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。     

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。     

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。     

负载氯己定的可注射水凝胶对粪肠球菌及生物膜的体外抑菌效果

目的:研究负载氯己定的结冷胶复合材料体外抗粪肠球菌及生物膜的效果。方法:通过添加钙离子交联剂,制备含有纳米羟基磷灰石的结冷胶复合材料,并有效负载氯己定。通过对细菌生长曲线测定、扫描电镜等评价水凝胶体外抗粪肠球菌的效果,结晶紫染色定量和共聚焦激光扫描显微镜观察水凝胶对生物膜的作用。结果:随着水凝胶中氯己定浓度的增加,抗菌活性逐渐增强。当氯己定浓度为50μg/mL时,可显著抑制细菌生长及生物膜形成;当浓度为250μg/mL时,可表现出显著杀菌性。结论:负载氯己定的结冷胶复合材料抑制粪肠球菌生长及清除生物膜作用明显,有望作为新型可注射的根管消毒材料,促进顽固性根尖周炎的愈合。     

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.     

法尼醇对白念珠菌和粪肠球菌混合生物膜体外抑制作用的研究

目的 探讨法尼醇对白念珠菌和粪肠球菌混合生物膜的抑制作用.方法 采用微量平板法制备24 h和48 h白念珠菌和粪肠球菌混合生物膜,每组膜分别加入不同浓度法尼醇(100～800μmol/L)培养24 h,XTT减低法检测法尼醇对白念珠菌和粪肠球菌混合生物膜的抑制作用,倒置显微镜下观察混合生物膜形态,激光共聚焦显微镜下观察...     

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。     

粪肠球菌生物膜的药物敏感性研究

目的 建立体外粪肠球菌生物膜模型,评价10%氢氧化钙溶液和2%洗必泰溶液去除粪肠球菌生物膜的效果.方法 在玻片上形成4、8、12、24、48 h粪肠球菌生物膜,荧光染色后,采用激光共聚焦扫描显微镜(CLSM)观察;用10%氢氧化钙溶液和2%洗必泰溶液分别处理24 h生物膜3、10、30 min,采用CLSM观察;ANO...     

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。     

根管治疗后疾病中粪肠球菌的致病性和检测及清除

粪肠球菌是根管治疗后疾病最常见的细菌,在再感染根管内的检出率为24%~77%。粪肠球菌的致病性与其形成的生物膜高度相关,而其耐药性也与其致病性密不可分,是引发根管内慢性感染的关键。粪肠球菌的检测以细菌培养和聚合酶链反应（PCR）为主,而PCR用于检测感染根管内粪肠球菌较细菌培养更为敏感。根管治疗后疾病中粪肠球菌的清除方法多种多样,结果各不相同。有研究显示抑菌率,质量分数2%的氯己定为100%,10%的盐酸氯丙嗪为88.8%,4%的利多卡因凝胶为76.4%和5%的盐酸阿米洛利为71.4%;有研究则显示,混合物-四环素-异构体-酸-去污剂、QMiX和次氯酸钠皆较2%氯己定更有效。还有研究显示,铒：钇-铝石榴石激光对粪肠球菌生物膜有清除作用。     

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.     

In vitro Enterococcus faecalis biofilm formation on five adhesive systems

Objective: To determine the E. faecalis biofilm formation on the surface of five adhesive systems (AS) and its relationship with roughness. Study Design: The formation of E. faecalis biofilms was tested on the surface of four dual-cure AS: AdheSE DC, Clearfil DC Bond, Futurabond DC and Excite DSC and one light-cure antimicrobial AS, Clearfil Protect Bond, after 24 hours of incubation, using the MBEC high-throughput device. Results: E. faecalis biofilms grew on all the adhesives. The least growth of biofilm was on Excite DSC, Clearfil Protect Bond, and the control. Futurabond DC resulted in the greatest roughness and biofilm amount. There was a close relationship between the quantity of biofilm and roughness, except for Clearfil Protect Bond, which showed little biofilm but high roughness. Conclusion: None of the tested AS prevented E. faecalis biofilm formation, although the least quantity was found on the surface of Clearfil Protect Bond. Key words:Adhesive systems, biofilm, Enterococcus faecalis, roughness.     

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.     

Effect of Long-term Exposure to Peptides on Mono- and Multispecies Biofilms in Dentinal Tubules

IntroductionThe aim of this study was to evaluate the antibiofilm effectiveness of 2% chlorhexidine (CHX) and peptides 1018 and DJK-5 used either alone or in a mixture (peptide and 2% CHX) against Enterococcus faecalis and multispecies biofilms in dentin canals after short-term and long-term exposure.MethodsOne hundred eighty dentin blocks were prepared and filled with E. faecalis or multispecies bacteria by centrifugation. Three-week-old biofilms in dentin were subjected to 2% CHX, DJK-5 (10 μg/mL), 1018 (10 μg/mL), DJK-5 + 2% CHX, or 1018 + 2% CHX for short-term (1 or 3 minutes), short-term exposure after 24 hours, and long-term exposure (24 hours of exposure). The antibacterial efficacy was determined by live/dead bacterial viability staining and confocal laser scanning microscopy.ResultsPeptide DJK-5 with or without CHX was the most effective agent against all the biofilms (P < .05), killing 77% of biofilm bacteria in 1 minute. No significant difference in bacterial killing was detected between the first 3 minutes of exposure (>81%) and after 24 hours of exposure (83%) to DJK-5 or DJK-5 + CHX. Chlorhexidine and peptide 1018 had a weaker antibiofilm effect than DJK-5, and their effect was time dependent (P < .05) with a maximum killing of 60% after 24 hours of exposure.ConclusionsPeptide DJK-5 alone and together with CHX had a rapid antibacterial effect against dentin infection. An additional antibacterial effect by CHX and peptide 1018 was achieved after a 24-hour long-term exposure.     

红芪多糖对脂多糖诱导的牙周膜细胞凋亡及对Wnt信号通路的影响

目的:探讨红芪多糖对脂多糖(LPS)诱导的牙周膜细胞凋亡及对Wnt信号通路的影响。方法:体外分离培养人牙周膜细胞。实验分为3组,依次为对照组、脂多糖组和红芪多糖组。对照组细胞用正常的细胞培养液培养细胞;脂多糖组和红芪多糖组细胞用含有脂多糖浓度为10 mg/L的培养液培养细胞;红芪多糖组细胞培养液中加入红芪多糖终浓度为10 mg/L的培养液。流式细胞仪检测细胞凋亡情况,探针法检测细胞内的活性氧(ROS)水平。试剂盒检测细胞中碱性磷酸酶活性和分泌的肿瘤坏死因子-α(TNF-α)水平。Western blot检测细胞中c-myc、β-连环蛋白(β-catenin)、Bcl-2相关X蛋白(Bax)单克隆抗体、B细胞淋巴瘤/白血病-2(Bcl-2)蛋白水平。结果:脂多糖组细胞存活率明显低于对照组(P<0.01)。脂多糖组细胞凋亡率和Bax水平、ROS水平明显高于对照组(P<0.01)。红芪多糖组细胞凋亡率和Bax水平、ROS水平明显低于脂多糖组(P<0.01)。脂多糖组细胞Bcl-2、c-myc、β-catenin水平和碱性磷酸酶活性明显低于对照组(P<0.01)。红芪多糖组细胞Bcl-2、c-myc、β-catenin水平和碱性磷酸酶活性明显高于脂多糖组(P<0.01)。脂多糖组细胞分泌的TNF-α水平明显高于对照组(P<0.01)。红芪多糖组细胞分泌的TNF-α水平明显低于脂多糖组(P<0.01)。结论:红芪多糖能够抑制脂多糖诱导的人牙周膜细胞凋亡,抑制细胞分泌TNF-α,作用机制可与Wnt信号通路、ROS水平有关。     

表没食子儿茶素没食子酸酯对牙龈卟啉单胞菌细菌毒力抑制作用的体外研究

目的 研究表没食子儿茶素没食子酸酯(epigallocatechin-3-gallate,EGCG)对牙龈卟啉单胞菌(Por-phyromonas gingivalis,P.gingivalis)体外抑菌活性及EGCG对P.gingivalis诱导人牙龈成纤维细胞(human gingi-val fibroblasts...     

Validation of Antibiotic Efficacy on In Vitro Subgingival Biofilms

Background: Systemic antibiotics are often used as adjunctive treatment modalities for periodontal diseases. Testing of antimicrobial efficacy can be relevant only if the bacteria are in the form of biofilms rather than the planktonic state, and at concentrations of physiologic relevance, i.e., reachable in the periodontal pocket. The aim of the present study is to test the antimicrobial efficacy of five common antibiotic schemes at physiologically relevant concentrations on a multispecies in vitro biofilm model. Methods: A 10‐species in vitro subgingival biofilm model was exposed to metronidazole (15 μg/mL), amoxicillin (15 μg/mL), metronidazole and amoxicillin in combination, doxycycline (2 μg/mL), and azithromycin (10 μg/mL) over 24 hours. Species‐specific bacterial numbers were determined by culture on selective agar media or by epifluorescence microscopy. Results: Metronidazole alone did not affect biofilm composition. Total bacterial counts were significantly reduced by doxycycline, azithromycin, and amoxicillin alone or in combination with metronidazole, albeit by less than 1 log. On the species‐specific level, these regimens significantly reduced the numbers of Streptococcus anginosus, Porphyromonas gingivalis, and Fusobacterium nucleatum, as well as Campylobacter rectus (except for amoxicillin alone). The strongest effects were displayed by the combination of amoxicillin and metronidazole. Conclusion: Antibiotics at concentrations detectable in gingival crevicular fluid do not dramatically reduce total bacterial loads in this in vitro biofilm model, but cause species‐specific reductions, which may disrupt the biofilm unity.     

氢氧化钙与二甲亚砜对粪肠球菌联合抗菌作用的实验观察

目的：研究氢氧化钙和二甲亚砜对粪肠球菌体外联合药敏实验中的相互作用及联合后化学性质的变化。方法：参照美国国家临床试验标准化委员会（NCCLS）提出的标准，采用棋盘试管稀释法对粪肠球菌进行了氢氧化钙与二甲亚砜体外联合药敏实验并检测了其pH值变化。结果：氢氧化钙与二甲亚砜联合时对粪肠球菌的抑菌浓度分数指数（FICI）=0．8，从羟基离子的角度考虑时，对粪肠球菌的FICI=0．35。氢氧化钙对粪肠球菌的最低抑菌浓度（MIC）=3．5mg／mL，相应pH=10．03，最低杀菌浓度（MBC）=4．5mg／mL，相应pH=11．05，二甲亚砜对粪肠球菌的MIC=20％，相应pH=7．50，MBC=40％，相应pH=8．20。结论：氢氧化钙与二甲亚砜体外联合对粪肠球菌的联合抗菌作用表现为协同相加作用，无拈抗作用。