Enterococcus Faecalis Biofilm Research Articles

Efficacy of chitosan root canal medicament against cross-kingdom dual-species biofilm of Candida albicans and Enterococcus faecalis in an in vitro root-canal model.

Infection of the root canals with cross-kingdom biofilm of Candida albicans (C. albicans) and Enterococcus faecalis (E. faecalis) causes more inflammation and is resistant to conventional medication. This study aimed to evaluate the efficacy of chitosan paste (chitosan + propylene glycol (PG)) against this dual-species biofilm in comparison to calcium hydroxide (Ca(OH)2) in the root canals of human teeth. Fifty extracted single-rooted human teeth were prepared and inoculated with C. albicans and E. faecalis, and incubated for 3days for biofilm formation. Four experimental groups (N = 10 each) were applied with different root canal medications for 7days, including (1) No medication (negative control); (2) Ca(OH)2 + distilled water; (3) PG (vehicle control) and (4) chitosan + PG. The efficacy of root canal medicaments was assessed by determining the percentage of remaining colony-forming units (CFUs) of viable microorganisms in intracanal dentin. The dentin was collected, serially diluted, and spread on solid media for colony count. The biofilm structure in root canals at 3days and 10days (ie. before and after treatments) were observed with scanning electron microscopy (SEM) at 5000× and 10,000× magnifications. Data were analyzed using Welch's ANOVA and Games-Howell post-hoc test (α = 5%). The results show that Chitosan + PG group had a significantly lower percentage of remaining microorganisms when compared to PG, Ca(OH)2, and negative control groups (P-value = 0.015, 0.005, and < 0.001, respectively). Consistently, SEM showed the lowest amount of remaining biofilm in Chitosan + PG group. Thus, Chitosan + PG paste was the most efficacious root canal medicament against dual-species biofilm of C. albicans and E. faecalis in this model.

Effectiveness of Activated Sodium Hypochlorite Irrigation by Shock Wave-Enhanced Emission Photoacoustic Streaming, Sonic and Ultrasonic Devices in Removing Enterococcus faecalis Biofilm From Root Canal System.

Aim: To compare shock wave-enhanced emission photoacoustic streaming (SWEEPS) with sonic- and ultrasonically activated irrigation systems in removing Enterococcus faecalis biofilm from the root canal system. Methodology: Fifty human single-canalled mandibular premolars were included in the study. After access cavity preparation, the root canals were prepared to a standardized size and taper. Then, the entire root surface was covered with two layers of resin, and the root apices were sealed before sterilization. All root canals were inoculated with E. faecalis biofilm, and the samples were incubated aerobically for 2 weeks at 37 °C. Biofilm formation was confirmed by scanning electron microscopy. All samples were randomly divided into five groups (n = 10 each) based on their irrigation activation method as A (no treatment or negative control), B (no irrigation or positive control), C (sonically activated irrigation (SAI)), D (ultrasonically activated irrigation (UAI)), and E (needle irrigation activated by an Er: YAG laser device using a SWEEPS quartz tip (SWEEPS)). Then, dentine chips were retrieved, vortexed, and diluted for colony-forming unit counts. Data were analysed using analysis of variance and post-hoc Tukey tests (α = 5%). Results: All methods could significantly reduce E. faecalis biofilm compared with control so that the UAI, SWEEPS, and SAI groups indicated a 23.54%, 14.89%, and 7.81% biofilm reduction, respectively. UAI demonstrated a significantly more effective reduction of E. faecalis biofilm than SAI (p = 0.004). Conclusions: All irrigation activation methods significantly reduced E. faecalis biofilm, with ultrasonic use being the most effective.

Comparison of a Novel Modality of Erbium-Doped Yttrium Aluminum Garnet Laser-Activated Irrigation and Ultrasonic Irrigation against Mature Enterococcus faecalis Biofilm-An In Vitro Study.

In this in vitro study, we aimed to evaluate and compare the antibacterial efficacy of a novel erbium-doped yttrium aluminum garnet laser modality, shock wave enhanced emission of photoacoustic streaming (SWEEPS), ultrasonically activated irrigation (UAI), and single needle irrigation (SNI) against old bacterial biofilm. A two-week-old Enterococcus faecalis biofilm was cultivated on transversal dentinal discs made from the middle third of the roots of single-rooted, single-canal premolars. Biofilm growth was confirmed using scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM). The dentine samples were randomly distributed into three experimental groups and one control group based on the irrigation protocol used: Group 1, SWEEPS; Group 2, UAI; and Group 3, SNI. The root canals were irrigated with a 3% sodium hypochlorite solution. Antibacterial efficacy was evaluated quantitatively through bacterial culture and qualitatively through CLSM and SEM. Both SWEEPS and UAI demonstrated a statistically significant reduction in Enterococcus faecalis colony-forming units (CFUs) (p < 0.001), while SNI did not show a statistically significant reduction (p = 0.553). No significant difference was observed between the efficacy of SWEEPS and UAI (p > 0.05). The SWEEPS and UAI techniques were equally effective in eliminating mature E. faecalis biofilm.

Antimicrobial efficacy of nanoparticle calcium hydroxide and silver nanoparticles on Enterococcus faecalis: An in vitro study

Abstract Background: In root canal disinfection, intracanal medicaments play a vital role. Typical intracanal medicament, calcium hydroxide (CH), has the lowest efficacy against the biofilm of Enterococcus faecalis. Nanoparticles and their combinations may increase antimicrobial activity, making them better than conventional CH. Aim: The aim is to determine the efficacy of nanoparticle CH (NPCH) and silver nanoparticles (AgNP) against E. faecalis. Materials and Methods: A total of 60 premolars were chosen. The samples were cleaned and decoronated. Cleaning and shaping were done. The apices were tightly sealed with modeling wax before being injected with 5 μl of E. faecalis (ATCC 29212) bacterial suspension, and then left to incubate. Samples were categorized into CH (G-I), NPCH (G-II), AgNP (G-III), and a combination of NPCH + AgNP (G-IV) according to the intracanal medication used. Eppendorf tubes were used to transfer dentin chips after they had been incubated for 7 days. The process of obtaining bacterial colony-forming units (CFUs) involves inoculating sediments onto blood agar. Results: The findings are statistically significant, number of CFUs seen with the Combination of NPCH + AgNP is lower than the other experimental groups, the group treated with CH had the largest number of CFUs. Conclusions: The highest antibacterial effect was seen in combination of NPCH + AgNP.

Antimicrobial Efficacy of a Novel Herbal Endodontic Irrigant Against Enterococcus Faecalis in Root Canals of Permanent Teeth: An in Vitro Study

Background: A successful endodontic treatment is aimed at the sterilization of the entire pulp space. The use of extracts from Rhamnus prinoides as a novel irrigating material for root canal has not been studied . Hence, the antimicrobial efficacy of the alcoholic extract of Rhamnus prinoides as an irrigation material against E. faecalis was evaluated in comparison with the 2.5% sodium hypochlorite (NaOCL) solution used for root canals of permanent teeth. Methods: A total of 30 single-rooted human permanent teeth were thoroughly cleaned, shaped, and disinfected. Then, each tooth was subjected to a two-week infection with Enterococcus faecalis at 37 °C . Afterward, the samples were divided into three groups (10 teeth per group): 0.9% normal saline, 2.5% sodium hypochlorite, and 250 µg/ml Rhamnus prinoides. Paper points were used before and after irrigation to collect samples. Bacterial growth was evaluated after 24 h. Bacterial colonies were counted. The data were examined statistically via one-way analysis of variance, Dunnett’s T3 post hoc test, and Shapiro–Wilk test. The level of significance was set at p&lt;0.05. Results: The 2.5% sodium hypochlorite and 250 µg/ml Rhamnus prinoides demonstrated higher effectiveness against biofilm of Enterococcus faecalis than normal saline (p&lt;0.05). The percentage of antibacterial effectiveness were 94.094 ±3.342, 93.685 ±5.280, and 25.603 ±3.912. Conclusions: Rhamnus prinoides, when used as root canal irrigant solution, was effective the sodium hypochlorite against the E. faecalis biofilm.

Lipoteichoic Acid from Lacticaseibacillus rhamnosus GG as a Novel Intracanal Medicament Targeting Enterococcus faecalis Biofilm Formation.

The demand for safe and effective endodontic medicaments to control Enterococcus faecalis biofilms, a contributor to apical periodontitis, is increasing. Recently, lipoteichoic acid (LTA) of family Lactobacillaceae has been shown to have anti-biofilm effects against various oral pathogens. Preliminary experiments showed that LTA purified from Lacticaseibacillus rhamnosus GG (Lgg.LTA) was the most effective against E. faecalis biofilms among LTAs from three Lactobacillaceae including L. rhamnosus GG, Lacticaseibacillus casei, and Lactobacillus acidophilus. Therefore, in this study, we investigated the potential of Lgg.LTA as an intracanal medicament in human root canals infected with E. faecalis. Twenty eight dentinal cylinders were prepared from extracted human teeth, where two-week-old E. faecalis biofilms were formed followed by intracanal treatment with sterile distilled water (SDW), N-2 methyl pyrrolidone (NMP), calcium hydroxide (CH), or Lgg.LTA. Bacteria and biofilms that formed in the root canals were analyzed by scanning electron microscopy and confocal laser scanning microscopy. The remaining E. faecalis cells in the root canals after intracanal medicament treatment were enumerated by culturing and counting. When applied to intracanal biofilms, Lgg.LTA effectively inhibited E. faecalis biofilm formation as much as CH, while SDW and NMP had little effect. Furthermore, Lgg.LTA reduced both live and dead bacteria within the dentinal tubules, indicating the possibility of minimal re-infection in the root canals. Collectively, intracanal application of Lgg.LTA effectively inhibited E. faecalis biofilm formation, implying that Lgg.LTA can be used as a novel endodontic medicament.

Photothermal Effect of 970 nm Diode Laser Irradiation on Enterococcus faecalis Biofilms in Single-Rooted Teeth Ex Vivo.

Objective: The aim of this study was to evaluate the photothermal effect of a 970 nm diode laser on Enterococcus faecalis biofilms. Methods: 72 extracted human single-rooted teeth were prepared, sterilized, and inoculated with Enterococcus faecalis to establish a two-week-old biofilm. The specimens were divided into six groups (n = 12): Group 1 (G1)-negative control (PBS-no laser), Group 2 (G2)-positive control (1% NaOCl rinse-no laser), Group 3 (G3)-a 970 nm laser in 1.5 W pulse mode, Group 4 (G4)-a 970 nm laser in 2 W pulse mode, Group 5 (G5)-a 970 nm laser in 1.5 W continuous mode, Group 6 (G6)-a 970 nm laser in 2 W continuous mode. Bacterial viability was evaluated using the LIVE/DEAD BacLight kit and analyzed by flow cytometry (FCM). Temperature changes on the root surface during irradiation were analyzed using a K-type thermocouple. Data were statistically analyzed using one-way ANOVA and Tukey's multiple comparison test (α = 0.05). Results: Bacterial viability was significantly reduced after laser irradiation in continuous mode using 1.5 W (21% of live bacteria) and 2 W (14% of live bacteria). When the pulsed mode was applied, the reduction in bacterial viability was less, with a mean survival of 53% (1.5 PF, whereas 29% of bacteria survived after 2 W irradiation). Conclusions: The 970 nm diode laser at 2 W continuous mode effectively reduced the viability of E. faecalis biofilms in root canals without causing unacceptable temperature rises at the root surface.

Effect of zinc oxide-eugenol endodontic paste on planktonic aggregates and biofilm of Enterococcus faecalis - An atomic force microscopy evaluation

ObjectiveThis work aimed to evaluate the in vitro effect of zinc oxide-eugenol paste (ZOE) on planktonic aggregates (EfPA) and biofilm (EfBio) of Enterococcus faecalis, focusing on their morphological aspects observed and analyzed using atomic force microscopy (AFM). DesignThe eugenol and paste were characterized by Gas Chromatography coupled with Mass Spectrometry (GC-MS) and Fourier Transform Infrared Spectroscopy (FTIR), respectively. The effect of ZOE on EfPA and EfBio was evaluated by a direct-contact test through colony counting and crystal violet staining protocol. AFM images of untreated and treated EfPA and EfBio growth on bovine dentin were obtained to analyze the morphological damage caused by the treatments. ResultsThe characterization showed high purity in the eugenol composition and chemical interaction between the components of the paste. A bactericidal effect on aggregates was observed after 6 h of exposure, and on biofilm after 24 h of treatment (p < 0.001). A disruptive effect on the biofilm was also evident. AFM images revealed the formation of EfPA, with a notable presence of an exopolysaccharide matrix. After 6 h of ZOE treatment, there was a significant increase in the size and surface roughness profile of treated cells (p < 0.05). Loss of typical cell morphology was observed after 24 h. The effect on the biofilm showed a tendency towards a less condensed biofilm pattern in the treated group, with no differences in surface roughness. ConclusionZOE presents bactericidal action on EfPA and EfBio, promoting significant morphological changes after treatment, especially in the aggregates.

Characterization of Two Novel Endolysins from Bacteriophage PEF1 and Evaluation of Their Combined Effects on the Control of Enterococcus faecalis Planktonic and Biofilm Cells.

Endolysin, a bacteriophage-derived lytic enzyme, has emerged as a promising alternative antimicrobial agent against rising multidrug-resistant bacterial infections. Two novel endolysins LysPEF1-1 and LysPEF1-2 derived from Enterococcus phage PEF1 were cloned and overexpressed in Escherichia coli to test their antimicrobial efficacy against multidrug-resistant E. faecalis strains and their biofilms. LysPEF1-1 comprises an enzymatically active domain and a cell-wall-binding domain originating from the NLPC-P60 and SH3 superfamilies, while LysPEF1-2 contains a putative peptidoglycan recognition domain that belongs to the PGRP superfamily. LysPEF1-1 was active against 89.86% (62/69) of Enterococcus spp. tested, displaying a wider antibacterial spectrum than phage PEF1. Moreover, two endolysins demonstrated lytic activity against additional gram-positive and gram-negative species pretreated with chloroform. LysPEF1-1 showed higher activity against multidrug-resistant E. faecalis strain E5 than LysPEF1-2. The combination of two endolysins effectively reduced planktonic cells of E5 in broth and was more efficient at inhibiting biofilm formation and removing biofilm cells of E. faecalis JCM 7783T than used individually. Especially at 4 °C, they reduced viable biofilm cells by 4.5 log after 2 h of treatment on glass slide surfaces. The results suggest that two novel endolysins could be alternative antimicrobial agents for controlling E. faecalis infections.

Molecular docking and antimicrobial activities of photoexcited inhibitors in antimicrobial photodynamic therapy against Enterococcus faecalis biofilms in endodontic infections

Antimicrobial photodynamic therapy (aPDT) is a promising approach to combat antibiotic resistance in endodontic infections. It eliminates residual bacteria from the root canal space and reduces the need for antibiotics. To enhance its effectiveness, an in silico and in vitro study was performed to investigate the potential of targeted aPDT using natural photosensitizers, Kojic acid and Parietin. This approach aims to inhibit the biofilm formation of Enterococcus faecalis, a frequent cause of endodontic infections, by targeting the Ace and Esp proteins. After determining the physicochemical characteristics of Ace and Esp proteins and model quality assessment, the molecular dynamic simulation was performed to recognize the structural variations. The stability and physical movement of the protein-ligand complexes were evaluated. In silico molecular docking was conducted, followed by ADME/Tox profiling, pharmacokinetics characteristics, and assessment of drug-likeness properties of the natural photosensitizers. The study also investigated the changes in the expression of genes (esp and ace) involved in E. faecalis biofilm formation. The results showed that both Kojic acid and Parietin complied with Lipinski’s rule of five and exhibited drug-like properties. In silico analysis indicated stable complexes between Ace and Esp proteins and the natural photosensitizers. The molecular docking studies demonstrated good binding affinity. Additionally, the expression of the ace and esp genes was significantly downregulated in aPDT using Kojic acid and Parietin with blue light compared to the control group. This investigation concluded that Kojic acid and Parietin with drug-likeness could efficiently interact with Ace and Esp proteins with a strong binding affinity. Hence, natural photosensitizers-mediated aPDT can be considered a promising adjunctive treatment against endodontic infections.

Antibiofilm effect of different concentrations of silver nanoparticles combined with calcium hydroxide against Enterococcus faecalis biofilm: An exvivo study.

This study aimed to evaluate the antibiofilm activity of different concentrations of silver nanoparticles (AgNPs) in combination with calcium hydroxide [Ca(OH)2] against Enterococcus faecalis biofilm. On an E. faecalis biofilm on dentin discs, the following medicaments were applied for 7 days (n = 13/group): 0.005% AgNPs+Ca(OH)2, 0.01% AgNPs+Ca(OH)2, 0.02% AgNPs + Ca(OH)2, Ca(OH)2 and saline/control. Specimens were stained with LIVE/DEAD® BacLight™ dye and analysed with confocal laser scanning microscopy. Proportion of dead bacteria was calculated and analysed. There was a significant reduction in E. faecalis biofilm in all medicament groups (43.5%, 49.1%, 69.1%, 48.7%) respectively, compared with control group (2.54%) (p < 0.001). The 0.02% AgNPs + Ca(OH)2 group demonstrated the most significantly superior antibiofilm effect, with no significant difference between remaining groups. In conclusion, combining 0.02% AgNPs enhanced the antibiofilm effect of Ca(OH)2 on E. faecalis biofilm compared with lower AgNPs concentrations.

Revision of ex vivo endodontic biofilm model using computer aided design

ObjectiveMost endodontic diseases are bacterium-mediated inflammatory or necrotic process induced by contaminated dental pulp. Although great advances are being performed to obtain more efficient antibacterial strategies for persistent infections, most studies lack of representative models to test their antibacterial effects and their outcomes cannot be promptly translated to clinical practice. Therefore, this study aimed to refine an ex vivo endodontic biofilm model combining human tooth, computer guided design and 3D printing to obtain a more reproducible and predictable model. MethodsMonoradicular teeth were cut using three different methods: hand-held (HCC), mechanical precision (MPC) and computer aid guided cutting (CGC). Then, blocks were reassembled. The different model preparations were assessed in terms of dimensional tolerance, surface analysis, liquid tightness and Enterococcus faecalis biofilm development for 21 days, which was studied by metabolic assays and confocal microscopy. Then, the proposed model was validated using different commercial disinfecting treatments. ResultsCGC exhibited significantly lower deviation and surface without defects compared to HHC and MPC, leading to superior liquid tightness. Similarly, mature biofilms with high metabolic activity and vitality were observed in all conditions, CGC showing the lowest variation. Regarding the model validation, all antibacterial treatments resulted in the complete eradication of bacteria in the standard 2D model, whereas commercial treatments exhibited varying levels of efficacy in the proposed ex vivo model, from moderately reduction of metabolic activity to complete elimination of biofilm. ConclusionsThe novel guided approach represents a more reliable, standardized, and reproducible model for the evaluation of endodontic disinfecting therapies. Clinical SignificanceDuring antibacterial treatment development, challenging 3D models using teeth substrates to test antibacterial treatments novel guided approach represents a more reliable, standardized, and reproducible model for the evaluation of endodontic disinfecting therapies.

Effect of Novel and Traditional Intracanal Medicaments on Biofilm Viability and Composition

IntroductionThe aim of this study was to test the hypothesis that a combination of D-amino acids (DAAs) and trans-cinnamaldehyde (TC) demonstrates superior antibiofilm activity to calcium hydroxide (CH) and untreated controls. MethodsIn this 3-part in vitro study, the concentration of DAAs (D-methionine, D-leucine, D-tyrosine, and D-tryptophan) that would significantly decrease Enterococcus faecalis and Actinomyces naeslundii biofilm biomass was first determined. Then, the effect of TC + selected DAAs on polymicrobial biofilms was characterized by quantifying the biomass and biofilm viability. Finally, the antibiofilm effects of TC + DAA was compared with CH and untreated controls by (i) determining bacterial viability and (ii) quantifying biofilm matrix composition using selective fluorescence-binding analysis. Statistical analysis was performed using one-way ANOVA and appropriate multiple comparisons test, with P < .05 considered as statistically significant. ResultsTC (0.06%) + D-tyrosine (1 mM) + D-tryptophan (25 mM) significantly reduced the biomass and biofilm viability compared to the control (P < .05). While no significant difference was observed between TC + DAA and CH in the cultivable bacterial counts (P > .05), confocal microscopy demonstrated a significantly greater percentage of dead bacteria in TC + DAA-treated biofilms compared to CH and the control (P < .05). TC + DAA significantly decreased the biovolume and all the examined components of the biofilm matrix quantity compared to the control, while CH significantly reduced only the exopolysaccharide quantity (P < .05). ConclusionThe combination of TC + D-tyrosine + D-tryptophan demonstrated superior antibiofilm activity (biofilm bacterial killing and reduction of matrix quantity) to CH and has potential to be developed as an intracanal medicament.

Cytocompatibility and Antibiofilm Activity of Calcium Hydroxide Mixed with Cyperus articulatus Essential Oil and Bio-C Temp Bioceramic Intracanal Medicament.

Calcium hydroxide represents the most commonly used intracanal dressing between sessions; however, it may not be effective against all types of microorganisms. Several compounds of plant origin have attracted increasing attention from researchers in recent years. The objective of this study was to evaluate the cytocompatibility and antimicrobial activity of calcium hydroxide associated with the essential oil of Cyperus articulatus and the new bioceramic intracanal medicament Bio-C Temp®. Five experimental groups were designed: group Ca-C. articulatus essential oil; group CHPG-calcium hydroxide associated with propylene glycol; group CHCa-essential oil of C. articulatus associated with calcium hydroxide; and group U-UltraCal® XS; group BCT-Bio-C Temp®. The control group was a culture medium. Cytocompatibility was assessed by the methyltetrazolium (MTT) assay after exposure of the Saos-2 human osteoblast-like cell line to dilutions of commercial products/associations for 24 h and 72 h. The antimicrobial activity against mature Enterococcus faecalis biofilm was evaluated by the crystal violet assay. All commercial products/associations showed a cell viability similar to or even higher than the control group (p > 0.05) for both periods evaluated. C. articulatus essential oil associated or not with calcium hydroxide showed better antibiofilm capacity. C. articulatus associated or not with calcium hydroxide showed superior cytocompatibility and antimicrobial capacity, representing a promissory intracanal medicament.

Comparative evaluation of antimicrobial efficacy of chitosan nanoparticles and calcium hydroxide against endodontic biofilm of Enterococcus faecalis: An in vitro study.

The aim of the study was to assess and evaluate the antimicrobial effectiveness of chitosan nanoparticles (CSNPs) with calcium hydroxide in the elimination of Enterococcus faecalis. Using the broth microdilution method, the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of calcium hydroxide and CSNPs were measured. The antibiofilm effect of calcium hydroxide and CSNPs against E. faecalis biofilm was qualitatively analyzed using a crystal violet assay. A 7-day-old biofilms of E. faecalis grown on dentine discs were assigned to the following three groups (n = 11 dentine discs), normal saline (group I), calcium hydroxide (group II), and CSNPs (group III). Quantification of live and dead cells using confocal microscopy was done to evaluate the antibiofilm efficacy of the medicaments included in the study. MIC of calcium hydroxide and CSNPs against E. faecalis was observed at 2.5 mg/mL and 0.31 mg/mL, respectively. MBC of calcium hydroxide and CSNPs was observed at 2.5 mg/mL and 0.31 mg/mL, respectively. Using Crystal Violet (CV) assay, calcium hydroxide and CSNPs showed biofilm inhibition at concentrations of 2.5 mg/mL and 0.625 mg/mL, respectively. Confocal laser scanning microscopy analysis found that both calcium hydroxide and CSNPs showed a significant decrease in viable cells at their MBC values compared to the control group's normal saline. CSNPs showed a significantly lower percentage of live cells than calcium hydroxide (P < 0.05). The study results reveal that the antimicrobial efficacy of CSNPs is better than calcium hydroxide and normal saline against E. faecalis biofilm.

Targeted metabolomics analysis approach to unravel the biofilm formation pathways of Enterococcus faecalis clinical isolates.

(i) To characterize Enterococcus faecalis biofilm formation pathways by semi-targeted metabolomics and targeted nitrogen panel analysis of strong (Ef63) and weak (Ef 64) biofilm forming E. faecalis clinical isolates and (ii) to validate the identified metabolic markers using targeted inhibitors. Previous proteomics profiling of E. faecalis clinical isolates with strong and weak biofilm formation revealed that differences in metabolic activity levels of small molecule, nucleotide and nitrogen compound metabolic processes and biosynthetic pathways, cofactor metabolic process, cellular amino acid and derivative metabolic process and lyase activity were associated with differences in biofilm formation. Hence, semi-targeted analysis of Ef 63, Ef 64 and ATC control strain Ef 29212 was performed by selecting metabolites that were part of both the previously identified pathways and a curated library with confirmed physical and chemical identity, followed by confirmatory targeted nitrogen panel analysis. Significantly regulated metabolites (p < .05) were selected based on fold change cut-offs of 1.2 and 0.8 for upregulation and downregulation, respectively, and subjected to pathway enrichment analysis. The identified metabolites and pathways were validated by minimum biofilm inhibitory concentration (MBIC) and colony forming unit (CFU) assays with targeted inhibitors. Metabolomics analysis showed upregulation of betaine, hypoxanthine, glycerophosphorylcholine, tyrosine, inosine, allantoin and citrulline in Ef 63 w.r.t Ef 64 and Ef 29212, and thesemetabolites mapped to purinemetabolism, urea cycle and aspartate metabolism pathways. MBIC and CFU assays using compounds against selected metabolites and metabolic pathways, namely glutathione against hypoxanthine and hydroxylamine against aspartate metabolism showed inhibitory effects against E. faecalis biofilm formation. The study demonstrated the importance of oxidative stress inducers such as hypoxanthine and aspartate metabolism pathway in E. faecalis biofilm formation. Targeted therapeutics against these metabolic markers can reduce the healthcare burden associated with E. faecalis infections.

Differences of protein expression in enterococcus faecalis biofilm during resistance to environmental pressures.

Enterococcus faecalis biofilm was frequently found on the failed treated root canal wall, which survived by resisting disinfectant during endodontic treatment.Many researches have been conducted to explore the mechanisms of persistence of this pathogen in unfavorable conditions. However, no comprehensive proteomics studies have been conducted to investigate stress response in Enterococcus faecalis caused by alkali and NaOCl. Enterococcus faecalis (E.f) has been recognized as a main pathogen of refractory apical periodontitis, its ability to withstand environmental pressure is the key to grow in the environment of high alkaline and anti-bacterial drug that causes chronic infection in the root canal. This study aims to focus on the protein expression patterns of E.f biofilm under extreme pressure environment". Enterococcus faecalis biofilm model was established in vitro. Liquid Chromatograph-Mass Spectrometer (LC-MS/MS)-based label free quantitative proteomics approach was applied to compare differential protein expression under different environmental pressures (pH 10 and 5% sodium hypochlorite (NaOCl)). And then qPCR and Parallel Reaction Monitoring Verification (PRM) were utilized to verify the consequence of proteomics. The number of taxa in this study was higher than those in previous studies, demonstrating the presence of a remarkable number of proteins in the groups of high alkaline and NaOCl. Proteins involved in ATP-binding cassette (ABC) transporter were significantly enriched in experimental samples. We identified a total of 15 highly expressed ABC transporters in the high alkaline environment pressure group, with 7 proteins greater than 1.5 times. This study revealed considerable changes in expression of proteins in E.f biofilm during resistance to environmental pressures. The findings enriched our understanding of association between the differential expression proteins and environmental pressures.

Development of Chlorhexidine-loaded Lipid Nanoparticles Incorporated in a Bioceramic Endodontic Sealer

IntroductionThis study aimed to assess BioRoot RCS (BR) incorporating liposomal chlorhexidine digluconate (CHX) for its antibacterial activity, drug release capacity, and physicochemical properties. MethodsDrug release of CHX liposomal formulations in combination with BR was evaluated spectrophotometrically and through mathematical release models for 30 days. A selected combination was evaluated for antimicrobial properties against Enterococcus faecalis biofilm growth on human dentin. Cytotoxicity was assessed following the ISO 10993-5:2019 standard on days 1, 3, and 7. Physicochemical properties were evaluated through setting time, Fourier transform infrared spectroscopy, solubility, contact angle, and film thickness. ResultsFrom BR, liposomal CHX released up to 7-fold higher CHX than CHX solution (P < .05), following a triphasic drug release pattern compared to the CHX solution, which followed a quasi-Fickian diffusion. BR combined with a selected liposomal CHX completely inhibited E. faecalis biofilm growth compared to the combination of BR with CHX solution and the control group (P < .05). Liposomal CHX decreased the contact angle (P < .05) and solubility but increased cytotoxicity (P < .05) of BR, staying above the ISO threshold. None of the other physicochemical characteristics tested differed from BR (P > .05). ConclusionThis liposomal formulation improved CHX release from BR, enhancing the antibacterial effectiveness. It presents a promising approach for local antibiofilm therapy in endodontics without substantially altering the physicochemical characteristics of BR.

Comparative Evaluation of Ultrasonic and Sonic Irrigant Activation Systems: Assessing Extrusion Risk, Debridement, and Biofilm Removal in Distinct Apical Preparation Sizes

This study aims to compare the effectiveness of ultrasonically and sonically activated irrigation in terms of extrusion risk, root canal debridement, and biofilm removal, considering distinct apical preparation sizes, through an ex vivo study in human teeth. Instrumented teeth, to an apical size of 35/.06 or 50/.06, were assigned to three different irrigation procedures: ultrasonically activated irrigation, sonically activated irrigation, and conventional manual irrigation. Apical extrusion risk was evaluated by quantifying irrigant and debris extrusion (n = 10/group). Debris evaluation and smear layer removal from the root canal wall were conducted by scanning electron microscopy (SEM) (n = 5/group), and the elimination of a mature biofilm of Enterococcus faecalis was assessed through resazurin assay and SEM (n = 10/group). For statistical analyses, Student’s paired t-test and the ANOVA with post-hoc Tukey were used. Activated irrigations exhibited a higher risk of extrusion for the larger apical size, while the risk for manual irrigation remained independent of the apical size. Substantially fewer residual debris and smear layers were observed after the activation of the irrigant, and there was a notable enhancement in biofilm elimination compared to manual irrigation (p &lt; 0.05). Notably, the effectiveness of both activated irrigations was more pronounced in root canals prepared to a size 50/.06, with ultrasonic activation showing enhanced improvements. The findings of this study underscore the substantial impact of both ultrasonically and sonically activated irrigation on the effectiveness of root canal disinfection and debridement. This impact is especially prominent with larger apical size, albeit accompanied by an increased risk of extrusion.

Diacetylcurcumin: a novel strategy against Enterococcus faecalis biofilm in root canal disinfection.

Aim: We evaluated Diacetylcurcumin (DAC), a derivative of curcumin, for its antibacterial and antibiofilm properties against Enterococcus faecalis. Methods: Minimum inhibitory concentration (MIC) and minimum bactericidal concentration were determined, along with antibiofilm potential and toxicity in Galleria mellonella. Additionally, in silico computational analysis was performed to understand its mechanisms of action. Results & conclusion: DAC demonstrated significant antibacterial effects, with MIC and MBC values of 15.6 and 31.25μg/ml, respectively, and reduced biofilm formation. A synergistic effect, reducing biofilm by 77%, was observed when combined with calcium hydroxide. G. mellonella toxicity tests confirmed DAC's safety at tested concentrations, suggesting its potential for use in root canal disinfection products.