影响变形链球菌生物膜形成因素的研究进展

龋病的发生发展过程中,致龋微生物生物膜(biofilm)形成是龋病发生的首要因素。变形链球菌(Streptococcus mutans)等在口腔生物膜内进行产酸代谢活动是产生龋病的直接原因。因此,控制S.mutans生物膜的形成可以减少龋病的发生、发展。本文就影响变形链球菌生物膜形成的因素研究进展做一综述。     

丹皮酚对变形链球菌生物膜影响的体外研究

目的 测定丹皮酚对变形链球菌的抑菌作用;共聚焦显微镜观察丹皮酚对变形链球菌生物膜结构和活性的影响。方法 梯度法测定丹皮酚对变形链球菌的MIC（最小抑菌浓度）、MBC（最小杀菌浓度）;体外构建变形链球菌生物膜模型,共聚焦显微镜观察不同浓度丹皮酚对变形链球菌生物膜作用后形态结构的影响并进行红绿荧光定量分析其活性变化。结果 丹皮酚对变形链球菌的MIC为6.25 mg/mL,MBC为25 mg/mL;激光共聚焦显微镜观察丹皮酚对变形链球菌生物膜作用后其生物膜结构变稀疏,细菌链变短,生物膜活性也随丹皮酚浓度的提高而逐渐降低。结论 丹皮酚对变形链球菌和变形链球菌生物膜结构及其活性均具抑制作用。     

替代失活法构建变形链球菌LuxS基因缺陷株

目的 LuxS基因是变形链球菌生物膜早期形成过程中的关键基因,构建该基因的缺陷菌。方法采用长臂同源多聚酶链反应（LFH-PCR）方法构建含红霉素耐药基因片段的LuxS基因上、下游同源序列的连接片段,转化到变形链球菌中,在红霉素的平板上筛选缺陷菌株,并采用PCR鉴定。结果对变形链球菌LuxS基因缺陷菌株进行PCR和DNA序列测定分析证实构建成功。结论成功构建出变形链球菌LuxS基因的缺陷菌株,为后期针对变形链球菌LuxS基因的相关研究奠定基础。     

变形链球菌UA159及其基因缺陷菌生长特性变化的初步研究

目的探讨变形链球菌密度信号系统相关基因缺陷后其生长特性的变化情况。方法分别配制BHI液体培养基,含2%葡萄糖的BHI液体培养基,含2%蔗糖的BHI液体培养基,然后将细菌接种于上述3个营养环境中生长,采用722S型可见光分光光度计,进行细菌生长曲线的测定。结果在3种不同的营养环境中,变形链球菌UA159△ComD基因缺陷菌株的生长速度最快,变形链球菌UA159菌株变形链球菌UA159△LuxS基因变异株次之,变形链球菌UA159野生菌株最慢。结论在本实验中,无论那种密度感应信号系统被阻断后,均会影响变形链球菌的生长。     

变形链球菌对不同牙科充填材料的粘附及早期生物膜的形成

目的探讨变形链球菌对不同牙科充填材料的粘附和早期生物膜的形成.方法比较经放射性同位素3H-TDR(3H-胸腺嘧啶核苷)标记的变形链球菌对3种唾液包被的充填材料的粘附.采用蛋白质测量试剂盒定量分析其对唾液蛋白的吸附量;采用凝胶电泳和图像分析系统定量分析其对唾液白蛋白和α-淀粉酶的吸收率.结果各种材料对变形链球菌的粘附能力,对唾液蛋白的吸附能力均随着材料的不同而不同.Fuji IX对细菌的粘附量很高,但是对蛋白的吸附量却很低;而F2000对细菌的粘附量很低,对蛋白的吸附量却很高.结论在不同充填材料表面形成的生物膜是不同的,提示早期生物膜的形成具有一定的特异性.这种生物膜的差异对口腔微生态环境及龋病和/或牙周病的发展具有重要意义.     

生物膜定量分析仪对不同细菌早期生物膜形成能力的比较研究

目的通过生物膜定量分析仪来观察铜绿假单胞菌(Pseudomonas aeruginosa PAO1),变形链球菌(Streptococcus mutans UA159)以及大肠埃希菌(Escherichia coli MG1655)生物膜形成能力的不同,并以各菌株的吸光度值A600为参考,对3种菌株早期生物膜形成能力进行比较。方法通过向生物膜培养悬液中加入与细菌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3种菌株在生物膜形成上的差别。结果实验发现铜绿假单胞菌PAO1和大肠埃希菌MG1655的细菌增长速度基本相同,但铜绿假单胞菌PAO1的生物膜形成明显快于大肠埃希菌MG1655。大肠埃希菌MG1655和变形链球菌UA159的生物膜形成速度基本相同,但大肠埃希菌MG1655的细菌增长速度明显高于变形链球菌UA159。结论不同细菌有各自的生物膜形成模式。生物膜定量分析仪作为一种高效简便的检测手段,可用于生物膜早期形成的动态分析。     

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.     

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（P<0.05）。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(P<0.05)。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。     

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。     

SYT09/PI荧光探针标记lasR rhlR基因缺陷对铜绿假单胞菌生物膜形成影响的体外实验研究

目的研究群体感应（QS）系统在铜绿假单胞菌生物膜（BF）形成中的作用。方法体外建立3 d QS系统完整的铜绿假单胞菌野生型PA01菌株与QS系统缺陷（lasRrhlR基因缺陷△lasR△rhlR）型菌株生物模型,通过SYT09/PI荧光探针标记,结合激光共聚焦显微镜摄取BF不同层面的图片,经图像结构分析（ISA）软件分析获得QS系统lasRrhlR缺陷株,PA01菌株BF相关空间结构参数。结果培养第3天PA01菌株可形成较厚、有孔状通道的成熟BF结构,而△lasR△rhlR菌株仅形成明显稀薄的早期BF结构,△lasR△rhlR菌株的3 d BF厚度为（7.36±0.2）μm,PA01菌株为（21.64±0.57）μm（P〈0.05）,区域孔率（areal porosity,AP）分别为：（0.902±0.006）、（0.928±0.002）;平均扩散距离（average diffusion distance,ADD）和结构熵（textural entropy,TE）在△lasR△rhlR菌株分别为：（1.503±0.029）和（5.706±0.190）;在PA01菌株分别为：（1.467±0.015）和（5.213±0.111）,△lasR△rhlR菌株AP较PA01菌株低（P〈0.05）;而ADD、TE较PA01菌株高（P〈0.05）。结论△lasR△rhlR基因缺陷明显影响铜绿假单胞菌的BF形成能力,QS系统lasRrhlR基因在铜绿假单胞菌BF形成中发挥重要作用。     

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（P<0.01）,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（P<0.01）。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。     

Protein expression by planktonic and biofilm cells of Streptococcus mutans

Streptococcus mutans, a major causal agent of dental caries, functions in nature as a component of a biofilm on teeth (dental plaque) and yet very little information is available on the physiology of the organism in such surface-associated communities. As a consequence, we undertook to examine the synthesis of proteins by planktonic and biofilm cells growing in a biofilm chemostat at pH 7.5 at a dilution rate of 0.1 h(-1) (mean generation time=7 h). Cells were incubated with (14)C-labelled amino acids, the proteins extracted and separated by two-dimensional electrophoresis followed by autoradiography and computer-assisted image analysis. Of 694 proteins analysed, 57 proteins were enhanced 1.3-fold or greater in biofilm cells compared to planktonic cells with 13 only expressed in sessile cells. Diminished protein expression was observed with 78 proteins, nine of which were not expressed in biofilm cells. The identification of enhanced and diminished proteins by mass spectrometry and computer-assisted protein sequence analysis revealed that, in general, glycolytic enzymes involved in acid formation were repressed in biofilm cells, while biosynthetic processes were enhanced. The results show that biofilm cells possess novel proteins, of as yet unknown function, that are not present in planktonic cells.     

群体感应抑制剂对海洋生态功能菌生物膜形成的影响

[目的]研究天然群体感应抑制剂(Quorum sensing inhibitors,QSI)分子对海洋生态功能菌生物膜形成的影响.[方法]以对污损生物幼虫附着具有诱导作用的海洋细菌为目标菌,通过在其生物膜的形成过程中添加天然群体感应抑制剂,研究其对目标菌成膜细菌数和浮游细菌数、生物膜形态以及生物膜表面胞外多糖含量的影响.[结果]呋喃酮和吡啶在50 mg/L时,对8株目标菌的成膜有显著的抑制作用,抑制率在80％左右,吲哚、青霉烷酸和香豆素在较高浓度800 mg/L才有比较好的抑制活性.生长抑制实验结果显示,同等浓度下,QSI分子对目标菌成膜的抑制活性明显高于其对浮游细菌生长的抑制活性.结果表明,QSI分子主要通过干扰目标菌群体感应系统以抑制生物膜的形成.[结论]研究证实QSI分子在海洋菌生物膜形成过程中具有一定的调控作用.通过添加QSI可能能够间接抑制由生物膜诱导的污损生物附着,从而以新的角度研制新型抗污损物质.     

Effects of quorum sensing on cell viability in Streptococcus mutans biofilm formation

Streptococcus mutans (S. mutans) uses a quorum sensing (QS) signaling system, which is dependent on competence stimulating peptide (CSP), to regulate diverse physiological activities including bacteriocin production, genetic transformation, and biofilm formation. However, the mechanism of the QS system-induced biofilm formation remains unclear. Here, we demonstrated that the late-stage biofilm formation was increased by the addition of exogenous CSP in S. mutans. The numbers of dead cells in biofilms formed in presence of CSP was 64.5% higher than that without CSP after 12 h (p < 0.05) and 76.3% higher after 24 h (p < 0.05), the numbers of live cells in biofilms formed in presence of CSP were 89.3% higher than that without CSP after 24 h (p < 0.01). The expression of QS-associated genes was increased 3.4-5.3-fold by CSP in biofilms. Our results revealed that cell viability of S. mutans grown in biofilms is affected by the CSP-dependent QS system.     

Signs of biofilm formation in the genome of Labrenzia sp. PO1

Labrenzia sp. are important components of marine ecology which play a key role in biochemical cycling. In this study, we isolated the Labrenzia sp. PO1 strain capable of forming biofilm, from the A. sanguinea culture. Growth analysis revealed that strain reached a logarithmic growth period at 24 hours. The whole genome of 6.21813 Mb of Labrezia sp. PO1 was sequenced and assembled into 15 scaffolds and 16 contigs, each with minimum and maximum lengths of 644 and 1,744,114 Mb. A total of 3,566 genes were classified into five pathways and 31 pathway groups. Of them, 521 genes encoded biofilm formation proteins, quorum sensing (QS) proteins, and ABC transporters. Gene Ontology annotation identified 49,272 genes that were involved in biological processes (33,425 genes), cellular components (7,031genes), and molecular function (7,816 genes). We recognised genes involved in bacterial quorum sensing, attachment, motility, and chemotaxis to investigate bacteria's ability to interact with the diatom phycosphere. As revealed by KEGG pathway analysis, several genes encoding ABC transporters exhibited a significant role during the growth and development of Labrenzia sp. PO1, indicating that ABC transporters may be involved in signalling pathways that enhance growth and biofilm formation.     

Streptococcus mutans glucan-binding protein-A affects Streptococcus gordonii biofilm architecture

The glucan-binding protein-A (GbpA) of Streptococcus mutans has been shown to contribute to the architecture of glucan-dependent biofilms formed by this species and influence virulence in a rat model. As S. mutans synthesizes multiple glucosyltransferases and nonglucosyltransferase glucan-binding proteins (GBPs), it is possible that there is functional redundancy that overshadows the full extent of GbpA contributions to S. mutans biology. Glucan-associated properties such as adhesion, aggregation, and biofilm formation were examined independently of other S. mutans GBPs by cloning the gbpA gene into a heterologous host, Streptococcus gordonii, and derivatives with altered or diminished glucosyltransferase activity. The presence of GbpA did not alter dextran-dependent aggregation nor the initial sucrose-dependent adhesion of S. gordonii. However, expression of GbpA altered the biofilm formed by wild-type S. gordonii as well as the biofilm formed by strain CH107 that produced primarily alpha-1,6-linked glucan. Expression of gbpA did not alter the biofilm formed by strain DS512, which produced significantly lower quantities of parental glucan. These data are consistent with a role for GbpA in facilitating the development of biofilms that harbor taller microcolonies via binding to alpha-1,6-linkages within glucan. The magnitude of the GbpA effect appears to be dependent on the quantity and linkage of available glucan.