且均高于阴性对照组,差异具有统计学意义(P0.05)。实验组变形链球菌的粘附率随椰子油浓度增高而降低,与阴性对照组相比差异有统计学意义(P0.05)。结论椰子油对变形链球菌有抑制作用,并能抑制其生物膜的活性、产酸及粘附等作用。     

苦参提取物对口腔主要致龋细菌作用的实验研究

目的观察苦参提取物对口腔主要致龋细菌及其生物膜生长、黏附、产酸和产糖的影响,探寻其防龋作用机制。方法将苦参提取物按照二倍梯度稀释法测定最低抑菌浓度,0.5 g/L氯己定为阳性对照,不含药液组为阴性对照组;采用紫外分光光度计测定细菌黏附能力;通过生物膜结晶紫染色法测定生物膜抑制浓度和生物膜清除浓度;通过ΔpH法和苯酚-硫酸法分别测定细菌的产酸和合成水不溶性胞外多糖情况。结果苦参提取物对口腔主要致龋细菌的最低抑菌浓度均为4 g/L;在4 g/L时,对变形链球菌、远缘链球菌、血链球菌、粘性放线菌和内氏放线菌黏附抑制率分别为(77.6%±1.2%)、(66.7%±1.8%)、(60.68%±2.9%)、(79.8%±1.2%)和(85.1%±1.3%)。2 g/L时能够显著抑制浮游菌产酸及合成水不溶性胞外多糖能力。4 g/L时对变形链球菌、远缘链球菌、血链球菌、粘性放线菌、内氏放线菌和嗜酸乳杆菌生物膜形成抑制率分别为(87.5%±1.3%)、(85.4%±0.5%)、(89.0%±0.3%)、(77.2%±0.7%)、(87.4%±1.1%)和(80.4%±1.3%);并对以上细菌生物膜的最低清除浓度分别为16、16、16、16、8和8 g/L。苦参提取物在50%的最小生物膜清除浓度下对单菌生物膜的产酸和合成水不溶性细胞外多糖的抑制率分别为67.5%～94.1%和42.3%～60.0%。结论苦参提取物能够抑制口腔主要致龋细菌浮游和生物膜状态下的生长、黏附、产酸和产糖,其有望成为一种龋齿预防制剂。     

厚朴酚对变形链球菌生物膜致龋毒力因子作用的研究

目的 通过激光共聚焦显微镜观察厚朴酚对变形链球菌生物膜的抑菌效果,并初步了解厚朴酚对变形链球菌生物膜的产酸、耐酸、胞外多糖形成及生物膜形成能力等相关致龋毒力因子的转录表达的影响,为进一步研究厚朴酚防龋的药理作用机制奠定基础.方法 建立变形链球菌生物膜体外模型,激光共聚焦显微镜观察不同药物浓度作用后效果,并进行红绿荧光定量分析;根据GenBank基因库查询ffh、gtfD、pdp等基因序列并设计引物,进行RT-PCR.结果 CLSM观察厚朴酚作用变形链球菌生物膜后可使膜内活菌比例明显下降;RT-PCR结果表明毒力因子ffh、gtfD、pdp的表达水平受到抑制.结论 厚朴酚对变形链球菌生物膜的致龋毒力因子ffh、gtfD、pdp的转录表达有明显的抑制作用.     

铜绿假单胞菌生物膜和浮游菌的毒力表达差异

目的探究铜绿假单胞菌生物膜和浮游菌状态下毒力因子的表达差异。方法使用铜绿假单胞菌标准菌株PAO1,分别在生物膜(静置)和浮游菌(摇床)状态下培养,收集上清液,检测总蛋白酶、LasA和LasB弹性蛋白酶、鼠李糖脂、绿脓素、溶血活性;通过荧光定量PCR检测群体感应(quorum sensing, QS)系统相关基因的表达;同时,通过活菌计数检测PAO1在生物膜和浮游菌状态下的生长曲线。结果生物膜状态下,铜绿假单胞菌PAO1的总蛋白酶、LasA、LasB弹性蛋白酶、鼠李糖脂、绿脓素表达均增高(均P0.05),溶血活性增高(P0.05),生物膜和浮游菌状态下细菌生长曲线差异无统计学意义,QS相关基因rhlI、rhlR、rhlA、lasI、lasR、pqsA、pqsR表达增高(均P0.05)。结论铜绿假单胞菌PAO1在生物膜状态下毒力因子表达较浮游菌状态下增高。     

盐酸小檗碱对变形链球菌生物膜及其致龋毒力因子作用的研究

目的研究盐酸小檗碱对变形链球菌生物膜的抑菌效果,并初步研究其抑菌作用是否为通过影响或干扰某些毒力因子的表达而实现的,为进一步研究盐酸小檗碱防龋的药理作用机制奠定基础。方法建立变形链球菌生物膜体外模型,MTT法评价盐酸小檗碱对变形链球菌生物膜的影响;然后用RT—PCR方法检测变形链球菌毒力因子cdsa、gbpD和ptsI受盐酸小檗碱作用后的表达水平的变化。结果盐酸小檗碱对变形链球菌生物膜的抑制作用随药物浓度增加而增加;RT—PCR结果表明毒力因子cdsa、gbpD和ptsI的表达水平受到抑制。结论盐酸小檗碱对变形链球菌生物膜起到了抑制作用,对其致龋毒力因子cdsa、gbpD和ptsI的转录表达有明显的抑制作用。     

大蒜素抗变异链球菌的致龋效果

目的研究大蒜素对口腔变异链球菌生长及其菌斑生物膜粘附的抑制作用。方法二倍稀释法梯度稀释测最小抑菌浓度(minimum inhibitory concentration,MIC),将MIC以上2个梯度浓度对应的培养物涂布于BHI培养基上进行次代培养获得最低杀菌浓度(minimum bactericidal concentration,MBC);酶标仪测A值观察不同浓度大蒜素抑菌效应;抑制产酸试验观察抑制细菌产酸效应;结晶紫法研究亚抑菌浓度提取物对变异链球菌粘附能力及生物膜总量的影响;采用激光共聚焦荧光显微镜(laser scanning confocal microscopy,LSCM)观察常态牙菌斑生物膜生长过程中及药物处理后牙菌斑生物膜中死菌和活菌的构成,研究其对牙菌斑生物膜结构和活性的影响。结果抑菌试验中,得到大蒜素MIC为12.8 mg/L,MBC为25.8 mg/L。MIC及亚抑菌浓度抑菌试验显示均有一定的抑菌性,抑制率为2.17%～67.12%,并且抑菌性与浓度梯度成正相关。产酸试验显示24 h内大蒜素明显抑制细菌产酸(P0.01),细菌粘附试验结果显示大蒜素在MIC时生物膜的生成速度最慢,生物膜的总量最低(P0.01)。共聚焦荧光显微镜可见大蒜素组随药物浓度增加,菌斑生物膜较薄,绿色的活菌及团块明显减少,抑制生物膜的生长。结论大蒜素对变异链球菌生长、产酸与粘附有一定抑制作用。     

维吾尔族不同龋敏感儿童变链菌临床分离株不同生存状态合成胞外多糖能力的研究

目的比较维吾尔族不同龋敏感儿童变链菌临床分离株生物膜状态和浮游状态致龋能力的差异。方法根据析因实验设计分组,选取课题组前期分离鉴定的维吾尔族儿童变链菌临床株27株,其中17株高龋菌株[龋失补牙数（dmft）≥5],10株无龋菌株[龋失补牙数（dmft）=0]。分别在24孔板静止培养以及在试管内摇动培养。采用蒽酮法测定高龋组与无龋组分别在生物膜状态和浮游状态下合成胞外多糖的量。使用SPSS 17.0软件包对实验结果进行析因分析。结果高龋组生物膜状态下合成水溶性与非水溶性葡聚糖量分别为（0.3011±0.0398）mg/mL、（0.3711±0.0372）mg/mL,高于浮游状态的（0.2300±0.0438）mg/mL、（0.2356±0.0568）mg/mL,无龋组生物膜状态下合成水溶性与非水溶性葡聚糖分别为（0.2067±0.0264）mg/mL、（0.3489±0.0537）mg/mL,高于浮游状态的（0.1489±0.0325）mg/mL、（0.1578±0.0227）mg/mL,差异有统计学意义（P〈0.05）。并且高龋组合成胞外多糖量均高于无龋组,差异有统计学意义（P〈0.05）。结论维吾尔族高龋儿童变链菌临床分离株的高致龋力可能与其携带有合成胞外多糖能力较强的菌株有关。     

L-(+)-lactate dehydrogenase deficiency is lethal in Streptococcus mutans.

The previously cloned gene for L-(+)-lactate dehydrogenase (LDH) from Streptococcus mutans was mutagenized in vitro. An Escherichia coli transformant which expressed a thermolabile LDH activity was identified. The ldh(Ts) gene was introduced into S. mutans on a suicide vector to create a heterodiploid expressing both wild-type and thermolabile LDH activities. Self-recombinants which had only one ldh gene were isolated. One of these clones expressed only the thermolabile LDH activity. This isolate grew well at 30 degrees C but did not grow at 42 degrees C under a variety of cultivation conditions, thereby proving that LDH deficiency is lethal in S. mutans in the absence of compensatory mutations.     

Non-disruptive, real-time analyses of the metabolic status and viability of Streptococcus mutans cells in response to antimicrobial treatments

Streptococcus mutans is one of a small number of recognized pathogens that lives among the hundreds of other bacterial species which comprise the oral flora. The virulence of this organism is intimately associated with its ability to live as an attached biofilm community on the tooth surface, and consequently, there is a great interest in its biofilm lifestyle. Currently, there are no established protocols that facilitate drug screening against this organism while it is entrenched in the biofilm. Furthermore, greater complications arise when attempting to perform these experiments in a multi-species setting. In an effort to circumvent these problems, we developed a quick, real-time, and non-disruptive method to probe the metabolic status of S. mutans growing as either a planktonic culture or a biofilm community. This assay takes advantage of the proven utility of luciferase measurements for drug screening. We placed the luciferase gene under the control of the S. mutans lactate dehydrogenase promoter (ldh) and integrated the construct onto its native position on the chromosome. We found this construct to be both highly expressed (<10000 cells easily detectable) and insensitive to many different growth parameters. When testing this reporter in both planktonic and biofilm cultures receiving either bacteriostatic or bactericidal antibiotics, we found the ldh-luc reporter to be a very accurate measurement of cell viability. Furthermore, we also demonstrated that this assay can generate useful information about the characteristics of intoxication caused by antibiotic activity. In addition, we modified the biofilm assay into the 96-well format and demonstrated the feasibility of high throughput drug screening of biofilm embedded S. mutans.     

LuxS基因缺陷对变形链球菌生物膜形成的影响

目的观察LuxS基因缺失后变形链球菌生物膜成熟初期的变化情况。方法通过扫描电镜观察标准菌和缺陷菌在不同营养环境中生物膜成熟初期的形成情况。结果对不同营养环境中形成的生物膜观察,发现在富含蔗糖的环境中,缺陷菌成熟初期的生物膜形成能力较标准菌弱。结论 LuxS基因缺失后变形链球菌在蔗糖环境中生物膜形成的能力减弱。     

Biofilm susceptibility to metal toxicity

This study compared bacterial biofilm and planktonic cell susceptibility to metal toxicity by evaluating the minimum inhibitory concentration (MIC), the planktonic minimum bactericidal concentration (MBC), and minimum biofilm eradication concentration (MBEC) using the MBEC device. In total, 17 metal cations and oxyanions, chosen to represent groups VIB to VIA of the periodic table, were each tested on biofilm and planktonic cultures of Escherichia coli JM109, Staphylococcus aureus ATCC 29213, and Pseudomonas aeruginosa ATCC 27853. In contrast to control antibiotic assays, where biofilm cultures were 2 to 64 times less susceptible to killing than logarithmically growing planktonic bacteria, metal compounds killed planktonic and biofilm cultures at the same concentration in the vast majority of combinations. Our data indicate that, under the conditions reported, growth in a biofilm does not provide resistance to bacteria against killing by metal cations or oxyanions.     

Effect of biofilm model, mode of growth, and strain on streptococcusmutans protein expression as determined by two-dimensional difference gel electrophoresis

The effect of biofilm model, strain and mode of growth (biofilm or planktonic) on protein expression in Streptococcus mutans, a dental pathogen, was determined by two-dimensional difference gel electrophoresis. The bacterial strain (21-28% differentially expressed proteins) and the biofilm model (0.3-7.8% differential expression) used have a much larger effect on protein expression than the mode of growth (0.2-0.7% differential expression), something that has been ignored in biofilm studies up to now.     

Chemical compositions and antimicrobial activities of four different Anatolian propolis samples

Propolis means a gum that is gathered by bees from various plants. It is known for its biological properties, having antibacterial, antifungal and healing properties. The aims of this study were to evaluate the antimicrobial activity of four different Anatolian propolis samples on different groups of microorganisms including some oral pathogens and comparison between their chemical compositions. Ethanol extracts of propolis (EEP) were prepared from four different Anatolian propolis samples and examined whether EEP inhibit the growth of the test microorganisms or not. For the antimicrobial activity assays, minimum inhibitory concentrations (MIC) were determined by using macrodilution method. The MIC values of the most effective propolis (TB) were 2 microg/ml for Streptococcus sobrinus and Enterococcus faecalis, 4 microg/ml for Micrococcus luteus, Candida albicans and C. krusei, 8 microg/ml for Streptococcus mutans, Staphylococcus aureus, Staphylococcus epidermidis and Enterobacter aerogenes, 16 microg/ml for Escherichia coli and C. tropicalis and 32 microg/ml for Salmonella typhimurium and Pseudomonas aeruginosa. The chemical compositions of EEP's were determined by high-temperature high-resolution gas chromatography coupled to mass spectrometry. The main compounds of four Anatolian propolis samples were flavonoids such as pinocembrin, pinostropin, isalpinin, pinobanksin, quercetin, naringenin, galangine and chrysin. Although propolis samples were collected from different regions of Anatolia all showed significant antimicrobial activity against the Gram positive bacteria and yeasts. Propolis can prevent dental caries since it demonstrated significant antimicrobial activity against the microorganisms such as Streptococcus mutans, Streptococcus sobrinus and C. albicans, which involves in oral diseases.     

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.