Visualization and characterization of Enterococcus faecalis biofilm structure in bovine dentin using 2D and 3D microscopic techniques.

Bacterial biofilms are related to various dental and periodontal infectious diseases, and the characterization of this biological structure with micro-computed tomography (micro-CT) may offer valuable information for clinical and research applications. In this study, we aimed to develop a model to visualize three-dimensionally the biofilm structure on dentin using micro-CT. Dentin blocks were prepared and incubated in tryptic soy broth with Enterococcus faecalis (ATCC 29212). The control group did not receive any staining procedure, while groups 1 and 2 were stained with 100% and 50% barium sulfate, respectively. Transmission electron microscopy (TEM) and confocal laser scanning microscopy (CLSM) were used to detect biofilm formation, barium sulfate penetration, and microbial cell density in the biofilm. Micro-computed tomography (micro-CT) (SkyScan 1172, Bruker Co., Belgium) was used to visualize biofilm formation on the dentin blocks. Biofilm thicknesses were measured from 10 different locations on the specimen surfaces, using CTAn v.1.14.4 software. Obtained data were statistically analyzed using Kruskal-Wallis and Dunn's tests. TEM photomicrographs showed that barium sulfate could penetrate the biofilm structure. CLSM analysis showed that viable and total cell densities were similar between the control and barium sulfate-treated groups (P > 0.05), indicating barium sulfate had no significant influence on cell density. In barium sulfate-treated blocks, biofilm could be discriminated from the dentin, and its thickness could be measured with micro-CT. This study showed that bacterial biofilm on dentin could be characterized by micro-CT after barium sulfate staining without causing any significant side effect on viable and total cell densities.

Cryo-electron microscopy structure of the 70S ribosome from Enterococcus faecalis.

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.

Inhibition of Enterococcus faecalis Growth and Cell Membrane Integrity by Perilla frutescens Essential Oil.

Some plant essential oils were reported to have antimicrobial activity and have the potential to replace chemical preservatives in food industry. In this study, the antibacterial activity and possible mechanism of Perilla frutescens essential oil (PEO) were evaluated using Enterococcus faecalis R612-Z1 as the target strain. The minimum inhibition concentration of PEO against E. faecalis was 0.5 µL/mL. The PEO solutions at the concentrations higher than minimum inhibition concentration had varying degrees of bactericidal effects against E. faecalis. With the addition of PEO, the cell membrane integrity was destroyed, the cell membrane potential was decreased, and the intracellular adenosine triphosphate loss was increased. By testing the bacterial counts and total volatile basic nitrogen contents in chicken breast meat, PEO can significantly inhibit the growth of E. faecalis. The results showed that PEO can be used as an effective natural food preservative during food storage.

Reagent controlled stereoselective synthesis of teichoic acid α-(1,2)-glucans.

The stereoselective construction of 1,2-cis-glycosidic linkages is key in the assembly of biologically relevant glycans, but remains a synthetic challenge. Reagent-controlled glycosylation methodologies, in which external nucleophiles are employed to modulate the reactivity of the glycosylation system, have become powerful means for the construction of 1,2-cis-glycosidic linkages. Here we establish that nucleophilic additives can support the construction of α-1,2-glucans, and apply our findings in the construction of a d-alanine kojibiose functionalized glycerol phosphate teichoic acid fragment. This latter molecule can be found in the cell wall of the opportunistic Gram-positive bacterium, Enterococcus faecalis and represents a structural element that can possibly be used in the development of therapeutic vaccines and diagnostic tools.

Remodeling of Cross-bridges Controls Peptidoglycan Cross-linking Levels in Bacterial Cell Walls.

Cell walls are barriers found in almost all known bacterial cells. These structures establish a controlled interface between the external environment and vital cellular components. A primary component of cell wall is a highly cross-linked matrix called peptidoglycan (PG). PG cross-linking, carried out by transglycosylases and transpeptidases, is necessary for proper cell wall assembly. Transpeptidases, targets of ß-lactam antibiotics, stitch together two neighboring PG stem peptides (acyl-donor and acyl-acceptor strands). We recently described a novel class of cellular PG probes that were processed exclusively as acyl-donor strands. Herein, we have accessed the other half of the transpeptidase reaction by developing probes that are processed exclusively as acyl-acceptor strands. The critical nature of the cross-bridge on the PG peptide was demonstrated in live bacterial cells, and surprising promiscuity in cross-bridge primary sequence was found in various bacterial species. Additionally, acyl-acceptor probes provided insight into how chemical remodeling of the PG cross-bridge (e.g., amidation) can modulate cross-linking levels, thus establishing a physiological role of PG structural variations. Together, the acyl-donor and -acceptor probes will provide a versatile platform to interrogate PG cross-linking in physiologically relevant settings.

The lytic bacteriophage vB_EfaH_EF1TV, a new member of the Herelleviridae family, disrupts biofilm produced by Enterococcus faecalis clinical strains.

OBJECTIVES: The aim of this study is to characterize a new bacteriophage able to infect Enterococcus faecalis, and to evaluate its ability to disrupt biofilm. METHODS: The vB_EfaH_EF1TV (EF1TV) host-range was determined by spot test and efficiency of plating using a collection of 15E. faecalis clinical strains. The phage genome was sequenced with a next generation sequencing approach. Anti-biofilm activity was tested by crystal violet method and confocal laser scanning microscopy. Phage-resistant mutants were selected and sequenced to investigate receptors exploited by phage for infection. RESULTS: EF1TV is a newly discoveredE. faecalis phage which belongs to the Herelleviridae family. EF1TV, whose genome is 98% identical to φEF24C, is characterized by a linear dsDNA genome of 143,507 bp with direct terminal repeats of 1,911 bp. The phage is able to infect E. faecalis and shows also the ability to degrade biofilm produced by strains of this species. The results were confirmed by confocal laser scanning microscopy analyzing the biofilm reduction in the same optical field before and after phage infection. CONCLUSIONS: The EF1TV phage shows promising features such as an obligatory lytic nature, an anti-biofilm activity and the absence of integration-related proteins, antibiotic resistance determinants and virulence factors, and therefore could be a promising tool for therapeutic applications.

Waveguiding and focusing in a bio-medium with an optofluidic cell chain.

Long-distance waveguiding and submicron focusing of light in a bio-medium are crucial for biomedical sensing and imaging. Disordered bio-mediums usually exhibit high scattering and absorption, which limits effective waveguiding and focusing. Here, we demonstrate an optofluidic cell chain, assembled via an optical trapping force from an optical fiber probe, to achieve long-distance waveguiding and submicron light focusing in a disordered bio-medium. By applying a trapping light at 980 nm to generate an optical force, stable binding of E. faecalis cells was achieved in a fluid to assemble cell chains of different lengths. The length could reach up to 360 µm and the incident light (at 675, 532 and 473 nm) could be focused into a beam with a waist radius of 400 nm. As a potential practical application, backscattered signals from human red blood cells were detected using the cell chains, which is expected to benefit biomedical sensing and single cell analysis. STATEMENT OF SIGNIFICANCE: With the assistance of optofluidic techniques, we assembled an E. faecalis cell chain with a length up to 360 µm to achieve long-distance waveguiding and submicron focusing at a propagation loss of 0.03 dB/µm in the bio-medium. Visible lights were launched into the cell chain and the incident lights can converge into a beam with a waist radius of 400 nm. The cell chain was further used to detect the backscattering signals from human red blood cells (RBCs), and the results indicate that the cell chain can be applied as a fully biocompatible extension of the probe for the real-time detection of RBCs in healthy and pathological states.

Antimicrobial activity of endodontic sealers and medications containing chitosan and silver nanoparticles against Enterococcus faecalis.

BACKGROUND: The main microorganism associated with the failure of endodontic treatments is Enterococcus faecalis. Although several endodontic therapeutics have demonstrated antimicrobial activity against E. faecalis, the antimicrobial effectiveness of chitosan (CsNPs) and silver nanoparticles (AgNPs) included into conventional endodontic sealers for endodontic therapies is still unclear. AIM: The objective of this study was to evaluate the antibacterial activity increment (AAI) of endodontic sealers containing CsNPs and AgNPs as well as some chemical components against E. faecalis by direct contact assays. METHODS: CsNPs and AgNPs were synthesized by reduction and ionic gelation methods, respectively. Nanoparticles were characterized by dynamic light scattering and energy dispersive X-ray analysis. The bactericidal activity was tested on monolayers on agar plates and collagen membrane surface assays against E. faecalis. RESULTS: The size of CsNPs was 70.6±14.8 nm and zeta potential was 52.0±5.4 mV; the size of AgNPs was 54.2±8.5 nm, and zeta potential was -48.4±6.9 mV. All materials, single or combined, showed an AAI, especially when CsNPs, chlorhexidine (Chx), and the combination of CsNPs-Chx were added. However, the combination of CsNPs-Chx showed the highest (55%) AAI, followed by Chx (35.5%) and CsNPs (11.1%), respectively. There was a significant statistical difference in all comparisons (p < 0.05). Tubliseal (40%) and AH Plus (32%) sealants showed a higher AAI on E. faecalis in the monolayer test and collagen membrane assay analyzed by scanning electron microscopy. CONCLUSIONS: Tubliseal and AH plus sealers combined with nanoparticles, especially CsNPs-Chx, could be used for conventional endodontic treatments in the control of E. faecalis bacteria.

Bactericidal efficacy of three parameters of Nd:YAP laser irradiation against Enterococcus faecalis compared with NaOCl irrigation.

The success of endodontic treatment depends on the thorough removal of microorganisms from the root canal system. The search for new ways to eliminate the microorganisms is therefore justified. Nd:YAP is a laser that uses yttrium aluminum perovskite, doped with neodymium crystal, as active laser medium. We used the Nd:YAP laser in an in vitro experiment to evaluate the bactericidal effect of three parameters of Nd:YAP laser-activated irrigation on biofilms of Enterococcus faecalis in root canals. The canals of 45 extracted human single-root teeth were prepared on a #35 Mtwo instrument and contaminated with E. faecalis for 14 days. Forty infected single-root teeth were then randomly divided into four groups according to the irrigation agitation protocols as follows: 5.25% sodium hypochlorite (NaOCl), Nd:YAP laser (180 mJ) + NaOCl, Nd:YAP laser (280 mJ) + NaOCl, and Nd:YAP laser (360 mJ) + NaOCl. The remaining bacteria were counted immediately using the cell count method. Teeth were firstly spilt and one half examined by scanning electron microscopy (SEM). The other half involved examination of bacterial colonization in dentinal tubules using confocal laser scanning microscopy (CLSM). Nd:YAP laser (280 mJ) + NaOCl and Nd:YAP laser (360 mJ) + NaOCl completely removed the E. faecalis biofilms from the root canal walls and made it the cleanest among the treatment groups. Bacterial reductions in the treatment groups for dentinal tubules are presented in a descending order as follows: Nd:YAP laser (360 mJ) (53.7%), Nd:YAP laser (280 mJ) (51.5%) > Nd:YAP laser (180 mJ) (45.3%) > 5.25% NaOCl (31.9%) > control (19.3%) (p < 0.05). Nd:YAP laser of 280 mJ and 360 mJ showed effective bactericidal effect in removing E. faecalis biofilm from the root canal walls and dentinal tubules.

Reverse rotary instrumentation in the apical third of the root canal system: An scanning electron microscope analysis.

AIMS: The aim of the present study was to evaluate the efficacy of reverse rotary instrumentation in disinfection of the root canal at the apical third and qualitative confirmatory analysis using the scanning electron microscope (SEM). SUBJECTS AND METHODS: Sixty single-rooted mandibular premolars were instrumented up to Protaper rotary file size F2 and contaminated with a known species of Enterococcus faecalis (ATCC 29212). The samples were then divided into three groups; Group 1: Experimental group-irrigation by agitation of 1% NaOCl with reverse rotary instrumentation; Group 2: Negative control-no irrigation; and Group 3 positive control-irrigation with 1% NaOCl using a 30-gauge needle. The colony forming units of all the groups were checked. SEM analysis of the samples was focused on the apical third to confirm the absence of E. faecalis biofilms. The data obtained were statistically analyzed by the Fisher's exact test and Pearson's Chi-square test. RESULTS: Group I and III showed significant reduction in the growth of E. faecalis (P ≤ 0.001). SEM confirmed dense bacterial colonies in the Group II consistent with biofilm formation and reduction in bacterial colonies in Group I and II. CONCLUSION: Agitation with reverse rotary instrumentation in the apical third of the root canal along with 1% sodium hypochlorite proved effective in disinfection of the apical third of the root canal, which was further confirmed by scanning electron microscopic analysis. Hence, it can be used as an adjunct during rotary instrumentation in efficient cleansing of the root canal system in the apical third of the root canal system.

Do different strains of E. faecalis have the same behavior towards intracanal medications in in vitro research?

The aim of this study was to evaluate the antimicrobial action of different endodontic pastes against Enterococcus faecalis ATCC 29212, isolated from the urinary tract, and compare the action with E. faecalis ATCC 4083, isolated from the root canal. For this purpose, dentin blocks were infected for 21 days with both bacteria at different time-intervals to ensure there would be no cross contamination. After this period, blocks were immersed in the test medications for 7 days, according to the following groups: CH/S, CH/P, CH/CMCP, CH/CHX, CH/DAP and TAP. Images of the samples were captured with a confocal microscope and the percentage of live cells was computed by means of the Bioimage program. The ATCC 29212 strain was shown to be more resistant to CH/SS, Calen, CH/DAP, and TAP than the ATCC 4083 strain. The antimicrobial action of the medications against each strain were divergent concerning the order of susceptibility. The authors concluded that the strains behaved in a different manner: in general, those extracted from the urinary tract were more resistant to the tested medications. Therefore, when E. faecalis must be used for in vitro research in endodontics, we suggest the use of ATCC 4083 strain to obtain results that are closer to the clinical reality.

Synergistic mechanism of Ag+-Zn2+ in anti-bacterial activity against Enterococcus faecalis and its application against dentin infection.

BACKGROUND: Ag+ and Zn2+ have already been used in combinations to obtain both enhanced antibacterial effect and low cytotoxicity. Despite this, it is still unclear how the Zn2+ co-works with Ag+ in the synergistic antibacterial activity. The main purposes of this study were to investigate the co-work pattern and optimum ratio between Ag+ and Zn2+ in their synergistic antibacterial activity against E. faecalis, the possible mechanisms behind this synergy and the primary application of optimum Ag+-Zn2+ co-work pattern against the E. faecalis biofilm on dentin. A serial of Ag+-Zn2+ atomic combination ratios were tested on both planktonic and biofilm-resident E. faecalis on dentin, their antibacterial efficiency was calculated and optimum ratio determined. And the cytotoxicity of various Ag+-Zn2+ atomic ratios was tested on MC3T3-E1 Cells. The role of Zn2+ in Ag+-Zn2+co-work was evaluated using a Zn2+ pretreatment study and membrane potential-permeability measurement. RESULTS: The results showed that the synergistically promoted antibacterial effect of Ag+-Zn2+ combinations was Zn2+ amount-dependent with the 1:9 and 1:12 Ag+-Zn2+ atomic ratios showing the most powerful ability against both planktonic and biofilm-resident E. faecalis. This co-work could likely be attributed to the depolarization of E. faecalis cell membrane by the addition of Zn2+. The cytotoxicity of the Ag+-Zn2+ atomic ratios of 1:9 and 1:12 was much lower than 2% chlorhexidine. CONCLUSIONS: The Ag+-Zn2+ atomic ratios of 1:9 and 1:12 demonstrated similar strong ability against E. faecalis biofilm on dentin but much lower cytotoxicity than 2% chlorhexidine. New medications containing optimum Ag+-Zn2+ atomic ratios higher than 1:6, such as 1:9 or 1:12, could be developed against E. faecalis infection in root canals of teeth or any other parts of human body.

Enterococcus faecalis Responds to Individual Exogenous Fatty Acids Independently of Their Degree of Saturation or Chain Length.

Enterococcus faecalis is a commensal of the human gastrointestinal tract that can persist in the external environment and is a leading cause of hospital-acquired infections. Given its diverse habitats, the organism has developed numerous strategies to survive a multitude of environmental conditions. Previous studies have demonstrated that E. faecalis will incorporate fatty acids from bile and serum into its membrane, resulting in an induced tolerance to membrane-damaging agents. To discern whether all fatty acids induce membrane stress protection, we examined how E. faecalis responded to individually supplied fatty acids. E. faecalis readily incorporated fatty acids 14 to 18 carbons in length into its membrane but poorly incorporated fatty acids shorter or longer than this length. Supplementation with saturated fatty acids tended to increase generation time and lead to altered cellular morphology in most cases. Further, exogenously supplied saturated fatty acids did not induce tolerance to the membrane-damaging antibiotic daptomycin. Supplementation with unsaturated fatty acids produced variable growth effects, with some impacting generation time and morphology. Exogenously supplied unsaturated fatty acids that are normally produced by E. faecalis and those that are found in bile or serum could restore growth in the presence of a fatty acid biosynthetic inhibitor. However, only the eukaryote-derived fatty acids oleic acid and linoleic acid provided protection from daptomycin. Thus, exogenous fatty acids do not lead to a common physiological effect on E. faecalis The organism responds uniquely to each, and only host-derived fatty acids induce membrane protection.IMPORTANCEEnterococcus faecalis is a commonly acquired hospital infectious agent with resistance to many antibiotics, including those that target its cellular membrane. We previously demonstrated that E. faecalis will incorporate fatty acids found in human fluids, like serum, into its cellular membrane, thereby altering its membrane composition. In turn, the organism is better able to survive membrane-damaging agents, including the antibiotic daptomycin. We examined fatty acids commonly found in serum and those normally produced by E. faecalis to determine which fatty acids can induce protection from membrane damage. Supplementation with individual fatty acids produced a myriad of different effects on cellular growth, morphology, and stress response. However, only host-derived unsaturated fatty acids provided stress protection. Future studies are aimed at understanding how these specific fatty acids induce protection from membrane damage.

Do different strains of E. faecalis have the same behavior towards intracanal medications in in vitro research?

Abstract: The aim of this study was to evaluate the antimicrobial action of different endodontic pastes against Enterococcus faecalis ATCC 29212, isolated from the urinary tract, and compare the action with E. faecalis ATCC 4083, isolated from the root canal. For this purpose, dentin blocks were infected for 21 days with both bacteria at different time-intervals to ensure there would be no cross contamination. After this period, blocks were immersed in the test medications for 7 days, according to the following groups: CH/S, CH/P, CH/CMCP, CH/CHX, CH/DAP and TAP. Images of the samples were captured with a confocal microscope and the percentage of live cells was computed by means of the Bioimage program. The ATCC 29212 strain was shown to be more resistant to CH/SS, Calen, CH/DAP, and TAP than the ATCC 4083 strain. The antimicrobial action of the medications against each strain were divergent concerning the order of susceptibility. The authors concluded that the strains behaved in a different manner: in general, those extracted from the urinary tract were more resistant to the tested medications. Therefore, when E. faecalis must be used for in vitro research in endodontics, we suggest the use of ATCC 4083 strain to obtain results that are closer to the clinical reality.

How proteases from Enterococcus faecalis contribute to its resistance to short α-helical antimicrobial peptides.

HYL-20 (GILSSLWKKLKKIIAK-NH2) is an analogue of a natural antimicrobial peptide (AMP) previously isolated from the venom of wild bee. We examined its antimicrobial activity against three strains of Enterococcus faecalis while focusing on its susceptibility to proteolytic degradation by two known proteases-gelatinase (GelE) and serine protease (SprE)-which are secreted by these bacterial strains. We found that HYL-20 was primarily deamidated at its C-terminal which made the peptide susceptible to consecutive intramolecular cleavage by GelE. Further study utilising 1,10-phenanthroline, a specific GelE inhibitor and analogous peptide with D-Lys at its C-terminus (HYL-20k) revealed that the C-terminal deamidation of HYL-20 is attributed to not yet unidentified protease which also cleaves internal peptide bonds of AMPs. In contrast to published data, participation of SprE in the protective mechanism of E. faecalis against AMPs was not proved. The resistance of HYL-20k to C-terminal deamidation and subsequent intramolecular cleavage has resulted in increased antimicrobial activity against E. faecalis grown in planktonic and biofilm form when compared to HYL-20.

Transcriptomics Analysis Reveals Putative Genes Involved in Biofilm Formation and Biofilm-associated Drug Resistance of Enterococcus faecalis.

INTRODUCTION: Enterococcus faecalis is a gram-positive bacterium associated with endodontic infections and is capable of forming biofilms that can confer drug resistance to the bacterium, resulting in treatment failure. Current knowledge on E. faecalis drug resistance is of a limited and conflicting nature. The present study examined the genetic basis of E. faecalis biofilm formation and drug resistance using a RNA sequencing (RNA-Seq)-based transcriptome approach. METHODS: Eighteen clinical isolates of E. faecalis were screened for their biofilm formation abilities using the crystal violet assay, colony counting, and confocal imaging. Selected isolates were then evaluated for antibiotic susceptibility in planktonic and biofilm growth modes followed by RNA-Seq analysis of E. faecalis planktonic, biofilm, and vancomycin-treated biofilm samples and Kyoto Encyclopedia of Genes and Genomes mapping in order to identify genes associated with biofilm formation and drug resistance of E. faecalis. RESULTS: All 18 clinical isolates retained biofilm formation ability and were classified as strong, weak, or laboratory American Type Culture Collection strainlike biofilm formers. Interestingly, both the strong and weak biofilm-forming isolates were uniformly resistant to ampicillin and vancomycin at the treated concentrations (256-4096 µg/mL). RNA-Seq analysis of these isolates identified a total of 163 and 101 differentially regulated genes in planktonic versus biofilm and vancomycin-treated biofilm versus biofilm comparisons, respectively, with significant differences in arsenic resistance operon genes arsR and arsD, sporulation regulatory gene paiA, ABC drug transporter classes, and penicillin-binding proteins. CONCLUSIONS: The present transcriptomic study revealed putative genes associated with E. faecalis biofilm formation and drug resistance, which will provide a foundation for improved therapeutic strategies against E. faecalis infections in the future.

Conjugative type IV secretion in Gram-positive pathogens: TraG, a lytic transglycosylase and endopeptidase, interacts with translocation channel protein TraM.

Conjugative transfer plays a major role in the transmission of antibiotic resistance in bacteria. pIP501 is a Gram-positive conjugative model plasmid with the broadest transfer host-range known so far and is frequently found in Enterococcus faecalis and Enterococcus faecium clinical isolates. The pIP501 type IV secretion system is encoded by 15 transfer genes. In this work, we focus on the VirB1-like protein TraG, a modular peptidoglycan metabolizing enzyme, and the VirB8-homolog TraM, a potential member of the translocation channel. By providing full-length traG in trans, but not with a truncated variant, we achieved full recovery of wild type transfer efficiency in the traG-knockout mutant E. faecalis pIP501ΔtraG. With peptidoglycan digestion experiments and tandem mass spectrometry we could assign lytic transglycosylase and endopeptidase activity to TraG, with the CHAP domain alone displaying endopeptidase activity. We identified a novel interaction between TraG and TraM in a bacterial-2-hybrid assay. In addition we found that both proteins localize in focal spots at the E. faecalis cell membrane using immunostaining and fluorescence microscopy. Extracellular protease digestion to evaluate protein cell surface exposure revealed that correct membrane localization of TraM requires the transmembrane helix of TraG. Thus, we suggest an essential role for TraG in the assembly of the pIP501 type IV secretion system.

Amyloidogenic amyloid-ß-peptide variants induce microbial agglutination and exert antimicrobial activity.

Amyloid-ß (Aß) peptides are the main components of the plaques found in the brains of patients with Alzheimer's disease. However, Aß peptides are also detectable in secretory compartments and peripheral blood contains a complex mixture of more than 40 different modified and/or N- and C-terminally truncated Aß peptides. Recently, anti-infective properties of Aß peptides have been reported. Here, we investigated the interaction of Aß peptides of different lengths with various bacterial strains and the yeast Candida albicans. The amyloidogenic peptides Aß1-42, Aß2-42, and Aß3p-42 but not the non-amyloidogenic peptides Aß1-40 and Aß2-40 bound to microbial surfaces. As observed by immunocytochemistry, scanning electron microscopy and Gram staining, treatment of several bacterial strains and Candida albicans with Aß peptide variants ending at position 42 (Aßx-42) caused the formation of large agglutinates. These aggregates were not detected after incubation with Aßx-40. Furthermore, Aßx-42 exerted an antimicrobial activity on all tested pathogens, killing up to 80% of microorganisms within 6 h. Aß1-40 only had a moderate antimicrobial activity against C. albicans. Agglutination of Aß1-42 was accelerated in the presence of microorganisms. These data demonstrate that the amyloidogenic Aßx-42 variants have antimicrobial activity and may therefore act as antimicrobial peptides in the immune system.

Evaluation of Cold Plasma Treatment and Safety in Disinfecting 3-week Root Canal Enterococcus faecalis Biofilm In Vitro.

INTRODUCTION: Although endodontic infection is caused by multi-bacteria species, Enterococcus faecalis is usually isolated in chronic apical periodontitis. The aim of this study was to evaluate the effectiveness and mechanical safety of cold plasma therapy in disinfecting 3-week E. faecalis biofilms. METHODS: Teeth with 3-week E. faecalis biofilm were treated with AC argon/oxygen (Ar/O2) cold plasma for various treatment times and compared with those treated with Ca(OH)2, 2% chlorhexidine gel, and Ca(OH)2/chlorhexidine for a week. Antimicrobial efficacy was assessed by colony-forming unit method. Scanning electron microscopy was used to assess the morphologic changes of E. faecalis biofilm by plasma. Confocal laser scanning microscopy was used to confirm the viability of the biofilm after the plasma treatment. Microhardness and roughness changes of root canal dentin caused by plasma were verified with Vickers Hardness Tester and 3D Profile Measurement Laser Microscope, respectively. RESULTS: There were no detectable live bacteria after 12 minutes of cold plasma treatment. This was further confirmed by scanning electron microscopy and confocal laser scanning microscopy results. Microhardness and roughness of root canal dentin showed no significant difference after plasma treatment. CONCLUSIONS: Atmospheric pressure cold plasma is an effective therapy in endodontics for its strong sterilization effect on fully matured biofilm within a few minutes. Meanwhile, it has an accepted mechanical safety for its low temperature and not affecting the microhardness and roughness of root canal dentin significantly.

Evaluation of the Effect of Enterococcus faecalis Biofilm on the 2% Chlorhexidine Substantivity: An In Vitro Study.

INTRODUCTION: The aim of this study was to correlate the bacterial viability and the presence of 2% chlorhexidine (CHX) solution on dentin by means of confocal laser scanning microscopy and high-performance liquid chromatography for 48 hours, 7 days, and 30 days. METHODS: One hundred twenty-three extracted human teeth were used. Samples were divided into 4 groups according to the solution (CHX or saline) and the presence of Enterococus faecalis biofilm. Samples were kept in contact with 5 mL of the solution for 5 minutes. Each group was divided into 3 subgroups according to the evaluation period (n = 10). Statistical analysis was performed by using the Kruskal-Wallis test, the Mann-Whitney U test (P < .05), and the Spearman rank correlation coefficient (P < .01). RESULTS: There was a negative correlation between the percentage of live cells and the amount of remaining CHX (P = .000). CHX significantly reduced the percentage of viable cells compared with saline after 48 hours (P = .007). Differences were maintained in the 7-day evaluation period (P = .001). After 30 days, the CHX group presented an increase of viable cells, thereby becoming similar to saline (P = .623). Simultaneously, the remaining CHX was significantly reduced in the 30-day specimens (P = .000). CONCLUSIONS: The results of this study indicate that 2% CHX solution was detected for 48 hours and 7 days with a low percentage of viable cells. The presence of microorganisms on human dentin did not affect 2% CHX maintenance.