Genome-wide analysis of acid tolerance genes of Enterococcus faecalis with RNA-seq and Tn-seq.

Enterococcus faecalis, a formidable nosocomial and community-acquired opportunistic pathogen, can persist a wide range of extreme environments, including low pH and nutrient deficiency. Clarifying the survival mechanism of E. faecalis in low-pH conditions is the key to combating the infectious diseases caused by E. faecalis. In this study, we combined transcriptome profiling (RNA-seq) and transposon insertion sequencing (TIS) to comprehensively understand the genes that confer these features on E. faecalis. The metadata showed that genes whose products are involved in cation transportation and amino acid biosynthesis were predominantly differentially expressed under acid conditions. The products of genes such as opp1C and copY reduced the hydrion concentration in the cell, whereas those of gldA2, gnd2, ubiD, and ubiD2 mainly participated in amino metabolism, increasing matters to neutralize excess acid. These, together with the folE and hexB genes, which are involved in mismatch repair, form a network of E. faecalis genes necessary for its survival under acid conditions.

IMPORTANCE: As a serious nosocomial pathogen, Enterococcus faecalis was considered responsible for large numbers of infections. Its ability to survive under stress conditions, such as acid condition and nutrient deficiency was indispensable for its growth and infection. Therefore, understanding how E. faecalis survives acid stress is necessary for the prevention and treatment of related diseases. RNA-seq and TIS provide us a way to analyze the changes in gene expression under such conditions.

Use of an autonomous robotic system for removal of fiber posts during endodontic retreatment: A clinical report.

The removal of fiber posts for the retreatment of root canals can be challenging. An autonomous robotic (ATR) system was used for accurate and minimally invasive endodontic retreatment in a patient who presented with symptomatic apical periodontitis and pain emanating from the mandibular left first molar, which had been previously restored with 3 fiber posts. Intraoral scanning data, radiographic data, and bur sizes were integrated into preoperative software program to design a treatment plan. The ATR system enabled precise and efficient post removal. The remnant filling material was removed before endodontic retreatment, and radiographs at 3 months after the procedure showed satisfactory healing. This treatment demonstrated the accuracy and efficiency of the ATR system for the removal of fiber posts from pretreated teeth, while minimizing the duration of the procedure and avoiding excessive tissue removal.

Pain sensitivity and quality of life of patients with burning mouth syndrome: a preliminary study in a Chinese population.

BACKGROUND: Burning mouth syndrome (BMS) is an oral-facial pain disorder involving the central and peripheral nervous systems, but the evidence for altered pain sensitivity remains inconclusive. The aim of this study was to investigate pain sensitivity and oral health-related quality of life (OHRQoL) in patients with BMS and to assess the relationship between them. METHODS: Fifty Chinese patients with BMS (57.82 ± 11.2 years) and fifty age- and gender-matched healthy subjects (55.64 ± 10.1 years) participated in the study. The Pain Sensitivity Questionnaire (PSQ) was used to assess participants' pain sensitivity. The Oral Health Impact Profile (OHIP-14) was used to evaluate participants' OHRQoL. RESULTS: The PSQ total score (p = 0.009), the PSQ minor score (p = 0.003) and the OHIP-14 score (p<0.05) of patients with BMS were significantly higher than those of the healthy subjects. Simple linear regression showed that the PSQ minor score was significantly associated with the OHIP-14 score in patients with BMS (ß = 0.338, p = 0.016). CONCLUSION: Patients with BMS have higher pain sensitivity than healthy subjects. Reducing pain sensitivity might help to improve the quality of life of patients with BMS.

Influence of silver nanoparticles on the resin-dentin bond strength and antibacterial activity of a self-etch adhesive system.

STATEMENT OF PROBLEM: The influence of silver nanoparticles on adhesive properties of Single Bond Universal (3M ESPE) and the antibacterial activity of silver nanoparticles-modified adhesives against Streptococcus mutans is unclear. PURPOSE: The purpose of this in vitro study was to evaluate the effect of silver nanoparticles on the dentin bond strength of modified adhesives and the antibacterial activity against the cariogenic pathogen S. mutans. MATERIAL AND METHODS: Single Bond Universal adhesive was used as the control. Silver nanoparticles were added to adhesives at 0.05% and 0.1% (by weight) (experimental groups), and scanning electron microscopy was used to observe the uniformity of the modified adhesives. The Single Bond Universal adhesive and the modified adhesives were then used to prepare dentin-composite resin blocks. The microtensile bond strength and microleakage of the prepared dentin-composite resin blocks were determined with or without thermocycling. The colony-forming units (CFU) of S. mutans cultured with the adhesives were evaluated, and the microtensile bond strength and microleakage of each group were tested after treatment with S. mutans. The differences in the microtensile bond strength or CFU were analyzed by using the 2-way analysis of variance and independent sample t test. The differences in microleakage between the groups were evaluated by using the Mann-Whitney test (α=.05). RESULTS: Silver nanoparticle-modified adhesives exhibited uniform morphologies without agglomeration and exhibited a homogeneous adhesive layer in dentin-composite resin blocks. The microtensile bond strength and microleakage of the modified adhesives were similar to those of the control group, with or without thermocycling (P>.05). However, thermocycling reduced the bond strength significantly (P<.001). Self-etch adhesives incorporated with silver nanoparticles showed significant antibacterial activities after less than 6 months of aging treatment. The modified adhesives did not exhibit a decreased bond strength after S. mutans exposure (P>.05), while the control group exhibited a markedly decreased bond strength after S. mutans exposure (P<.05). CONCLUSIONS: Silver nanoparticle-modified adhesives showed excellent antibacterial activities against S. mutans and resisted the destruction of dentin bond strength caused by S. mutans while not compromising the bonding properties of Single Bond Universal self-etch adhesives.

Enterococcus Faecalis activates NLRP3 inflammasomes leading to increased interleukin-1 beta secretion and pyroptosis of THP-1 macrophages.

OBJECTIVES: Enterococcus faecalis is the bacterial species closely related to persistent infection in root canals. Interleukin-1 beta (IL-1ß) is the most commonly detected proinflammatory cytokine in periapical granulation tissue and plays a critical role in host defenses against microbial infection. The synthesis and secretion of IL-1ß are mediated mainly by Toll-like receptors and inflammasome activation. The previous study found that the nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) and the absent in Melanoma 2 (AIM2) inflammasomes are positively expressed in periapical granulation tissue. The aim of this study was to investigate the pathogenicity of E. faecalis and the molecular mechanisms of IL-1ß secretion by THP-1 macrophages infected with E. faecalis. METHODS: The IL-1ß and lactate dehydrogenase (LDH) levels induced by E. faecalis were investigated with enzyme-linked immunosorbent assay (ELISA) kit and cytotoxicity assay kit, caspase-1 and inflammasome expression levels were investigated using real time PCR and Western blot analysis. Then the effect of caspase-1, NLRP3, adenosine triphosphate (ATP), and extracellular K+ on IL-1ß and LDH secretion, Gasdermin-D (GSDMD) cleavage induced by E. faecalis were analyzed. RESULTS: E. faecalis significantly increased IL-1ß and LDH release, caspase-1 and GSDMD cleavage, and NLRP3 inflammasome activation. It also showed that IL-1ß and LDH release, GSDMD cleavage required caspase-1 and NLRP3 activation. Furthermore, the expression and activation of caspase-1 and NLRP3 were blocked by oxidized ATP and extracellular K+. CONCLUSION: E. faecalis infection activated caspase-1 and the NLRP3 inflammasome to induce IL-1ß secretion and inflammatory cell death (pyroptosis). Furthermore, the activation and expression of NLRP3 induced by E. faecalis required P2X7R and K+ efflux. This study furthers our understanding of the inflammatory response mechanism induced by E. faecalis indicates that NLRP3 may be a potential target for treatment and prevention of persistent periodontitis caused by E. faecalis.

Human Dental Pulp Stem Cells via the NF-κB Pathway.

BACKGROUND/AIMS: Odontogenic differentiation of human dental pulp stem cells (HDPSCs) is regulated by multiple factors and signaling molecules. However, their regulatory mechanisms are not completely understood. In this study, we investigated the role of Zinc finger and BTB domain-containing 20 (ZBTB20) in odontoblastic differentiation of HDPSCs. METHODS: HDPSCs were obtained from human third molars and ZBTB20 expression was examined by qRT-PCR and western blot. Their osteo/odontogenic differentiation and the involvement of NF-κB pathway were subsequently investigated. RESULTS: The expression of ZBTB20 is upregulated in a time-dependent manner during odontogenic differentiation of hDPSCs. Inhibition of ZBTB20 reduced osteogenic medium (OM)-induced odontogenic differentiation, reflected in decreased alkaline phosphatase (ALP) activity, mineralized nodule formation and mRNA expression of odonto/osteogenic marker genes. In contrast, overexpression of ZBTB20 enhanced ALP activity, mineralization and the expression of differentiation marker genes. Furthermore, the expression of IκBα was increased by ZBTB20 silencing in HDPSCs, whereas ZBTB20 overexpression decreased IκBα and enhanced nuclear NF-κB p65. Inhibition of the NF-κB pathway significantly suppressed the odontogenic differentiation of HDPSCs induced by ZBTB20. CONCLUSION: This study shows for the first time that ZBTB20 plays an important role during odontoblastic differentiation of HDPSCs and may have clinical implications for regenerative endodontics.

Dentin tubule invasion by Enterococcus faecalis under stress conditions ex vivo.

Enterococcus faecalis is the species most frequently isolated from failed endodontic treatments because it can survive under stress conditions imposed by root canal treatment. The objective of this study was to determine the ability of E. faecalis to invade dentine tubules under alkaline and energy-starvation stress and to explore the potential mechanisms. Roots from single-rooted human teeth were infected with E. faecalis under alkaline and energy-starvation stress conditions. After 4 wk of culture, samples were processed to establish the tubule-penetration distance. In addition, the hydrophobicity of E. faecalis cells under these conditions was analysed and the expression of genes involved in adhesion was quantified by real-time quantitative PCR. Culture of E. faecalis under alkaline and energy-starvation stress conditions resulted in a marked reduction of tubule-penetration distance, a significant increase in hydrophobicity of the bacterial surface, and marked down-regulation of most adhesin genes compared with E. faecalis cultured in tryptic soy broth. The results indicate that the dentine tubule invasion ability of E. faecalis was markedly decreased under alkaline and glucose-starvation stress conditions, possibly because of the increase of hydrophobicity and down-regulation of some adhesion genes.

Proinflammatory Effect of Membrane Vesicles Derived from Enterococcus faecalis at Neutral and Alkaline pH.

INTRODUCTION: The present study explored the proinflammatory impact of Enterococcus faecalis membrane vesicles (MVs) derived from culture medium at pH 7.4 and 9.0. METHODS: E. faecalis MVs were obtained by centrifugation and purified by size exclusion chromatography. Proteomic analyses were carried out on E. faecalis MVs to investigate their components. THP-1 macrophages were exposed to E. faecalis MVs, and the inflammatory cytokines and proteins were evaluated using ELISA and immunoblotting. The inflammatory cytokines in the serum of mice with intraperitoneal injection of E. faecalis MVs were evaluated by ELISA, and immunophenotyping of spleen cells was investigated with flow cytometry. RESULTS: Proteomic analysis revealed 196 proteins in E. faecalis MVs obtained under neutral and alkali conditions, 110 proteins were upregulated and 79 proteins were downregulated by alkaline pH. E. faecalis MVs induced secretion of inflammatory factors interleukin (IL)-1ß, IL-6 and tumor necrosis factor-α in a concentration-dependent manner. Immunoblotting revealed that E. faecalis MVs increased expression of pro-IL-1ß, nuclear factor-κBp65, and Toll-like receptor 2. In vivo studies demonstrated that E. faecalis MVs significantly promoted secretion of IL-1ß in mouse serum, while inflammatory cells were activated in the spleen. E. faecalis MVs obtained at pH 9.0 showed stronger proinflammatory effects than those obtained under neutral pH. CONCLUSION: E. faecalis produce MVs that carry specific proteins associated with virulence factors, and these MVs can promote inflammation in vitro and in vivo. E. faecalis MVs obtained under alkaline conditions have a stronger proinflammatory effect.

Endodontic management considerations for Type III dens invaginatus based on anatomical characteristics: A case series.

Dens invaginatus (DI) is a developmental anomaly of the teeth characterised by the in-folding of the enamel into the dentin. Oehlers' Type III DI is the most serious form, in which the inherently invaginated channels communicate with periodontal and dental pulp tissue, increasing the risk of bacterial contamination. However, varying and complex anatomical features make diagnosis and treatment challenging. Conventional endodontic therapies promote healing by avoiding unnecessary interventions (e.g., surgical or other invasive treatments). Radiographic examination can reveal the structural details of such malformations. We obtained multiple procedural details for treating Type III DI based on radiographic analyses from our clinical experience. In addition, we introduce a new classification strategy for the management of Type III DI that is more applicable to treatment needs. This study aimed to discuss the anatomical features and current treatment considerations of Type III DI.

Antibacterial tellurium-containing polycarbonate drug carriers to eliminate intratumor bacteria for synergetic chemotherapy against colorectal cancer.

Intratumor microbes have attracted great attention in cancer research due to its influence on the tumorigenesis, progression and metastasis of cancer. However, the therapeutic strategies targeting intratumoral microbes are still in their infancy. Specific microorganisms, such as Fusobacterium nucleatum (F. nucleatum), are abundant in various cancer and always result in the CRC progression and chemotherapy resistance. Here, a combined anticancer and antibacterial therapeutic strategy is proposed to deliver antitumor drug to the tumors containing intratumor microbiota by the antibacerial polymeric drug carriers. We construct oral tellurium-containing drug carriers using a complex of tellurium-containing polycarbonate with cisplatin (PTE@CDDP). The results show that the particle size of the prepared nanoparticles could be maintained at about 105 nm in the digestive system environment, which is in line with the optimal particle size of oral nanomedicine. In vitro mechanism study indicates that the tellurium-containing polymers are highly effective in killing F.nucleatum through a membrane disruption mechanism. The pharmacokinetic experiments confirmed that PTE@CDDP has the potential function of enhancing the oral bioavailability of cisplatin. Both in vitro and in vivo studies show that PTE@CDDP could inhibit intratumor F.nucleatum and lead to a reduction in cell proliferation and inflammation in the tumor site. Together, the study identifies that the CDDP-loaded tellurium-containing nanoparticles have great potential for treating the F.nucleatum-promoted colorectal cancer (CRC) by combining intratumor microbiota modulation and chemotherapy. The synergistic therapeutic strategy provide new insight into treating various cancers combined with bacterial infection. STATEMENT OF SIGNIFICANCE: The synthesized antibacterial polymer was first employed to remodel the intratumor microbes in tumor microenvironment (TME). Moreover, it was the first report of tellurium-containing polymers against F.nucleatum and employed for treatment of the CRC. A convenient oral dosage form of cisplatin (CDDP)-loaded tellurium-containing nanoparticles (PTE@CDDP) was adopted here, and the synergistic antibacterial/chemotherapy effect occurred. The PTE@CDDP could quickly and completely eliminate F.nucleatum in a safe dose. In the CRC model, PTE@CDDP effectively reversed the inflammation level and even restored the intestinal barrier damaged by F.nucleatum. The ultrasensitive ROS-responsiveness of PTE@CDDP triggered the fast oxidation and efficient drug release of CDDP and thus a highly efficient apoptosis of the tumors. Therefore, the tellurium-containing polymers are expected to serve as novel antibacterial agents in vivo and have great potential in the F.nucleatum-associated cancers. The achievements provided new insight into treating CRC and other cancers combined with bacterial infection.

Variations in protein expression associated with oral cancer.

BACKGROUND: Differential protein expression of the oral microbiome is related to human diseases, including cancer. OBJECTIVE: In order to reveal the potential relationship between oral bacterial protein expression in oral squamous cell carcinoma (OSCC), we designed this study. METHODS: We obtained samples of the same patient from cancer lesion and anatomically matched normal site. Then, we used the label free quantitative technique based on liquid chromatography tandem mass spectrometry (LC-MS/MS) to analyze the bacteria in the samples of oral squamous cell carcinoma at the protein level, so as to detect the functional proteins. RESULTS: Protein diversity in the cancer samples was significantly greater than in the normal samples. We identified a substantially higher number of the taxa than those detected in previous studies, demonstrating the presence of a remarkable number of proteins in the groups. In particular, proteins involved in energy production and conversion, proton transport, hydrogen transport and hydrogen ion transmembrane transport, ATP-binding cassette (ABC) transporter, PTS system, and L-serine dehydratase were enriched significantly in the experimental group. Moreover, some proteins associated with Actinomyces and Fusobacterium were highly associated with OSCC and provided a good diagnostic outcome. CONCLUSION: The present study revealed considerable changes in the expression of bacterial proteins in OSCC and enrich our understanding in this point.

Prevalence of taste and smell dysfunction in mild and asymptomatic COVID-19 patients during Omicron prevalent period in Shanghai, China: a cross-sectional survey study.

OBJECTIVES: COVID-19, which is caused by SARS-CoV-2, is a severe threat to human health and the economy globally. This study aimed to investigate the prevalence of taste and/or smell dysfunction and associated risk factors in mild and asymptomatic patients with Omicron infection in Shanghai, China.DesignThis was a questionnaire-based cross-sectional study. SETTING: COVID-19 patients at the makeshift hospital in the Shanghai World Expo Exhibition and Convention Centre were recruited from March to April 2022. PARTICIPANTS: In total, 686 COVID-19-infected patients who were defined as mild or asymptomatic cases according to the diagnostic criteria of New Coronavirus Pneumonia Prevention and Control Programme ninth edition (National Health Commission of China, 2022) were enrolled. MEASURES: Data to investigate taste and smell loss and to characterise other symptoms were collected by the modified Chemotherapy-induced Taste Alteration Scale and Sino-Nasal Outcome Test-22 questionnaires. The risk factors for the severity of taste/smell dysfunction were analysed by binary logistic regression models. RESULTS: 379 males (379/686, 55.2%) and 307 females (307/686, 44.8%) completed the questionnaires to record recent changes in taste and smell ability. A total of 302 patients (44%) had chemosensory dysfunction with Omicron infection, of which 22.7% (156/686) suffered from both taste and smell dysfunction. In addition, cough (60.2%), expectoration (40.5%), fever (33.2%) and sore throat (32.5%) were common symptoms during Omicron infection. The quality-of-life-related indicators were negatively associated with participants' self-reported taste and smell dysfunction. CONCLUSIONS: The prevalence of taste or/and smell dysfunction in patients with Omicron infections was 44%. Individuals with chemosensory dysfunction had significantly higher rates of various upper respiratory influenza-like symptoms, xerostomia and bad breath. Moreover, smell dysfunction was a risk factor for the prevalence of taste dysfunction in patients with Omicron infection. TRIAL REGISTRATION NUMBER: ChiCTR 2200059097.

Fluoride resistance capacity in mammalian cells involves global gene expression changes associate with ferroptosis.

OBJECTIVE: The purpose of this study was to understand mouse osteoblast ferroptosis under high fluoride environment by stimulating fluoride levels to corresponding levels. In order to define the underlying mechanism of fluoride resistance in mammals and provide a theoretical basis for fluorosis treatment, high-throughput sequencing was applied to map the genetic changes of fluoride-resistant mouse osteoblasts and analyze the role of ferroptosis-related genes. METHODS: Cell Counting Kit-8, Reactive Oxygen Species Assay Kit and C11 BODIPY 581/591 were used to monitor proliferation and ferroptosis of mouse osteoblasts MC3T3-E1 under high fluoride environment. Fluoride-tolerant MC3T3-E1 cells were developed by gradient fluoride exposure. The differentially expressed genes of fluorine-resistant MC3T3-E1 cells were identified by high-throughput sequencing. RESULTS: MC3T3-E1 cells cultured in medium containing 20, 30, 60, 90 ppm F- exhibited decreased viability and increased reactive oxygen species and lipid peroxidation levels in correlation with F- concentrations. High-throughput RNA sequencing identified 2702 differentially expressed genes (DEGs) showed more than 2-fold difference in 30 ppm FR MC3T3-E1 cells, of which 17 DEGs were associated with ferroptosis. CONCLUSION: High fluoride environment affected the content of lipid peroxides in the body and increased the level of ferroptosis, further, ferroptosis-related genes played specific roles in the fluoride resistance of mouse osteoblasts.

Proteomic study of plaque fluid in high caries and caries free children.

BACKGROUND: The occurrence and development of caries is a complex process affected by multiple factor. OBJECTIVE: The present study was envisaged to evaluate the plaque fluid in caries free children and children with high caries, in order to establish a data set of bacterial secretion proteomics. A non-labeled quantitative technique based on two-dimensional liquid chromatography-series mass spectroscopy was employed to detect plaque fluid. Based on the proteomics data, the database search, data processing and pathway analysis illuminated the function of these proteins, and clarified the role of plaque microecology in caries occurrence and development. METHODS: The study enrolled 8 caries free (CF) children, whose decayed-missed-filled surface of teeth is 0 (dmfs = 0), and caries sensitive (CS) children, whose decayed-missed-filled surface of teeth is > 10(dmfs > 10) (3 ∼ 5 years old) for the smooth tooth plaque and the plaque in the high caries group. The plaque protein was extracted using the unlabeled quantitative technique like liquid chromatography-series mass spectrometry, using DeCyderTM MS Differential Analysis Software (version 1.0, GE Healthcare) that detected and compared the spectra, and quantified the full scanning before series mass spectroscopy. After obtaining all peptides with quantitative information, significantly differential polypeptide molecules were obtained (p< 0.05), and a metabolic pathway analysis was performed. RESULTS: We identified 1,804 peptides with quantitative information, including 39 in CF, 30 in CS, and 1,735 similarly expressing peptides. After statistical analysis, 603 statistically different expression peptide data sets were obtained, including 202 high-expressed peptides in Group CF, 33 greater than 1.5 fold peptides, 401 high-expressed in Group CS and 199 greater than 1.5 fold peptide (173 nonredundant proteins). CONCLUSION: Our study obtained the largest known dataset of the bacterial secretion protein in children with high caries, and screened the data set of high caries state. 603 peptides were statistically rich in 101 pathways, including glycolysis pyruvate acid metabolism, tricarboxylic acid cycle, pentyl phosphate pathway, fructose mannose metabolism, starch and sucrose metabolism, and ABC transporters.

Monitoring of canonical BMP and Wnt activities during postnatal stages of mouse first molar root formation.

OBJECTIVE: This study aimed to explore the precise temporospatial distributions of bone morphogenetic protein (BMP) and Wnt signaling pathways during postnatal development of mammalian tooth roots after the termination of crown morphogenesis. METHODOLOGY: A total of two transgenic mouse lines, BRE-LacZ mice and BAT-gal mice, were undertaken. The mice were sacrificed on every postnatal (PN) day from PN 3d up to PN 21d. Then, the first lower molars were extracted, and the dissected mandibles were stained with 5-bromo-4-chloro-3-indolyl-ß-d-galactopyranoside (X-gal) and fixed. Serial sections at 10 µm were prepared after decalcification, dehydration, and embedding in paraffin. RESULTS: We observed BMP/Smads and Wnt/ß-catenin signaling activities in the dental sac, dental pulp, and apical papilla with a certain degree of variation. The position of activation of the BMP/Smad signaling pathway was located more coronally in the early stage, which then gradually expanded as root elongation proceeded and was associated with blood vessels in the pulp and developing complex apical tissues in the later stage. However, Wnt/ß-catenin signaling was highly concentrated in the mesenchyme below the cusps in the early stage, gradually expanded to regions around the root in the transition/root stage, and then disappeared entirely in the later stage. CONCLUSIONS: These results further confirmed the participation of both BMP and Wnt canonical signaling pathways in tooth root development, as well as formed the basis for future studies on how precisely integrated signaling pathways regulate root morphogenesis and regeneration.

Phosphate transport system mediates the resistance of Enterococcus faecalis to multidrug.

Enterococcus faecalis, a severe nosocomial and community opportunistic pathogen, is difficult to control due to its multidrug resistance. Through heredity and the recombination of intrinsic resistance genes and horizontally acquired resistance genes, E. faecalis can rapidly evolve drug resistance. Nisin, an important antimicrobial peptide, is extensively employed in the healthcare and food industries to inhibit Gram-positive bacteria and may induce the emergence of nisin-resistant bacteria worldwide. However, the mechanism governing nisin resistance in E. faecalis has not been fully elucidated. This study utilizes transposon insertion sequencing (TIS) to comprehensively explore novel genes related to nisin resistance. According to the analysis of TIS results, hundreds of genes appear to be essential for nisin resistance in E. faecalis. The phosphate transport system (OG1RF_10018-10021, named PTS), which is screened by TIS results, enhances the resistance of E. faecalis to nisin, the mechanism of which may be involved in potA and/or OG1RF_10526 (hypothetical gene). Meanwhile, PTS also strongly represses the biosynthesis of ribosomes to increase the sensitivity of E. faecalis to gentamycin. In addition, the overexpression of PTS increases the sensitivity of E. faecalis to daptomycin, the mechanism of which is independent of the LiaFSR system. This study first demonstrated that E. faecalis utilizes PTS to mediate the resistance to multidrug, which may help to elucidate the mechanism governing drug resistance and to establish guidelines for the treatment of infectious diseases caused by E. faecalis.

Carbohydrate Metabolism Affects Macrophage-Mediated Killing of Enterococcus faecalis.

Enterococcus faecalis, an opportunistic pathogen that causes severe community-acquired and nosocomial infections, has been reported to resist phagocyte-mediated killing, which enables its long-term survival in the host. Metabolism, especially carbohydrate metabolism, plays a key role in the battle between pathogens and hosts. However, the function of carbohydrate metabolism in the long-term survival of E. faecalis in phagocytes has rarely been reported. In this study, we utilized transposon insertion sequencing (TIS) to investigate the function of carbohydrate metabolism during the survival of E. faecalis in RAW264.7 cells. The TIS results showed that the fitness of carbohydrate metabolism-related mutants, especially those associated with fructose and mannose metabolism, were significantly enhanced, suggesting that the attenuation of carbohydrate metabolism promotes the survival of E. faecalis in macrophages. The results of our investigation indicated that macrophages responded to carbohydrate metabolism of E. faecalis and polarized to M1 macrophages to increase nitric oxide (NO) production, leading to the enhancement of macrophage-mediated killing to E. faecalis. Meanwhile, E. faecalis automatically decreased carbohydrate metabolism to escape from the immune clearance of macrophages during intracellular survival. The shift of primary carbon resources for macrophages affected the ability to clear intracellular E. faecalis. In summary, the results of the present study demonstrated that carbohydrate metabolism affects the macrophage-mediated killing of E. faecalis. IMPORTANCE E. faecalis has become a major pathogen leading to a variety of infections around the world. The metabolic interaction between E. faecalis and its host is important during infection but is rarely investigated. We used transposon insertion sequencing coupled with transcriptome sequencing to explore the metabolic interaction between E. faecalis and macrophages and uncovered that the shift of carbohydrate metabolism dramatically affected the inflammatory response of macrophages. In addition, E. faecalis attenuated carbohydrate metabolism to avoid the activation of the immune response of macrophages. This study provides new insights for the reason why E. faecalis is capable of long-term survival in macrophages and may facilitate the development of novel strategies to treat infectious diseases.

miR-200a contributes to the migration of BMSCs induced by the secretions of E. faecalis via FOXJ1/NFκB/MMPs axis.

BACKGROUND: Upon migrating to the injured sites, bone marrow mesenchymal stem cells (BMSCs) play critical roles in the repair of bone lesion caused by chronic apical periodontitis. Emerging evidences have shown that Enterococcus faecalis is always associated with apical periodontitis, especially refractory apical periodontitis. But the mechanism underlying how Enterococcus faecalis affects the migration of BMSCs remains unclear. METHODS: The effects of Enterococcus faecalis supernatants on the migration of BMSCs were determined by transwell migration assays. miRNA sequencing was performed to detect the significantly differentially expressed miRNAs of BMSCs. Proteomics analysis was used to detect the protein expression alterations of BMSCs. Luciferase report assays were deployed to verify the targets of miRNA. Western blot analysis was performed to examine the expressions of matrix metalloproteinases-3, matrix metalloproteinases-9, Forkhead Box Protein J1 (FOXJ1), and nuclear factor kappa B (NFκB). The activations of NFκB were detected by luciferase assays with NFκBluc reporter. RESULTS: We found that Enterococcus faecalis supernatants could promote the migration of BMSCs. The upregulation of miR-200a-3p in this process contributed to BMSC migration through downregulating its target Forkhead Box Protein J1. Moreover, FOXJ1/ NFκB axis was found to regulate matrix metalloproteinases (MMPs) in this process. CONCLUSIONS: These results above suggest that miR-200a contributes to the migration of BMSCs induced by the secretions of E. faecalis via FOXJ1/NFκB/MMPs axis.

[In vitro study of antimicrobial efficacy of different irrigations on Enterococcus faecalis biofilm formation in root canal].

PURPOSE: To compare the antimicrobial effect of different irrigations on Enterococcus faecalis biofilms in extracted teeth and evaluate the antimicrobial activity of irrigating solutions residual against E. faecalis biofilms formation, in order to provide a better strategy for clinician. METHODS: Extracted human premolar teeth with single root canal were clearly autoclaved. These teeth were contaminated with E. faecalis（ATCC33186） and incubated for 60 days. The samples were randomly assigned to 4 experimental groups. During biomechanical instrumentation, the root canal was irrigated with different irrigating agents. The bacteria samples were collected with sterile paper points before and after instrumentation to F2. Then, samples that had been instrumented and autoclaved again were randomly divided into 2 groups treated with normal saline and 1%NaOCl for 30 min. E. faecalis was used to contaminate these root canals. The bacteria samples were collected with sterile paper points after 2, 6, 24, 48 h. SPSS19.0 software package was used for statistical analysis. RESULTS: Group using 1% NaOCI with ultrasound devices was significantly more effective than NS alone groups. 1% NaOCI groups showed a better residual activity than NS group. CONCLUSIONS: NaOCl is still the most important irrigating solutions, and it could be a better choice after biomechanical instrumentation, because of its long time substantivity achieves residual antimicrobial activity. Ultrasound devices is recommended to coordinate with irrigation.

Analysis of the expression of NLRP3 and AIM2 in periapical lesions with apical periodontitis and microbial analysis outside the apical segment of teeth.

OBJECTIVE: To detect the distribution and expression levels of the nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) and the absent in Melanoma 2 (AIM2) inflammasomes in periapical lesions and to analyse the possible microbial stimuli outside of teeth. DESIGN: The distribution of NLRP3 and AIM2 inflammasomes in sixteen periapical lesions was investigated by immunohistochemistry. Meanwhile, the relative gene expression levels of NLRP3 and AIM2 in sixteen periapical lesions and three health periodontal tissue were quantified by real-time polymerase chain reaction (PCR). Moreover, forty-seven teeth without sinus tracts were obtained in the clinic and included in bacterial analysis using PCR. Then, the mRNA levels of apoptosis-associated speck-like protein (ASC), caspase-1, interleukin-1ß (IL-1ß), NLRP3 and AIM2 in THP-1-derived macrophages treated with lipopolysaccharides (LPS) of Porphyromonas were also quantified by real-time PCR, and the IL-1ß secretion level was investigated using enzyme-linked immunosorbent assay (ELISA). RESULTS: NLRP3 and AIM2 were positively expressed in periapical lesions and were mainly distributed in inflammatory cells. Most of the samples that demonstrated up-regulation of NLRP3 mRNA also demonstrated up-regulation of caspase-1 mRNA. Microbial analysis revealed that Porphyromonas endodontalis was the most commonly detected species and was detected in 27 of 47 cases (57.4%), followed by Fusobacterium nucleatum (20/47, 42.6%), Porphyromonas gingivalis (19/47, 40.4%), Tannerella forsythia (19/47, 40.4%), Actinomyces sp. (17/47, 36.17%), Treponema denticola (10/47,21.28%), Actinomyces israelii (9/47,19.15%), Prevotella intermedia (6/47, 12.77%), Enterococcus faecalis (1/47,2.13%) and Enterococcus faecium (0/47,0). Furthermore, we found that LPS of P. gingivalis induced THP-1 cells to produce IL-1ß and to activate NLRP3 and AIM2 inflammasomes. CONCLUSIONS: Our results suggest that the NLRP3 and AIM2 proteins play a part in the pathogenesis of periapical periodontitis. Anaerobes, such as P. endodontalis, P. gingivalis, F. nucleatum and T. forsythia, were the main detected microbial stimuli that might activate inflammasomes in periapical tissues.