原子力显微镜观察粪肠球菌的超微结构及生物膜的动态形成过程

目的应用原子力显微镜（AFM）观察生理状态下粪肠球菌的表面超微结构及粪肠球菌生物膜的动态形成过程。方法利用AFM观察生理状态下粪肠球菌的超微结构并进行三维成像。利用硝酸纤维素薄膜在体外构建粪肠球菌生物膜模型,用AFM观察粪肠球菌生物膜形成过程中的表面变化。结果粪肠球菌菌体呈球状,表面凹凸不平,被颗粒状物包绕。初步确定了6 h粪肠球菌生物膜逐渐形成,24 h生物膜维持稳定状态,观察到细菌表面局部特征的变化及细菌胞外的多聚体物质。结论应用AFM能够在生理条件下清晰地观察粪肠球菌表面超微结构及粪肠球菌生物膜的整个动态形成过程。     

难治性根尖周炎根尖生物膜内粪肠球菌的检出及临床意义

目的探讨难治性根尖周炎根尖生物膜内的粪肠球菌的检出率,分析粪肠球菌的检出率与临床表现的关系。方法按纳入标准收集需要进行根尖外科手术的单根管患牙42颗,记录症状和体征,采集根尖生物膜样本,各样本均采用生化鉴定和聚合酶链反应(PCR)两种方法检测粪肠球菌。统计学分析两种方法对粪肠球菌的检出率差异及其与患者症状体征之间的关系。结果生化鉴定和PCR检测难治性根尖周炎根尖生物膜内粪肠球菌的检出率分别为52.4%和71.4%,差异有统计学意义(P<0.05)。PCR检测发现在疼痛组粪肠球菌的检出率高于无疼痛组(P<0.05)。结论 PCR检测难治性根尖周炎根尖生物膜内粪肠球菌检出率较高,且粪肠球菌的检出与疼痛存在相关性。     

粪肠球菌生物膜细胞外聚合物的研究进展

粪肠球菌是根管治疗后再感染的主要致病菌,常以生物膜的形式存在。包绕在细菌周围或黏附在细菌表面的糖类、核酸和分泌蛋白对粪肠球菌的初始黏附、生物膜空间结构和细菌间信息交流等起重要的作用。本文就粪肠球菌生物膜及其细胞外聚合物组分和去除方法作一综述。     

胞外多糖对粪肠球菌单菌种生物膜形成的影响

目的:观察粪肠球菌单菌种生物膜的动态形成过程,探讨胞外多糖在粪肠球菌生物膜形成过程中的分布和作用。方法:粪肠球菌在盖玻片上形成单菌种生物膜,采用荧光技术标记胞外多糖和细菌,荧光显微镜观察生物膜结构和胞外多糖的分布;蒽酮法测定粪肠球菌在游离和粘附状态下产生水溶性胞外多糖(Water soluble exopolysaccharides,WSE)和水不溶性胞外多糖(Water insoluble exopolysaccharides,WIE)的能力变化。结果:粪肠球菌能在玻片表面粘附形成生物膜,生物膜中胞外多糖分布与菌落中的细菌分布一致,相互包裹成熟形成网络状结构。不同培养时间、不同生存状态粪肠球菌合成WSE和WIE的能力不同,差异有显著性(P<0.05)。粘附菌产生WSE的能力往往低于浮游菌,但是粘附菌产生WIE均高于浮游菌,差异具有统计学意义(P<0.05)。结论:胞外多糖在粪肠球菌生物膜形成过程中发挥重要作用。     

不同冲洗方法对粪肠球菌感染根管的抗菌性研究

目的比较超声、Er,Cr：YSGG激光及Er：YAG激光辅助1%次氯酸钠（NaOCl）溶液冲洗对人离体牙根管表面粪肠球菌生物膜及根尖区不同深度牙本质小管内粪肠球菌的杀灭效果。 方法单直根管下颌前磨牙42颗,建立粪肠球菌生物膜感染的根管模型,采用随机字表法随机抽取2颗确定建成粪肠球菌生物膜感染根管模型,剩余40颗牙采用随机数字表法按随机对照原则分为5组,每组8颗。A组：5.25% NaOCl联合17%乙二胺四乙酸（EDTA）注射器冲洗;B组：0.9%氯化钠溶液注射器冲洗;C组：1% NaOCl溶液超声荡洗;D组：1% NaOCl溶液Er：YAG激光活化冲洗;E组：1% NaOCl溶液Er,Cr：YSGG激光活化冲洗。按分组进行处理后取样菌落计数,计算灭菌率。使用SPSS 17.0统计软件对实验数据进行方差齐性及正态性检验比较各组间和组内的灭菌率。 结果（1）组间比较：对根管壁表面,各组冲洗方法灭菌率比较,B组（5.74%）相似文献   

不同方法建立实验性狗根尖周炎模型的比较

目的：对比研究不同根尖周炎致病菌、内毒素分组注入根管内制备动物根尖周炎模型的效果。方法：将80个犬牙根管失活牙髓后分成四个组,分别封混合菌（A组）,细菌内毒素（B组）,粪肠球菌（C组）,生理盐水（D组）,术后不同阶段拍摄根尖周X线片观察分析。结果：内毒素组术后30 d出现根尖周阴影;混合菌组术后45d出现根尖周阴影,进展迅速;粪肠球菌组术后90d有阴影出现;对照组在实验观察期间均未见根尖周异常表现。结论：犬牙失活牙髓后封内毒素或混合菌能较快有效形成根尖周炎症。     

碱性pH对浮游状态和生物膜状态粪肠球菌活性的影响

目的:比较生物膜状态与浮游状态下粪肠球菌对碱的耐受性。方法:制备浮游状态和生物膜状态的粪肠球菌细胞,用pH值分别为7、8、9、10、11和12的培养液作用2h,利用MTT比色法比较2种状态下细菌细胞活性变化。采用SAS6.12软件包对数据进行统计学分析。结果:低碱性环境(pH值7～9)对粪肠球菌的生长无明显影响,高碱性环境(pH值>10)下存活细菌的比例明显减少;高pH环境下,生物膜状态的粪肠球菌存活细菌比例显著高于浮游状态下细菌的存活比例。结论:粪肠球菌对碱性环境具有强耐受性,形成生物膜是粪肠球菌抵抗高碱性环境的一个重要原因。     

粪肠球菌和变异链球菌脂磷壁酸的生物学活性

粪肠球菌是难治性根尖周炎根管内的主要致病菌。变异链球菌是引起龋齿的主要致病菌。粪肠球菌和变异链球菌的毒力因子脂磷壁酸（LTA）皆可促进细胞分泌炎症因子,引发炎症反应,促进根尖周牙槽骨吸收;有助于细菌黏附于牙体表面和生物膜的形成。粪肠球菌和变异链球菌LTA在细菌致病性方面起着重要的作用,但其机制尚不清楚。因此,研究LTA的致病机制及分子结构,旨在实现阻断其致病通路、降低其致病性。     

17％乙二胺四乙酸冲洗液对牙本质粪肠球菌黏附的影响

目的 研究17%乙二胺四乙酸（EDTA）冲洗液对促进牙本质粪肠球菌黏附的影响。方法 将48例半劈样本及12例牙本质片样本随机分为3个实验组和1个对照组,实验组分别经17%EDTA处理1、3、5 min,对照组经生理盐水处理5 min,接种粪肠球菌后通过扫描电子显微镜、激光共聚焦电子显微镜（CLSM）、菌落形成单位及组织学方法进行细菌黏附量的评价。结果 除组织学革兰染色显示,1 min组与对照组、3 min组的细菌侵入深度无明显差异（P>0.05）外,其余结果显示,实验组细菌生物膜厚度、侵入牙本质小管深度（CLSM测量）及菌落计数均大于对照组,差异具有统计学意义（P<0.05）;3个实验组之间比较,差异也具有统计学意义（P< 0.05）。结论 17%EDTA冲洗液可促进粪肠球菌对牙本质的黏附,且黏附量随处理时间的延长而增加。     

17%乙二胺四乙酸冲洗液对牙本质粪肠球菌黏附的影响

目的 研究17%乙二胺四乙酸（EDTA）冲洗液对促进牙本质粪肠球菌黏附的影响。方法 将48例半劈样本及12例牙本质片样本随机分为3个实验组和1个对照组,实验组分别经17%EDTA处理1、3、5 min,对照组经生理盐水处理5 min,接种粪肠球菌后通过扫描电子显微镜、激光共聚焦电子显微镜（CLSM）、菌落形成单位及组织学方法进行细菌黏附量的评价。结果 除组织学革兰染色显示,1 min组与对照组、3 min组的细菌侵入深度无明显差异（P>0.05）外,其余结果显示,实验组细菌生物膜厚度、侵入牙本质小管深度（CLSM测量）及菌落计数均大于对照组,差异具有统计学意义（P<0.05）;3个实验组之间比较,差异也具有统计学意义（P< 0.05）。结论 17%EDTA冲洗液可促进粪肠球菌对牙本质的黏附,且黏附量随处理时间的延长而增加。     

Confocal laser scanning microscopy is appropriate to detect viability of Enterococcus faecalis in infected dentin

The purpose of this study was to explore the potential of confocal laser scanning microscopy (CLSM) for in situ identification of live and dead Enterococcus faecalis in infected dentin. Eight cylindrical dentin specimens were infected with Enterococcus faecalis in BHI for 21 days. After the experimental period, the specimens were stained with fluorescein diacetate (FDA) and propidium iodide (PI) or acridine orange (0.01%) and analyzed by CLSM. Two noninfected dentin specimens were used as negative controls. CLSM analysis shows that the discrimination between viable (green) and dead (red) bacteria in infected dentinal tubules could be observed after staining with FDA/PI. Acridine orange was able to show metabolic activity of the E. faecalis cells inside the dentinal tubules showed by its red fluorescence. The viability of bacteria in infected dentin can be determined in situ by CLSM. FDA/PI and acridine orange are useful for this technique.     

纳米银干预牙本质表面粪肠球菌粘附的实验研究

目的:研究纳米银对牙本质表面粪肠球菌粘附的干预作用。方法:制备牙本质片42个,随机均分成3组,分别置于0.1%纳米银溶液、1.313%次氯酸钠溶液和生理盐水内浸泡30min后,于200μL粪肠球菌悬液中培养1h,用扫描电镜观察牙本质表面形态和粪肠球菌粘附情况,荧光显微镜分析牙本质表面粘附的粪肠球菌数量并拍照记录,Image pro plus 6.0软件计算粪肠球菌总菌数、活菌数和活菌百分比。结果:0.1%纳米银溶液组和生理盐水组牙本质表面形态清晰无破坏,1.313%次氯酸钠溶液组牙本质溶融坍塌,胶原溶解破坏。牙本质经0.1%纳米银溶液处理后,其表面粘附的粪肠球菌总菌数、活菌数和活菌百分比均低于1.313%次氯酸钠溶液组和生理盐水组(P﹤0.01)。结论:0.1%纳米银溶液可有效减少粪肠球菌对牙本质的粘附,对牙本质表面形态无明显破坏。     

激光扫描共聚焦显微镜观察碱性环境对粪肠球菌生物膜的影响

目的:比较不同碱性条件对生物膜状态粪肠球菌的影响。方法:制备粪肠球菌生物膜,分别用pH值为7、9和11的TSB培养液作用2 h后,用激光扫描共聚焦显微镜观察粪肠球菌生物膜的变化。结果:pH值由7升到9时,生物膜内、中、外各层活菌比例虽有所减少,但差异无统计学意义(P>0.05);当pH值11时,各层活菌比例虽然均较pH值为7和9时明显减少(P<0.05),但仍有大量活菌存在。结论:粪肠球菌对碱性环境具有强的耐受性,pH值为7～9时,对生物膜状态粪肠球菌无明显影响。     

洗必泰葡萄糖酸盐对感染根管细菌生物膜的实验研究

目的：探讨洗必泰葡萄糖酸盐作为根管冲洗药物,对粪肠球菌生物膜的灭菌作用。方法：在无菌盖玻片上制备粪肠球菌生物膜,应用0．2％和5％洗必泰葡萄糖酸盐冲洗,分别作用1min和5min,激光扫描共聚焦显微镜观察灭菌效果。结果：0．2％或5％洗必泰葡萄糖酸盐的灭菌效果随作用时间的延长而增加（P＜0．05）;5％洗必泰葡萄糖酸盐作用1min的灭菌效果明显优于0．2％洗必泰葡萄糖酸盐（P＜0．05）,而其作用5min的灭菌效果与0．2％洗必泰葡萄糖酸盐者无差异（p＞0．05）。结论：洗必泰葡萄糖酸盐具有明显的杀灭粪肠球菌生物膜的作用,其不同浓度和不同作用时间的灭菌效果不同,然而尚不能达到完美的根管冲洗消毒目的。     

Antimicrobial activity and enterococcus faecalis biofilm formation on chlorhexidine varnishes

Objective: To evaluate, in vitro, the antimicrobial activity and biofilm formation of three chlorhexidine varnishes in four Enterococcus faecalis strains: E. faecalis ATCC 29212, E. faecalis EF-D1 (from failed endodontic treatment), E. faecalis 072 (cheese) and E. faecalis U-1765 (nosocomial infection), and one Enterococcus durans strain (failed endodontic treatment). Study Design: The direct contact test was used to study the antimicrobial activity. Bacterial suspensions were exposed for one hour to EC40, Cervitec (CE) and Cervitec Plus (CEP) varnishes. “Eradication” was defined as 100% bacterial kill. The formation of enterococci biofilms was tested on the surface of the varnishes after 24 hours of incubation and expressed as percentage of biofilm reduction. Results: EC40 eradicated all strains except E. faecalis ATCC 29212, where 98.78% kill was achieved. CE and CEP showed antimicrobial activity against all the strains, but most clearly against E. durans and E. faecalis 072. EC40 completely inhibited the formation of biofilm of E. faecalis ATCC 29212, E. faecalis 072 and E. durans. CE and CEP led to over 92% of biofilm reduction, except in the case of E. faecalis U-1765 on CEP (76.42%). Conclusion: The three varnishes studied were seen to be effective in killing the tested strains of enterococci and in inhibiting the formation of biofilm, the best results being observed with EC40. Key words:Biofilm, chlorhexidine varnish, direct contact test, Enterococcus durans, Enterococcus faecalis, intracanal medication.     

三种无机纳米抗菌剂对粪肠球菌的抗菌作用

目的:研究纳米银、载银纳米二氧化钛、掺氮纳米二氧化钛对粪肠球菌的抗菌作用。方法:将粪肠球菌在MBECTMP&G Assay中培养24 h,构建粪肠球菌感染模型,扫描电镜观察MBECTMP&G Assay上粪肠球菌感染情况。应用MBECTMP&G Assay以连续稀释法检测纳米银、载银纳米二氧化钛、掺氮纳米二氧化钛的最小抑菌浓度(MIC)和最小生物膜清除浓度(MBEC)。结果:扫描电镜下见粪肠球菌在MBECTM P&G Assay上形成生物膜。纳米银对粪肠球菌的MIC和MBEC分别为62.5 mg/L和1 000 mg/L,载银纳米二氧化钛MIC为64 g/L,但在所用浓度范围内均不能清除生物膜细菌,即未测得MBEC;掺氮纳米二氧化钛的MIC、MBEC均未测得;次氯酸钠的MIC和MBEC分别为6.56 g/L和13.13 g/L。结论:纳米银对粪肠球菌有较强抗菌作用,能清除粪肠球菌生物膜;粪肠球菌对载银纳米二氧化钛和掺氮纳米二氧化钛有较强抵抗力。     

Single species biofilm-forming ability of root canal isolates on gutta-percha points

The participation of bacterial biofilms in the over-filled gutta-percha points associated with refractory periapical periodontitis has recently been reported. This study investigated the initial biofilm-forming ability of root canal isolates (Enterococcus faecalis, Streptococcus sanguis, Strep. intermedius, Strep. pyogenes, Staphylococcus aureus, Fusobacterium nucleatum, Propionibacterium acnes, Porphyromonas gingivalis and Prevotella intermedia) on gutta-percha points in vitro. Each bacterial strain was suspended in 100% cell culture medium or in culture medium containing 4.5, 45 or 90% (vol/vol) serum. The bacterial suspensions were then co-incubated anaerobically with gutta-percha points for 7 d. The gutta-percha points were processed for scanning electron microscopic observation and examined for biofilm presence and thickness. E. faecalis, Strep. sanguis, Strep. intermedius, Strep. pyogenes and Staph. aureus biofilms were generated on the surfaces of the specimens incubated in culture medium supplemented with 45 or 90% (vol/vol) serum. The E. faecalis and Strep. sanguis biofilms were significantly thicker than those of Strep. intermedius, Strep. pyogenes and Staph. aureus. No biofilms were detected on the specimens incubated with F. nucleatum, Prop. acnes, Porph. gingivalis and Prev. intermedia. These findings suggest that Gram-positive facultative anaerobes have the ability to colonize and form extracellular matrices on gutta-percha points, while serum plays a crucial role in biofilm formation.     

The effect of dental restorative materials on dental biofilm

To investigate the arrangement of biofilms formed in vivo, volunteers wore splints with slabs of six different dental materials inserted to collect smooth surface plaque. After 5 d of undisturbed plaque accumulation, the specimens were vital stained and analyzed by the confocal laser scanning microscopy (CLSM) to evaluate the percentage of vital biofilm microflora (VF percentage). Further parameters were the area of the specimens covered by plaque (surface coating; SC, %) and the height of the biofilms (BH, pm). The metals amalgam and gold, the compomer, as well as the glass-ionomer cement harboured an almost entirely dead biofilm (VF <8%). Resin composite led to vitality values between 4 and 21%, while a very thin biofilm on ceramic revealed the highest vitality values (34-86%). SC varied from 6% on glass-ionomer cement to 100% on amalgam. BH reached its highest value on amalgam and gold of 17 and 11 microm, respectively, while heights of between 1 and 6 microm were found on the ceramic, resin composite, compomer and the glass-ionomer cement. Within their limits, the present findings indicate that amalgam, gold, compomer and glass-ionomer cement exert an influence against the adhering biofilm. No general relationship could be established between the different parameters VF percentage, SC percentage and BH (microm).     

纳米银对多菌种生物膜中粪肠球菌的杀菌作用

目的:研究纳米银对多菌种生物膜中粪肠球菌的杀菌作用.方法:用粪肠球菌、产黑色素普雷沃菌以及具核梭杆菌构建多菌种生物膜85 个.分别用0.1%(12~15 nm、100 nm)纳米银悬浊液以及2%的次氯酸钠溶液处理,然后通过qPCR方法检测样本中粪肠球菌的活菌量和粪肠球菌的细菌总量.配对t检验比较各组细菌样本临界循环数(Ct).结果:各个实验组使用和不使用PMA之间粪肠球菌的细菌计数有着差别(P=0.000);将3 个实验组使用和不使用PMA qPCR检测到的Ct值的差值进行比较:纳米银(12~15 nm)实验组最大,纳米银(100 nm)实验组次之,次氯酸钠实验组最小(P<0.05).结论:纳米银对于多菌种生物膜中的粪肠球菌有着较强的杀菌作用,直径较小的纳米银杀菌作用较强.     

Histological assessment of periodontally involved cementum

Contamination of periodontally involved cementum by bacterial substances such as lipopolysaccharide (LPS) is considered a major reason for root planing. The purpose of the present study was to investigate the presence and location of lipid and polysaccharide within involved cementum as compared with uninvolved cementum. Frozen sections were prepared from the decalcified roots of 36 periodontally diseased and two control teeth. Serial sections were stained for either lipid (Oil-Red-O) or polysaccharide (Alcian Blue - PAS) and also with haematoxylin & eosin (H & E) or Huberstone's gram stain. Specimens of involved and uninvolved cementum were then examined under the light microscope for assessment of differences. Involved cementum from 12 of the periodontally diseased teeth exhibited strongly PAS-positive stained processes penetrating 3-7 mum into the surface of cementum from overlying plaque. Such processes were not observed in uninvolved cementum, suggesting a possible bacterial origin. Lipid granules were noted in only one involved specimen where they were situated up to 10 mum beneath the cemental surface. Similar granules were observed within plaque deposits but never in uninvolved cementum, again suggesting a possible bacterial origin. H & E and gram-stained specimens revealed the presence of microbial deposits in surface defects and within defects at the cemento-dentinal junction (CDJ), as well as penetration of micro-organisms into cementum in the absence of any surface defects. The results indicate that although lipid and polysaccharide of possible bacterial origin may be present within the 10 mum surface zone of involved cementum, the finding of microbial deposits down to the level of the CDJ suggests that all periodontally involved cementum should be removed during root planing, in order to achieve a root surface free of bacterial contamination.