Biofilm formation of oral and endodontic Enterococcus faecalis

Biofilms are complex aggregations of microorganisms attached to a surface. The formation of biofilms might facilitate certain survival and virulence characteristics under some situations. This study tested the hypothesis that the ability of Enterococcus faecalis to form biofilms is related to the source of the strains. E. faecalis strains recovered from root canals (n = 33), the oral cavity (n = 21), and non-oral/non-endodontic sources (n = 16) were studied. Biofilms were grown in tryptic soy broth in 96-well plates for 24 hours at 37 degrees C, fixed with Bouin's fixative, and stained with 1% crystal violet. Optical density at 570 nm (OD(570)) was measured by using a microtiter plate reader. Experiments were performed in quadruplicate on three occasions and mean OD(570) readings determined for each strain. There were no statistically significant differences between groups (p = 0.066, Kruskal-Wallis). Within the root canal and oral isolates there were no significant associations between biofilm formation and the presence of the virulence determinants asa, cylA, esp, and gelE.     

根管治疗后疾病与粪肠球菌

根管治疗后疾病中最常发现的细菌是粪肠球菌,有效清除粪肠球菌与提高根管治疗治疗术成功率存在着联系。本文就这方面的研究现状进行综述。     

Quantitative real-time PCR detection of oral Enterococcus faecalis in humans

OBJECTIVE: Enterococcus faecalis is consistently associated with recurrent root canal infections. Only low concentrations of E. faecalis in the human mouth have been demonstrated by culture techniques. Quantitative detection strategies more sensitive than culturing, such as quantitative PCR (qPCR), could provide more illuminating data. DESIGN: Thirty outpatients attending the University of Michigan Graduate Endodontic Clinic for endodontic treatment provided oral rinse samples that were analysed for E. faecalis using qPCR and microbiological culturing. A SYBR Green I qPCR protocol was developed for the quantifiable detection of E. faecalis and total bacteria in oral rinse samples using primers designed to target the 16S rRNA gene. Annealing temperature and primer, magnesium ion, and dimethyl sulfoxide concentrations were investigated for optimisation of the protocol; a minimum sensitivity limit of 23 rRNA copies (an estimated six E. faecalis cells) was established for E. faecalis in pure culture, and 104 rRNA copies (an estimated 26 E. faecalis cells) in mixed culture. RESULTS: In qPCR assays, based on extrapolation from estimated rRNA gene copy numbers, E. faecalis comprised 0.0006-0.0047% of a total bacteria load that ranged from 5.92 x 10(5) to 5.69 x 10(7) cells/ml of oral rinse. E. faecalis was detected in five (17%) samples in concentrations from 114 to 490 cells/ml. In parallel culture assays E. faecalis were detected in only two samples (7%) of the five identified by qPCR and in concentrations 30 and 240 CFU/ml. CONCLUSIONS: qPCR reported a higher incidence of E. faecalis in oral rinse samples than culture techniques and afforded greater sensitivity.     

Antimicrobial activity of endodontic sealers on Enterococcus faecalis

Enterococcus faecalis has been shown to be highly resistant once established in the root canal system and may play an important role in endodontic failures. The purpose of this study was to evaluate the antimicrobial activity of four root canal sealers on E. faecalis. Seventeen blood-agar plates were inoculated with E. faecalis using the Lawn technique. Five discs were placed on each plate, four with sealers--Sealapex, Roth 811, Kerr EWT, and AH-Plus--and an ampicillin disc as the control. The plates were incubated at 37 degrees C. The zones of inhibition were measured at 24 and 48 h. Analysis using a one-way ANOVA and Tukey test showed a statistically significance difference (p < 0.05) between all four groups of sealers. Roth 811 showed the largest zone of inhibition (1.1 mm), followed by Sealapex (0.8 mm) and Kerr EWT (0.5 mm), whereas AH-Plus had no antimicrobial activity. There was no difference in the zones of inhibition between the 24- and 48-h time periods.     

Comparative evaluation of endodontic irrigants against Enterococcus faecalis biofilms

The aim of this study was to compare the efficacy of root canal irrigants against E. faecalis biofilms using a novel in vitro testing system. Biofilms grown in a flow cell system were submerged in test irrigants for either 1 or 5 minutes. Statistical analysis revealed a significant relationship between test agent and percentage kill of the biofilm bacteria (P < 0.05). No statistically significant relationship between time and percentage kill was found. The percentage kill of the biofilm bacteria was: 6% NaOCl (>99.99%), 1% NaOCl (99.78%), Smear Clear (78.06%), 2% chlorhexidine (60.49%), REDTA (26.99%), and BioPure MTAD (16.08%). Post-hoc analysis showed a significant difference between 1% and 6% NaOCl, and all other agents including Smear Clear, 2% chlorhexidine, REDTA, and BioPure MTAD (P < 0.05). Within the parameters of this study, both 1% NaOCl and 6% NaOCl were more efficient in eliminating E. faecalis biofilm than the other solutions tested.     

Coaggregation interactions between oral and endodontic Enterococcus faecalis and bacterial species isolated from persistent apical periodontitis

Interactions between Enterococcus faecalis and other species found in root canal infections might be important for the development and persistence of periapical disease. The aim of this study was to investigate the coaggregation interactions between E. faecalis clinical isolates and species previously shown to survive and induce apical periodontitis in monkeys: Peptostreptococcus anaerobius, Prevotella oralis, Fusobacterium nucleatum, and Streptococcus anginosus. Intergeneric coaggregation assays were conducted in duplicate with observations scored immediately at 0 h, 1 h and 24 h after mixing of combinations of strains. All E. faecalis strains (n = 53) coaggregated with F. nucleatum; E. faecalis did not coaggregate with P. anaerobius or S. anginosus. One strain, E. faecalis E1, coaggregated with P. oralis, with aggregates visible at 1 h. Coaggregation interactions between E. faecalis and F. nucleatum observed in this study suggest a potential role for this combination in endodontic infections.     

完善根管充填后治疗失败感染根管中粪肠球菌的活菌研究

目的:将叠氮溴化丙锭(PMA)与定量PCR(q PCR)技术结合,用于计算根管治疗失败感染根管中粪肠球菌活菌数量。方法:选取34名根管治疗失败患者,提取感染根管内细菌DNA,每个样本分成2份,一份经PMA处理(实验组),一份未用PMA处理(对照组),然后通过q PCR方法检测样本中粪肠球菌的活菌数量和细菌总量。结果:20例患者(58.8%)细菌样本中检测出粪肠球菌。实验组和对照组Ct值分别为25.12±2.04和24.62±2.02(P=0.001)。结论:对于根管治疗失败感染根管内活菌的研究使用PMA结合定量PCR可能是一种有效的方法。     

Isolation and identification of Enterococcus faecalis from necrotic root canals using multiplex PCR

This study was designed to survey the incidence of Enterococcus faecalis infection in symptomatic and asymptomatic root canals of necrotic teeth using PCR and to isolate the bacterium for further screening. Sixty patients categorized according to their clinical symptoms were used for sampling by insertion of paper points into the root canals and absorbing all the fluids present within them. The samples were incubated in 1.0 ml 2xYT (containing 16 g bacto tryptone, 10 g yeast extract and 5.0 g NaCl per liter) for 24 h at 37 degrees C without aeration prior to multiplex PCR analysis. To assist the isolation of E. faecalis, sub-samples were further grown in the same medium supplemented with 6.5% NaCl and back-inoculated into bile esculin. Using multiple cultivation-dependent and PCR analyses, 6 cases (10%) of E. faecalis were identified. Four isolates were obtained from asymptomatic cases of chronic apical periodontitis, and the other two were associated with phoenix abscess and acute apical abscess, respectively. No E. faecalis infection was found in 5 patients with acute apical periodontitis or in 9 with chronic suppurative periodontitis. Our results indicate that there is no significant difference in the incidence of E. faecalis between symptomatic and asymptomatic necrotic dental root canals (P > 0.05).     

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.     

Investigation of the interactions between neutrophils and endodontic isolates of Enterococcus faecalis

Aim The primary aim of this study was to investigate the effect of phagocytosis by neutrophils on the antimicrobial sensitivity of Enterococcus faecalis strains. A secondary aim was to determine whether carriage of a plasmid encoding aggregation substance (AS), which has been reported to increase the survival of some strains inside neutrophils, affected the antimicrobial susceptibility of E. faecalis after phagocytosis by neutrophils. Methodology An assay was carried out to identify isolates of E. faecalis which demonstrated pheromone‐responsive clumping caused by the production of aggregation substance (AS). Four E. faecalis strains grown to both logarithmic and stationary phases were exposed to sodium hypochlorite (NaOCl) and chlorhexidine gluconate (CHX) to determine the minimum inhibitory concentrations of these two agents. The antimicrobial susceptibility tests were repeated with E. faecalis strains which survived phagocytosis by neutrophils for 18 h. Results As expected a laboratory strain of E. faecalis OG1RF which was AS negative became AS positive after introduction of the pheromone responsive plasmid pCF10 into the bacterium to give strain OG1RF(pCF10). These two strains and two endodontic isolates, E08‐584 which demonstrated pheromone‐responsive clumping and E08‐398 which did not, were selected for further study All the test E. faecalis strains were inhibited by low concentrations of sodium hypochlorite (MIC range 0.02–0.3%) and chlorhexidine gluconate (MIC range 0.0004–0.004%). Bacteria recovered from inside neutrophils after 18 h following phagocytosis were susceptible to both ¼MIC and MIC of CHX and NaOCl. Conclusions Aggregation substance did not appear to affect the antimicrobial susceptibility of any of the strains to CHX or NaOCl. All of the E. faecalis strains examined were capable of survival for 18 h inside the neutrophils following phagocytosis; regardless of their capacity to produce aggregation substance. In addition, all strains of E. faecalis had enhanced susceptibilities to the antimicrobial agents after residence inside neutrophils for 18 h.     

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。     

根管治疗后疾病相关性粪肠球菌生物膜的形成机制

粪肠球菌生物膜持续向牙根尖释放致病因子,导致根管治疗后相关疾病发生.有效控制粪肠球菌生物膜的形成,是提高根管治疗成功率的关键.本文就根管内粪肠球菌生物膜的结构、生物学特性以及影响其生物膜形成的黏附因子和环境因子等作一综述.     

Relationship of biofilm formation and gelE gene expression in Enterococcus faecalis recovered from root canals in patients requiring endodontic retreatment

Introduction

Enterococcus faecalis is known to be the most frequently detected species in root canals with failed endodontic treatment. Many studies are available on biofilm formation and the expression of virulence factors such as gelatinase (gelE) in E. faecalis. However, the relationship of biofilm formation and the expression of gelE in E. faecalis recovered from root canals undergoing orthograde retreatment is not well understood.

Methods

E. faecalis was isolated from clinical samples of root canal retreatment, and the expression of gelE in E. faecalis was assessed. Automatic microplate reader and scanning electron microscopy were used to investigate the biofilm formation ability of E. faecalis isolates. Real-time quantitative polymerase chain reaction was used for detecting the expression of gelE in biofilm-positive and biofilm-negative E. faecalis isolates.

Results

The detection rate of E. faecalis in the root canal retreatment cases was 39.26%. An automatic microplate reader showed that most isolates were able to form biofilms, and the biofilm formation ability of strains isolated from the teeth without a sinus tract was better than that with a sinus tract (P < .05). The expression of gelE was stronger in the cases of apical radiolucency than in those without the symptom (P < .05). The expression of gelE was higher in the biofilm-positive than in biofilm-negative strains (P < .05).

Conclusions

Biofilm formation in E. faecalis was facilitated in the cases without a sinus tract. In the cases of apical radiolucency and in the biofilm-positive strains, the expression of gelE was higher.     

Antimicrobial susceptibility and molecular analysis of Enterococcus faecalis originating from endodontic infections in Finland and Lithuania

BACKGROUND/AIMS: Enterococcus faecalis strains with multiple antibiotic resistances can cause infections that are difficult to treat. The microbial flora in treatment-resistant apical periodontitis is dominated by E. faecalis, and is a potential source of infections at other sites. MATERIAL AND METHODS: Sensitivities to a range of antibiotics were determined for 59 endodontic E. faecalis isolates from Finland and Lithuania. The DNA sequence of the gene responsible for the species' intrinsic quinupristin-dalfopristin resistance, lsa, was determined from two isolates with diminished resistance. Four pairs of isolates from the same root canal were typed by pulsed-field gel electrophoresis. RESULTS: A high prevalence of resistance to rifampicin was found, whereas all isolates were susceptible or showed intermediate susceptibility to penicillin and ampicillin and four isolates were unusually susceptible to cefotaxime. No vancomycin or high-level gentamicin resistance was detected. Nine of 59 isolates were susceptible to quinupristin-dalfopristin. A fully quinupristin-dalfopristin-susceptible isolate also susceptible to clindamycin produced a truncated Lsa polypeptide, and an isolate with borderline quinupristin-dalfopristin-susceptibility had mutations proximal to the predicted ribosomal binding site. Pulsed-field gel electrophoresis showed that the same root canal could harbor two different strains of E. faecalis during the course of the same infection. CONCLUSION: Despite the differing antibiotic usage in Finland and Lithuania, E. faecalis from endodontic infections in these countries showed similar susceptibility patterns with levels of resistance considered typical for the species, and decreased resistance to clindamycin and quinupristin-dalfopristin as well as lesions in the lsa gene which were similar to those described in other clinical isolates.     

An in vitro comparison of the antimicrobial effects of various endodontic medicaments on Enterococcus faecalis

The purpose of this in vitro study was to investigate the antimicrobial action of Dermacyn (Oculus Innovative Sciences, Petaluma, CA), BioPure MTAD (Dentsply Tulsa Dental, Johnson City, TN), 2% chlorhexidine (CHX; Ultradent, West Jordan, UT), and 5.25% sodium hypochlorite (NaOCl) against Enterococcus faecalis (American Type Culture Collection 4082). Eighteen Petri dishes of BHI agar were inoculated with E faecalis. Each Petri dish had five saturated paper disks placed. Four of the disks were saturated with a different test solution, and the last paper disk served as the control and was saturated with sterile distilled water. The plates were randomly distributed into two groups. Group one (n=9) was incubated aerobically and group 2 (n=9) was incubated anaerobically for 48 hours at 37 degrees C. The largest diameter of the zones of microbial inhibition was measured in millimeters and recorded. Statistical analysis was performed with repeated-measures analysis of variance. BioPure MTAD showed significantly (p<0.05) more zones of microbial inhibition than 5.25% NaOCl, 2% CHX, and Dermacyn. Sodium hypochlorite and CHX showed significantly (p<0.05) more zones of microbial inhibition than Dermacyn. The zone of inhibition between NaOCl and CHX was not significant (p>0.05). The control group showed no microbial inhibition.     

Antibacterial activity of Propolis versus conventional endodontic disinfectants against Enterococcus faecalis in infected dentinal tubules

Introduction

The antibacterial activity against Enterococcus faecalis of 2 propolis samples was investigated in a dentin block model, and their effectiveness was compared with that of established endodontic disinfectants, chlorhexidine (CHX) and calcium hydroxide [Ca(OH)₂].

Methods

Standardized dentin blocks were infected with E. faecalis ATCC 29212. The root canal space was filled with one of the ethanolic extracts of propolis (Artvin or Tekirda? mix [TM]), CHX 2%, Ca(OH)₂, or ethanol or phosphate-buffered saline for control. Canal dentin was sampled after 1 or 7 days by using a standard-size bur. The dentinal shavings were vortexed vigorously in phosphate-buffered saline, and aliquots were cultured on tryptone soy agar plates. Colonies were counted after 2 days of incubation. Statistical significance was set to 5%.

Results

All experimental agents significantly reduced the number of the cultivable bacteria. CHX was the most potent disinfectant at both times. Compared with the ethanol control, no significant reduction in the number of colonies was found for the propolis extracts at day 1; however, significant reduction was found at day 7. The 2 propolis samples were statistically similar to each other and to Ca(OH)₂, but the TM sample was also similar to CHX at day 7. This has been linked to the greater concentration of flavonoids, a group of antibacterially active compounds, in the TM sample as determined by gas chromatography-mass spectrometry analysis.

Conclusions

The antimicrobial activity of the propolis samples tested in this study was between Ca(OH)₂ and CHX. Both propolis samples were antimicrobially effective; however, their activity did not exceed CHX.