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。     

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。     

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.     

持续性根尖周炎优势菌粪肠球菌生物膜形成能力的体外研究

目的：比较持续性根尖周炎优势细菌粪肠球菌( Enterococcus faecalis,E． faecalis)标准菌株与临床菌株生物膜体外形成能力。方法 E． faecalis 标准菌株 ATCC 29212及持续性根尖周炎分离的 E． faecalis 临床株lcl1709,体外形成生物膜,采用激光扫描共聚焦荧光显微镜(LSCM)观察并对比在12、24、36、48h时2种生物膜形成面积;使用96微孔板结晶紫染色法检测并比较在12、24、36、48h时两者生物膜形成量。结果 ATCC 29212菌株培养12、24、36、48h在离体牙根尖表面生物膜形成面积(μm2)分别为47．577±45．221,206．935±122．596,558．782±330．877,1865．023±702．539;lcl1709的为62．227±33．040,461．578±285．281,1211．077±515．262,2632．515±1332．914。36h与48h lcl1709形成的生物膜面积显著高于ATCC 29212(P<0．05)。12、24、36、48h的生物膜OD590值ATCC 29212菌株分别为0．048±0．001,0．130±0．025,0．148±0．022,0．137±0．021;lcl1709菌株分别为0．096±0．029,0．162±0．015,0．201±0．042,0．235±0．078。 lcl1709生物膜OD590值显著高于ATCC 29212(P<0．05),随时间延长,各组OD590值显著增大( P<0．05)。结论 E． faecalis可形成生物膜,持续性根尖周炎分离的E． faecalis菌株生物膜形成能力强于标准菌株,可能与其致病性密切相关。     

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。     

Starvation survival,growth and recovery of Enterococcus faecalis in human serum

The ability of Enterococcus faecalis to survive starvation for long periods in the obturated root canal is likely to be an important factor in the pathogenesis and maintenance of a persistent infection after endodontic treatment. The response of E. faecalis to starvation survival in water and glucose-, phosphate- or amino acid-limited chemically defined medium was studied, along with the capacity for growth and recovery of starved cells of E. faecalis in pooled human serum. After an initial rapid fall in cell numbers, a small remaining population of E. faecalis was able to survive in water for over 4 months and in nutrient-limited media for extended periods. A high cell density at the onset of starvation was critical for the ability of E. faecalis to endure prolonged nutrient limitation. Upon starvation, a static population of starved cells developed and were apparently in a minimal metabolic state, since blocking cell wall synthesis with penicillin G or inhibiting DNA synthesis with norfloxacin during starvation resulted in limited change in the rate of loss of viable cells. In 50% serum, E. faecalis grew, then stabilized at a relatively constant population of 106 colony-forming units/ml for 4 months, irrespective of the initial cell density. In summary, E. faecalis is capable of withstanding prolonged periods of starvation in a minimal metabolic state provided that there is a high cell density at the onset of starvation. Starved cells were capable of recovery upon addition of human serum.     

Starvation survival,growth and recovery of Enterococcus faecalis in human serum

The ability of Enterococcus faecalis to survive starvation for long periods in the obturated root canal is likely to be an important factor in the pathogenesis and maintenance of a persistent infection after endodontic treatment. The response of E. faecalis to starvation survival in water and glucose‐, phosphate‐ or amino acid‐limited chemically defined medium was studied, along with the capacity for growth and recovery of starved cells of E. faecalis in pooled human serum. After an initial rapid fall in cell numbers, a small remaining population of E. faecalis was able to survive in water for over 4 months and in nutrient‐limited media for extended periods. A high cell density at the onset of starvation was critical for the ability of E. faecalis to endure prolonged nutrient limitation. Upon starvation, a static population of starved cells developed and were apparently in a minimal metabolic state, since blocking cell wall synthesis with penicillin G or inhibiting DNA synthesis with norfloxacin during starvation resulted in limited change in the rate of loss of viable cells. In 50% serum, E. faecalis grew, then stabilized at a relatively constant population of 10⁶ colony‐forming units/ml for 4 months, irrespective of the initial cell density. In summary, E. faecalis is capable of withstanding prolonged periods of starvation in a minimal metabolic state provided that there is a high cell density at the onset of starvation. Starved cells were capable of recovery upon addition of human serum.     

再感染根管内粪肠球菌生物膜的研究进展

粪肠球菌是顽固性和继发性根管感染中最易分离到的细菌,其主要致病机制之一是形成生物膜.笔者下面就再感染根管内粪肠球菌的分离与鉴定、影响粪肠球菌生物膜形成的相关因素等作一综述.     

Resistance to acidic and alkaline environments in the endodontic pathogen Enterococcus faecalis

BACKGROUND/AIMS: This study aimed to investigate the biochemical mechanisms employed by the endodontic pathogen Enterococcus faecalis to confer acid- and alkali-resistance and to compare these with the mechanisms of representative oral streptococci. METHODS: E. faecalis JCM8728, Streptococcus mutans NCTC10449 and Streptococcus sanguinis ATCC10556 were used to assess both acid- and alkali-resistance by examining: (i) growth in complex media; (ii) stability of intracellular pH (pH(in)); (iii) cell durability to leakage of preloaded BCECF (2',7'-bis-(2-carboxyethyl)-5,6-carboxy-fluorescein); and (iv) cell permeability to SYTOX-Green. RESULTS: Growth was initiated by E. faecalis at pH 4.0-11.0, by S. mutans at pH 4.0-9.0 and by S. sanguinis at pH 5.0-9.0. The pH(in) was similar to the extracellular pH in S. mutans and S. sanguinis at pH 5-10, while the pH(in) of E. faecalis was maintained at approximately 7.5-8.5 when extracellular pH was 7.5-10 and was maintained at levels equivalent to the extracellular pH when pH < 7.5. Cell membranes of E. faecalis were resistant to BCECF leakage when extracellular pH was 2.5-12 and to SYTOX-Green permeability at pH 4-10. The cell membrane durability to extracellular pH in E. faecalis was higher than that observed in the Streptococcus strains. CONCLUSION: Compared to S. mutans, E. faecalis was found to be equally resistant to acid and more resistant to alkalis. The results suggest that pH-resistance in E. faecalis is attributed to membrane durability against acid and alkali, in addition to cell membrane-bound proton-transport systems. These characteristics may account for why E. faecalis is frequently isolated from acidic caries lesions and from persistently infected root canals where calcium hydroxide medication is ineffective.     

Effectiveness of curcumin against Enterococcus faecalis biofilm

Abstract

Objectives. To evaluate the antimicrobial efficacy of curcumin against Enterococcus faecalis bio?lm formed on tooth substrate in vitro. Sodium hypochlorite (NaOCl) and chlorhexidine (CHX) served as standards for comparison. Materials and methods. Biofilms of E.faecalis were formed on instrumented, extracted human teeth (n = 96). At the end of the 2nd day, 2nd and 8th weeks, specimens were treated for 30 min with one of the test solutions or saline (control) and the surviving colony-forming units (CFU/mL) was recorded. Results were analyzed by Kruskal-Wallis test and Dunnet test for pair-wise comparison with Bonferroni correction (p = 0.05). Results. Only NaOCl showed complete eradication of bacteria at all time periods. In the 2-day and 2nd week biofilms, curcumin and NaOCl showed complete inhibition, which was significantly lower than the CFU recovered in the CHX and saline groups (p < 0.05). In 8 week biofilms, samples treated with curcumin showed 553 ± 137.6 CFU/mL, which was significantly higher than NaOCl (0 CFU/mL), but significantly lower than CHX (2551 ± 129.8) and saline control (1.42 × 1011 ± 2.12 × 1010; p < 0.05). Conclusions. Sodium hypochlorite (3%) showed maximum antibacterial activity against E.faecalis bio?lm formed on the tooth substrate, followed by curcumin and CHX. Considering the potential for undesirable properties of NaOCl, the use of herbal alternatives in endodontics might prove to be advantageous.     

不同根管冲洗方式对粪肠球菌生物膜清除效果评价

目的 比较不同根管冲洗方式对粪肠球菌感染根管预备、冲洗的清除效果,评价不同根管冲洗液残余药量的抗菌效果,为临床上选择根管冲洗方法提供参考。方法 将32颗离体牙（前磨牙、单根管）消毒,接种粪肠球菌60 d,随机分为4组（第1组：生理盐水;第2组：生理盐水+超声1 min;第3组：1%NaOCl;第4组：1%NaOCl+超声1 min）,使用ProTaper器械按照冠向下法进行根管预备,并在预备冲洗前及预备至F2进行冲洗后分别取样、计数,进行统计学分析。将预备好的离体牙再次消毒,随机分为2组,分别浸泡生理盐水和1%NaOCl;浸泡30 min后取出,置于接种粪肠球菌标准菌株（ATCC33186）的培养液内,分别在培养2、6、24、48 h时取样、计数。采用SPSS19.0软件包对数据进行统计学分析。结果 4组在预备至F2冲洗结束后,根管中的细菌数量均不同程度降低,其中,使用1% NaOCl结合超声冲洗1 min,几乎可完全去除根管内的细菌。经1% NaOCl浸泡的牙根样本内的细菌,在培养48 h时后总量少于生理盐水。结论 1%NaOCl是有效的根管冲洗液,用于根管化学预备后的残余液体,也可发挥有效的抗菌效果。联合使用超声器械,可以使其抗菌效果最大化。     

再治疗根管粪肠球菌生物膜形成与临床表现相关性分析

目的检测粪肠球菌形成生物膜的能力,探讨其生物膜形成能力与临床表现之间的关系。方法采用96孔板法形成生物膜,结合结晶紫染色,检测临床样本中分离的53株粪肠球菌形成生物膜的能力,分析其生物膜形成能力与患牙临床表现之间的关系。结果53株粪肠球菌中,40株（75.47%）具有生物膜形成能力;在患牙的多种临床表现中,瘘道与再治疗根管粪肠球菌生物膜形成具有相关性,结果具有统计学意义（P＜0.05）。结论再治疗根管中,无瘘道的患牙分离出来的粪肠球菌生物膜形成能力强于有瘘道的患牙,临床治疗中应予以注意。     

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。     

根管内粪肠球菌感染的研究进展

粪肠球菌（E.faecalis）在治疗失败根管内的检出率较高,是根管持续感染和再感染的重要微生物之一。粪肠球菌在治疗前后根管内的感染特点不同,并且对抗菌药物有较强耐药性。目前的根管清理和消毒方法难以将定植于根管中的粪肠球菌彻底清除。本文就有关根管内粪肠球菌的感染特点及其对抗菌剂的敏感性研究进展作一综述。     

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。     

Dentinal tubule invasion and adherence by Enterococcus faecalis

Aim To investigate dentinal tubule invasion and the predilection of Enterococcus faecalis for dentinal tubule walls. Methodology The invasion of dentinal tubules in extracted human teeth by E. faecalis was measured ex vivo after 8 weeks of incubation. The canal walls of 16 root sections were either intact or instrumented with or without smear layer present. Extent and maximum depth of tubule invasion were assessed histologically and compared between groups. In the adherence study, 44 vertically split root samples were prepared to expose longitudinally aligned dentinal tubules and fractured orthodentine (OD). Surfaces were exposed to E. faecalis (erythromycin resistant strain, JH2‐2 carrying plasmid pGh9:ISS1) and incubated aerobically for 2 h. Samples were processed for analysis using scanning electron microscopy. Bacterial adhesion to tubule walls versus fractured OD was calculated as number of cells per 100 μm². Results The strain of E. faecalis used in this study showed moderate to heavy tubule invasion after 8 weeks. In the adhesion studies, significantly more bacteria adhered to fractured OD than to dentinal tubule walls (anova , P < 0.001). With respect to the tubule wall, adherence was greater in inner versus outer dentine (P = 0.02) and greater when bacterial adhesion was tested in chemically defined medium than in phosphate‐buffered saline (anova , P < 0.001). Conclusions Although E. faecalis readily invaded tubules, it did not adhere preferentially to tubule walls. Initial colonization of dentinal tubules by E. faecalis may depend primarily on other factors.     

粪肠球菌及其在牙本质小管内的检测和鉴定

粪肠球菌的抗饥饿能力较强，可在非常苛刻的环境下生存。在根管内的不同部位，粪肠球菌的定植密度不同。粪肠球菌具有的多种毒力因子，可在肠球菌菌种之间转移扩散，耐受宿主的非特异性免疫应答，增强其致病性和生存能力。本文就粪肠球菌的特点，牙本质小管内粪肠球菌的检测与鉴定等研究进展作一综述。     

粪肠球菌在口腔及全身系统性疾病中的致病相关因素及其机制的研究进展

作为人类口腔中根管再感染和难治性根尖周炎中的主要致病菌,粪肠球菌能够在根管内恶劣的环境中长期生存,对大多数根管治疗药物和清理消毒的方法都具有一定的抗性,是目前根管治疗的棘手之处。除此之外,它还与一些全身系统性感染,如胃肠道感染、尿道感染等有关。粪肠球菌的致病性与其对宿主的初始黏附、生物膜形成和入侵感染有关。粪肠球菌主要通过表达各种蛋白和糖脂等黏附相关因子实现初始黏附,随后通过调节各种生物膜相关基因的表达形成成熟的生物膜,以对抗机体的杀伤并实现细胞间的交流,最终定植到人体各个部位乃至引起全身感染。本文就粪肠球菌的致病相关因素及其机制的研究进展进行综述。