Bacteriophages induced from lysogenic root canal isolates of Enterococcus faecalis

Introduction:  Bacterial viruses play crucial roles in the pathogenesis of many systemic diseases. They are known to inhabit the oral cavity, both as free virions and as prophages in lysogenic bacterial strains; however, there has been no report of bacteriophages in endodontic infections. In this study, we sought to detect, isolate, and describe temperate bacteriophages harbored by Enterococcus faecalis strains isolated from endodontic infections.
Methods:  Ten E. faecalis strains were isolated from root canals of teeth undergoing retreatment following unsuccessful endodontic therapy. Mitomycin C was used to induce any prophages present in the bacterial isolates. The induced phages were purified and examined using electron microscopy. The DNA extracted from one of the phage isolates was subjected to restriction endonuclease digestion and agarose electrophoresis analysis.
Results:  Lysogeny was demonstrated in 4 of the 10 E. faecalis strains. Three of the lysogenic strains yielded phages exhibiting a Siphoviridae morphology, with long, non-contractile tails 130 nm in length, and spherical/icosahedral heads 41 nm in diameter. The virus induced from the fourth lysogenic E. faecalis strain had a contractile tail characteristic of Myoviridae. Restriction endonuclease analysis of Nsi I and Nde I DNA fragments from one of the Siphoviridae phage isolates (phage φEf11) indicated a genome size of approximately 41 kbp.
Conclusion:  This is the first report of lysogenic bacteria and their inducible viruses in infected root canals.     

Loop-mediated isothermal amplification method for the rapid detection of Enterococcus faecalis in infected root canals

INTRODUCTION: Enterococcus faecalis is a major pathogen in the etiology of apical periodontitis after root canal treatment. A loop-mediated isothermal amplification method, which amplifies DNA with high specificity, efficiency and rapidity under isothermal conditions using a set of four specially designed primers and a DNA polymerase with strand-displacement activity, was developed for the rapid detection of E. faecalis in clinical specimens from root canals. METHODS: Primers for detecting E. faecalis from the azoA gene were designed. The specificity of this assay was evaluated using various oral bacteria and the sensitivity was evaluated using serially diluted E. faecalis chromosomal DNA. In addition, loop-mediated isothermal amplification assays were applied to the rapid detection of E. faecalis from endodontic samples. RESULTS: The loop-mediated isothermal amplification products amplified with the primer set were specific for E. faecalis. To confirm the specificity of the amplicon, the amplified products were digested with the restriction endonuclease Sau3AI. The lower detection limit of the E. faecalis primer set without the loop primer set was 10 microg/tube for a 50-min loop-mediated isothermal amplification reaction. Using loop primers increased the detection sensitivity by several orders of magnitude. Furthermore, E. faecalis was detected with the loop-mediated isothermal amplification assay in four root canals from 18 individuals and the detection results were consistent with those of conventional polymerase chain reactions. CONCLUSION: These results indicate that the loop-mediated isothermal amplification assay is very useful for rapid detection of E. faecalis and diagnosis of endodontic infection.     

顽固感染根管内粪肠球菌的致病机制及其控制方法进展

根管治疗是治疗牙髓感染的常用方法。成功的根管治疗依赖于良好的根管预备、冲洗消毒以及对三维根管系统的严密充填,其中通过清除根管系统内感染生物膜、细菌毒素以防止高度复杂的根管系统再次感染是根管治疗成功的关键。本文从根管内严苛环境下粪肠球菌的致病机制、根管顽固感染的根尖周组织炎症与局部免疫、感染根管控制方法进展等方面,对这一主题进行阐述。     

Recovery of Enterococcus faecalis after single- or multiple-visit root canal treatments carried out in infected teeth ex vivo

AIM: To assess the presence of Enterococcus faecalis after root canal treatment in single or multiple visits in an ex vivo model. METHODOLOGY: Forty-five premolar teeth were infected ex vivo with E. faecalis for 60 days. The canals were then prepared using a crowndown technique with System GT and Gates-Glidden burs and irrigated with 2% chlorhexidine gel. The specimens were divided into five groups (G1, G2, G3, G4 and G5) according to the time elapsed between chemical-mechanical preparation and root canal filling, the irrigant solution used and the use or nonuse of a calcium hydroxide intra-canal medicament. The teeth were then root-filled and incubated for 60 days at 37 degrees C. Dentine chips were removed from the canal walls with sequential sterile round burs at low speed. The samples obtained with each bur were immediately collected in separate test tubes containing Brain-Heart Infusion broth. These samples were placed onto agar plates and colony forming units were counted after 24 h at 37 degrees C. Data were ranked and analysed using the Kruskal-Wallis statistical test. RESULTS: Enterococcus faecalis was recovered from 20% (three of 15 specimens) of G1 (chlorhexidine irrigation and immediate root filling in a single visit), 25% (four of 15 specimens) of G2 (chlorhexidine irrigation and filling after 14 days use of a calcium hydroxide dressing in multiple visits), 40% (two of five specimens) of G3 (chlorhexidine irrigation and filling after 7 days), 60% (three of five specimens) of G4 (saline irrigation and filling after 7 days) and from 100% (five of five specimens) of G5 (saline irrigation and immediate filling without sealer). CONCLUSIONS: Neither single- nor multiple-visit root canal treatment ex vivo, eliminated E. faecalis completely from dentinal tubules. Up to 60 days after root filling, E. faecalis remained viable inside dentinal tubules. When no sealer was used, E. faecalis presented a higher growth rate.     

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.     

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。     

根管冲洗液对粪肠球菌作用的研究进展

粪肠球菌是一种最常见的导致根管治疗失败的细菌,如何控制根管内的粪肠球菌是研究的热点。根管冲洗剂是杀灭和抑制粪肠球菌的有效方法之一,本文就根管内粪肠球菌的感染特点、常用根管冲洗液以及冲洗剂联合使用对其的作用等方面作一综述。     

次氯酸钠对根管内粪肠球菌杀菌效果的体外实验

目的评价次氯酸钠对根管内粪肠球菌的杀菌效果。方法将45个离体前磨牙的感染根管标本分为6组，1、2组用5．25％及2．5％次氯酸钠冲洗，3组用0．9％NaCl冲洗，4、5组在根管预备时辅以5．25％及2．5％次氯酸钠冲洗，6组在根管预备时辅以0．9％Nacl冲洗。冲洗前、冲洗后即刻及冲洗后72h分别取样培养。结果6组根管内的细菌量均显著下降。1、2组差异无统计学意义（P〉0．05），但均好于3组（P〈0．05）。4、5和6组差异均有统计学意义（P〈0．05）。根管冲洗后培养72h均有细菌生长。结论2．5％次氯酸钠基本可达到更高浓度的灭菌效果，但是经过机械预备和次氯酸钠化学消毒后的根管内仍有细菌残留。     

Loop‐mediated isothermal amplification method for the rapid detection of Enterococcus faecalis in infected root canals

Introduction: Enterococcus faecalis is a major pathogen in the etiology of apical periodontitis after root canal treatment. A loop‐mediated isothermal amplification method, which amplifies DNA with high specificity, efficiency and rapidity under isothermal conditions using a set of four specially designed primers and a DNA polymerase with strand‐displacement activity, was developed for the rapid detection of E. faecalis in clinical specimens from root canals. Methods: Primers for detecting E. faecalis from the azoA gene were designed. The specificity of this assay was evaluated using various oral bacteria and the sensitivity was evaluated using serially diluted E. faecalis chromosomal DNA. In addition, loop‐mediated isothermal amplification assays were applied to the rapid detection of E. faecalis from endodontic samples. Results: The loop‐mediated isothermal amplification products amplified with the primer set were specific for E. faecalis. To confirm the specificity of the amplicon, the amplified products were digested with the restriction endonuclease Sau3AI. The lower detection limit of the E. faecalis primer set without the loop primer set was 10 μg/tube for a 50‐min loop‐mediated isothermal amplification reaction. Using loop primers increased the detection sensitivity by several orders of magnitude. Furthermore, E. faecalis was detected with the loop‐mediated isothermal amplification assay in four root canals from 18 individuals and the detection results were consistent with those of conventional polymerase chain reactions. Conclusion: These results indicate that the loop‐mediated isothermal amplification assay is very useful for rapid detection of E. faecalis and diagnosis of endodontic infection.     

利用粪肠球菌感染根管模型评价不同根管封闭剂的抗菌效果

目的 体外建立粪肠球菌根管感染模型,比较3种生物陶瓷类根管封闭剂的抗菌性能.方法 选择人单直根管的离体前牙48颗,接种粪肠球菌并孵育4周以构建体外根管感染模型.完成根管成形和清理后,随机将样本分为3个实验组和2个对照组,每组按照如下方法进行根管充填:A组,Biodentine+牙胶;B组,iRoot BP+牙胶;C组,...     

Octenidine in root canal and dentine disinfection ex vivo

AIM: The aim of the present study was to investigate the antimicrobial activity of octenidine on Enterococcus faecalis ATCC 29212 in a dentine block model. METHODOLOGY: Fifty-six root segments of extracted human teeth were infected with E. faecalis for 4 weeks. Octenidine-phenoxyethanol gel (1 : 1) was applied for different timing: 1 min, 10 min, 7 days and in a different formula (1 : 3) for 10 min. Three samples were chosen for the group with placebo gel and for the group without infection (negative control). Dentine samples were collected, and the total count of bacteria and colony-forming units were determined. In addition, for controls and the 10 min group with 1 : 1 gel, the proportion of viable bacteria (PVB) was assessed. RESULTS: Octenidine was particularly effective after incubation periods of 10 min and 7 days. The mean PVB decreased significantly from 57.2% to 5.7% after 10 min application. After 7 days, only one of 10 samples showed positive culture. CONCLUSION: The present study showed the effectiveness of octenidine against E. faecalis in dentine disinfection. Further laboratory and clinical studies are required.     

四种冲洗剂对根管内粪肠球菌清除效果的体外实验

目的比较常用的根管冲洗剂对根管内粪肠球菌感染的清除效果。方法建立粪肠球菌根管内感染模型，实验组用4种常用的化学冲洗剂、对照组用0．9％NaCl溶液冲洗根管。冲洗前、后计数根管内的细菌量，检测残余细菌并观察72h细菌复苏情况。结果化学冲洗剂的杀菌效果明显好于0．9％NaCl溶液（P〈0．05），2．5％次氯酸钠及2％氯己定明显好于3％H2O2（P〈0．05）。结论2％氯己定、2％氯胺-T的杀菌效果与2．5％次氯酸钠相似，3％H2O2杀菌效果较弱。     

Antibacterial activity of various root canal sealers and root-end filling materials in dentin blocks infected ex vivo with Enterococcus faecalis

Objective. The aim was to investigate the antibacterial activity of the root-end filling materials MTA and IRM, different endodontic sealers and calcium hydroxide [Ca(OH)₂] in experimentally infected dentinal tubules. Materials and methods. Ninety-four human root segments were prepared and the root canals were enlarged to ISO size 90. After smear removal, the specimens were infected with Enterococcus faecalis for 3 weeks. The roots were divided into eight groups and filled either with MTA, IRM, Ca(OH)₂, gutta-percha and EndoRez (ER)/GuttaFlow (GF)/AH Plus (AH+) or with Resilon and Epiphany (EpRe). One group of specimens was left unfilled for control. Half of the specimens were treated for 1 day and the other half for 7 days in humid conditions at 37°C. Dentin samples from each canal were collected by enlarging the canals to ISO size 150; thus a dentinal depth of 300 µm was sampled. The number of cultivable bacteria was determined for each specimen. Statistical significance was set to 5%. Results. After 1-day or 7-days of treatment, compared to control, all materials (except ER and GF at day 7) significantly reduced the number of bacteria. At day 1 and day 7, no significant difference was found between ER and GF and between Ca(OH)₂, AH+, EpRe, IRM and MTA. However, a significant difference was found between these two groups of materials (except between GF and EpRe at day 7). Significantly more bacteria were cultured in the ER, GF, EpRe and IRM groups at day 7 compared to day 1. Conclusions. All materials exerted varying degrees of antibacterial activity which generally tended to decrease with time. The most stable antibacterial effect throughout the 7-day period was for Ca(OH)₂, AH+ and MTA.     

Short-term antibacterial activity of root canal sealers towards Enterococcus faecalis

AIM: To investigate the antimicrobial activity of root canal sealers on Enterococcus faecalis, either allowing or avoiding direct contact between sealers and bacteria. METHODOLOGY: Filter paper discs were immersed in standardized E. faecalis suspensions and exposed to freshly mixed sealers (MCS, AH Plus, Grossman's sealer, Sealapex, Apexit) in teflon wells for 30 min, with or without a filter membrane placed between filter paper discs and sealers (membrane-restricted contact test and direct contact test, respectively). After exposure, the filter paper discs were transferred to vials containing phosphate-buffered saline (PBS) and glass beads, and vigorously vortexed. PBS with resuspended bacterial cells was serially diluted and 25 microL droplets were seeded on TSA plates. The plates were incubated in air at 37 degrees C for 24 h and colony-forming units were counted. Using alpha = 0.05 as level for statistical significance, the data obtained were analysed using Student's t-test. RESULTS: In the direct contact test, MCS and AH Plus killed the bacteria to a level below the detection limit. They were followed in decreasing order of efficacy by Grossman's sealer, Sealapex and Apexit. In the membrane-restricted contact test, the sealers ranked: MCS, AH Plus, Grossman's sealer, Apexit and Sealapex, in descending order of antibacterial potency. MCS, AH Plus and Grossman's sealer significantly reduced the number of viable bacteria in both tests. Sealapex and Apexit were not statistically different from control. CONCLUSIONS: MCS, AH Plus and Grossman's sealer were effective in reducing the number of cultivable cells of E. faecalis. Calcium hydroxide-based sealers, Sealapex and Apexit were ineffective in this short-term experiment.     

KaVo KEY激光对感染根管和根面粪肠球菌杀菌效果的体外研究

目的评价KaVo KEY激光对感染根管和根面粪肠球菌的杀菌效果。方法选用50颗人离体单根牙,建立粪肠球菌感染模型,随机分为3组,即空白对照组、激光组1和激光组2。激光组1和激光组2分别使用80、140 mJ能量激光照射整个根面,照射后再分别照射根管15、30 s。标本处理前与处理后即刻取样进行细菌培养计数。分别取激光处理后根管标本3个不同深度（100、200、300 μm）的牙本质碎屑培养24 h,比浊计数。结果激光处理后根管内和根面各组的细菌量均显著下降（P<0.05）,但各组间灭菌效果差异无统计学意义（P>0.05）。各个深度的牙本质碎屑培养24 h比浊显示80 mJ能量激光照射可清除100 μm处牙本质小管内的细菌,且照射15 s时差异有统计学意义（P<0.05）,其余组与空白对照组比较差异无统计学意义。结论KaVo KEY激光能有效杀灭感染根管内壁和根面的粪肠球菌;用低能量、短时间照射根管可有效清除浅层牙本质小管内的粪肠球菌。     

Effectiveness of KTP laser versus 980 nm diode laser to kill Enterococcus faecalis in biofilms developed in experimentally infected root canals

This study aimed to evaluate the antibacterial action of KTP (potassium‐titanyl‐phosphate) laser irradiations (compared with 980 nm diode laser), associated with conventional endodontic procedures, on Enterococcus faecalis biofilms. Fifty‐six dental roots with single canals were prepared with Ni‐Ti rotary instruments, autoclaved, inoculated with an E. faecalis suspension and incubated for 72 h. They were randomly allocated to control and treatment groups. Laser parameters were as follows: power 2.5 W, Ton 35 ms, Toff 50 ms (KTP laser); power 2.5 W, Ton 30 ms, Toff 30 ms (980 nm diode laser). To evaluate the residual bacterial load, BioTimer Assay was employed. The chemo‐mechanical treatment together with laser irradiations (KTP and 980 nm diode lasers) achieved a considerable reduction of bacterial load (higher than 96% and 93%, respectively). Regarding both laser systems, comparisons with conventional endodontic procedures (mortality rate of about 67%) revealed statistically highly significant differences (P ≤ 0.01). This study confirms that laser systems can provide an additional aid in endodontic disinfection.     

评价两种低温等离子体对粪肠球菌生物膜作用的体外实验研究

目的 比较辉光放电和介质阻挡放电两种低温等离子体装置产生的大气压低温等离子体对根管内粪肠球菌生物膜的杀菌效果。方法 在120颗离体牙的根管内部培养粪肠球菌生物膜,培养时间为7 d。将离体牙随机分为12个组,其中,10组分别接受介质阻挡放电和辉光放电这两种大气压低温等离子体装置处理离体牙根管,每种装置各处理5组,每组处理时间分别为2、4、6、8、10 min;另外2组为两种不同装置的单纯气体对照组。采用菌落形成单位计数法比较两种装置对根管内生物膜的杀菌效果,通过光谱测量仪分析两种装置的等离子体活性成分。结果 介质阻挡放电装置比辉光放电装置对根管内粪肠球菌生物膜的杀菌效果更好,不同时间段二者存活的细菌数量均有统计学差异（P<0.05）,而且随着处理时间延长优势更加明显。发射光谱显示两种装置的低温等离子体活性物质成分一致,但激发态Ar原子的群峰总体上表现为介质阻挡放电装置是辉光放电装置的2倍。结论 介质阻挡放电装置产生的低温等离子体杀灭根管内粪肠球菌生物膜更具优势。     

Bacterial leakage in root canals filled with AH Plus and dentine bonding agents

Objective. The aim of this study was to compare the efficacy of different dentine adhesives in delaying the coronal bacterial leakage of Enterococcus faecalis in filled root canals. Materials and methods. Ninety-five lower incisors of patients >65 years of age were instrumented using the ProTaper® system and were irrigated with 1 mL of 2.5% sodium hypochlorite (NaOCl) alternated with 1 mL 17% EDTA between each file change. Final irrigation was performed with 5 mL of 17% EDTA and then flushed with 5 mL of distilled water. The teeth were randomly divided into five experimental groups (n = 15/group) and one of the following dentine adhesives was applied: (1) AdheSE®; (2) Excite® DSC; (3) Clearfil^? Protect Bond; (4) One Coat 7.0; or (5) Control group without adhesive. After filling the root canals, the samples were mounted on a double chamber device to evaluate the bacterial filtration of E. faecalis during a period of 240 days. The results underwent non-parametric Kaplan-Meier survival analysis and comparisons among groups were done using the Log-Rank test. Results. At 240 days, E. faecalis was detected in samples of all groups in the lower chamber. The highest survival value was obtained by One Coat 7.0, giving statistically significant differences from the other groups, whereas Clearfil^? Protect Bond, AdheSE® and Excite® DSC showed similar behaviours, likewise similar to the Control group. Conclusions. One Coat 7.0 adhesive system provides the longest survival value to delay E. faecalis coronal leakage in filled root canals.     

Antimicrobial susceptibility of Enterococcus faecalis isolated from canals of root filled teeth with periapical lesions

AIM: To test, in vitro, the susceptibility to different antibiotics of Enterococcus faecalis isolates from canals of root filled teeth with periapical lesions. METHODOLOGY: Twenty-one E. faecalis isolates, from canals of root filled teeth with persisting periapical lesions, were tested for their antibiotic susceptibilities. The following antibiotics were used: benzylpenicillin, amoxicillin, amoxicillin-clavulanic acid, erythromycin, azithromycin, vancomycin, chloramphenicol, tetracycline, doxycycline, ciprofloxacin and moxifloxacin. Minimal inhibitory concentrations (MICs) for the antimicrobial agents were determined using the E-test System (AB BIODISK, Solna, Sweden), and the E. faecalis strains classified as susceptible or resistant according to the guidelines of National Committee for Clinical Laboratory Standards (NCCLS). The strains were also tested for beta-lactamase production with nitrocefin (Oxoid, Basingstoke, UK). RESULTS: All strains were susceptible to penicillins in vitro, however, the MICs of amoxicillin and amoxicillin-clavulanic acid (MIC(90) = 0.75 microg mL(-1)) were lower than for benzylpenicillin (MIC(90) = 3.0 microg mL(-1)). All strains studied were also susceptible to vancomycin and moxifloxacin, whilst 95.2% were susceptible to chloramphenicol. Amongst the isolates, 85.7% were susceptible to tetracycline and doxycycline and 80.9% to ciprofloxacin. The MIC of erythromycin ranged from 0.38 to >256 microg mL(-1); only 28.5% of the strains were susceptible (MIC < or = 0.5 microg mL(-1)). Limited susceptibility was also observed with azithromycin which was active against only 14.2% of isolates. No strains produced beta-lactamase. CONCLUSION: Enterococcus faecalis isolates were completely susceptible, in vitro, to amoxicillin, amoxicillin-clavulanic acid, vancomycin and moxifloxacin. Most isolates were susceptible to chloramphenicol, tetracycline, doxycycline or ciprofloxacin. Erythromycin and azithromycin were least effective.