Evaluating the Depth of Penetration of Calcium Hydroxide Mixed With Three Different Herbal Essential Oils Using a Confocal Laser Scanning Microscope.

Introduction The goal of endodontic therapy is to completely eliminate the infection and stop microbes from infecting or reinfecting the root canal and the periradicular tissues. Amongst the primary microorganisms, Enterococcus faecalis (E. faecalis), a Gram-positive anaerobe, is the main cause of pulpal and periapical inflammation causing root canal failure. Literature evidence shows that the gold-standard calcium hydroxide is ineffective against E. faecalis due to its resistance to the alkaline pH and proton pump mechanism. Herbal essential oils such as oregano, basil, and thyme are known to possess antimicrobial properties against E. faecalis. However, their combination with calcium hydroxide as an intracanal medicament and the depth of penetration is still unknown. Aim To evaluate the depth of penetration of calcium hydroxide mixed with three different herbal essential oils using a confocal laser scanning microscope. Material and methods Fifty single-rooted premolars were decoronated and randomly divided into five groups. Group 1 - Oregano oil with calcium hydroxide, Group 2 - Basil oil with Calcium hydroxide, Group 3 - Thyme oil with calcium hydroxide, Group 4 - Calcium hydroxide with saline, Group 5 - Negative control. The teeth were instrumented and inoculated with E. faecalis and incubated for 21 days. Calcium hydroxide mixed with respective oils or saline and 0.1% rhodamine B dye was placed in the canals and again incubated for 7 days. Two sections each of 1 mm were horizontally cut at 3 mm and 5 mm from the apex and later subjected to a confocal laser scanning microscope to evaluate the depth of penetration. One-way ANOVA, post-hoc Tukey test, and student t-test were performed. Results At the middle third, basil oil had the maximum depth of penetration (1377.47±14.1 µm) followed by oregano oil (1345.4±26.5 µm) and thyme oil (1160.4±24.6 µm). At apical third, basil oil (1152.4±31.6 µm) showed maximum depth of penetration, followed by thyme (988.3±26.2 µm) and oregano oils (419.5±19.8 µm). The depth of penetration of these oils was greater at the middle third than at the apical third. Conclusion Basil, oregano, and thyme oil have good penetration depth into the dentinal tubules and can be successfully used in root canal procedures as intracanal medicaments.

Assessment of Long-Term Success and Survival Rates of Different Types of Intracanal Medicaments.

Background: Antimicrobial endodontic intracanal medicaments play a vital role along with biomechanical preparation in the elimination of bacteria present in the pulp area to achieve success in endodontic therapy. Aim: The present research was conducted with the objective of evaluating and comparing intracanal medicaments regarding antibacterial properties against Enterococcus faecalis. Materials and Methods: One hundred and twenty healthy human premolars of the mandible were used. All premolar crowns were removed, keeping the standardized length of the root as 15 mm. Category 1 received triple antibiotic paste (TAP) (n = 30), Category 2 received double antibiotic paste (DAP) (n = 30), Category 3 received augmentin (n = 30), and Category 4 received calcium hydroxide (n = 30). Hygienic paper points were placed in each root canal, then they were moved to a tube containing 1 mL of brain-heart infusion medium and left there for bacteriological testing. Colony-forming units (CFUs) of E. faecalis were then observed and counted after the plates had been incubated overnight at a temperature of 37°C. Results: During intracategory comparisons, there was a significant reduction in colonies of E. feacilis in each category (P ≤ 0.001). When there were intercategory comparisons, there was a maximum reduction in CFU in specimens of Category 1 (TAP) followed by Category 3 (Augmentin) and Category 2 (DAP). Conclusion: Intracanal medicaments like TAP, DAP, and augmentin are effective against root canal E. faecalis.

Combinatory Effect of Nitroxoline and Gentamicin in the Control of Uropathogenic Enterococci Infections.

Enterococcus faecalis, responsible for a majority of human and nosocomial enterococcal infections, is intrinsically resistant to aminoglycoside antibiotics (such as gentamicin, GEN), which must be used in a combined therapy to be effective. Nitroxoline (NTX) is an old antibiotic, underused for decades, but rediscovered now in an era of growing antibiotic resistance. In this in vitro study, the types of interactions between NTX and GEN on 29 E. faecalis strains were analyzed with an aim to find synergistic antimicrobial and antiadhesion combinations. Transmission electron microscopy (TEM) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) were used to analyze changes in cell morphology and bacterial proteome after monotreatments and combined treatments. The results showed the synergistic effect for six combinations on eight strains, including the ATCC29212, and an additive effect for most strains. Combinations causing a complete inhibition of adhesion were established. Cell membrane integrity was affected by NTX, while combined NTX/GEN treatment caused dramatic changes in cell morphology. Upregulation of the expression of many proteins was established, with some emerging only after combined treatment. The results strongly imply that NTX has the potential for use in combined therapy with GEN against enterococci and it could further provide a substantial contribution to an ongoing fight against antimicrobial resistance and nosocomial infections.

A Comparative Analysis of the Antimicrobial Efficacy of Nisin in Different Vehicles Against Enterococcus faecalis: An In Vitro Study.

Aim To evaluate and compare the antimicrobial efficacy of nisin in different carriers against Enterococcus faecalis. Materials and methods Test materials were divided into four groups of five samples each as follows: group 1 = nisin + 17% ethylenediaminetetraacetic acid (EDTA); group 2 = nisin + 2% chitosan; group 3 = nisin + 2% chlorhexidine; group 4 = nisin + distilled water (control). The antimicrobial effectiveness was assessed using the direct contact method, where a standardized E. faecalis suspension was applied to the test materials. Optical density (OD) was assessed using enzyme-linked immunosorbent assay (ELISA) at the end of days one and seven. Data were analyzed using ANOVA and Tukey's post hoc analysis. The level of significance was set at p < 0.05. Results On day one, there was a significant difference in the mean OD values (p < 0.001) with group 3 showing the highest, followed by groups 1, 2, and 4. On day seven, all groups demonstrated antibacterial activity (group 1 > group 3 > group 4 > group 2) but the differences were not statistically significant (p = 0.393). Intragroup analysis showed a decrease in the OD values from day one to day seven, the difference of which was not significant in all groups except group 1, which showed a significant difference (p = 0.035). Conclusion The antibacterial efficacy of nisin was synergistically enhanced with the addition of 17% EDTA and 2% chlorhexidine over seven days against E. faecalis.

Antimicrobial Susceptibility Patterns of Enterococcus Species and Molecular Detection of Enterococcus faecalis Isolated from Patients with Urinary Tract Infection in a Tertiary Care Hospital in Bangladesh.

One of the most prevalent infectious diseases identified in both communities and hospitalized patients is urinary tract infection (UTI). Enterococcus is evolved into a clinically pertinent uropathogen due to its evolving resistance to multiple antimicrobial agents.This study, detects antimicrobial susceptibility patterns of Enterococcus species and molecular detection of Enterococcus faecalis from patients with urinary tract infections. In this cross-sectional observational study, 165 urine samples were obtained from clinically diagnosed patients with UTIs of different ages and gender. Enterococcus species were identified by standard microbiological procedure and PCR (by using species-specific primers for Enterococcus faecalis). A modified Kirby Bauer Disc diffusion method was used to identify the antimicrobial susceptibility pattern following Clinical and Laboratory Standards Institute (CLSI) guidelines. Out of 165 urine samples, 134 samples yielded positive cultures. Enterococcus species were isolated from 23 (17.1%) urine samples. Among all Enterococcus, 16 (69.6%) isolates were E. faecalis, detected by PCR assay. A higher (30.4%) proportion of Enterococcus-positive patients were from the age group 48-57 years and female patients (78.2%) had a higher prevalence. Enterococcal infection was found in 56.5% of non-catheterized patients and 43.5% of catheterized patients. Vancomycin and linezolid (78.3%) and meropenem (73.9%) sensitivity was prevalent among all Enterococcus species. They showed 100% resistance towards ceftriaxone, cefixime 95.7%, cefuroxime 91.3%, azithromycin 82.6%. This research indicated the occurrence of Enterococcus species and the advent of multidrug-resistant E. faecalis in patients with UTIs. Routine speciation and antimicrobial susceptibility testing of Enterococcus in various clinical samples is encouraged.

Identification of Enterococcus spp. by MALDI-TOF mass spectrometry isolated from clinical mastitis and bulk tank milk samples.

BACKGROUND: Throughout a three-year study period, 1,577 bovine clinical mastitis samples and 302 bulk tank samples were analyzed from ten Brazilian dairy herds. Enterococcus spp. was isolated and identified in 93 (5.9%) clinical mastitis samples. In addition, 258 Enterococcus spp. were isolated from the bulk tank samples of the same herds. The identification of Enterococcus spp. isolated from bulk tanks and milk samples of clinical mastitis were accomplished by phenotypic characteristics and confirmed by MALDI-TOF Mass Spectrometry (MS). Fisher test was performed to verify the difference between bulk tanks and mastitis samples. RESULTS: The following species were identified from clinical mastitis: E. saccharolyticus (62.4%), E. faecalis (19.4%), E. faecium (15.1%), E. hirae (1.1%), E. mundtii (1.1%), E. durans (1.1%). Furthermore, from 258 bulk tank milk samples, eight enterococci species were isolated: E. faecalis (67.8%), E. hirae (15.1%), E. faecium (4.6%), E. saccharolyticus (4.6%), E. mundtii (3.1%), E. caseliflavus ( 2.7%), E. durans (1.2%), E. galinarum (0.8%). CONCLUSIONS: The difference in species predominance in bulk tank samples (67.8% of E. faecalis) and clinical mastitis (62.4% of E. saccharolyticus) was unexpected and caught our attention. Although Enterococcus spp. are traditionally classified as an environmental mastitis agent, in the present study, E. saccharolyticus behaved as a contagious agent of mastitis, which consequently changed the control patterns to be implemented.

Evaluation of the pH and Antibacterial Efficacy of Mineral Trioxide Aggregate With and Without the Incorporation of Titanium Tetrafluoride.

Introduction Microorganisms play an important role in causing inflammation in the pulp and periapical regions. Even after undergoing chemo-mechanical procedures during root canal treatment, bacteria may persist within dentinal tubules, posing a risk of disease recurrence. Mineral trioxide aggregate (MTA), introduced as a dental material, has been investigated as a potential antibacterial agent since its early use. Calcium and phosphorus are the primary ions in MTA, and their antibacterial characteristics are attributed to the release of calcium hydroxide through surface hydrolysis of calcium silicate components. Previous studies have shown that MTA has limited antimicrobial properties. Several alterations have been made to enhance the biological properties of MTA, such as incorporating nanoparticles made from silver, zinc, gold, and titanium. Therefore, in this study, titanium tetrafluoride (TiF4) was added to MTA in an effort to enhance its antimicrobial properties. Aim To compare and evaluate the antibacterial efficacy of MTA after the incorporation of TiF4. Materials and methods A total of 56 samples were made by mixing MTA with different weight proportions of TiF4 (1 wt%, 2 wt%, and 3 wt%). Out of these, 28 samples were taken to test each of the following properties: antibacterial efficacy and pH. The specimens were prepared using stainless steel molds of recommended dimensions for testing the pH. The pH was evaluated using a pH meter, and the antibacterial efficacy was assessed using the direct contact test. Data regarding the antibacterial efficacy and pH of MTA with various proportions of TiF4 were investigated for normality using the Kolmogorov-Smirnov test and assessed for normal distribution. The antibacterial properties among the four groups were analyzed using one-way analysis of variance (ANOVA), followed by pairwise multiple comparisons using Tukey's Honest Significant Difference test. The level of statistical significance was determined at p ≤ 0.05. MTA, when incorporated with TiF4, showed enhanced antibacterial properties. Results On day 1, the group treated with MTA containing 3% TiF4 demonstrated the strongest antibacterial effectiveness, with a mean of 4.67 ± 0.04 colony-forming units (CFU)/mL × 10^8. However, the group treated with plain MTA had the lowest mean values, at 5.67 ± 0.25 CFU/mL × 10^8. On day 1, the MTA group with 3% TiF4 also had the highest mean pH values (11.90 ± 0.05), while the plain MTA group had the lowest mean pH values (11.64 ± 0.78). On day 7, the MTA group with 3% TiF4 had the highest pH value (12.85 ± 0.08), whereas the plain MTA group had the lowest pH value (11.92 ± 0.09). Conclusion The inclusion of TiF4 resulted in an augmentation of the antibacterial efficacy of MTA against Enterococcus faecalis (E. faecalis). Hence, the integration of TiF4 into MTA can be considered a promising development against E. faecalis during endodontic procedures.

Totarol exhibits antibacterial effects through antibiofilm and combined interaction against vancomycin-resistant Enterococcus faecalis.

The rise of vancomycin-resistant enterococci (VRE) due to antibiotic overuse poses a significant threat to long-term care patients and those with impaired immune systems. Therefore, it is imperative to seek alternatives to overcome multidrug resistance. This study aimed to evaluate totarol, a natural compound derived from Podocarpus totara, for its antibacterial activity against vancomycin-resistant Enterococcus faecalis (VREF). Totarol exhibited potent antibacterial activity at a very low concentration of 0.25 µg/mL and demonstrated antibiofilm effects through biofilm inhibitory concentration and biofilm eradication concentration assays. Confocal laser scanning microscopy confirmed that totarol inhibited not only biofilm mass but also bacterial cell viability. The combinatorial use of sublethal concentrations of totarol and vancomycin showed antibacterial activity, as observed in the time-kill assay. Quantitative polymerase chain reaction assays revealed a concentration-dependent downregulation of key virulence genes (vanA, ace, asa, efaA, and esp) in VREF when exposed to totarol. In summary, totarol emerges as a promising adjuvant with vancomycin for inhibiting VREF, addressing vancomycin resistance and biofilm formation-critical challenges associated with VRE infection. Since this was an in vitro study, the role of totarol in the clinical implications of VREF treatment remains to be demonstrated.

A Rare Case of Enterococcus faecalis Keratitis in a Neurotrophic Cornea Successfully Treated With Topical Antibiotics and Amniotic Membrane.

A patient in his 60s presented with severe keratitis in his right eye. He had a background of diabetes, high body mass index, arthritis and limited mobility, and high alcohol intake. Examination showed lower lid tarsal ectropion, floppy eyelid syndrome, advanced meibomian gland dysfunction, moderate neurotrophia, and large inferior keratitis with hypopyon. Corneal scrapes revealed Enterococcus faecalis, sensitive to vancomycin and ciprofloxacin only. Due to poor compliance with vancomycin, he was started on topical ciprofloxacin resulting in partial improvement but a persistent epithelial defect. Inserting a dry patch of amniotic membrane on the cornea accelerated epithelialization, and 11 weeks from presentation, complete corneal healing was noted. In the presence of multiple systemic and ocular risk factors like diabetes, high body mass index, high alcohol intake, tarsal ectropion, floppy eyelid syndrome, neurotrophic cornea, blepharitis, and ocular surface inflammation, atypical keratitis, like this rare infection, should be suspected. The use of dry amniotic membrane has a role in epithelial healing in patients with neurotrophia.

Antibacterial Property and Mechanisms of Au@Ag Core-Shell Nanoparticles with Near-Infrared Absorption Against E. faecalis Infection of Dentin.

Background: Enterococcus faecalis (E. faecalis) is one of the main pathogens responsible for refractory root canal infections in the teeth and shows resistance against various antibacterial managements. Effective control of E. faecalis infection is a prerequisite for successful treatment of refractory apical periodontitis. This study aimed to analyze the antibacterial activity and mechanisms of Au@Ag nanoparticles (NPs) combined with photothermal therapy (PTT) against the original and Ag+-resistant E. faecalis. Methods: Au@AgNPs with optimal shell thicknesses were synthesized and characterized. The antibacterial activity of Au@AgNPs with PTT against the original or Ag+-resistant E. faecalis was evaluated, and the antibiofilm activity was tested on E. faecalis biofilm on the dentin of teeth. The potential antibacterial mechanisms of Au@AgNPs combined with PTT against E. faecalis have also been studied. Moreover, its influence on dentin microhardness and cytotoxicity was assessed. Results: This study revealed that Au@AgNPs combined with PTT showed enhanced antibacterial and antibiofilm effects, no negative effects on dentin microhardness, and low cytotoxicity toward human periodontal ligament cells (hPDLCs). Moreover, Au@AgNPs combined with PTT effectively inhibited the growth of Ag+-resistant E. faecalis. Its antibacterial effects may be exerted through the release of silver ions (Ag+), destruction of the cell membrane, production of reactive oxygen species (ROS) and inhibition of adenosine triphosphate (ATP) production. Hyperthermia generated by Au@AgNPs with PTT reduced membrane fluidity and enhanced Ag+ sensitivity by downregulating fabF expression. The upregulated expression of heat shock genes demonstrated that the Ag+ released from Au@AgNPs compromised the heat adaptation of E. faecalis. Conclusion: PTT significantly enhanced Ag+ sensitivity of the original and Ag+-resistant E. faecalis. Au@AgNPs combined with PTT may have the potential to be developed as a new antibacterial agent to control E. faecalis infections in teeth.

Fe-S biogenesis by SMS and SUF pathways: A focus on the assembly step.

FeS clusters are prosthetic groups present in all organisms. Proteins with FeS centers are involved in most cellular processes. ISC and SUF are machineries necessary for the formation and insertion of FeS in proteins. Recently, a phylogenetic analysis on more than 10,000 genomes of prokaryotes have uncovered two new systems, MIS and SMS, which were proposed to be ancestral to ISC and SUF. SMS is composed of SmsBC, two homologs of SufBC(D), the scaffolding complex of SUF. In this review, we will specifically focus on the current knowledge of the SUF system and on the new perspectives given by the recent discovery of its ancestor, the SMS system.

Biological Traits and Comprehensive Genomic Analysis of Novel Enterococcus faecalis Bacteriophage EFP6.

Enterococcus faecalis is a prevalent opportunistic pathogen associated with chicken embryonic and neonatal chick mortality, posing a significant challenge in poultry farming. In the current study, E. faecalis strain EF6, isolated from a recent hatchery outbreak, served as the host bacterium for the isolation of a novel phage EFP6, capable of lysing E. faecalis. Transmission electron microscopy revealed a hexagonal head and a short tail, classifying EFP6 as a member of the Autographiviridae family. EFP6 showed sensitivity to ultraviolet radiation and resistance to chloroform. The lytic cycle duration of EFP6 was determined to be 50 min, highlighting its efficacy in host eradication. With an optimal multiplicity of infection of 0.001, EFP6 exhibited a narrow lysis spectrum and strong specificity towards host strains. Additionally, EFP6 demonstrated optimal growth conditions at 40 °C and pH 8.0. Whole genome sequencing unveiled a genome length of 18,147 bp, characterized by a GC concentration of 33.21% and comprising 25 open reading frames. Comparative genomic assessment underscored its collinearity with related phages, notably devoid of lysogenic genes, thus ensuring genetic stability. This in-depth characterization forms the basis for understanding the biological attributes of EFP6 and its potential utilization in phage therapy, offering promising prospects for mitigating E. faecalis-associated poultry infections.

Novel antimicrobial applications of copper oxide nanoparticles after combination with tissue conditioner used in complete prostheses.

BACKGROUND: Tissue conditioners are used for treating and improving the tissues supporting complete dentures. On the other hand, recent advances in nanotechnology have revolutionized various fields of science, including dentistry. The present study aimed to investigate novel antimicrobial applications of copper oxide nanoparticle-based tissue conditioner used in complete prostheses. METHODS: The present experimental study included 126 tissue conditioner samples with different concentrations of copper oxide nanoparticles (20%, 10%, 5%, 2.5%, 1.25%, 0.625%, and 0% w/w). The samples were incubated with Enterococcus faecalis, Pseudomonas aeruginosa, and Candida albicans in 24-well plates for 24 h. Then, samples from the wells were re-incubated for 24 h, and the microorganisms were counted. RESULTS: The culture media containing E. faecalis and P. aeruginosa showed significantly different growth between different nanoparticle concentrations following 24 h (P < 0.001), showing a reduction in bacterial growth with increased nanoparticle concentration. Both bacteria did not show any growth at the 20% concentration. However, C. albicans showed significant differences in growth between different nanoparticle concentrations following 48 h (P < 0.001), showing a reduction in growth with increased nanoparticle concentration. Also, the least growth was observed at the 20% concentration. CONCLUSIONS: In conclusion, the CuO nanoparticles were prepared using a green synthesis methon in the suitable sizes. Moreover, the tissue conditioners containing CuO nanoparticles showed acceptable antimicrobial properties against E. faecalis, P. aeruginosa, and C. albicans.

Comparative Evaluation of Antibacterial Efficacy, Molecular Docking of Ethanolic Extract of Blackseed, Seaweed and Calcium Hydroxide Intracanal Medicament with Enterococcus Faecalis Antigens.

Aim: To evaluate the inhibitory effect of ethanolic extract blackseed, seaweed, and calcium hydroxide intracanal medicament with Enterococcus faecalis biofilm. To study the binding interaction between the active components of blackseed and seaweed against the enterococcal surface protein of (E. faecalis) by molecular docking. Materials and Methods: The ethanolic extracts of blackseed and seaweed were prepared using the Soxhlet apparatus. They were divided into three groups, namely, |Group I: Calcium hydroxide, Group II: Blackseed, and Group III: Seaweed. The antibacterial activity of the three groups was detected employing various concentrations ranging from 250, 125, and 62.5 µg/ml and based on the zone of inhibition. The inhibitory potential of medicaments to inhibit E. faecalis growth at various stages and kinetics plate were assessed following biofilm architecture evaluation by crystal violet biofilm assay. With the Swissdock suite, the molecular docking procedure was carried out. PyMOL version 4.1.5 was the program used for visualization. Since enterococcal surface protein (Esp) is primarily involved in the formation of biofilms, it was chosen as the target protein of E. faecalis. Based on their chromatographic investigations, Group II Thymoquinone (TQ) and Group III Ledenoxide were chosen as ligands. Results: The percentage of inhibition of E. faecalis biofilm was analyzed as statistically significant observed within groups. On post-hoc analysis, significant differences were present between the groups (P < 0.05). Molecular docking reveals binding energies of thymoquinone (Group II) and ledenoxide (Group III) against the enterococcal surface protein of E. faecalis were -6.90 Kcal/mol and -6.44 Kcal/mol, respectively. Conclusion: Compared to seaweed, black seed extract exhibited higher antibacterial activity against the E. faecalis biofilm in microbial inhibition and molecular interaction.

Decoding the complexity of delayed wound healing following Enterococcus faecalis infection.

Wound infections are highly prevalent and can lead to delayed or failed healing, causing significant morbidity and adverse economic impacts. These infections occur in various contexts, including diabetic foot ulcers, burns, and surgical sites. Enterococcus faecalis is often found in persistent non-healing wounds, but its contribution to chronic wounds remains understudied. To address this, we employed single-cell RNA sequencing (scRNA-seq) on infected wounds in comparison to uninfected wounds in a mouse model. Examining over 23,000 cells, we created a comprehensive single-cell atlas that captures the cellular and transcriptomic landscape of these wounds. Our analysis revealed unique transcriptional and metabolic alterations in infected wounds, elucidating the distinct molecular changes associated with bacterial infection compared to the normal wound healing process. We identified dysregulated keratinocyte and fibroblast transcriptomes in response to infection, jointly contributing to an anti-inflammatory environment. Notably, E. faecalis infection prompted a premature, incomplete epithelial-mesenchymal transition in keratinocytes. Additionally, E. faecalis infection modulated M2-like macrophage polarization by inhibiting pro-inflammatory resolution in vitro, in vivo, and in our scRNA-seq atlas. Furthermore, we discovered macrophage crosstalk with neutrophils, which regulates chemokine signaling pathways, while promoting anti-inflammatory interactions with endothelial cells. Overall, our findings offer new insights into the immunosuppressive role of E. faecalis in wound infections.

If wounds get infected, they heal much more slowly, sometimes leading to skin damage and other complications, including disseminated infections or even amputation. Infections can happen in many types of wounds, ranging from ulcers in patients with diabetes to severe burns. If infections are not cleared quickly, the wounds can become 'chronic' and are unable to heal without intervention. Enterococcus faecalis is a type of bacteria that normally lives in the gut. Within that environment, in healthy people, it is not harmful. However, if it comes into contact with wounds ­ particularly diabetic ulcers or the site of a surgery ­ it can cause persistent infections and prevent healing. Although researchers are beginning to understand how E. faecalis initially colonises wounds, the biological mechanisms that transform these infections into chronic wounds are still largely unknown. Celik et al. therefore set out to investigate exactly how E. faecalis interferes with wound healing. To do this, Celik et al. looked at E. faecalis-infected wounds in mice and compared them to uninfected ones. Using a genetic technique called single-cell RNA sequencing, Celik et al. were able to determine which genes were switched on in individual skin and immune cells at the site of the wounds. This in turn allowed the researchers to determine how those cells were behaving in both infected and uninfected conditions. The experiments revealed that when E. faecalis was present in wounds, several important cell types in the wounds did not behave normally. For example, although the infected skin cells still underwent a change in behaviour required for healing (called an epithelial-mesenchymal transition), the change was both premature and incomplete. In other words, the skin cells in infected wounds started changing too early and did not finish the healing process properly. E. faecalis also changed the way macrophages and neutrophils worked within the wounds. These are cells in our immune system that normally promote inflammation, a process involved in both uninfected wounds or during infections and is a key part of wound healing when properly controlled. In the E. faecalis-infected wounds, these cells' inflammatory properties were suppressed, making them less helpful for healing. These results shed new light on how E. faecalis interacts with skin cells and the immune system to disrupt wound healing. Celik et al. hope that this knowledge will allow us to find new ways to target E. faecalis infections, and ultimately develop treatments to help chronic wounds heal better and faster.

Antibacterial Activity and Action Mechanism of Bacteriocin Paracin wx7 as a Selective Biopreservative against Vancomycin-Resistant Enterococcus faecalis in Fresh-Cut Lettuce.

Fresh-cut vegetables are widely consumed, but there is no food preservative available to selectively inhibit vancomycin-resistant E. faecalis, which is a serious health menace in fresh-cut vegetables. To develop a promising food biopreservative, a bacteriocin, paracin wx7, was synthesized, showing selective inhibition against E. faecalis with MIC values of 4-8 µM. It showed instant bactericidal mode within 1 h at high concentrations with concomitant cell lysis against vancomycin-resistant E. faecalis. Its lethal effect was visualized in a dose-dependent manner by PI/SYTO9 staining observation. The results of an in vivo control experiment carried out on E. faecalis in fresh-cut lettuce showed that 99.97% of vancomycin-resistant E. faecalis were dead after 64 µM paracin wx7 treatment for 7 days without influencing total bacteria. Further, the action mechanism of paracin wx7 was investigated. Confocal microscopy showed that paracin wx7 was located both on the cell envelope and in cytoplasm. For the cell envelope, the studies of membrane permeability using SYTOX Green dyeing and DNA leakage revealed that paracin wx7 damaged the membrane integrity of E. faecalis. Simultaneously, it exhibited membrane depolarization after analysis using DiSC3(5). Damage to the cell envelope resulted in cell deformation observed by scanning electron microscopy. On entering the cytoplasm, the paracin wx7 induced the production of endogenous reactive oxygen species.

Multispecies bacterial invasion of human host cells.

Urinary tract infection (UTI), one of the most common bacterial infections worldwide, is a typical example of an infection that is often polymicrobial in nature. While the overall infection course is known on a macroscale, bacterial behavior is not fully understood at the cellular level and bacterial pathophysiology during multispecies infection is not well characterized. Here, using clinically relevant bacteria, human epithelial bladder cells and human urine, we establish co-infection models combined with high resolution imaging to compare single- and multi-species bladder cell invasion events in three common uropathogens: uropathogenic Escherichia coli (UPEC), Klebsiella pneumoniae and Enterococcus faecalis. While all three species invaded the bladder cells, under flow conditions the Gram-positive E. faecalis was significantly less invasive compared to the Gram-negative UPEC and K. pneumoniae. When introduced simultaneously during an infection experiment, all three bacterial species sometimes invaded the same bladder cell, at differing frequencies suggesting complex interactions between bacterial species and bladder cells. Inside host cells, we observed encasement of E. faecalis colonies specifically by UPEC. During subsequent dispersal from the host cells, only the Gram-negative bacteria underwent infection-related filamentation (IRF). Taken together, our data suggest that bacterial multispecies invasions of single bladder cells are frequent and support earlier studies showing intraspecies cooperation on a biochemical level during UTI.

Engineering of chimeric enzymes with expanded tolerance to ionic strength.

Antimicrobial resistance poses a significant global threat, reaching dangerously high levels as reported by the World Health Organization. The emergence and rapid spread of new resistance mechanisms, coupled with the absence of effective treatments in recent decades, have led to thousands of deaths annually from infections caused by drug-resistant microorganisms. Consequently, there is an urgent need for the development of new compounds capable of combating antibiotic-resistant bacteria. A promising class of molecules exhibiting potent bactericidal effects is peptidoglycan hydrolases. Previously, we cloned and characterized the biochemical properties of the M23 catalytic domain of the EnpA (EnpACD) protein from Enterococcus faecalis. Unlike other enzymes within the M23 family, EnpACD demonstrates broad specificity. However, its activity is constrained under low ionic strength conditions. In this study, we present the engineering of three chimeric enzymes comprising EnpACD fused with three distinct SH3b cell wall-binding domains. These chimeras exhibit enhanced tolerance to environmental conditions and sustained activity in bovine and human serum. Furthermore, our findings demonstrate that the addition of SH3b domains influences the activity of the chimeric enzymes, thereby expanding their potential applications in combating antimicrobial resistance.IMPORTANCEThese studies demonstrate that the addition of the SH3b-binding domain to the EnpACD results in generation of chimeras with a broader tolerance to ionic strength and pH values, enabling them to remain active over a wider range of conditions. Such approach offers a relatively straightforward method for obtaining antibacterial enzymes with tailored properties and emphasizes the potential for proteins' engineering with enhanced functionality, contributing to the ongoing efforts to address antimicrobial resistance effectively.

Complete genome sequence of the linezolid-resistant clinical Enterococcus faecalis N23-3408 linked to a livestock lineage in Switzerland.

Here, we report the complete genome of human clinical linezolid-resistant Enterococcus faecalis N23-3408. N23-3408 harbored a 59.5 kb plasmid with antimicrobial resistance genes cat, erm(B), fexA, optrA, tet(L), and tet(M). Closely related E. faecalis harboring this plasmid was previously obtained from livestock animals and pet food in Switzerland.

Epidemiological trends and susceptibility patterns of bloodstream infections caused by Enterococcus spp. in six German university hospitals: a prospectively evaluated multicentre cohort study from 2016 to 2020 of the R-Net study group.

PURPOSE: To analyse recent epidemiological trends of bloodstream infections (BSI) caused by Enterococcus spp. In adult patients admitted to tertiary care centres in Germany. METHODS: Epidemiological data from the multicentre R-NET study was analysed. Patients presenting with E. faecium or E. faecalis in blood cultures in six German tertiary care university hospitals between October 2016 and June 2020 were prospectively evaluated. In vancomycin-resistant enterococci (VRE), the presence of vanA/vanB was confirmed via molecular methods. RESULTS: In the 4-year study period, 3001 patients with BSI due to Enterococcus spp. were identified. E. faecium was detected in 1830 patients (61%) and E. faecalis in 1229 patients (41%). Most BSI occurred in (sub-) specialties of internal medicine. The pooled incidence density of enterococcal BSI increased significantly (4.0-4.5 cases per 10,000 patient days), which was primarily driven by VRE BSI (0.5 to 1.0 cases per 10,000 patient days). In 2020, the proportion of VRE BSI was > 12% in all study sites (range, 12.8-32.2%). Molecular detection of resistance in 363 VRE isolates showed a predominance of the vanB gene (77.1%). CONCLUSION: This large multicentre study highlights an increase of BSI due to E. faecium, which was primarily driven by VRE. The high rates of hospital- and ICU-acquired VRE BSI point towards an important role of prior antibiotic exposure and invasive procedures as risk factors. Due to limited treatment options and high mortality rates of VRE BSI, the increasing incidence of VRE BSI is of major concern.