变形链球菌菌斑生物膜相关基因突变的研究进展

变形链球菌能生成菌斑生物膜,后者与龋病的发生密切相关.随着现代口腔医学和分子生物学的发展,菌斑生物膜相关基因的研究也越来越多,发现了许多与之相关的基因,主要包括与黏附力相关的基因、与群体感应信号系统相关的基因等.对变形链球菌菌斑生物膜相关基因的研究可以更深入地了解其致龋机制,并对寻找防龋新途径作出有益的探索.本文就近年来与变形链球菌菌斑生物膜相关的基因突变研究作一综述.     

变形链球菌抗氧化相关基因研究进展

变形链球菌的粘附、产酸、耐酸能力和能在氧化环境及其他恶劣环境中生存的能力对于其致龋性至关重要,其抗氧化机制成为目前研究的热点。本文就目前研究较多的有关变形链球菌中与耐氧性相关的基因作一综述。     

变形链球菌luxS基因缺失对生物膜早期形成的影响

目的 利用变形链球菌luxS基因敲除突变株,研究这一种属间密度感应系统对生物膜早期形成的影响.方法通过在生物膜培养悬液中加入与菌细胞直径相近的磁性小珠,利用这些小珠在磁场中受到生物膜的位移约束力的原理,采用生物膜定量分析仪,定量比较、分析变形链球菌luxS基因knockout突变株与野生株在生物膜形成上的差异.结果变形链球菌luxS基因突变株与野生株在生物膜形成模式上有显著差别,突变株生物膜自第6小时起开始形成,生物膜形成指数(biofilm index,BFI)差值ABFI=2.015,约第10小时突变株形成的生物膜可完全限制磁珠在磁场中的位移(ABFI=7.025);而野生株生物膜约第10小时开始形成(ABFI=1.875),明显晚于突变株.12h后两菌株生物膜形成未见明显差异(P>0.05).结论变形链球菌luxS基因的缺失可影响生物膜的早期形成.     

luxS基因在变形链球菌生物膜形成中的硫代谢作用

目的 通过比较在不同条件培养基中变形链球菌Ingbritt C国际标准株及luxS基因缺陷株24 h生物膜形成的厚度及平均荧光强度,探讨luxS基因在变形链球菌生膜形成中的硫代谢作用.方法 建立以圆形玻片为载体的生物膜模型,通过营养补充实验培养两菌株生物膜,利用激光共聚焦扫描显微镜观察测定两菌株生物膜厚度和平均荧光强度.结果 当分别加入一定浓度半胱氨酸、蛋氨酸时,缺陷株生物膜厚度有明显增长,但未恢复至标准株水平;加入半胱氨酸,标准株生物膜厚度有所增加;当加入S-腺苷甲硫氨酸时,缺陷株与标准株生物膜的厚度均降低.结论 在变形链球菌生物膜形成中,luxS基因不但具有密度感应功能,在硫代谢中也起着重要作用.     

变形链球菌抗氧化相关基因研究进展

变形链球菌的粘附、产酸、耐酸能力和能在氧化环境及其他恶劣环境中生存的能力对于其致龋性至关重要，其抗氧化机制成为目前研究的热点。本文就目前研究较多的有关变形链球菌中与耐氧性相关的基因作一综述。     

变形链球菌粘附调节基因研究进展

变形链球菌是主要致龋菌之一。粘附是变链菌形成生物膜的初期阶段,也是必经阶段,是公认的细菌四大致龋条件(细菌的粘附、生物膜形成能力、菌细胞代谢碳水化合物能力及细菌对不断波动环境的适应能力)之一。分析与粘附相关的变链菌基因,将有助于我们从分子水平上探索变链菌在生物膜环境中的致龋机理,为从基因水平探索和丰富龋病病因学及龋病防治提供理论依据。     

变形链球菌luxS基因突变对口腔混合细菌生物膜的影响

目的 研究致龋菌变形链球菌luxS基因在口腔细菌混合培养形成牙菌斑生物膜中的作用。方法 将变形链球菌野生株(UA159)及其2种luxS基因突变株（luxS基因高表达株和luxS基因缺陷株）分别与口腔细菌嗜酸乳杆菌（ATCC4356）按照1∶1比例接种于牛心脑浸液培养基,体外混合培养不同时间,包括生物膜形成过程中的初期（4 h）、中期（14 h）、晚期（24 h）,通过MTT法检测混合菌在生物膜形成的量。通过激光共聚焦显微镜观察混合细菌24 h形成的生物膜结构,实时定量PCR检测变形链球菌相关基因（ftf, smu630, brpA, gbpB, gtfB, vicR, comDE和relA）的表达。采用SPSS17.0软件包对数据进行统计学分析。结果 变形链球菌野生株及其2种luxS基因突变株与嗜酸乳杆菌混合培养14 h后,生物膜的量分别为0.481±0.024、0.591±0.023和0.279±0.019;24 h后,混合细菌形成生物膜的量趋势与该时间点一致,变形链球菌高表达株高于野生株,而缺陷株明显降低;但4 h后形成的生物膜组间无显著差异。激光共聚焦显微镜结果表明,高表达株和野生株的集聚程度更高,形成生物膜的结构更加紧密;而缺陷株生物膜菌间结构比较稀疏。以变形链球菌野生株和嗜酸乳杆菌混合形成的生物膜中相关基因的表达为标准,高表达株相关基因的表达均增加,缺陷株表达均降低,且各组间存在显著差异（P<0.05）。结论 变形链球菌luxS基因影响与具核梭杆菌混合培养形成的牙菌斑生物膜,为进一步研究该基因在生物膜中的作用及其调控机制提供了依据。     

变形链球菌耐酸性相关基因的研究

耐酸性是变形链球菌重要的致龋因素之一，虽然其分子机制并不明了，但目前已分离，测序并克隆了几个相关基因，本文对已发现的，影响变形链球菌耐酸性的基因及其结构。功能和可能的作用机制作一综述。     

变形链球菌基因转化相关DNA片段的初步研究

目的 :研究与变链菌基因转化相关的cslA基因在临床分离株中的检出情况。方法 :实验菌株源自前期工作所获得的不同黏附力及合成IG能力的变链菌临床分离株 ,提取全菌DNA并鉴定浓度和纯度 ,PCR扩增cslA基因 ,10g/L琼脂糖电泳观察结果。结果 :6 6株变链菌临床分离株中有 5 2株扩增出cslA基因 ,检出率为 79%。cslA基因的检出率在不同黏附力及合成IG能力的菌株间无统计学差异 (P >0 .0 5 )。结论 :cslA基因在变链菌临床分离株中的分布较广泛 ;尚不能认为cslA基因在毒力不同的菌株间的分布不同。     

不同pH对变异链球菌体外生物膜形成的影响

目的：了解变异链球菌临床株体外生物膜形成规律以及不同pH对生物膜形成的影响。方法：采用微孔板培养,染色、分光光度测定法（A630）绘制体外不同pH条件下（pH=7.0～5.0）593号、18号菌株以及变异链球菌标准株（ATCC25175）的生物膜生长曲线。结果：体外变异链球菌各株在pH=5.0时均不能形成生物膜;pH=7.0时细菌生物膜形成表现为缓慢的非线性生长,12～24h生物膜开始成熟,24～36h出现一相对的生长停滞期;pH=5.0时对已形成12h的变链菌生物膜生长有明显的抑制作用,但经历12h的酸休克后各菌株的生物膜均能恢复生长。结论：变异链球菌在体外pH=7.0时于12～24h形成稳定的生物膜,该生物膜能抵抗一定程度的酸（pH=5.0）攻击,而浮游状态的细菌则不能。     

变异链球菌LuxS基因缺陷突变株的构建及其生物膜结构的初步观察研究

目的:通过同源重组法构建变异链球菌LuxS基因缺陷突变菌株,比较其与变异链球菌UA159标准株在生物膜形成上的差异。方法:运用基因同源重组方法将氯霉素抗性基因(Cmr)与LuxS基因上下游区域的2个基因片段按一定顺序重组到PUC载体的多克隆位点中,构建出带氯霉素抗性标志的重组质粒,将载体质粒转化到含完整LuxS基因的变异链球菌UA159中,利用氯霉素抗性筛选出LuxS基因缺陷的突变株,并利用聚合酶链式反应(PCR)和哈氏弧菌发光实验进行检测。以釉质磨片为载体,在扫描电镜下观察上述两菌株含有20 g/L葡萄糖、20 g/L蔗糖的乳酪消化胨酵母(TPY)液体培养基中形成24 h的生物膜。结果:PCR基因扩增结果显示:突变株LuxS基因已被Cmr基因完全替换,成功的构建了变异链球菌LuxS基因突变株,并且突变株不能诱导哈氏弧菌BB170的生物发光。当用葡萄糖作为补充糖源时,变异链球菌UA159标准株和LuxS基因突变株所形成的生物膜表现型未见明显差异;而用蔗糖作为补充糖源时,两菌株所形成的生物膜有明显不同,UA159生物膜相对平滑均质,而LuxS基因突变株生物膜呈松散的蜂房状,基质间有较大的间隙,形成较大的团簇状菌落。结论:成功构建出LuxS基因缺陷的变异链球菌突变株,与变异链球菌UA159标准株其生物膜结构发生改变,提示LuxS会影响变异链球菌生物膜的形成。     

Effects of recombinase A deficiency on biofilm formation by Streptococcus mutans

Background/aim: Recombinase A (RecA) is essential for the transformation of both plasmid and chromosomal DNA in Streptococcus pneumoniae and is considered to be related to the SOS‐response in Streptococcus mutans. Methods: In the present study, a RecA‐deficient mutant strain (RAD) was constructed by insertional inactivation of the recA gene encoding the RecA protein in strain MT8148 of S. mutans, after which the biological functions of acid tolerance and biofilm formation were investigated. Results: RAD showed reduced acid tolerance and produced lower density biofilm compared with the wild‐type strain. In addition, confocal microscopic observation indicated that the biofilm produced by RAD was composed of cells with significantly lower viability compared with that produced by strain MT8148. Conclusion: These results suggest that RecA has a relationship with biofilm formation.     

Influence of starvation and biofilm formation on acid resistance of Streptococcus mutans

The aim of this study was to investigate acid resistance induced by starvation or biofilm formation in Streptococcus mutans ATCC 25175. The artificial biofilms were made on cover glasses, starved for 24 h and immersed in 0.1 M lactate buffer at pH 3.8 for 10 min. The biofilms were also exposed to 5% sucrose solution for 20 min to simulate acid shock produced by sucrose metabolism. Confocal laser scanning microscopy with fluorescein isothiocyanate staining measured the resultant minimum pH in biofilms. Live and dead organisms in biofilms were differentiated by confocal laser scanning microscopy with proidium iodide and SYTO9 staining. The same processes were used to treat planktonic organisms. The results showed that starved biofilms or planktonic cells showed significantly more viable bacteria after acid shock induced either by lactic acid or during sucrose consumption than non-starved biofilms or planktonic cells. In addition, biofilms showed greater resistance to acid shock induced by lactic acid than planktonic cells, whereas similar results were obtained where sucrose was used as a carbon source to reduce pH in biofilms and planktonic cells. Thus, it is suggested that starvation protects both biofilm and planktonic S. mutans from acid shock induced either by lactic acid or during sucrose consumption, while biofilm formation seemed to protect bacteria only from acid shock induced by pH 3.8 lactate buffer but not the acid shock of a slightly higher pH produced during sucrose consumption.     

表面蛋白抗原P及其在变异链球菌生物膜形成中的作用

变异链球菌表面蛋白抗原(SPA)P是一类介导细菌黏附和生物膜形成的重要的黏附毒力因子,具有高度保守性.SPAP含有1 561个氨基酸残基,其线性结构氨基酸序列由前导肽区、N端、A区、V区、P区、C端和细胞壁锚着端组成.SPAP具有的淀粉样纤维特性,在细菌生物膜形成中十分重要.SPAP可与牙面获得性膜中的唾液成分结合,介导变异链球菌对牙面的初始黏附.SPAP通过分选酶转移肽共价结合到细菌胞壁表面,在内源性的表面蛋白释放酶作用下又可从细菌胞壁表面释放出来,从而使变异链球菌生物膜降解.本文就SPAP的结构、淀粉样纤维特性,变异链球菌黏附,SPAP各区在生物膜形成中的作用等研究进展作一综述,明确其生物学特性,对于龋病病因学和龋病防治的意义不言而喻.     

密度感应拮抗剂C-30对变异链球菌生物膜的影响

目的:研究密度感应拮抗剂呋喃C-30对变异链球菌生物膜形成的影响。方法:将已合成的密度感应拮抗剂呋喃C-30用BHI培养基配制至终浓度分别为2.0,4.0μg/mL,再加入新鲜培养的变异链球菌液,37℃微需氧培养24 h,在96孔板上形成体外生物膜,用MTT法检测生物膜的量。激光共聚焦显微镜观察不同浓度呋喃C-30处理后的生物膜结构。实验中以不含呋喃C-30的BHI培养基作为阴性对照。结果:实验发现呋喃C-30不会对变异链球菌的生长产生影响,但随着呋喃C-30的浓度增加,变异链球菌形成生物膜的量显著降低(P<0.05);激光共聚焦显微镜的结果也表明呋喃C-30浓度的增加使生物膜菌间结构变得稀疏,集聚程度明显减轻。结论:密度感应拮抗剂呋喃C-30能有效抑制变异链球菌生物膜的形成。     

Removal of Streptococcus mutans biofilm by bubbles

BACKGROUND: Air bubbles had been shown to remove particles and bacteria from surfaces, but they had not yet been studied regarding the removal of mature biofilm from a surface. METHODS: Streptococcus mutans were grown as a biofilm on glass coverslips and were exposed to a fluid stream with or without bubbles. Three parameters (stream velocity, gas fraction, and bubble size) were varied in the bubble stream to determine which conditions best remove the biofilm. RESULTS: At low velocities bubbles enhance biofilm removal compared with the liquid alone. Stream conditions that were shown to be the most effective in removing biofilm were large bubbles at low gas fractions. CONCLUSIONS: These results suggest that flowing bubble streams may be a desirable feature to incorporate into oral hygiene products to remove accumulated biofilms such as dental plaque.     

The effects of orthodontic bonding steps on biofilm formation of Streptococcus mutans in the presence of saliva

Abstract

Objective. To investigate the effects of various orthodontic bonding steps on biofilm formation of Streptococcus mutans in the presence of saliva. Materials and methods: Hydroxyapatite (HA) and orthodontic adhesive (AD) disks were prepared to a uniform size. HA disks were etched with 37% phosphoric acid gel in the etched group (HE). In the primed group (HP), Transbond XT primer was applied to the etched HA surface and light-cured. For biofilm formation, Streptococcus mutans was grown on each specimen in a biofilm medium with either glucose or sucrose in the presence of fluid-phase UWS (F-UWS) or surface adsorbed saliva (S-UWS). The adherent bacteria were quantified by enumeration of the total viable counts of bacteria. Biofilms formed on each surface were examined by scanning electron microscopy. Results. When glucose was used, both F-UWS and S-UWS suppressed biofilm formation of S. mutans. Compared to HA and HE, biofilm formation was significantly inhibited on HP and AD in the presence of glucose. Biofilm-forming patterns that were inhibited by saliva were restored in a sucrose-containing medium. F-UWS promoted biofilm formation on HA and HE, while S-UWS significantly promoted biofilm formation on HP. S. mutans developed biofilm better on HA and HE than on AD when sucrose was used as the sole carbohydrate source. Conclusions. This study suggests that the biofilm development by S. mutans is significantly influenced by the orthodontic bonding procedure. Biofilm formation of S. mutans was inhibited on AD more than other surfaces, irrespective of the presence of saliva or a carbohydrate source.