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.

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.

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.

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.

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.

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.

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.

New role for human α-defensin 5 in the fight against hypervirulent Clostridium difficile strains.

Clostridium difficile infection (CDI), one of the most common hospital-acquired infections, is increasing in incidence and severity with the emergence and diffusion of hypervirulent strains. CDI is precipitated by antibiotic treatment that destroys the equilibrium of the gut microbiota. Human α-defensin 5 (HD5), the most abundant enteric antimicrobial peptide, is a key regulator of gut microbiota homeostasis, yet it is still unknown if C. difficile, which successfully evades killing by other host microbicidal peptides, is susceptible to HD5. We evaluated, by means of viability assay, fluorescence-activated cell sorter (FACS) analysis, and electron microscopy, the antimicrobial activities of α-defensins 1 and 5 against a panel of C. difficile strains encompassing the most prevalent epidemic and hypervirulent PCR ribotypes in Europe (012, 014/020, 106, 018, 027, and 078). Here we show that (i) concentrations of HD5 within the intestinal physiological range produced massive C. difficile cell killing; (ii) HD5 bactericidal activity was mediated by membrane depolarization and bacterial fragmentation with a pattern of damage peculiar to C. difficile bacilli, compared to commensals like Escherichia coli and Enterococcus faecalis; and (iii) unexpectedly, hypervirulent ribotypes were among the most susceptible to both defensins. These results support the notion that HD5, naturally present at very high concentrations in the mucosa of the small intestine, could indeed control the very early steps of CDI by killing C. difficile bacilli at their germination site. As a consequence, HD5 can be regarded as a good candidate for the containment of hypervirulent C. difficile strains, and it could be exploited in the therapy of CDI and relapsing C. difficile-associated disease.

Assessment of dentinal tubule invasion capacity of Enterococcus faecalis under stress conditions ex vivo.

AIM: To investigate the dentinal tubule invasion capacity of Enterococcus faecalis under alkaline and energy starvation stress conditions. METHODOLOGY: The root canals from human single-rooted teeth (n = 40) were infected with E. faecalis under alkaline (pH 9, 10, 11 and 12) and energy starvation (no glucose, 0.05% glucose and 0.15% glucose) stress conditions. The root canals were prepared in a standard manner and treated to remove the smear layer before incubation. After 4 weeks of cultivation, the roots were split vertically into two halves: one half was processed for biofilm formation analysis using a scanning electron microscope; the other half was stained with fluorescent DNA-binding reagents, washed thoroughly and sectioned (100 µm thick), and the depth of tubule invasion by the microorganism was examined by confocal laser-scanning microscopy. The extent of dentine tubule invasion was analysed statistically. RESULTS: The E. faecalis strain resulted in biofilm formation and dentine tubules invasion under all of the stress conditions, except for pH 11 and 12 conditions. However, the tubule penetration distance was markedly reduced in these stress conditions (P < 0.01) compared with in tryptic soy broth (TSB) or pH 7 medium. The invasion depth in the middle root dentine was significantly higher than in the apical sections in TSB and energy starvation medium (P < 0.01). CONCLUSIONS: Ex vivo E. faecalis formed biofilms and colonized dentine under alkaline and glucose starvation stress conditions, but its ability to invade dentine tubules was significantly decreased.

New insights into the antimicrobial effect of mast cells against Enterococcus faecalis.

Enterococcus faecalis has emerged as an important cause of life-threatening multidrug-resistant bacterial infections in the hospital setting. The pathogenesis of enterococcal infections has remained a relatively neglected field despite their obvious clinical relevance. The objective of this study was to characterize the interactions between mast cells (MCs), an innate immune cell population abundant in the intestinal lamina propria, and E. faecalis. This study was conducted with primary bone marrow-derived murine MCs. The results demonstrated that MCs exerted an antimicrobial effect against E. faecalis that was mediated both by degranulation, with the concomitant discharge of the antimicrobial effectors contained in the granules, and by the release of extracellular traps, in which E. faecalis was snared and killed. In particular, the cathelicidin LL-37 released by the MCs had potent antimicrobial effect against E. faecalis. We also investigated the specific receptors involved in the recognition of E. faecalis by MCs. We found that Toll-like receptors (TLRs) are critically involved in the MC recognition of E. faecalis, since MCs deficient in the expression of MyD88, an adaptor molecule required for signaling by most TLRs, were significantly impaired in their capacity to degranulate, to reduce E. faecalis growth as well as to release tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6) after encountering this pathogen. Furthermore, TLR2 was identified as the most prominent TLR involved in the recognition of E. faecalis by MCs. The results of this study indicate that MCs may be important contributors to the host innate immune defenses against E. faecalis.

Genetic basis for in vivo daptomycin resistance in enterococci.

BACKGROUND: Daptomycin is a lipopeptide with bactericidal activity that acts on the cell membrane of enterococci and is often used off-label to treat patients infected with vancomycin-resistant enterococci. However, the emergence of resistance to daptomycin during therapy threatens its usefulness. METHODS: We performed whole-genome sequencing and characterization of the cell envelope of a clinical pair of vancomycin-resistant Enterococcus faecalis isolates from the blood of a patient with fatal bacteremia; one isolate (S613) was from blood drawn before treatment and the other isolate (R712) was from blood drawn after treatment with daptomycin. The minimal inhibitory concentrations (MICs) of these two isolates were 1 and 12 µg per milliliter, respectively. Gene replacements were made to exchange the alleles found in isolate S613 with those in isolate R712. RESULTS: Isolate R712 had in-frame deletions in three genes. Two genes encoded putative enzymes involved in phospholipid metabolism, GdpD (which denotes glycerophosphoryl diester phosphodiesterase) and Cls (which denotes cardiolipin synthetase), and one gene encoded a putative membrane protein, LiaF (which denotes lipid II cycle-interfering antibiotics protein but whose exact function is not known). LiaF is predicted to be a member of a three-component regulatory system (LiaFSR) involved in the stress-sensing response of the cell envelope to antibiotics. Replacement of the liaF allele of isolate S613 with the liaF allele from isolate R712 quadrupled the MIC of daptomycin, whereas replacement of the gdpD allele had no effect on MIC. Replacement of both the liaF and gdpD alleles of isolate S613 with the liaF and gdpD alleles of isolate R712 raised the daptomycin MIC for isolate S613 to 12 µg per milliliter. As compared with isolate S613, isolate R712--the daptomycin-resistant isolate--had changes in the structure of the cell envelope and alterations in membrane permeability and membrane potential. CONCLUSIONS: Mutations in genes encoding LiaF and a GdpD-family protein were necessary and sufficient for the development of resistance to daptomycin during the treatment of vancomycin-resistant enterococci. (Funded by the National Institute of Allergy and Infectious Diseases and the National Institutes of Health.).

Continuous drip flow system to develop biofilm of E. faecalis under anaerobic conditions.

PURPOSE: To evaluate a structurally mature E. faecalis biofilm developed under anaerobic/dynamic conditions in an in vitro system. METHODS: An experimental device was developed using a continuous drip flow system designed to develop biofilm under anaerobic conditions. The inoculum was replaced every 24 hours with a fresh growth medium for up to 10 days to feed the system. Gram staining was done every 24 hours to control the microorganism purity. Biofilms developed under the system were evaluated under the scanning electron microscope (SEM). RESULTS: SEM micrographs demonstrated mushroom-shaped structures, corresponding to a mature E. faecalis biofilm. In the mature biofilm bacterial cells are totally encased in a polymeric extracellular matrix. CONCLUSIONS: The proposed in vitro system model provides an additional useful tool to study the biofilm concept in endodontic microbiology, allowing for a better understanding of persistent root canal infections.

Reversible monensin adaptation in Enterococcus faecium, Enterococcus faecalis and Clostridium perfringens of cattle origin: potential impact on human food safety.

OBJECTIVES: To determine the stability/reversibility and mechanism of monensin adaptation in monensin-treated cattle isolates compared with reference bacterial isolates, exposed in vitro to high monensin concentrations. METHODS: We evaluated the potential for cattle-origin strains of Clostridium perfringens, Enterococcus faecium and Enterococcus faecalis exposed to monensin in vivo (in vivo monensin-exposed isolates) to maintain or achieve the ability to grow in the presence of high monensin concentrations (in vitro monensin-adapted isolates). Twenty-one consecutive subcultures of the in vitro monensin-adapted strains were performed, and monensin MICs were determined for the 3rd, 7th, 14th and 21st subcultures (subcultured isolates). SDS-PAGE and transmission electron microscopy (TEM) were used to determine protein expression and visualize extracellular morphology changes. RESULTS: Monensin-non-exposed isolates did not display monensin adaptation during in vitro monensin exposure. In contrast, in vivo monensin-exposed isolates displayed monensin adaptation enabling growth at 32× MIC. Upon consecutive subculturing, monensin MICs returned to baseline, or one dilution above, for the monensin-adapted strains. SDS-PAGE identified overexpression of a 14 kDa protein (C. perfringens) and a 20.5 kDa protein (E. faecium and E. faecalis) in the monensin-adapted isolates. TEM demonstrated that in vitro monensin-adapted strains had a significantly thicker cell wall or glycocalyx compared with in vivo monensin-exposed or subcultured isolates. CONCLUSIONS: In vivo monensin-exposed isolates of C. perfringens, E. faecium and E. faecalis have the ability to grow in the presence of high monensin concentrations in vitro. This is associated with an increased thickening of the cell wall or glycocalyx that is reversible upon serial passage, suggesting a phenotypically expressed, but not genetically stable, trait.

Substantiation in Enterococcus faecalis of dose-dependent resistance and cross-resistance to pore-forming antimicrobial peptides by use of a polydiacetylene-based colorimetric assay.

A better understanding of the antimicrobial peptide (AMP) resistance mechanisms of bacteria will facilitate the design of effective and potent AMPs. Therefore, to understand resistance mechanisms and for in vitro assessment, variants of Enterococcus faecalis that are resistant to different doses of the fungal AMP alamethicin (Alm(r)) were selected and characterized. The resistance developed was dose dependent, as both doses of alamethicin and degrees of resistance were colinear. The formation of bacterial cell aggregates observed in resistant cells may be the prime mechanism of resistance because overall, a smaller cell surface in aggregated cells is exposed to AMPs. Increased rigidity of the membranes of Alm(r) variants, because of their altered fatty acids, was correlated with limited membrane penetration by alamethicin. Thus, resistance developed against alamethicin was an adaptation of the bacterial cells through changes in their morphological features and physiological activity and the composition of membrane phospholipids. The Alm(r) variants showed cross-resistance to pediocin, which indicated that resistance developed against both AMPs may share a mechanism, i.e., an alteration in the cell membrane. High percentages of colorimetric response by both AMPs against polydiacetylene/lipid biomimetic membranes of Alm(r) variants confirmed that altered phospholipid and fatty acid compositions were responsible for acquisition of resistance. So far, this is the only report of quantification of resistance and cross-resistance using an in vitro colorimetric approach. Our results imply that a single AMP or AMP analog may be effective against bacterial strains having a common mechanism of resistance. Therefore, an understanding of resistance would contribute to the development of a single efficient, potent AMP against resistant strains that share a mechanism of resistance.

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.

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.

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.

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.

Mechanism for sortase localization and the role of sortase localization in efficient pilus assembly in Enterococcus faecalis.

Pathogenic streptococci and enterococci primarily rely on the conserved secretory (Sec) pathway for the translocation and secretion of virulence factors out of the cell. Since many secreted virulence factors in gram-positive organisms are subsequently attached to the bacterial cell surface via sortase enzymes, we sought to investigate the spatial relationship between secretion and cell wall attachment in Enterococcus faecalis. We discovered that sortase A (SrtA) and sortase C (SrtC) are colocalized with SecA at single foci in the enterococcus. The SrtA-processed substrate aggregation substance accumulated in single foci when SrtA was deleted, implying a single site of secretion for these proteins. Furthermore, in the absence of the pilus-polymerizing SrtC, pilin subunits also accumulate in single foci. Proteins that localized to single foci in E. faecalis were found to share a positively charged domain flanking a transmembrane helix. Mutation or deletion of this domain in SrtC abolished both its retention at single foci and its function in efficient pilus assembly. We conclude that this positively charged domain can act as a localization retention signal for the focal compartmentalization of membrane proteins.

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.