Confocal laser scanning, scanning electron, and transmission electron microscopy investigation of Enterococcus faecalis biofilm degradation using passive and active sodium hypochlorite irrigation within a simulated root canal model.
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ABSTRACT:
SUBMITTER: Mohmmed SA
PROVIDER: S-EPMC5552959 | biostudies-other | 2017 Aug
REPOSITORIES: biostudies-other
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Project description:Background: Sodium hypochlorite (NaOCl) is the most commonly used irrigant in endodontics . The purpose of this study was to evaluate the effect of NaOCl solution (2.5%) and gel (3%) with/without passive ultrasonic irrigation (PUI) on Enterococcus faecalis, Escherichia coli, and their endotoxins, lipopolysaccharide (LPS) and lipoteichoic acid (LTA). Methods: 40 human lower premolars were contaminated with E. coli (ATCC 25922) for 28 days and E. faecalis (ATCC 29212) for 21 days. Specimens were randomly divided into four groups: (1) 2.5% NaOCl irrigating the canals without PUI activation; (2) 2.5% NaOCl with PUI; (3) 3% NaOCl gel irrigating the canals without PUI; and (4) 3% NaOCl gel with PUI. 40 mL of irrigant was used for each group. PUI activation was carried out using E1-Irrisonic stainless-steel tip at 10% frequency. After treatment, all specimens were filled with 3mL of 17% ethylenediaminetetraacetic acid (EDTA) for 3min and then washed with nonpyrogenic saline solution. Three samples were collected from the canals: S1, at baseline to confirm biofilm formation; S2 after treatment; and S3 after EDTA. Samples were assessed for E. coli and E. faecalis colony forming units, and LPS and LTA were assessed using chromogenic kinetic LAL assay and ELISA, respectively. Data were analyzed by Kruskal-Wallis, Friedmann and Dunn tests with α≤0.05. Results: All groups were effective in reducing the microbial load of E. coli and E. faecalis after treatment without a significant difference among the groups. NaOCl and NaOCl gel groups had no significant difference in reducing LPS and LTA. Statistically increased reduction was seen for NaOCL + PUI and NaOCl gel + PUI compared for groups without PUI. Conclusions: NaOCl gel has the same antimicrobial action of NaOCl solution and can partially detoxify endotoxins. PUI improves NaOCl (gel or solution) action over E. faecalis and E. coli and their endotoxins.
| S-EPMC7573735 | biostudies-literature
Project description:The aim of the study was to compare the antimicrobial efficacy of three irrigation techniques after the use of standardized volume of NaOCl and with standardized time and irrigation.Forty-eight single rooted teeth were inoculated with an Enterococcus faecalis suspension for 24 h. The remaining six canals served as negative controls. The 36 root canals were randomly distributed into three experimental groups; group 1, conventional syringe irrigation; group 2, automated-dynamic irrigation (RinsEndo); group 3, passive ultrasonic irrigation (PUI). In the first protocol, the standardized volume of 3% NaOCl (20 mL) was used and in the second protocol, and standardized irrigation time (45 seconds) was used. Samples from root canals were cultured and the colony-forming units (CFUs) were counted.When the volume of the irrigant was standardized, RinsEndo was more effective than PUI (p<0.01). When the irrigation time was standardized, there were no significant differences between any irrigation techniques (p>0.05). The RinsEndo group had the highest percentage of minimal counts of E. faecalis CFUs.RinsEndo was more effective than PUI only when the volume of the irrigant was standardized. However, the RinsEndo provided higher bacterial reduction in both protocols when using the least amount of the irrigant and providing longer contact time.
| S-EPMC5017279 | biostudies-literature
Project description:Enterococcus faecalis cells are known to have ferric reductase activity and the ability to transfer electrons generated in metabolism to the external environment. We have isolated mutants defective in ferric reductase activity and studied their electron transfer properties to electrodes mediated by ferric ions and an osmium complex-modified redox polymer (OsRP). Electron transfer mediated with ferric ions and ferric reductase activity were both found to be dependent on the membrane-associated Ndh3 and EetA proteins, consistent with findings in Listeria monocytogenes In contrast, electron transfer mediated with OsRP was independent of these two proteins. Quinone in the cell membrane was required for the electron transfer with both mediators. The combined results demonstrate that extracellular electron transfer from reduced quinone to ferric ions and to OsRP occurs via different routes in the cell envelope of E. faecalisIMPORTANCE The transfer of reducing power in the form of electrons, generated in the catabolism of nutrients, from a bacterium to an extracellular acceptor appears to be common in nature. The electron acceptor can be another cell or abiotic material. Such extracellular electron transfer contributes to syntrophic metabolism and is of wide environmental, industrial, and medical importance. Electron transfer between microorganisms and electrodes is fundamental in microbial fuel cells for energy production and for electricity-driven synthesis of chemical compounds in cells. In contrast to the much-studied extracellular electron transfer mediated by cell surface exposed cytochromes, little is known about components and mechanisms for such electron transfer in organisms without these cytochromes and in Gram-positive bacteria such as E. faecalis, which is a commensal gut lactic acid bacterium and opportunistic pathogen.
| S-EPMC7167473 | biostudies-literature
Project description:To determine the antibacterial effect of propolis nanoparticles (PNs) as an endodontic irrigant against Enterococcus faecalis biofilm inside the endodontic root canal system. Two-hundred-ten extracted human teeth were sectioned to obtain 6 mm of the middle third of the root. The root canal was enlarged to an internal diameter of 0.9 mm. The specimens were inoculated with E. faecalis for 21 days. Following this, specimens were randomly divided into seven groups, with 30 dentinal blocks in each group including: group I-saline; group II-propolis 100 µg/mL; group III-propolis 300 µg/mL; group IV-propolis nanoparticle 100 µg/mL; group V-propolis nanoparticle 300µg/mL; group VI-6% sodium hypochlorite; group VII-2% chlorhexidine. Dentin shavings were collected at 200 and 400 ?m depths, and total numbers of CFUs were determined at the end of one, five, and ten minutes. The non-parametric Kruskal-Wallis and Mann-Whitney tests were used to compare the differences in reduction in CFUs between all groups, and probability values of p < 0.05 were set as the reference for statistically significant results. The antibacterial effect of PNs as an endodontic irrigant was also assessed against E. faecalis isolates from patients with failed root canal treatment. Scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) were also performed after exposure to PNs. A Raman spectroscope, equipped with a Leica microscope and lenses with curve-fitting Raman software, was used for analysis. The molecular interactions between bioactive compounds of propolis (Pinocembrin, Kaempferol, and Quercetin) and the proteins Sortase A and ?-galactosidase were also understood by computational molecular docking studies. PN300 was significantly more effective in reducing CFUs compared to all other groups (p < 0.05) except 6% NaOCl and 2% CHX (p > 0.05) at all time intervals and both depths. At five minutes, 6% NaOCl and 2% CHX were the most effective in reducing CFUs (p < 0.05). However, no significant difference was found between PN300, 6% NaOCl, and 2% CHX at 10 min (p > 0.05). SEM images also showed the maximum reduction in E. faecalis with PN300, 6% NaOCl, and 2% CHX at five and ten minutes. CLSM images showed the number of dead cells in dentin were highest with PN300 compared to PN100 and saline. There was a reduction in the 484 cm-1 band and an increase in the 870 cm-1 band in the PN300 group. The detailed observations of the docking poses of bioactive compounds and their interactions with key residues of the binding site in all the three docking protocols revealed that the interactions were consistent with reasonable docking and IFD docking scores. PN300 was equally as effective as 6% NaOCl and 2% CHX in reducing the E. faecalis biofilms.
| S-EPMC7866495 | biostudies-literature
Project description:Background. The aim of this study was to evaluate the effectiveness of different concentrations of sodium hypochlorite (NaOCl) solution in reducing bacterial growth in Enterococcus faecalis biofilms in root canals. Methods. The root canals of maxillary central incisors of 104 subjects underwent chemomechanical debridement. In order to remove the smear layer, 5.25% sodium hypochlorite solution was used for 3 minutes in the root canals. Then, the samples were immersed in 1 mL of 17% EDTA for 3 minutes. Finally, the root canals were irrigated with phosphate-buffered saline (PBS) solution. After removing the smear layer, the samples were sterilized. Then E. faecalis biofilms formed within the root canals at 4-, 6-, and 10-week intervals were evaluated. Each group was divided into 4 subgroups in terms of the antibacterial treatment: group 1: 1% NaOCl solution; group 2: 2.5% NaOCl solution; group 3: 5.25% NaOCl solution; and group 4: PBS solution. After preparation of root canal filings, the counts of live bacteria were calculated through the classic method of counting, i.e. colony forming units (CFU), followed by the analysis of data. Results. In groups 2 and 3, there was no bacterial growth due to complete removal of E. faecalis biofilms (P<0001), while the bacterial counts in group 1 at 4-, 6- and 10-week intervals decreased compared to the control group. Conclusion. The bacterial cells in mature and old biofilms have higher resistance to 1% NaOCl solution compared to the young biofilms. However, the 2.5% and 5.25% NaOCl solutions caused complete inhibition of the growth of E. faecalis biofilm in all the stages of development.
| S-EPMC5768953 | biostudies-other
Project description:Given the increasing prevalence of antibiotic resistance among bacterial strains and the side effects caused by synthetic drugs, it is increasingly important to investigate potential herbal alternatives. In the present study, antimicrobial, cell cytotoxicity, and cleaning tests were performed to evaluate the potential of Fufang Bingpeng irrigant as a root canal irrigant, in addition to q-PCR and high-throughput sequencing analyses. Our in vitro results showed a low minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of Fufang Bingpeng irrigant against Porphyromonas gingivalis ATCC 33277 (6.25 and 12.5%, respectively), Prevotella intermedius ATCC 25611 (6.25 and 6.25%, respectively), Fusobacterium nucleatum ATCC 25286 (6.25 and 6.25%, respectively), Enterococcus faecalis ATCC 19433 (25 and 25%, respectively), and Bacteriodes fragilis ATCC 25285 (12.5 and 12.5%, respectively). Furthermore, it effectively removed the remaining debris and increased the number of open dentinal tubules in root canals compared to the NaCl irrigant (p < 0.05). Fufang Bingpeng irrigant also presented low cytotoxicity to L929 cells compared to the NaClO irrigant. The in vivo results indicated that all irrigants used significantly reduced the number of bacteria compared to the number prior to treatment, and only 1/104.95 bacteria remained in the root canal following the use of Fufang Bingpeng irrigant (p < 0.001). Moreover, the high-throughput sequencing results indicated that all irrigants markedly enhanced the α diversity in the root canal compared to the before preparation control group, while Fufang Bingpeng maintained better microbial diversity than other groups. Therefore, Fufang Bingpeng irrigant presents a promising alternative for use as a root canal irrigant in clinical settings.
| S-EPMC6522938 | biostudies-literature
Project description:Enterococcus faecalis is a gram-positive organism responsible for serious infections in humans, but as with many bacterial pathogens, resistance has rendered a number of commonly used antibiotics ineffective. Here, we report the cryo-EM structure of the E. faecalis 70S ribosome to a global resolution of 2.8 Å. Structural differences are clustered in peripheral and solvent exposed regions when compared with Escherichia coli, whereas functional centres, including antibiotic binding sites, are similar to other bacterial ribosomes. Comparison of intersubunit conformations among five classes obtained after three-dimensional classification identifies several rotated states. Large ribosomal subunit protein bL31, which forms intersubunit bridges to the small ribosomal subunit, assumes different conformations in the five classes, revealing how contacts to the small subunit are maintained throughout intersubunit rotation. A tRNA observed in one of the five classes is positioned in a chimeric pe/E position in a rotated ribosomal state. The 70S ribosome structure of E. faecalis now extends our knowledge of bacterial ribosome structures and may serve as a basis for the development of novel antibiotic compounds effective against this pathogen.
| S-EPMC7530986 | biostudies-literature
Project description:The enterococci comprise a genus of 49 low-GC content Gram-positive commensal species within the Firmicutes phylum that are known to occupy diverse habitats, notably the gastrointestinal core microbiota of nearly every phylum, including human. Of particular clinical relevance are two rogue species of enterococci, Enterococcus faecalis and the distantly related Enterococcus faecium, standing among the nefarious multi-drug resistant and hospital-acquired pathogens. Despite increasing evidence for RNA-based regulation in the enterococci, including regulation of virulence factors, their transcriptome structure and arsenal of regulatory small sRNAs (sRNAs) are not thoroughly understood. Using dRNA-seq, we have mapped at single-nucleotide resolution the primary transcriptomes of E. faecalis V583 and E. faecium AUS0004. We identified 2517 and 2771 transcription start sites (TSS) in E. faecalis and E. faecium, respectively. Based on the identified TSS, we created a global map of s70 promoter motifs. We also revealed features of 5’ and 3’UTRs across the genomes. The transcriptome maps also predicted 150 and 128 sRNA candidates in E. faecalis and E. faecium, respectively, some of which have been identified in previous studies and many of which are new. Finally, we validated several of the predicted sRNAs by Northern Blot in biologically relevant conditions. Comprehensive TSS mapping of two representative strains will provide a valuable resource for the continued development of RNA biology in the Enterococci.
2020-11-25 | GSE115009 | GEO
Project description:Enterococcus faecalis, a ubiquitous member of mammalian gastrointestinal flora, is a leading cause of nosocomial infections and a growing public health concern. The enterococci responsible for these infections are often resistant to multiple antibiotics and have become notorious for their ability to acquire and disseminate antibiotic resistances. In the current study, we examined genetic relationships among 106 strains of E. faecalis isolated over the past 100 years, including strains identified for their diversity and used historically for serotyping, strains that have been adapted for laboratory use, and isolates from previously described E. faecalis infection outbreaks. This collection also includes isolates first characterized as having novel plasmids, virulence traits, antibiotic resistances, and pathogenicity island (PAI) components. We evaluated variation in factors contributing to pathogenicity, including toxin production, antibiotic resistance, polymorphism in the capsule (cps) operon, pathogenicity island (PAI) gene content, and other accessory factors. This information was correlated with multi-locus sequence typing (MLST) data, which was used to define genetic lineages. Our findings show that virulence and antibiotic resistance traits can be found within many diverse lineages of E. faecalis. However, lineages have emerged that have caused infection outbreaks globally, in which several new antibiotic resistances have entered the species, and in which virulence traits have converged. Comparing genomic hybridization profiles, using a microarray, of strains identified by MLST as spanning the diversity of the species, allowed us to identify the core E. faecalis genome as consisting of an estimated 2057 unique genes.
| S-EPMC1899230 | biostudies-literature
Project description:Enterococcus faecium clinical isolates A902 and BM4538, which were resistant to relatively high levels of vancomycin (128 and 64 microg/ml, respectively) and to low levels of teicoplanin (4 microg/ml), and Enterococcus faecalis clinical isolates BM4539 and BM4540, which were resistant to moderate levels of vancomycin (16 microg/ml) and susceptible to teicoplanin (0.25 microg/ml), were studied. They were constitutively resistant by synthesis of peptidoglycan precursors ending with d-alanyl-d-lactate and harbored a chromosomal vanD gene cluster which was not transferable by conjugation to other enterococci. VanX(D) activity, which is not required in the absence of d-Ala-d-Ala, was low in the four strains, although none of the conserved residues was mutated; and the constitutive VanY(D) activity in the membrane fractions was inhibited by penicillin G. The mutations E(13)G in the region of d-alanine:d-alanine ligase (which is implicated in d-Ala1 binding in A902) and S(319)N of the serine involved in ATP binding in BM4538 and a 7-bp insertion at different locations in BM4539 and BM4540 (which led to putative truncated proteins) led to the production of an impaired enzyme and accounted for the lack of d-Ala-d-Ala-containing peptidoglycan precursors. The same 7-bp insertion in vanS(D) of BM4539 and BM4540 and a 1-bp deletion in vanS(D) of A902, which in each case led to a putative truncated and presumably nonfunctional protein, could account for the constitutive resistance. Strain BM4538, with a functional VanS(D), had a G(140)E mutation in VanR(D) that could be responsible for constitutive glycopeptide resistance. This would represent the first example of constitutive van gene expression due to a mutation in the structural gene for a VanR transcriptional activator. Study of these four additional strains that could be distinguished on the basis of their various assortments of mutations confirmed that all VanD-type strains isolated so far have mutations in the ddl housekeeping gene and in the acquired vanS(D) or vanR(D) gene that lead to constitutive resistance to vancomycin.
| S-EPMC521886 | biostudies-literature