Effect of Laurus nobilis on bacteria and human transforming growth factor-ß1.

OBJECTIVE: In this study, we aimed to determine the phenolic compounds, the antibacterial activity of extract from Laurus nobilis leaves, and its possible effect on transforming growth factor-ß1 expression level in peripheral blood mononuclear cells. METHODS: The phenolic components of Laurus nobilis were identified by the high-performance liquid chromatography method. The antibacterial activity of this extract was determined by disk diffusion and broth microdilution methods. The transforming growth factor-ß1 expression was analyzed using the RT-qPCR method. RESULTS: Epicatechin was found in the highest amount and o-coumaric acid in the lowest amount. The half-maximal inhibitory concentration (IC50) was determined to be 55.17 µg/mL. The zones of inhibition and minimum inhibitory concentration for Staphylococcus aureus, Enterococcus faecalis, and Klebsiella pneumoniae were 15, 14, and 8 mm and 125, 250, and 1000 µg/mL, respectively. The change in transforming growth factor-ß1 expression levels was found to be statistically significant compared with the control groups (p<0.0001). CONCLUSION: Laurus nobilis extract was found to be effective against bacteria and altered the expression level of transforming growth factor-ß1 in peripheral blood mononuclear cells.

In Vitro Antibacterial and Phytochemical Screening of Hypericum perforatum Extract as Potential Antimicrobial Agents against Multi-Drug-Resistant (MDR) Strains of Clinical Origin.

Background: The perennial plant Hypericum perforatum is widely distributed around the world. It has been used for many years in conventional medicine to treat a variety of illnesses, including stress, mild to moderate depression, and minor injuries. This study examined the antimicrobial activity of the H. perforatum total extract and its fractions (n-hexane, ethyl acetate, chloroform, and aqueous) against multi-drug-resistant (MDR) isolates that were gathered from clinical samples, including methicillin-resistant Staphylococcus aureus (MRSA), Enterococcus faecalis, Escherichia coli, and Klebsiella pneumonia. Materials and Methods: Aerial parts of H. perforatum were collected and extracted using various solvents and were tested versus different isolated bacterial species. The inhibition zone of tested extracts was detected using an agar diffusion assay, and MICs were measured. Phytochemical analysis of promising H. perforatum extract was done using LC-ESI-MS/MS. Ultrastructure examination for the most altered bacteria used transmission electron microscopy. Antioxidant assays were done using DPPH and ABTS scavenging capacity methods. Cytotoxicity was reported versus Vero cells. Results: Different extracts of H. perforatum showed promising antibacterial activity against the pathogens. While the subfractions of the total extract were observed to show lesser inhibition zones and higher MIC values than the total extract of H. perforatum against MDR strains, the total extract of H. perforatum demonstrated the most potent antimicrobial action with an inhibition zone range of 17.9-27.9 mm. MDR-K. pneumoniae was discovered to be the most susceptible strain, which is consistent with the antibacterial inhibitory action of H. perforatum whole extract. Additionally, after treatment at the minimum inhibitory concentration (MIC 3.9 µg/ml), the transmission electron microscope showed alterations in the ultrastructure of the K. pneumoniae cells. Methanol extract from H. perforatum has a CC50 value of 976.75 µg/ml. Conclusion: Future inhibitors that target MDR strains may be revealed by these findings. Additionally, the extracts that were put to the test demonstrated strong antioxidant effects as shown by DPPH or ABTS radical-scavenging assays.

The Mechanism of Action of Ginkgolic Acid (15:1) against Gram-Positive Bacteria Involves Cross Talk with Iron Homeostasis.

With the increasing reports of community-acquired and nosocomial infection caused by multidrug-resistant Gram-positive pathogens, there is an urgent need to develop new antimicrobial agents with novel antibacterial mechanisms. Here, we investigated the antibacterial activity of the natural product ginkgolic acid (GA) (15:1), derived from Ginkgo biloba, and its potential mode of action against the Gram-positive bacteria Enterococcus faecalis and Staphylococcus aureus. The MIC values of GA (15:1) against clinical E. faecalis and S. aureus isolates from China were ≤4 and ≤8 µg/mL, respectively, from our test results. Moreover, GA (15:1) displayed high efficiency in biofilm formation inhibition and bactericidal activity against E. faecalis and S. aureus. During its inhibition of the planktonic bacteria, the antibacterial activity of GA (15:1) was significantly improved under the condition of abolishing iron homeostasis. When iron homeostasis was abolished, inhibition of planktonic bacteria by GA (15:1) was significantly improved. This phenomenon can be interpreted as showing that iron homeostasis disruption facilitated the disruption of the functions of ribosome and protein synthesis by GA (15:1), resulting in inhibition of bacterial growth and cell death. Genetic mutation of ferric uptake regulator (Fur) led to GA (15:1) tolerance in in vitro-induced resistant derivatives, while overexpression of Fur led to increased GA (15:1) susceptibility. Additionally, GA (15:1) significantly decreased the bacterial loads of S. aureus strain USA300 in the lung tissues of mice in a pneumonic murine model. Conclusively, this study revealed an antimicrobial mechanism of GA (15:1) involving cross talk with iron homeostasis against Gram-positive pathogens. In the future, the natural product GA (15:1) might be applied to combat infections caused by Gram-positive pathogens. IMPORTANCE The increasing emergence of infectious diseases associated with multidrug-resistant Gram-positive pathogens has raised the urgent need to develop novel antibiotics. GA (15:1) is a natural product derived from Ginkgo biloba and possesses a wide range of bioactivities, including antimicrobial activity. However, its antibacterial mechanisms remain unclear. Our current study found that the function of ferric uptake regulator (Fur) was highly correlated with the antimicrobial activity of GA (15:1) against E. faecalis and that the antibacterial activity of GA (15:1) could be strengthened by the disruption of iron homeostasis. This study provided important insight into the mode of action of GA (15:1) against Gram-positive bacteria and suggested that GA (15:1) holds the potential to be an antimicrobial treatment option for infection caused by multidrug-resistant Gram-positive pathogens.

Evaluation of the antibacterial effect of tea tree oil on Enterococcus faecalis and biofilm in vitro.

ETHNOPHARMACOLOGICAL RELEVANCE: Tea tree essential oil (TTO) is extracted from the leaves of Melaleuca alternifolia by steam distillation. It is well known for its traditional medicinal uses, particularly for the treatment of bruises, insect bites, skin infections, vertigo, convulsions, toothache, and rheumatism. Earlier research has shown that TTO can effectively inhibit oral microorganisms in the root canals. Enterococcus faecalis (E. faecalis) has been considered to be associated with persistent root canal infections and root canal treatment failure. The biofilm of E. faecalis makes it more vigorous, toxic, and resistant to antibiotics. AIM OF THE STUDY: In this study, our aim was to evaluate the antimicrobial effects of TTO on planktonic E. faecalis and biofilms compared with 0.2% CHX. MATERIALS AND METHODS: We explored the minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC), the bacteriostatic rate by MTT assay, the antimicrobial time by time-kill assay, and the effects on cell integrity, the biomass, and bacterial activity of E. faecalis biofilms. Finally, we investigated the microstructure changes of E. faecalis biofilms using scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM). RESULTS: The MIC and MBC values were 0.25% and 0.5%, the bacterial inhibition rate, time-kill was dosage dependent, and TTO can effectively destroy membrane integrity. SEM CLSM images revealed that TTO could reduce bacterial aggregation, biofilm thickness and inhibited biofilm formation. The effect of TTO was the same as that of 0.2% CHX at some specific concentrations. In summary, TTO has the potential to be effective against E. faecalis infections. CONCLUSIONS: TTO was able to inhibit E. faecalis by destroying cell membrane, inhibiting the formation of E. faecalis biofilms, and eliminating mature formed biofilms. In this study, TTO has the potential to be further developed as a novel antibacterial drug.

Cymbopogon citratus essential oil: an active principle of nanoemulsion against Enterococcus faecalis root canal biofilm.

Aim: The objective was to formulate and characterize the nanoemulsion based on Cymbopogon citratus oil, intended for use in infected teeth root canal therapy. The investigation of the antioxidant and antibiofilm potential toward Enterococcus faecalis was aimed as well. Materials & methods: Characterization of oil (by GC/MS analysis) and nanoemulsion (by dynamic light scattering instrument), and determination of antibacterial (by microdilution assay), antibiofilm (by crystal violet assay) and antioxidant properties (by 2,2-diphenyl-1-picryl-hydrazyl-hydrate and thiobarbituric acid assay methods) were provided. Antibiofilm efficacy of irrigation procedure including nanoemulsion was screened on extracted teeth (by CFU-counting assay). Results: Notable antibacterial and antibiofilm activity, both against forming and preformed biofilms of oil, was observed. Irrigation involved nanoemulsion showed remarkable antibiofilm potential. Both substances induced some antioxidant activity. Conclusion: Results encourage further research with the aim of application of the nanoemulsion in dental practice.

Application of Green Algal Planktochlorella nurekis Biomasses to Modulate Growth of Selected Microbial Species.

As microalgae are producers of proteins, lipids, polysaccharides, pigments, vitamins and unique secondary metabolites, microalgal biotechnology has gained attention in recent decades. Microalgae can be used for biomass production and to obtain biotechnologically important products. Here, we present the application of a method of producing a natural, biologically active composite obtained from unicellular microalgae of the genus Planktochlorella sp. as a modulator of the growth of microorganisms that can be used in the cosmetics and pharmaceutical industries by exploiting the phenomenon of photo-reprogramming of metabolism. The combination of red and blue light allows the collection of biomass with unique biochemical profiles, especially fatty acid composition (Patent Application P.429620). The ethanolic and water extracts of algae biomass inhibited the growth of a number of pathogenic bacteria, namely Enterococcus faecalis, Staphylococcus aureus PCM 458, Streptococcus pyogenes PCM 2318, Pseudomonas aeruginosa, Escherichia coli PCM 2209 and Candida albicans ATCC 14053. The algal biocomposite obtained according to our procedure can be used also as a prebiotic supplement. The presented technology may allow the limitation of the use of antibiotics and environmentally harmful chemicals commonly used in preparations against Enterococcus faecalis, Staphylococcus aureus, Streptococcus pyogenes, Pseudomonas aeruginosa, Escherichia coli or Candida spp.

Metabolic Profiling of VOCs Emitted by Bacteria Isolated from Pressure Ulcers and Treated with Different Concentrations of Bio-AgNPs.

Considering the advent of antibiotic resistance, the study of bacterial metabolic behavior stimulated by novel antimicrobial agents becomes a relevant tool to elucidate involved adaptive pathways. Profiling of volatile metabolites was performed to monitor alterations of bacterial metabolism induced by biosynthesized silver nanoparticles (bio-AgNPs). Escherichia coli, Enterococcus faecalis, Klebsiella pneumoniae and Proteus mirabilis were isolated from pressure ulcers, and their cultures were prepared in the presence/absence of bio-AgNPs at 12.5, 25 and 50 µg mL-1. Headspace solid phase microextraction associated to gas chromatography-mass spectrometry was the employed analytical platform. At the lower concentration level, the agent promoted positive modulation of products of fermentation routes and bioactive volatiles, indicating an attempt of bacteria to adapt to an ongoing suppression of cellular respiration. Augmented response of aldehydes and other possible products of lipid oxidative cleavage was noticed for increasing levels of bio-AgNPs. The greatest concentration of agent caused a reduction of 44 to 80% in the variety of compounds found in the control samples. Pathway analysis indicated overall inhibition of amino acids and fatty acids routes. The present assessment may provide a deeper understanding of molecular mechanisms of bio-AgNPs and how the metabolic response of bacteria is untangled.

Wound Infections from Taiwan Cobra (Naja atra) Bites: Determining Bacteriology, Antibiotic Susceptibility, and the Use of Antibiotics-A Cobra BITE Study.

Wound necrosis and secondary infection are common complications after Naja atra bites. Clinical tools to evaluate the infection risk after Taiwan cobra bites are lacking. In this Cobra BITE study, we investigated the prevalence of wound infection, bacteriology, and corresponding antibiotic usage in patients presenting with Taiwan cobra snakebites. Patients with wound infection lacking tissue necrosis were included in developing Cobra BITE score utilizing univariate and multiple logistic regression, as patients with wound necrosis require antibiotics for infection treatment. 8,295,497 emergency department visits occurred in the span of this study, with 195 of those patients being diagnosed as having cobra bites. Of these patients, 23 had wound necrosis, and 30 had wound infection, resulting in a wound infection rate of 27.2% (53/195). Enterococcus faecalis and Morganella morganii were the main bacteria identified in the culture report regardless of whether patients' wounds had necrosis. As per our Cobra BITE score, the three factors predicting secondary wound infection after cobra bites are hospital admission, a white blood cell count (in 103/µL) × by neu-trophil-lymphocyte ratio value of ≥114.23, and the use of antivenin medication. The area under the receiver operating characteristic curve for the Cobra BITE score system was 0.88; ideal sensitivity and specificity were 0.89 and 0.76. This scoring system enables the assessment of wound infections after N. atra bites, and it could be modified and improved in the future for other Naja spp. bites.

Antimicrobial and physical properties of experimental endodontic sealers containing vegetable extracts.

To assess the antimicrobial activity and the physical properties of resin-based experimental endodontic sealers with the incorporation of vegetable extracts obtained from Bixa orellana, Mentha piperita, and Tagetes minuta species. The extracts were obtained and characterized by gas chromatography-mass spectrometry (GC-MS), and minimum inhibitory concentration (MIC) against Streptococcus mutans, Enterococcus faecalis, and Candida albicans. The extracts were individually incorporated into a dual-cure experimental sealer at a mass concentration of 0.5%. A commercial reference RealSeal was used. The sealers were evaluated by measuring the setting time, degree of conversion, dimensional stability, radiopacity, flow, and film thickness of these materials, also and its antimicrobial effect was evaluated using the direct contact test. Data were statistically analyzed by analysis of variance and Tukey's post-hoc test at α = 0.05 significance level. The physical properties were not influenced by the addition of the vegetable extracts (p > 0.05). For S. mutans, only T. minuta and B. orellana groups presented antibacterial activity after 24 h of contact (p < 0.05). All extracts evidenced an antibacterial effect against E. faecalis (p < 0.05). The experimental sealers hold promise as a novel vegetable sealer with great antimicrobial activity and also great physical-mechanical properties. Nonetheless, more studies are needed.

Essential Oil Analysis and Antimicrobial Evaluation of Three Aromatic Plant Species Growing in Saudi Arabia.

Arabian flora is a rich source of bioactive compounds. In this study, we investigated three aromatic plant species with the aim of finding valuable sources of antimicrobial agents against common pathogenic microorganisms. We focused especially on microorganisms, which cause outbreaks of infectious disease during mass gatherings and pilgrimages season in Saudi Arabia. The essential oils of three aromatic plant species were hydrodistilled from flowering aerial parts of Lavandula pubescens Decne. and Pulicaria incisa subsp. candolleana E.Gamal-Eldin, and from leaves, stems, ripe and unripe fruits of Juniperus procera Hochst. Ex Endl. They were subsequently analyzed by gas chromatography-mass spectrometry (GC-MS). The main constituents of L. pubescens were found to be carvacrol (55.7%), methyl carvacrol (13.4%), and ß-bisabolene (9.1%). P. incisa subsp. Candolleana essential oil was rich in linalool (33.0%), chrysanthenone (10.3%), eugenol (8.9%), and cis-chrysanthenol (8.0%); the major components of J. procera essential oil were α-pinene (31.3-62.5%) and δ-3-carene (7.3-30.3%). These essential oils were tested against thirteen American Type Culture Collection (ATCC) strains of Gram-positive and Gram-negative bacteria using the agar diffusion assay. The only effective essential oil was that of L. pubescens and the most sensitive strains were Acinetobacter baumannii, Salmonella typhimurium, Shigella sonnei, Enterococcus faecalis and Staphylococcus epidermidis. Carvacrol, the major constituent of L. pubescens, was tested on these strains and was compared with vancomycin, amikacin, and ciprofloxacin. The Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) assays of L. pubescens essential oil and carvacrol revealed that Gram-negative strains were more susceptible than the Gram-positive ones.

Polyphenolic characteristics, antimicrobial and antioxidant activity of Zingiber neesanum (Graham) Ramamoorthy rhizomes and identification of volatile metabolites by GC-MS analysis.

Rhizomes of Zingiber neesanum (Graham) Ramamoorthy, endemic to Western Ghats and subject to few studies, were analysed for bioactivity and phytochemical composition. High total tannin (TT) content in isopropanol extract [55.261 ± 6.623 mg TAE.100 g-1 DW] contributed to antimicrobial activity against gram negative Enterococcus fecalis (21.7 ± 0.6 cm) while high total flavonoid (TF) content in ethyl acetate extract [681.94 ± 33.87 mg CE.100 g-1 DW] accounted for the antifungal activity against Mucor rouxii (9.7 ± 0.6). Isopropanol extracts also showed high anti-oxidant activity as determined by DPPH and FRAP assays. Major bioactive phytochemical constituents in Z. neesanum rhizome identified by GC-MS analysis included 2-Methyl-7-nonadecene (13.99%; antimicrobial), Actinomycin C2 (8.57%; antineoplastic) and Deoxyspergualin (12.55%; immunosuppressive).

Chemical composition and antimicrobial activity of Osage orange (Maclura pomifera) leaf extracts.

The main goal of this study was to establish the chemical profile of Osage orange (Maclura pomifera) leaf extracts, obtained by conventional maceration technique, and to examine its antimicrobial activity. The identification and quantification of the extract compounds were done using ultra-high-performance liquid chromatography, with a diode array detector coupled with triple-quadrupole mass spectrometer and gas chromatography-mass spectrometry techniques. Thirty-one polyphenolic compounds were detected and identified in the ethanolic extracts, whereby 5-O-caffeoylquinic acid was found to be the dominant compound. Among other compounds, pentacosane and palmitic acid were the most abundant compounds in the dichloromethane extract. The preliminary antimicrobial activity screening shows that Gram-positive bacteria tend to be more sensitive to the investigated extracts. The highest antimicrobial activity was determined against Enterococcus faecalis ATCC 19433 and Listeria monocytogenes ATCC 35152. From these results, Osage orange leaves can be considered as plant material with significant antimicrobial properties.

Realgar increases defenses against infection by Enterococcus faecalis in Caenorhabditis elegans.

ETHNOPHARMACOLOGICAL RELEVANCE: Realgar has been used in traditional remedies for a long history in China and India. It is clinically used to treat diverse cancers, especially acute promyelocytic leukemia (APL), chronic myelogenous leukemia (CML) in China. However, paradoxic roles of realgar to increase or decrease immunity are reported. It is urgent to address this question, due to immune depression can be strongly benefit to cancer development, but detrimental to patients. AIM OF THE STUDY: This present work is to explore whether realgar promote or suppress immune responses, and shed light on its mode of action. Our results should provide cues for rational strategy to explore realgar for clinical use. MATERIAL AND METHODS: Infection model in vivo was established by using Enterococcus faecalis to attack Caenorhabditis elegans, then realgar was used to treat the infected worms to investigate its effects on infectivity and the underlying mechanism. Killing analysis was carried out to test whether realgar can mitigate worm infection. Thermotolerance resistance analysis was used to evaluate if realgar functions hormetic effect. Quantification of live E. faecalis in nematode intestine was employed to ascertain if realgar alleviate the bacterial load in worm gut. Quantitative real-time PCR (qRT-PCR) was used to test the expression of antibacterial effectors. Western blot was used to test the effect of realgar on the expressions of p38 and phospho-p38 in worms infected by E. faecalis. RESULTS: Realgar alleviated the infected worms in strains of N2, glp-4, and daf-2, but failed in sek-1, glp-4; sek-1, and daf-2; daf-16 when p38 MAPK or daf-16 was blocked or inactivated. Western blot assay demonstrated that realgar increased the expression of phosph-p38. Thermotolerance assay showed that realgar played a hormetic role on nemtodes, triggered protective response and reduced bacterial load after realgar treatment for 120 h qRT-PCR demonstrated that realgar significantly increased antibacterial effectors, thus leading to pathogen elimination. CONCLUSION: Realgar increased defenses against E. faecalis in C. elegans by inducing both immune responses and protective responses. It was regulated by p38 MAPK pathway and DAF-16.

Development of Chlorhexidine Digluconate and Lippia sidoides Essential Oil Loaded in Microemulsion for Disinfection of Dental Root Canals: Substantivity Profile and Antimicrobial Activity.

The dental intracanal disinfection is crucial to achieve the success of endodontic treatment, avoiding the maintenance of endodontic infections. Chlorhexidine digluconate can act as an irrigating agent for it. However, it can cause tissue irritation in high concentrations. Therefore, combinations with other antimicrobial agents and more efficient therapeutic alternatives are studied, which make it possible to administer drugs more safely and with minimal adverse effects. Thus, the objective of this study was the development of a microemulsion containing chlorhexidine digluconate and essential oil of Lippia sidoides to be used for disinfection of dental root canals and to evaluate its profile of substantivity and antimicrobial activity. The microemulsions were obtained through phase diagrams, using the spontaneous formation method. We completed a physical-chemical characterization and evaluate the stability of the microemulsions, in addition to the substantivity profile in a bovine root dentin model, and in vitro antibacterial effect on Enterococcus faecalis. A method for quantifying chlorhexidine was developed using UV-Vis spectroscopy. The microemulsions showed acid pH, conductivity above 1.3 µScm-1, and dispersion index similar to water. The microemulsions showed antimicrobial inhibition halos similar to the commercial gel conventionally used, but with four times more substantivity to dentinal tissues. Microemulsions were obtained as a therapeutic alternative to formulations available on the market, presenting themselves as a system with great potential for the administration of drugs for disinfection of root canals.

Development, characterization and photobiological activity of nanoemulsion containing zinc phthalocyanine for oral infections treatment.

Nanotechnology, when applied to PDT's, allows the encapsulation of ZnPc in nanocarriers, producing thus nanoemulsions that permit the use of ZnPc as photosensitizers. The Enterococcus faecalis and methicillin-resistant Staphylococcus aureus (MRSA) are microorganisms present in biofilms which can cause resistant endodontic infections. The objective of this work is the development and characterization of clove essential oil nanoemulsions containing ZnPc. The formulations were developed according to factorial experimental planning and characterized by the determination of the mean drop size, Polydispersity Index (PdI), content, organoleptic characteristics, stability, morphology, cytotoxicity in the dark and evaluation of the photobiological activity. The experimental planning was able to indicate the maximum amount of ZnPc that could be encapsulated in the nanoemulsion while maintaining droplet size <50 nm and PdI < 0.2. The surface plots for the response variables indicated a robust region for the combination of Pluronic® F-127 and clove oil factors. The result of this study was the choice of the nanoemulsion containing ZnPc solution at 5%, clove oil at 5%, Pluronic® F-127 at 10% and will be codified as ZnPc-NE. The nanoemulsion presented a mean diameter of 30.52 nm, PDI < 0.2 and a concentration of 17.5 µg/mL, as well as stability at room temperature for 180 days. TEM showed that the drops are spherical with nanometric size, which corroborates the results of dynamic light scattering. Concerning the photobiological activity, the ZnPc-NE exhibited MIC 1.09 µg/mL for Enterococcus faecalis and 0.065 µg/mL for MRSA (Methicillin-resistant Staphylococcus aureus). ZnPc-NE showed higher photobiological activity than free ZnPc. Besides, cytotoxicity studies showed that blank-NE (nanoemulsions without PS) showed good antimicrobial activity. Thus, clove oil nanoemulsion is an excellent nanocarrier to promote the photobiological activity of the ZnPc against pathogenic microorganisms.

Antimicrobial Activity of Schinopsis brasiliensis Engler Extract-Loaded Chitosan Microparticles in Oral Infectious Disease.

Enterococcus faecalis infections represent a health concern, mainly in oral diseases, in which treatments with chlorhexidine solution (0.2%) are often used; however, it presents high toxicity degree and several side effects. Based on this, the use of natural products as an alternative to treatment has been explored. Nonetheless, plant extracts have poor organoleptic characteristics that impair theirs in natura use. Therefore, this work aimed to evaluate the analytical profile, biological activity, and cytotoxicity in vitro of S. brasiliensis-loaded chitosan microparticles (CMSb) produced using different aspersion flow rates. The analytical fingerprint was obtained by FTIR and NIR spectra. Principal components analysis (PCA) was used to verify the similarity between the samples. The crystallinity degree was evaluated by X-ray diffraction (XRD). Phytochemical screening (PS) was performed to quantify phytocompounds. Antimicrobial activity was evaluated by minimum inhibitory concentration (MIC). Antibiofilm activity and bactericidal kinetics against E. faecalis (ATCC 29212 and MB 146-clinical isolated) were also assessed. The hemolytic potential was performed to evaluate the cytotoxicity. Data provided by FTIR, NIR, and PCA analyses revealed chemical similarity between all CMSb. Furthermore, the results from XRD analysis showed that the obtained CMSb present amorphous characteristic. Tannins and polyphenols were accurately quantified by the PS, but methodology limitations did not allow the flavonoid quantification. The low hemolytic potential assay indicates that all samples are safe. Antimicrobial assays revealed that CMSb were able to inhibit not only the E. faecalis ATCC growth but also the biofilm formation. Only one CMSb sample was able to inhibit the clinical strain. These results highlighted the CMSb antimicrobial potential and revealed this system as a promising product to treat infections caused by E. faecalis.

Induction of Daptomycin Tolerance in Enterococcus faecalis by Fatty Acid Combinations.

Enterococcus faecalis is a Gram-positive bacterium that normally exists as an intestinal commensal in humans but is also a leading cause of nosocomial infections. Previous work noted that growth supplementation with serum induced tolerance to membrane-damaging agents, including the antibiotic daptomycin. Specific fatty acids found within serum could independently provide tolerance to daptomycin (protective fatty acids), yet some fatty acids found in serum did not and had negative effects on enterococcal physiology (nonprotective fatty acids). Here, we measured a wide array of physiological responses after supplementation with combinations of protective and nonprotective fatty acids to better understand how serum induces daptomycin tolerance. When cells were supplemented with either nonprotective fatty acid, palmitic acid, or stearic acid, there were marked defects in growth and morphology, but these defects were rescued upon supplementation with either protective fatty acid, oleic acid, or linoleic acid. Membrane fluidity decreased with growth in either palmitic or stearic acid alone but returned to basal levels when a protective fatty acid was supplied. Daptomycin tolerance could be induced if a protective fatty acid was provided with a nonprotective fatty acid, and some specific combinations protected as well as serum supplementation. While cell envelope charge has been associated with tolerance to daptomycin in other Gram-positive bacteria, we concluded that it does not correlate with the fatty acid-induced protection we observed. Based on these observations, we conclude that daptomycin tolerance by serum is driven by specific, protective fatty acids found within the fluid.IMPORTANCE With an increasing prevalence of antibiotic resistance in the clinic, we strive to understand more about microbial defensive mechanisms. A nongenetic tolerance to the antibiotic daptomycin was discovered in Enterococcus faecalis that results in the increased survival of bacterial populations after treatment with the drug. This tolerance mechanism likely synergizes with antibiotic resistance in the clinic. Given that this tolerance phenotype is induced by incorporation of fatty acids present in the host, it can be assumed that infections by this organism require a higher dose of antibiotic for successful eradication. The mixture of fatty acids in human fluids is quite diverse, with little understanding between the interplay of fatty acid combinations and the tolerance phenotype we observe. It is crucial to understand the effects of fatty acid combinations on E. faecalis physiology if we are to suppress the tolerance physiology in the clinic.

Development of an antibacterial nanocomposite hydrogel for human dental pulp engineering.

Hydrogel-based regenerative endodontic procedures (REPs) are considered to be very promising therapeutic strategies to reconstruct the dental pulp (DP) tissue in devitalized human teeth. However, the success of the regeneration process is limited by residual bacteria that may persist in the endodontic space after the disinfection step and contaminate the biomaterial. The aim of this work was to develop an innovative fibrin hydrogel incorporating clindamycin (CLIN)-loaded Poly (d,l) Lactic Acid (PLA) nanoparticles (NPs) to provide the hydrogel with antibacterial properties. CLIN-PLA-NPs were synthesized by a surfactant-free nanoprecipitation method and their microphysical properties were assessed by dynamic light scattering, electrophoretic mobility and scanning electron microscopy. Their antimicrobial efficacy was evaluated on Enteroccocus fæcalis by the determination of the minimal inhibitory concentration (MIC) and the minimal biofilm inhibition and eradication concentrations (MBIC and MBEC). Antibacterial properties of the nanocomposite hydrogel were verified by agar diffusion assays. NP distribution into the hydrogel and release from it were evaluated using fluorescent PLA-NPs. NP cytotoxicity was assessed on DP mesenchymal stem cells (DP-MSCs) incorporated into the hydrogel. Type I collagen synthesis was investigated after 7 days of culture by immunohistochemistry. We found that CLIN-PLA-NPs displayed a drug loading of 10 ± 2 µg per mg of PLA polymer and an entrapment efficiency of 43 ± 7%. Antibiotic loading did not affect NP size, polydispersity index and zeta potential. The MIC for Enterococcus fæcalis was 32 µg mL-1. MBIC50 and MBEC50 were 4 and 16 µg mL-1, respectively. CLIN-PLA-NPs appeared homogenously distributed throughout the hydrogel. CLIN-PLA-NP-loaded hydrogels clearly inhibited E. faecalis growth. DP-MSC viability and type I collagen synthesis within the fibrin hydrogel were not affected by CLIN-PLA-NPs. In conclusion, CLIN-PLA-NP incorporation into the fibrin hydrogel gave the latter antibacterial and antibiofilm properties without affecting cell viability and function. This formulation could help establish an aseptic environment supporting DP reconstruction and, accordingly, might be a valuable tool for REPs.

Efeito in vitro da laserterapia e da terapia fotodinâmica na redução de bactérias presentes em canais radiculares / In vitro effect of lasertherapy and photodynamic therapy in bacterial reduction presents in root canals

Este estudo tem por objetivo verificar in vitro o efeito bactericida da laserterapia e da terapia fotodinâmica com laser de baixa potência (660 nm e 808 nm) em bactérias presentes nos canais radiculares. Métodos: foram preparadas 60 placas de Petri com bactérias: 20 placas com Enterococcus faecalis, 20 placas com Staphylococcus aureus e 20 com Pseudomonas aeruginosa. Aleatoriamente, dividiu-se cada grupo em 10 subgrupos (duas placas cada): três subgrupos tratados com laserterapia 660 nm em doses de 150, 225 e 300J/ cm², três subgrupos tratados com terapia fotodinâmica (azul de metileno 0,2% e laser 660 nm) em doses de 150, 225 e 300J/cm²; um subgrupo tratado com laserterapia 808 nm na dose de 225J/cm², um subgrupo com terapia fotodinâmica e laser 808 nm, em dose 225J/cm²; um subgrupo tratado apenas com fotossensibilizante (FS), e um não tratado (controle). Os tratados com laserterapia e terapia fotodinâmica foram irradiados uma única vez e incubados por 24 horas. Os últimos dois não receberam irradiação. As culturas foram analisadas visualmente para verificação do halo de inibição. Nos grupos submetidos somente à laserterapia, para o grupo FS e para o grupo controle, não foram observados halos de inibição, já onde houve aplicação da TFD, tanto com L1 quanto com L2, observaram-se halos de inibição em todas as espécies bacterianas estudadas. Conclui-se que a laserterapia, não produziu efeitos bactericidas e/ou bacteriostáticos, enquanto a terapia fotodinâmica nos dois comprimentos de onda produziu halos significativos de inibição de crescimento nas três bactérias do estudo.(AU)

This study aims to verify in vitro the bactericidal effect of laser therapy and photodynamic therapy with low power laser (660 nm and 808 nm), in bacteria present in the root canals.Methods: 60 Petri dishes were prepared with bacteria: 20 plates with Enterococcus faecalis, 20 plates with Staphylococcus aureus and 20 with Pseudomonas aeruginosa. At random, each group was divided into 10 subgroups (two plates each): three subgroups treated with 660nm laser therapy at doses of 150, 225 and 300J / cm², three subgroups treated with photodynamic therapy, (0.2% methylene blue and laser 660nm) in doses of 150, 225 and 300J / cm²; a subgroup treated with 808nm laser therapy at a dose of 225J / cm², a subgroup with (photodynamic therapy and 808nm laser) at a dose of 225J / cm²; a subgroup treated only with photosensitizer(FS), and an untreated (control). Those treated with laser therapy and photodynamic therapy were irradiated only once and incubated for 24 hours. The last two received no radiation. The cultures were analyzed visually to check the inhibition zone. In the groups submitted to laser therapy only, for the FS group and for the Control group, no inhibition halos were observed, since PDT was applied, with both L1 and L2, inhibition halos were observed in all studied bacterial species. It was concluded that laser therapy did not produce bactericidal and / or bacteriostatic effects, while photodynamic therapy at both wavelengths produced significant growth inhibition halos in the three studied bacteria.(AU)

Natural antioxidant pterostilbene as an effective antibiofilm agent, particularly for gram-positive cocci.

Pterostilbene (PTE), a dimethylated analogue of resveratrol, mostly contained in Vitis vinifera leaves or in other plant sources is well-known for its antioxidant activity. Due to its bioavailability, low hydrophilicity and thus ability to penetrate hydrophobic biological membranes it was found to be an antimicrobial agent. These properties of PTE offer the possibility of its use in the treatment of microbial infections. The emergence of antibiotic resistance of microorganisms is often caused by their ability to form biofilm; new substances with antibiofilm activity are therefore sought. The representatives of opportunistic pathogenic gram-positive and gram-negative bacteria as well as fungi were used for the determination of minimum inhibitory concentrations (MIC50 and MIC80), minimum biofilm inhibitory concentrations (MBIC50 and MBIC80) and minimum biofilm eradication concentrations (MBEC50 and MBEC80) of PTE and commonly used antibiotics erythromycin, polymyxin B or antimycotic amphotericin B. Total biofilm biomass was investigated by crystal violet staining, and the results were confirmed using microscopic techniques. The most significant antibiofilm action was proved for gram-positive cocci, e.g., MBEC50 of PTE for all strains of Staphylococcus epidermidis tested was 25 mg/L. By contrast, the antibiotic ERM did not exhibit antibiofilm activity in most cases. The permeabilization of cell membranes of gram-positive cocci biofilm by MBIC50 and MBEC50 of PTE was confirmed by LIVE/DEAD staining using spinning disc confocal microscopy. PTE significantly influenced the ability of gram-positive cocci to form biofilm and it effectively eradicated pre-formed biofilm in vitro; its potential for the treatment of biofilm-associated infections of Staphylococcus spp. or Enterococcus faecalis is thus apparent.