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。     

电解质水对粪肠球菌生物膜的杀菌作用研究

目的:观察强酸性电解质水(SAEW)和碱性电解质水(AEW)对粪肠球菌生物膜的杀菌作用。方法:利用BioFluxTM200微流体系统建立粪肠球菌生物膜模型,并分别用SAEW、AEW、52.5 g/L次氯酸钠液(阳性对照)和生理盐水(阴性对照)处理1、5、10 min后,激光共聚焦显微镜(CLSM)观察并采集荧光图像;ImageJ图像分析软件分析并计算各处理组不同时间点的活菌比例。结果:SAEW、AEW和52.5 g/L次氯酸钠液处理1、5、10 min各时间点的活菌比例都显著低于生理盐水组(P<0.05),其中AEW组活菌比例下降幅度最小,与SAEW和52.5 g/L次氯酸钠液处理组相比,各时间点均有统计学差异(P<0.05);而SAEW与52.5 g/L次氯酸钠液处理组相比,各时间点的活菌比例均无统计学差异(P>0.05)。结论:SAEW和AEW对粪肠球菌均有杀菌作用,其中SAEW的作用与52.5 g/L次氯酸钠液相当;而AEW的杀菌效果较弱,且需要延长作用时间。     

茶多酚、MTAD和次氯酸钠液抑制粪肠球菌及其生物膜的作用比较

目的:检测中药茶多酚、MTAD、52.5 g/L次氯酸钠抑制根管壁粪肠球菌生物膜的能力,并分析中药茶多酚作为根管冲洗液的可行性。方法:采用微孔板滴定法检测茶多酚,MTAD和52.5 g/L次氯酸钠液对粪肠球菌的最小抑菌浓度(MIC)和最小杀菌浓度(MBC),采用琼脂扩散实验检测3种抗菌剂对粪肠球菌的抑菌圈。通过建立粪肠球菌感染根管3周和6周的模型,分别用茶多酚、MTAD和52.5 g/L次氯酸钠液对粪肠球菌感染后的根管进行处理,然后对根管壁生物膜中的细菌生存情况进行定性定量分析,比较这3种根管冲洗液抗粪肠球菌生物膜的能力。结果:MIC、MBC和琼脂扩散实验显示:3组冲洗剂对粪肠球菌均有抑制作用。在粪肠球菌感染根管3周的生物膜中,MTAD和次氯酸钠液可以完全抑制粪肠球菌生物膜,茶多酚处理后虽仍有粪肠球菌生长,但明显低于生理盐水处理组的细菌生存数,差异有统计学意义(P<0.05)。在粪肠球菌感染根管6周的生物膜中,次氯酸钠液可以完全抑制细菌生长,茶多酚和MTAD作用后仍有活菌生长,但与对照组相比,细菌生存数均显示了8个log值的下降,差异有统计学意义(P<0.05)。结论:52.5 g/L次氯酸钠液具有很强的抑制根管内粪肠球菌生物膜作用,而茶多酚和MTAD也具有一定的抑制粪肠球菌生物膜的能力。     

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。     

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.     

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。     

再治疗根管内粪肠球菌的分离鉴定及其相关特性研究

目的:研究粪肠球菌在再治疗根管内的检出率及其相关特性(生长情况、生物膜形成能力及耐药性)。方法:收集临床病例样本54例,使用粪肠球菌选择性培养基和胆盐七叶苷琼脂进行分离培养。分离株通过16SrRNA测序法鉴定。将鉴定正确的粪肠球菌临床分菌株和作为对照的粪肠球菌标准株(EfATCC29212)用BHI培养基进行培养,分别对比其生长曲线、生物膜形成能力及耐药性。结果:收集临床病例样本54例,分离鉴定为粪肠球菌的有18例,检出率为33.33%。经统计分析:粪肠球菌在再治疗根管内的检出率在病人性别、年龄、患牙尖周稀疏大小及距离上次根管治疗时间长短等方面无统计学差异(P>0.05);但与上次根管治疗根充是否到位有关,欠填根管中粪肠球菌的检出率明显高于恰填根管(P<0.05),但不同欠填长度组间粪肠球菌的检出率无统计学差异(P>0.05)。此外,粪肠球菌临床分离株与对照菌株在生长曲线、生物膜形成能力及耐药性等方面均无统计学差异(P>0.05)。结论:粪肠球菌在再治疗根管内的检出率为33.33%,其检出率与上次根管治疗根充情况有关。同时,粪肠球菌临床分离株与标准株的生长情况、生物膜形成能力及耐药性基本一致。     

纳米银颗粒联合氢氧化钙对饥饿期粪肠球菌生物膜的作用

目的:将氢氧化钙和纳米银联合,探讨其对饥饿期粪肠球菌生物膜的抑制效果.方法:使用256个牙根样本建立饥饿期粪肠球菌生物膜体外模型,使用平板菌落计数法和结晶紫染色法测定不同药物[Ca(OH)2+AgNP,Ca(OH)2,AgNPy不同作用时间(1、7d)对饥饿期粪肠球菌生物膜的抑制效果.以灭菌水作为实验对照组.采用SPSS13.0软件包对数据进行统计学分析.结果:在1d和7d时,Ca (OH)2+AgNP对粪肠球菌生物膜的抑制效果强于Ca (OH)2和AgNP;同时,AgNP对粪肠球菌生物膜的抑制效果显著强于Ca(OH)2.Ca(OH)2在7d时对粪肠球菌生物膜的抑制效果强于1d时;Ca(OH)2+AgNP和AgNP在7d和1d时对粪肠球菌生物膜的抑制效果无显著差异.结论:纳米银颗粒联合氢氧化钙对饥饿期粪肠球菌生物膜具显著的抑制作用.     

饥饿状态粪肠球菌生物膜胞外多糖的合成能力

目的探讨饥饿环境对粪肠球菌生物膜胞外多糖合成能力的影响。方法体外培养饥饿状态粪肠球菌形成48h生物膜,凝集素标记结合倒置荧光显微镜观察胞外多糖分布情况,提取饥饿状态浮游粪肠球菌和对数期、稳定期、饥饿状态粪肠球菌48h生物膜胞外多糖,蒽酮法定量分析比较胞外多糖合成能力。结果饥饿状态粪肠球菌形成的48h生物膜内胞外多糖呈云絮状,与细菌分布区域不完全一致;生物膜细菌合成水溶性和水不溶性胞外多糖的能力均高于浮游细菌(P<0.05);其合成水溶性胞外多糖的能力与对数期和稳定期细菌无显著差别(P>0.05),合成水不溶性胞外多糖的能力高于后两者(P<0.05)。结论饥饿状态粪肠球菌合成生物膜胞外多糖的能力增强。     

饥饿状态的粪肠球菌的研究进展

作为顽固性或继发性根管内感染的主要致病菌,粪肠球菌可以饥饿状态在恶劣的环境中长期生存。在饥饿状态下,粪肠球菌可保持较低的代谢水平并形成生物膜,从而提高其对外界刺激的耐受水平。本文就饥饿状态与活的不可培养状态,饥饿状态下的粪肠球菌的生长状况,饥饿状态下的粪肠球菌的生物膜形成能力,饥饿状态下的粪肠球菌对外界刺激的耐受水平等研究进展作一综述。     

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。     

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.     

粪肠球菌体外根尖生物膜模型的建立及形态学研究

目的 通过体外实验建立根尖生物膜模型,观察粪肠球菌根尖生物膜模型的形态及结构。方法 选取人新鲜拔除的单根离体牙24颗,随机分为8组,每组3颗,浸泡于粪肠球菌 ATCC 29212菌悬液中,分别于1、2、7d建立根尖生物膜模型。使用扫描电子显微镜（ SEM）观察生物膜形成过程;使用碘化丙啶（PI）和刀豆球蛋白 A异硫氰酸荧光素（ConA-FITC）对生物膜细菌及细胞外基质进行双染色,通过激光共聚焦扫描电子显微镜（CLSM）观察其形态和结构;计算生物膜覆盖率。结果 随时间的增加,根尖样本表面细菌覆盖面积增加,7d组与1、2d组间的差异存在统计学意义（P<0.05）, 1d与2d组间的差异无统计学意义（P>0.05）。 7d组形成生物膜结构,其样本表面的覆盖率为 17.23%±1.52%,样本表面有大量细菌附着,细菌被细胞外基质包绕,并呈群落分布。生物膜中的细菌被 PI染成红色,细胞外基质被 ConA-FITC染成绿色,可见多层次的空间结构以及亲水性通道。结论 粪肠球菌根尖生物膜于培养7d时形成完整的生物膜结构,由细菌群落及其周围大量的不定型基质组成。     

Biofilm formation in medicated root canals

The hypothesis that Enterococcus faecalis resists common intracanal medications by forming biofilms was tested. E. faecalis colonization of 46 extracted, medicated roots was observed with scanning electron microscopy (SEM) and scanning confocal laser microscopy. SEM detected colonization of root canals medicated with calcium hydroxide points and the positive control within 2 days. SEM detected biofilms in canals medicated with calcium hydroxide paste in an average of 77 days. Scanning confocal laser microscopy analysis of two calcium hydroxide paste medicated roots showed viable colonies forming in a root canal infected for 86 days, whereas in a canal infected for 160 days, a mushroom-shape typical of a biofilm was observed. Analysis by sodium dodecyl sulfate polyacrylamide gel electrophoresis showed no differences between the protein profiles of bacteria in free-floating (planktonic) and inoculum cultures. Analysis of biofilm bacteria was inconclusive. These observations support potential E. faecalis biofilm formation in vivo in medicated root canals.     

大蒜素标准品DATS体外抗粪肠球菌生物膜作用的研究

目的:探讨不同浓度大蒜素对照品二烯丙基化三硫(dially trisulfide,DATS)体外对粪肠球菌生物膜的作用。方法:体外培养形成粪肠球菌生物膜,分别经1280、2560、5120 mg/L浓度的DATS作用后,采用MTT法观察DATS对生物膜粪肠球菌的抑菌率。结果:1280 mg/L DATS对对数期和饥饿期的生物膜粪肠球菌抑菌率约71%,显著高于对稳定期生物膜粪肠球菌的抑菌率58%,差异具有统计学意义(P<0.05),随着药物浓度的增高,抑菌率增高。结论:DATS对生物膜中的粪肠球菌具有明显杀灭或抑制作用。     

变形链球菌生物膜对抗菌剂敏感性的研究

目的用激光共聚焦扫描显微镜（CLSM）观察变形链球菌生物膜对抗菌剂的敏感性.方法在体外形成变形链球菌生物膜,用不同浓度青霉素分别作用于生物膜3 h.生物膜进行荧光染色,CLSM扫描观察.结果变形链球菌生物膜在青霉素浓度为2 500mg/L时作用3 h,未被完全杀死,随着药物浓度增加,生物膜对青霉素的抵抗力逐渐减弱.结论变形链球菌生物膜比浮游状态变形链球菌对青霉素具有更高的抵抗力,提示临床应该以新的思路来研究控制生物膜造成的感染.     

An in vitro evaluation of the ability of ozone to kill a strain of Enterococcus faecalis

AIM: To evaluate the potential of ozone as an antibacterial agent using Enterococcus faecalis as the test species. METHODOLOGY: Ozone was produced by a custom-made bench top generator and its solubility in water determined by ultraviolet (258 nm) spectrophotometric analysis of solutions through which ozone was sparged for various time-periods. The antibacterial efficacy of ozone was tested against both broth and biofilm cultures. Ozone was sparged for 30, 60, 120 and 240 s, through overnight broth cultures of a strain of E. faecalis (E78.2) and compared with those that were centrifuged, washed and resuspended in water. Enterococcus faecalis (E78.2) biofilms were grown on cellulose nitrate membrane filters for 48 h and suspended in water through which ozone gas was sparged with stirring for 60, 120 and 240 s in a standard fashion. In a separate test, biofilms were also exposed to gaseous ozone. Sodium hypochlorite (NaOCl) was used as a positive control. All experiments were repeated four times. RESULTS: There were significant (P < 0.05) reductions of bacteria in the unwashed (2 log(10) reductions) and washed (5 log(10) reductions) broth cultures following 240 s applications. Biofilms incubated for 240 s with ozonated water showed no significant reduction in cell viability attributable to ozone alone, whereas with NaOCl no viable cells were detected over the same time. Gaseous ozone applied for 300 s had no effect on these biofilms. CONCLUSIONS: Ozone had an antibacterial effect on planktonic E. faecalis cells and those suspended in fluid, but little effect when embedded in biofilms. Its antibacterial efficacy was not comparable with that of NaOCl under the test conditions used.     

Eradication of enterococci biofilms by lactic acid alone and combined with chlorhexidine and cetrimide

Objective: The antimicrobial activity of lactic acid (LA) alone or in combination with chlorhexidine (CHX) and cetrimide (CTR) against three Enterococcus faecalis strains, E. faecalis ATCC 29212, E. faecalis EF-D1 and E. faecalis U-1765, one Enterococcus durans strain and one dual-species biofilm was investigated. Study Design: The irrigating solutions tested were 20%, 15%, 10%, 5% and 2.5% LA, alone and in combination with 2% CHX and with 0.2% CTR. The biofilms were grown in the MBECTM high-throughput device for 24 hours and exposed to the solutions for 30 seconds and 1 minute. "Eradication" was defined as 100% bacterial kill. Results: Twenty percent LA eradicated all enterococci biofilms after 30 seconds contact time. The association of LA + 0.2% CTR achieved better results than LA alone, in contrast with the results obtained using LA + 2% CHX. E. durans was eradicated by all the tested solutions at 1 minute. The dual-species biofilm, E. faecalis ATCC 29212 + E. durans, gave intermediate values of the pure cultures. Conclusions: LA is capable of eradicating enterococci biofilm at a concentration of 20%. The combination of lower concentrations with 0.2% CTR achieved eradication after 1 minute.     

饥饿期粪肠球菌生物膜毒力因子明胶酶E表达研究

目的:研究粪肠球菌生物膜形成相关毒力因子明胶酶E(gelE)在饥饿期及药物作用后的表达情况。方法:体外建立对数期、稳定期、饥饿期粪肠球菌生物膜模型,分别以1%、2.5%、5.25%次氯酸钠溶液作用于各时期粪肠球菌生物膜后,用Real-time PCR对gelE的基因表达相对量进行检测。结果:gelE基因表达相对量,饥饿期高于稳定期及对数期(P<0.05);用药后,3个期gelE基因表达相对量,5.25%次氯酸钠溶液组低于2.5%次氯酸钠溶液组和1%次氯酸钠溶液组(P<0.05)。结论:饥饿期粪肠球菌生物膜形成相关毒力因子gelE较对数期及稳定期表达增强。次氯酸钠浓度依赖性可抑制粪肠球菌毒力因子gelE的表达。     

Eradication of enterococci biofilms by lactic acid alone and combined with chlorhexidine and cetrimide

Objective: The antimicrobial activity of lactic acid (LA) alone or in combination with chlorhexidine (CHX) and cetrimide (CTR) against three Enterococcus faecalis strains, E. faecalis ATCC 29212, E. faecalis EF-D1 and E. faecalis U-1765, one Enterococcus durans strain and one dual-species biofilm was investigated. Study Design: The irrigating solutions tested were 20%, 15%, 10%, 5% and 2.5% LA, alone and in combination with 2% CHX and with 0.2% CTR. The biofilms were grown in the MBECTM high-throughput device for 24 hours and exposed to the solutions for 30 seconds and 1 minute. “Eradication” was defined as 100% bacterial kill. Results: Twenty percent LA eradicated all enterococci biofilms after 30 seconds contact time. The association of LA + 0.2% CTR achieved better results than LA alone, in contrast with the results obtained using LA + 2% CHX. E. durans was eradicated by all the tested solutions at 1 minute. The dual-species biofilm, E. faecalis ATCC 29212 + E. durans, gave intermediate values of the pure cultures. Conclusions: LA is capable of eradicating enterococci biofilm at a concentration of 20%. The combination of lower concentrations with 0.2% CTR achieved eradication after 1 minute. Key words:Biofilm, cetrimide, chlorhexidine, enterococcus durans, enterococcus faecalis.