激光共聚焦显微镜观察维拉帕米对HMME-PDT抑制混合菌菌斑生物膜作用的影响

目的 探讨细菌外排泵抑制剂(EPI)——维拉帕米对以血卟啉单甲醚(HMME)为光敏剂的光动力疗法(PDT)抵抗牙菌斑生物膜内主要致龋菌作用的影响.方法 以变形链球菌、血链球菌、嗜酸性乳杆菌和黏性放线菌为实验菌株,建立牙菌斑生物膜模型.以维拉帕米作为细菌外排泵抑制剂,根据在PDT过程中加入维拉帕米和光敏剂的先后顺序,实验分为5组:A组为同时加入光敏剂+维拉帕米组,B组为维拉帕米(先)+光敏剂组(后)组,C组为光敏剂(先)+维拉帕米(后)组,D组为单纯PDT处理组,E组为生理盐水处理组.激光照射后以激光共聚焦显微镜观察PDT作用后生物膜的变化.结果 生理盐水处理组:生物膜内活菌数量占据大多数.以此组为正常细菌活力组作对比,单纯PDT处理后,红色荧光显著增加,细菌失去了积聚能力,多呈散乱分散状态;与单纯PDT组相比,加入维拉帕米的PDT组绿色荧光染色增多,细菌活力增加,其中先加入维拉帕米后加入光敏剂组,绿色荧光染色明显增多,红色荧光减少,说明活菌相对活力较高.结论 实验表明,HMME-PDT在对混合菌菌斑生物膜中的细菌有抑制效应,细菌外排泵抑制剂对HMME-PDT抑制牙菌斑生物膜内主要致龋菌有抑制作用;且前期维拉帕米给药能够显著抑制PDT治疗龋病的效果.     

光动力疗法防龋对釉质微量元素的影响

目的 将通体发光光纤应用于光动力疗法(PDT)防龋,探讨其对大鼠磨牙牙釉质中Ca、P含量的影响.方法 接种变形链球菌(S.mutans)制备大鼠致龋模型.将80只Wistar大白鼠随机分成5组:17mW(8 mW/cm2)PDT组(A组)、34 mW(15 mW/cm2)PDT组(B组)、68 rnW(30 mW/cm2)PDT组(C组)、20 g/LNaF溶液组(阳性对照组,D组)和生理盐水组(阴性对照组,E组),每组16只.采用650 nm半导体激光器,以质量浓度为40 μg/ml的血卟啉单甲醚为光敏剂,连续进行4周实验.应用电感耦合等离子体发射光谱法检测各组大鼠磨牙牙釉菌中的Ca、P含量.结果 实验后B、C、D组Ca、P含量明显高于A、E组,差异均具有统计学意义(均P＜0.05).A组Ca、P含量实验前后差异均无统计学意义(均P＞0.05);实验后B、C组Ca、P含量分别高于实验前,差异均具有统计学意义(均P＜0.05).A组Ca增量低于D组,差异具有统计学意义(P＜0.05);B、C两组Ca、P增量明显高于D组,差异均具有统计学意义(均P＜0.05);而B、C两组间Ca、P增量差异均无统计学意义(均P＞0.05).结论 在设定的参数范围内,PDT促进牙齿再矿化效果优于20 g/L NaF溶液.采用PDT防龋可增加大鼠磨牙牙釉质中的Ca、P含量,且Ca、P含量与PDT的功率有关.当PDT功率较低时,对釉质再矿化作用不明显;随着PDT功率的增加,Ca、P含量亦增加;当功率增加至一定数值时,两元素含量增量变化不明显,表明PDT可维持牙齿再矿化微环境.     

DOX联合HMME-PDT对人肝癌细胞HepG2联合作用的研究

目的:初步探讨阿霉素(DOX)联合血卟啉单甲醚介导的光动力疗法(HMME-PDT)对人肝癌细胞(HepG2)的作用及可能的机制.方法:实验分对照组、单独PDT组、单独DOX组和联合作用组.PDT组所用的光敏剂剂量为2.5μg/mL,能量密度为0.2 J/cm2、0.4 J/cm2、0.8 J/cm2、1.6 J/cm2和3.2 J/cm2.DOX组所用的DOX浓度为0.1 μg/mL、0.2μg/mL和0.4μg/mL.联合作用为不同剂量单独作用的组合,联合时序为PDT后立即加入阿霉素(PDT DOX)或阿霉素先于PDT24h加入(DOX PDT).MTT法检测24h后的细胞活性抑制率,并用金氏公式分析联合效应.荧光显微镜观察不同处理组阿霉素的摄取情况.结果:MTT法结果表明,联合作用的细胞抑制率高于单独作用,这在PDT DOX的联合时序作用中表现更为明显.金氏公式分析表明,PDT DOX的联合作用相对DOX PDT的联合作用产生更明显的协同或相加效应.荧光显微镜实验可以观察到联合作用明显提高了细胞对阿霉素的摄取,并且随着联合剂量的加大摄取加强.结论:相对单独作用,联合作用有更高的细胞活性抑制率,联合效应与联合时序有关,PDT DOX的联合作用的联合效果好.联合作用产生相加或协同效应的机制之一可能是因为PDT处理改变了细胞膜通透性从而更有利于细胞对阿霉素的摄取,最终提高作用效果.     

变异链球菌生物膜结构观察

目的　建立变异链球菌生物膜模型 ,用激光共聚焦扫描显微镜 (CLSM)观察变异链球菌生物膜结构。方法　在盖玻片上分别形成 6、12、18、2 4、4 8、72h变异链球菌生物膜 ,将得到的各时段生物膜荧光染色后 ,用CLSM观察生物膜的断层扫描图像、生物膜厚度、每层红光绿光的面积 ,计算生物膜中细菌密度和活菌百分比 ,用软件处理扫描数据 ,得到生物膜的三维重建图像。结果　变异链球菌生物膜具有空间立体结构 ,形态多样 ,其中细菌密集 ,由死细菌和活细菌组成 ,还有丰富的基质和管道系统。 2 4h生物膜平均厚度最大 ,生物膜内层、中间层的细菌密度相对较大 ,而外层较低 ,72h内各时间段生物膜中活菌百分比由内往外逐渐增加。结论　变异链球菌生物膜有一定的厚度 ,具有三维立体空间结构 ,结构形态具有多样、不均质、开放的特点。     

胞外聚合物葡聚糖和DNA对粪肠球菌根管生物膜形成的影响

目的 研究胞外多糖和胞外DNA对粪肠球菌在人牙本质片上黏附并形成生物膜过程中的作用.方法 制备牙本质片,分成葡聚糖酶(Dextranase)组、DNA酶(DNaseI)组和对照组,体外培养获得粪肠球菌的牙本质生物膜标本.采用活菌菌落计数法、激光共聚焦扫描显微镜结合COMSTAT软件分析胞外聚合物对牙本质黏附菌量和生物膜三维结构的影响.结果 相同生物膜膜龄,Dextranase组和DNaseI组粪肠球菌牙本质黏附菌量均少于对照组,差异具有统计学意义(P＜0.05),其中1 h黏附菌量分别下降至对照组的15.61%、18.39%.激光共聚焦显微镜观察到Dextranase和DNaseI对粪肠球菌牙本质生物膜的形成有影响,处理后生物膜结构松散,生物量、底层覆盖率及平均厚度均下降,具有统计学意义(P＜0.0167),其中生物量由(3.95±0.09)μm3/μm2下降至(0.36±0.08)μm3/μm2、(1.05±0.06)μm3/μm2.DNaseI对成熟牙本质生物膜产生显著降解作用,而Dextranase对成熟牙本质生物膜作用不明显.结论 胞外多糖和胞外DNA是粪肠球菌生物膜的重要成分,对其初始黏附和生物膜三维结构起重要作用.     

噬菌体PaP3多糖解聚酶对铜绿假单胞菌生物膜的作用研究

目的 检测重组铜绿假单胞菌噬菌体PaP3多糖解聚酶对铜绿假单胞菌生物膜的降解活性.方法 经FTTCConA/PI荧光双染色后,采用激光扫描共聚焦显微镜观察重组多糖解聚酶处理前后PA3生物膜的形态变化,以测定多糖解聚酶对生物膜的降解活性;测定多糖解聚酶作用前、后铜绿假单胞菌对4种敏感抗生素(乳酸环丙沙星、加替沙星、头孢他啶、硫酸庆大霉素)的MIC、MBC变化情况,从而判断多糖解聚酶对抗生素的协同杀菌作用.结果 FTTC-ConA/PI荧光双染色结果显示.经重组多糖解聚酶处理后,铜绿假单胞菌生物膜的胞外多糖组分少而分散,包裹在胞外多糖组分里的细菌数量也大大减少,表明多糖解聚酶确实能够特异性的降解生物膜的胞外多糖组分.多糖解聚酶作用后四种抗生素相应的MIC、MBC都出现了不同程度的降低,其中以头孢他啶降低最为明显,表明多糖解聚酶对抗生素具有协同杀菌活性.结论 重组噬菌体PaP3多糖解聚酶在体外可以特异性的降解铜绿假单胞菌生物膜的胞外多糖,有利于抗生素通透生物膜,作用于菌体,具有协同抗菌作用.     

pH值对血型链球菌和变形链球菌生长的影响

血型链球菌（血链菌）是牙菌斑形成过程中的先锋定殖菌，由于它与龋病的发生没有密切的关系，人们对它的研究尚不多。本文采用连续培养方法来观察血链菌３４和变型链球菌（变链菌）Ｉｎｇｂｒｉｔ（ｃ）在不同ｐＨ值时的生长状态，探讨血链菌对牙菌斑形成的作用及其与主要...     

变形链球菌gcp基因失活菌株生物膜形成能力及在唾液包被羟基磷灰石表面的黏附率

背景：前期实验发现外源性单磷酸鸟苷环二聚体能够抑制变形链球菌生物膜的形成及其体外黏附能力。变形链球菌内部存在的单磷酸鸟苷环二聚体是否具有同样作用。 目的：成功构建了变形链球菌内部单磷酸鸟苷环二聚体相关基因gcp的失活菌株,观察gcp基因失活后变形链球菌生物学特性的改变。 方法：将gcp失活菌和野生菌菌悬液在96孔酶标板中厌氧培养48 h,用结晶紫染色,乙醇/丙酮混合液显色,测量A575 nm值,以定量反映生物膜形成量;荧光标记gcp失活菌和野生菌,与唾液包被的羟基磷灰石粉末共同孵育后,测定羟基磷灰石沉淀的荧光值,比较黏附率的差异。 结果与结论：gcp失活菌生物膜形成量低于野生菌,gcp失活菌在唾液包被羟基磷灰石表面的黏附率低于野生菌(P < 0.05)。提示gcp基因的失活可抑制变形链球菌生物膜形成能力及在生物体表面的黏附。     

外源性单磷酸鸟苷环二聚体抑制变形链球菌生物膜的形成能力*☆

背景：有研究发现外源性的单磷酸鸟苷环二聚体(bis-(3'-5')-cyclic dimeric guanosinemonophosphate,c-di-GMP)能够抑制金黄色葡萄球菌生物膜的形成,且作用呈剂量依赖性。 目的：观察外源性c-di-GMP对变形链球菌生物膜形成能力的影响。 方法：将不同浓度(0,2,20,200,400 µmol/L)外源性c-di-GMP作用于变形链球菌生物膜48 h,使用酶标仪测定吸光度值,观测生物膜形成量的改变,以生理盐水作为阴性对照。同时在离体牙的新鲜釉质片上形成变形链球菌生物膜,以      200 μmol/L的c-di-GMP与生理盐水分别作用于生物膜48 h,扫描电镜观察结构的改变。 结果与结论：与阴性对照组比较,c-di-GMP明显抑制了变形链球菌生物膜的形成,而且这种抑制成剂量依赖关系,当c-di-GMP浓度为200 µmol/L时,变形链球菌生物膜的形成能力下降了65%左右,达到400 µmol/L时,生物膜的形成能力几乎被完全抑制(P < 0.05)。扫描电镜结果显示,c-di-GMP处理组细菌排列无明显规律,细胞外基质减少。表明c-di-GMP可以抑制变形链球菌的生物膜形成能力。      

克拉霉素对金黄色葡萄球菌生物膜的影响

目的:研究克拉霉素对金黄色葡萄球菌生物膜的影响及与氧氟沙星合用后的抗菌协同作用。方法:用0.22μm微孔滤膜构建金黄色葡萄球菌生物膜模型;测定克拉霉素、氧氟沙星的MIC值;银染金黄色葡萄球菌生物膜,扫描电镜(SEM)观察生物膜形态结构,连续稀释法进行活菌计数。结果:生物膜(BF)可在七天内稳定形成,1/16MIC、1/4MIC浓度克拉霉素显著减少单用氧氟沙星组中的BF活菌数(P<0.05)。结论:克拉霉素能显著提高氧氟沙星对BF中金黄色葡萄球菌的抗菌活性。     

Drug resistance of bacterial dental biofilm and the potential use of natural compounds as alternative for prevention and treatment

Oral diseases, such as dental caries and periodontal disease are directly linked with the ability of bacteria to form biofilm. The development of dental caries involves acidogenic and aciduric Gram-positive bacteria colonizing the supragingival biofilm (Streptococcus, Lactobacillus and Actinomycetes). Periodontal diseases have been linked to anaerobic Gram-negative bacteria forming a subgingival plaque (Porphyromonas gingivalis, Actinobacillus, Prevotella and Fusobacterium). Cells embedded in biofilm are up to 1000-fold more resistant to antibiotics compared to their planctonic ones. Several mechanisms have been proposed to explain biofilms drug resistance. Given the increased bacterial resistance to antibiotics currently used in dentistry, a great importance is given to natural compounds for the prevention of oral bacterial growth, adhesion and colonization. Over the past decade, interest in drugs derived from medicinal plants has markedly increased. It has been well documented that medicinal plants and natural compounds confer considerable antibacterial activity against various microorganisms including cariogenic and periodontal pathogens.This paper provides a review of the literature focusing on the studies on (i) biofilm in the oral cavity, (ii) drug resistance of bacterial biofilm and (iii) the potential use of plant extracts, essential oils and natural compounds as biofilm preventive agents in dentistry, involving their origin and their mechanism of biofilm inhibition.     

天然菌斑生物被膜中变形链球菌、远缘链球菌和血链球菌的荧光原位检测

目的 检测菌斑生物被膜初期形成过程中的变形链球菌(Streptpcoccus mutans)、远缘链球菌(Streptpcoccus sobrinus)和血链球菌(Streptpcoccus sanguis).方法 从植入釉质磨片表面获得完整的菌斑生物被膜标本,应用激光共聚焦扫描显微镜和荧光原位杂交技术相结合的方法,对天然菌斑生物被膜形成初期的变形链球菌、远缘链球菌和血链球菌进行原位的、实时的动态观察,通过连续断层扫描及三维重建,观察这3种菌在天然菌斑生物被膜中的空间分布,测量3种细菌的扫描厚度.实验中的扫描厚度结果采用方差分析方法进行统计处理,SPSS11.5统计软件辅助完成,α设在0.05.结果 在激光共聚焦扫描显微镜下观察生物被膜呈三维立体结构,形态多样,生物被膜内层和外层细菌较稀疏,中间层较密集,细菌之间可见许多黑色空隙,贯穿整个生物被膜.菌斑生物被膜变形链球菌、远缘链球菌和血链球菌在菌斑生物被膜形成初期的平均扫描厚度随时间延长而增加,1 h时平均扫描厚度分别为20.43、11.50、14.76 μm,到24 h时平均厚度最大,分别为70.25、75.40、79.98 μm.结论 应用荧光原位杂交技术结合激光共聚焦扫描显微镜,可以快速、灵敏地检测出菌斑生物被膜变形链球菌、远缘链球菌和血链球菌.     

The virulence of Streptococcus mutans and the ability to form biofilms

In some diseases, a very important role is played by the ability of bacteria to form multi-dimensional complex structure known as biofilm. The most common disease of the oral cavity, known as dental caries, is a top leader. Streptococcus mutans, one of the many etiological factors of dental caries, is a microorganism which is able to acquire new properties allowing for the expression of pathogenicity determinants determining its virulence in specific environmental conditions. Through the mechanism of adhesion to a solid surface, S. mutans is capable of colonizing the oral cavity and also of forming bacterial biofilm. Additional properties enabling S. mutans to colonize the oral cavity include the ability to survive in an acidic environment and specific interaction with other microorganisms colonizing this ecosystem. This review is an attempt to establish which characteristics associated with biofilm formation—virulence determinants of S. mutans—are responsible for the development of dental caries. In order to extend the knowledge of the nature of Streptococcus infections, an attempt to face the following problems will be made: Biofilm formation as a complex process of protein–bacterium interaction. To what extent do microorganisms of the cariogenic flora exemplified by S. mutans differ in virulence determinants “expression” from microorganisms of physiological flora? How does the environment of the oral cavity and its microorganisms affect the biofilm formation of dominant species? How do selected inhibitors affect the biofilm formation of cariogenic microorganisms?     

左氧氟沙星浸涂生物材料防止生物膜形成的体外研究

目的观察左氧氟沙星（LFX）浸涂生物材料在体外对铜绿假单胞菌黏附、定植及生物膜形成的影响。方法配置LFX与外消旋聚乳酸（PDLLA）比值（w／w）为1：4、1：32的丙酮溶液,分别浸涂聚氯乙烯（PVC）材料10min制备LFX浸涂生物材料,按LFX浓度分为LFX高浓度组和LFX低浓度组,以PVC材料（PVC组）作为对照。3组材料分别浸没在含铜绿假单胞菌PA01（细菌浓度为1X108CFU／m1）的LB培养液中,37℃孵育24、72h,分别进行各组材料表面细菌计数及扫描电子显微镜观察。结果孵育24h,LFX高浓度组、LFX低浓度组和PVC组材料表面细菌计数分别为8．02（7．93～8．08）、8．10（8．03～8．20）和8．36（8．26～8．49）logloCFU／ml;孵育72h,LFX高浓度组、LFX低浓度组和PVC组材料表面细菌计数分别为7．77（7．64～7．87）、8．31（8．14—8．44）和8．74（8．50～8．99）logloCFU／ml,LFX浸涂材料表面细菌计数均显著低于PVC组（均P〈O．05）。扫描电子显微镜观察显示,孵育24h,LFX高浓度组和LFX低浓度组材料表面见散在单个细菌,PVC组材料表面大量细菌分散存在;孵育72h,LFX高浓度组和LFX低浓度组材料表面未见细菌,PVC组材料表面可见微菌落及“珊瑚状”生物膜形成。结论LFX浸涂生物材料可抑制铜绿假单胞菌黏附、定植及生物膜的形成。     

Characterization of recombinant, ureolytic Streptococcus mutans demonstrates an inverse relationship between dental plaque ureolytic capacity and cariogenicity

Dental caries results from prolonged plaque acidification that leads to the establishment of a cariogenic microflora and demineralization of the tooth. Urease enzymes of oral bacteria hydrolyze urea to ammonia, which can neutralize plaque acids. To begin to examine the relationship between plaque ureolytic activity and the incidence of dental caries, recombinant, ureolytic strains of Streptococcus mutans were constructed. Specifically, the ureABCEFGD operon from Streptococcus salivarius 57.I was integrated into the S. mutans chromosome in such a way that the operon was transcribed from a weak, cognate promoter in S. mutans ACUS4 or a stronger promoter in S. mutans ACUS6. Both strains expressed NiCl(2)-dependent urease activity, but the maximal urease levels in ACUS6 were threefold higher than those in ACUS4. In vitro pH drop experiments demonstrated that the ability of the recombinant S. mutans strains to moderate a decrease in pH during the simultaneous metabolism of glucose and urea increased proportionately with the level of urease activity expressed. Specific-pathogen-free rats that were infected with ACUS6 and fed a cariogenic diet with drinking water containing 25 mM urea and 50 microM NiCl(2) had relatively high levels of oral urease activity, as well as dramatic decreases in the prevalence of smooth-surface caries and the severity of sulcal caries, relative to controls. Urease activity appears to influence plaque biochemistry and metabolism in a manner that reduces cariogenicity, suggesting that recombinant, ureolytic bacteria may be useful to promote dental health.     

Effect of exopolysaccharides from cariogenic bacteria on human gingival fibroblasts

Bacterial biofilm (dental plaque) plays a key role in caries etiopathogenesis and chronic periodontitis in humans. Dental plaque formation is determined by exopolysaccharides (EPSs) produced by cariogenic and periopathogenic bacteria. The most frequent cariogenic bacteria include oral streptococci (in particular S. mutans) and lactobacilli (most frequently L. acidophilus). In turn, the dominant periopathogen in periodontitis is Porphyromonas gingivalis. Development of dental caries is often accompanied with gingivitis constituting the mildest form of periodontal disease. Basic cellular components of the gingiva tissue are fibroblasts the damage of which determines the progression of chronic periodontitis. Due to insufficient knowledge of the direct effect of dental plaque on metabolic activity of the fibroblasts, this work analyses the effect of EPSs produced by S. mutans and L. acidophilus strains (H₂O₂-producing and H₂O₂-not producing) on ATP levels in human gingival fibroblasts (HGF-1) and their viability. EPSs produced in 48-hours bacterial cultures were isolated by precipitation method and quantitatively determined by phenol - sulphuric acid assay. ATP levels in HGF-1 were evaluated using a luminescence test, and cell viability was estimated using fluorescence test. The tests have proven that EPS from S. mutans did not affect the levels of ATP in HGF-1. Whereas EPS derived from L. acidophilus strains, irrespective of the tested strain, significantly increased ATP levels in HGF-1. The analysed EPSs did not affect the viability of cells. The tests presented in this work show that EPSs from cariogenic bacteria have no cytotoxic effect on HGF-1. At the same time, the results provide new data indicating that EPSs from selected oral lactobacilli may have stimulating effect on the synthesis of ATP in gingival fibroblasts which increases their energetic potential and takes a protective effect.     

HMME-PDT对牙骨质片及种植体表面Pg菌斑生物膜的影响

目的 探讨光动力疗法（PDT）对牙骨质片及种植体表面牙龈卟啉单胞菌（Pg）菌斑生物膜的杀灭效果,获得有效杀灭Pg的光敏剂和激光照射的合理剂量.方法 选用Pg标准菌株分别联合经全唾液处理后的无菌牙骨质片和无菌种植体共同培养,建立简易的体外人工牙骨质片和种植体表面细菌附着模型.以血卟啉单甲醚（HMME）为光敏剂,波长630 nm半导体激光为光源,照射牙骨质片表面细菌附着模型60 s后,计算菌落形成单位（CFU）,筛选出最佳激光能量密度（EDL）和HMME剂量,以筛选的合理剂量照射种植体表面细菌附着模型,并用扫描电镜观察PDT对种植体表面菌斑生物膜的影响.结果 当EDL为12J/cm2、HMME质量浓度为25 μg/ml时,PDT可有效杀灭Pg菌斑生物膜（（13.00±5.00） CFU）,且扫描电镜观察到种植体表面无损伤.结论 630nm半导体激光联合HMME介导的光动力疗法（HMME-PDT）对Pg菌斑生物膜有良好的杀灭效果,本实验使用的EDL与HMME剂量较小,有望作为临床治疗剂量.     

Symbiotic Relationship between Streptococcus mutans and Candida albicans Synergizes Virulence of Plaque Biofilms In Vivo

Streptococcus mutans is often cited as the main bacterial pathogen in dental caries, particularly in early-childhood caries (ECC). S. mutans may not act alone; Candida albicans cells are frequently detected along with heavy infection by S. mutans in plaque biofilms from ECC-affected children. It remains to be elucidated whether this association is involved in the enhancement of biofilm virulence. We showed that the ability of these organisms together to form biofilms is enhanced in vitro and in vivo. The presence of C. albicans augments the production of exopolysaccharides (EPS), such that cospecies biofilms accrue more biomass and harbor more viable S. mutans cells than single-species biofilms. The resulting 3-dimensional biofilm architecture displays sizeable S. mutans microcolonies surrounded by fungal cells, which are enmeshed in a dense EPS-rich matrix. Using a rodent model, we explored the implications of this cross-kingdom interaction for the pathogenesis of dental caries. Coinfected animals displayed higher levels of infection and microbial carriage within plaque biofilms than animals infected with either species alone. Furthermore, coinfection synergistically enhanced biofilm virulence, leading to aggressive onset of the disease with rampant carious lesions. Our in vitro data also revealed that glucosyltransferase-derived EPS is a key mediator of cospecies biofilm development and that coexistence with C. albicans induces the expression of virulence genes in S. mutans (e.g., gtfB, fabM). We also found that Candida-derived β1,3-glucans contribute to the EPS matrix structure, while fungal mannan and β-glucan provide sites for GtfB binding and activity. Altogether, we demonstrate a novel mutualistic bacterium-fungus relationship that occurs at a clinically relevant site to amplify the severity of a ubiquitous infectious disease.     

Effective immunity to dental caries: dose-dependent studies of secretory immunity by oral administration of Streptococcus mutans to rats.

Rats (COBS/CD) provided Formalin-killed Streptococcus mutans 6715, C211 in their drinking water (10(8) to 10(9) equivalent colony-forming units [CFU] per ml) had high levels of specific antibodies in saliva, colostrum, and milk. Rats provided a lower concentration of S. mutans antigen (10(7) CFU per ml) in water had agglutinin titers in secretions that were similar to those in controls. Gnotobiotic rats provided S. mutans antigen in food (10(7) to 10(8) equivalent CFU per g of diet) manifested a secretory immune response as evidenced by the presence of specific immunoglobulin A antibodies in saliva, colostrum, and milk. Gnotobiotic rats provided a higher concentration of antigen (10(9) CFU per g) in food had levels of specific antibodies in their secretions that were similar to those in controls. No significant antibody activity to S. mutans was observed in sera of any group of animals. Furthermore, the presence of specific salivary immunoglobulin A antibodies in gnotobiotic rats correlated with a reduction in the level of plaque, numbers of viable S. mutans in plaque, and levels of S. mutans-induced dental caries. This paper discusses the importance of antigen dosage for induction of a secretory immune response that is protective against S. mutans-induced dental caries.     

Mutation of luxS affects biofilm formation in Streptococcus mutans

Quorum sensing is a bacterial mechanism for regulating gene expression in response to changes in population density. Many bacteria are capable of acyl-homoserine lactone-based or peptide-based intraspecies quorum sensing and luxS-dependent interspecies quorum sensing. While there is good evidence about the involvement of intraspecies quorum sensing in bacterial biofilm, little is known about the role of luxS in biofilm formation. In this study, we report for the first time that luxS-dependent quorum sensing is involved in biofilm formation of Streptococcus mutans. S. mutans is a major cariogenic bacterium in the multispecies bacterial biofilm commonly known as dental plaque. An ortholog of luxS for S. mutans was identified using the data available in the S. mutans genome project (http://www.genome.ou.edu/smutans.html). Using an assay developed for the detection of the LuxS-associated quorum sensing signal autoinducer 2 (AI-2), it was demonstrated that this ortholog was able to complement the luxS negative phenotype of Escherichia coli DH5alpha. It was also shown that AI-2 is indeed produced by S. mutans. AI-2 production is maximal during mid- to late-log growth in batch culture. Mutant strains devoid of the luxS gene were constructed and found to be defective in producing the AI-2 signal. There are also marked phenotypic differences between the wild type and the luxS mutants. Microscopic analysis of in vitro-grown biofilm structure revealed that the luxS mutant biofilms adopted a much more granular appearance, rather than the relatively smooth, confluent layer normally seen in the wild type. These results suggest that LuxS-dependent signal may play an important role in biofilm formation of S. mutans.