Cytotoxicity evaluation of fungal-derived silver nanoparticles on human gingival fibroblast cell line: An in vitro study.

BACKGROUND: Biosynthesized silver nanoparticles (AgNPs) have been proposed as effective antimicrobial agents against endo-perio pathogens. Determination of cytotoxicity is important for effective clinical use. AIM: The aim is to determine the cytotoxicity of fungal-derived AgNPs on human gingival fibroblast (HGF) cell line using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. MATERIALS AND METHODS: HGF cell cultures were trypsinized and adjusted to 5 × 103 cells/ml and 100-µl cell suspension (50,000 cells/well) and were added to 96-well plate. After 24 h, 100 µl of AgNPs (8-512-µg/ml concentrations) was added and incubated at 37°C for 24 h in 5% CO2 atmosphere. Controls were used without AgNPs. MTT (1 mg/ml) was added and incubated for 4 h at 37°C in 5% CO2 atmosphere. Microscopic examination was done, and absorbance was measured using a microplate reader at a wavelength of 540 nm. Percentage growth inhibition was calculated, and the concentration of AgNPs needed to inhibit cell growth by 50% (CTC50) was generated. RESULTS: CTC50 was found at a concentration of 260 µg/ml. AgNPs exerted less cytotoxicity against HGF cell line and increased with increase in the concentration of AgNPs. CONCLUSION: Fungal-derived AgNPs are safe to healthy cells at a concentration <260 µg/ml. Therefore, they can be effectively used for the treatment of endo-perio lesions.

Antibiofilm efficacy of biosynthesized silver nanoparticles against endodontic-periodontal pathogens: An in vitro study.

BACKGROUND: Endodontic-periodontal pathogens exist as biofilms which are difficult to eliminate. Biosynthesized silver nanoparticles (AgNPs) emerged as newer antimicrobial agents with potential benefits. AIM: The aim of this study is to determine the minimum inhibitory concentration (MIC); evaluate the antibiofilm efficacy of fungal-derived AgNPs against Porphyromonas gingivalis, Bacillus pumilus, and Enterococcus faecalis. MATERIALS AND METHODS: MIC of AgNPs against test pathogens was determined using micro broth dilution method. Serial dilutions of AgNPs ranging from 80 to 1 µg/ml concentration were added to wells containing 10 µl of bacterial inoculum in culture media and control group without AgNPs. For biofilm models, 120 dentin blocks were prepared, sterilized, and contaminated for 2 weeks with (n = 40 each). Group 1: B. pumilus, Group 2: E. faecalis, and Group 3: P. gingivalis and each group is divided into four subgroups (n = 10 each) and treated with distilled water, AgNPs, 2% and 0.2% chlorhexidine (CHX). Colonies counted after 24 h of incubation and statistically analyzed using one-way ANOVA and post hoc Tukey tests. RESULTS: MIC for B. pumilus was determined as 20 µg/ml and 30 µg/ml for E. faecalis and P. gingivalis. AgNPs were effective as 2% CHX against all biofilms compared to control group. Post hoc Tukey test (P < 0.0001) shows no significant difference between groups. CONCLUSION: Fungal-derived AgNPs are effective against endo-perio pathogens.

Antibacterial Efficacy of Biosynthesized Silver Nanoparticles against Enterococcus faecalis Biofilm: An in vitro Study.

AIM: This study aims to evaluate the antibacterial efficacy of biosynthesized silver nanoparticles (AgNPs) produced using the fungi against Enterococcus faecalis biofilm model on root dentin. MATERIALS AND METHODS: AgNPs were biosynthesized using the fungi Fusarium semitectum isolated from healthy leaves of Withania somnifera. Minimum inhibitory concentration (MIC) of AgNPs was determined by microbroth dilution method using series of dilutions. MIC dose was standardized to evaluate the antibacterial efficacy. For biofilm model, thirty root dentin blocks prepared using human extracted single-rooted teeth were inoculated with E. faecalis in Trypticase soy agar broth for 2 weeks with alternate day replenishment and randomly divided into three groups (n = 10 each) and treated as: Group I: Sterile distilled water, Group II: AgNPs, and Group III: 2% chlorhexidine gluconate (CHX) and incubated at 37°C for 24 h. Each dentin block was rinsed in saline, vortex shaken for 60 s, and serial decimal dilutions were prepared and plated on trypticase soy agar plates and incubated for 24 h followed by CFU colony counting and statistically analyzed using one-way ANOVA followed by post hoc Tukey honestly significant difference test. RESULTS: MIC of AgNPs for E. faecalis was determined as 30 mg/ml. No significant difference was seen between AgNPs and 2% CHX when compared to the control group with mean colony counts being 2.4, 2.5, and 6.77 CFU/ml (107), respectively (P < 0.0001), against E. faecalis biofilm. CONCLUSION: Biosynthesized AgNPs exhibit effective antimicrobial activity against E. faecalis biofilm on root dentin. Therefore, it can be employed as antimicrobial agent for root canal disinfection.

Evaluation of Antibacterial Efficacy of Fungal-Derived Silver Nanoparticles against Enterococcus faecalis.

BACKGROUND: The main objective of endodontic therapy is complete elimination and prevention of bacteria from the root canal system; however, it is difficult due to anatomical ramifications of root canal system and growing resistant microbes to available disinfectants. Therefore, to overcome this problem, newer antimicrobial agents have to be developed. AIMS: The aim of the study was to evaluate the antibacterial efficacy of fungal-derived biosynthesized silver nanoparticles (AgNPs) against Enterococcus faecalis. MATERIALS AND METHODS: Freshly prepared silver nanoparticles using the endophytic fungi Fusarium semitectum, characterized by different techniques were used to evaluate the antibacterial efficacy against E. faecalis by agar well diffusion method measuring the zone of inhibition using different concentrations of nanoparticles (AgNPs) (A [20 µl], B [40 µl], C [60 µl], D [80 µl], and E [100 µl]), F (0.2% chlorhexidine [CHX]), G (2% CHX), H (ampicillin), and I (distilled water) were used as control groups. Statistical Analysis Used: One-way ANOVA followed by post hoc Tukey multiple comparison test was done. RESULTS: AgNPs (100 ml) showed highest zone of inhibition 19.5 mm against E. faecalis. CHX (0.2%) 14.52 mm, CHX (2%) 20.02 mm, and ampicillin showed highest mean zone of inhibition 20.5 mm and distilled water showed no zone of inhibition. Results indicate no significant difference between E (100 µl), G (2% CHX), and H (ampicillin) (P < 0.0001). CONCLUSIONS: Biosynthesized AgNPs exhibit efficient antibacterial activity against E. faecalis and therefore can be used as root canal irrigant or intracanal medicament for root canal disinfection.

Biosynthesis, Characterization and Antibacterial Efficacy of Silver Nanoparticles Derived from Endophytic Fungi against P. gingivalis.

INTRODUCTION: Microbial resistance to existing antimicrobial agents in periodontal therapy is a growing problem. Therefore, there is a need for development of new antimicrobial agents. AIM: To biosynthesize and characterize Silver Nanoparticles (AgNPs) using endophytic fungi and to evaluate the antibacterial efficacy against P. gingivalis. MATERIALS AND METHODS: Cut leaf segments of Withania Somnifera (Ashwagandha) were used to isolate the fungi. Fresh cultures of fungi were inoculated in Erlenmeyer flask of 100 ml Malt Glucose Yeast Peptone (MGYP) broth and incubated at 29°C for 72 hours for the biomass to grow. Biomass was filtered and cell free fungal filtrate was used further. Biosynthesized AgNPs were characterized by visual observation, Ultraviolet-Visible (UV-Vis) spectrophotometer, Transmission Electron Microscopy (TEM), Selected Area Electron Diffraction Analysis (SAED) and Fourier Transform Infrared Spectroscopy (FTIR). Antibacterial efficacy was evaluated by agar diffusion method measuring the zone of inhibition. The study groups included different concentrations of AgNPs: A (20 µl), B (40 µl), C (60 µl), D (80 µl) and E (100 µl) of AgNPs, F (0.2% CHX), G (2% CHX), H (Ampicillin) and I (sterile distilled water). The data collected for inhibition zones were statistically analysed using One-way Anova followed by Tukey post-hoc multiple comparison tests. RESULTS: The fungi were identified as Fusarium semitectum. Characterization studies showed the colour change from colourless to reddish brown; U-V spectrum showed peak 420 nm, TEM revealed the particles spherical in shape and 10-20 nm in size. FTIR analysis revealed the presence of functional groups. AgNPs 80 µl and 100 µl showed mean zone of inhibition 17.33 and 18 mm against P. gingivalis. CHX (0.2%) 17.85 and CHX (2%) 19.97 mm, Ampicillin 20.5 mm and no zone for sterile distilled water. CONCLUSION: Biosynthesized AgNPs showed efficient antibacterial efficacy against P. gingivalis hence, creates a new horizon in periodontal therapy.

Evaluation of antibacterial efficacy of biosynthesized silver nanoparticles derived from fungi against endo-perio pathogens Porphyromonas gingivalis, Bacillus pumilus, and Enterococcus faecalis.

BACKGROUND: Even after rapid progress in contemporary dental practice, we encounter the failures due to endodontic, periodontal, or combined lesions. Complex anatomy of tooth and resistant microbes demands the development of new treatment strategies. AIM: The aim of this study is to biosynthesize silver nanoparticles (AgNPs) using fungi and determine the antibacterial efficacy against Porphyromonas gingivalis, Bacillus pumilus, and Enterococcus faecalis. MATERIALS AND METHODS: Fungi isolated from healthy leaves of Withania somnifera were used to biosynthesize AgNPs. The biosynthesized AgNPs were characterized by different methods, and antibacterial efficacy was evaluated by agar well diffusion method measuring the zone of inhibition. Test microorganisms were divided as Group 1: B. pumilus 27142 (American Type Culture Collection [ATCC]), Group 2: E. faecalis 29212 (ATCC), and Group 3: P. gingivalis 33277 (ATCC). Agents used for antibacterial efficacy were grouped as: AgNPs: A (20 µl), B (40 µl), C (60 µl), D (80 µl), E (100 µl), F (0.2% chlorhexidine [CHX]), G (2% CHX), H (Ampicillin), and I (sterile distilled water). RESULTS: Characterization studies showed the color change from colorless to reddish brown color; ultraviolet spectrum showed peak at 420 nm, transmission electron microscope revealed the particles spherical in shape and 10-20 nm size. Fourier transform infrared spectroscopy analysis revealed the presence of functional groups. Data collected for antibacterial efficacy were analyzed using one-way ANOVA and post hoc Tukey's multiple shows no significant difference among three groups (P < 0.0001). AgNPs were as effective as CHX and positive control ampicillin. No zones were seen for I (distilled water). CONCLUSION: Biosynthesized AgNPs showed efficient antibacterial efficacy. Therefore, it creates a new horizon in the management of endodontic, periodontal, and combined lesions.

Growth kinetics and mechanistic action of reactive oxygen species released by silver nanoparticles from Aspergillus niger on Escherichia coli.

Silver Nanoparticles (AgNPs), the real silver bullet, are known to have good antibacterial properties against pathogenic microorganisms. In the present study AgNPs were prepared from extracellular filtrate of Aspergillus niger. Characterization of AgNPs by UV-Vis spectrum reveals specific surface plasmon resonance at peak 416 nm; TEM photographs revealed the size of the AgNPs to be 20-55 nm. Average diameter of the produced AgNPs was found to be 73 nm with a zeta potential that was -24 mV using Malvern Zetasizer. SEM micrographs showed AgNPs to be spherical with smooth morphology. EDS revealed the presence of pure metallic AgNPs along with carbon and oxygen signatures. Of the different concentrations (0, 2.5, 5, 10, and 15 µg/mL) used 10 µg/mL were sufficient to inhibit 10(7) CFU/mL of E. coli. ROS production was measured using DCFH-DA method and the the free radical generation effect of AgNPs on bacterial growth inhibition was investigated by ESR spectroscopy. This paper not only deals with the damage inflicted on microorganisms by AgNPs but also induces cell death through the production of ROS released by AgNPs and also growth kinetics of E. coli supplemented with AgNPs produced by A. niger.

Optimization and Characterization of Silver Nanoparticle by Endophytic Fungi Penicillium sp. Isolated from Curcuma longa (Turmeric) and Application Studies against MDR E. coli and S. aureus.

Development of ecofriendly and reliable processes for the synthesis of nanoparticles has attracted considerable interest in nanotechnology because of its tremendous impetus in modulating metals into nanosize to their potential use for human benefits. In this study an endophytic fungus, Penicillium sp., isolated from healthy leaves of Curcuma longa (turmeric) was subjected to extracellular biosynthesis of silver nanoparticles (AgNps) and their activity against MDR E. coli and S. aureus. The biosynthesized AgNps optimization was studied and characterized by UV-visible spectroscopy, Fourier transform infrared spectroscopy (FTIR), and transmission electron microscopy (TEM). Then produced AgNps were tested against MDR E. coli and S. aureus. The endophytic fungus Penicillium sp. from healthy leaves of C. longa (turmeric) was found to be a good producer of AgNps. Parametric optimization showed maximum absorbance of 420-425 nm at pH-7, 25°C with 1 mM AgNO3 concentration and 15-20 g of wet biomass. Further TEM revealed the formation of spherical, well-dispersed nanoparticles with size ranging between 25 and 30 nm and FTIR shows the bands at 1644 and 1538 cm(-1) corresponding to the binding vibrations of amide I and II bands of proteins, respectively. Antibacterial activity against MDR E. coli and S. aureus showed good results showing maximum zone of inhibition of 17 mm and 16 mm, respectively, at 80 µL of AgNps.

Transformation of L-tyrosine to L-dopa by a novel fungus, Acremonium rutilum, under submerged fermentation.

The present study deals with the transformation of L-tyrosine to L-dopa by Acremonium rutilum, a fungal tyrosinase producer, isolated from decomposed banana stud. This appears to be the first report on A. rutilum as a polyphenoloxidase producer with both cresolase and catecholase activity. Enriched Czapek-Dox agar was used for plate assay screening. Enriched potato dextrose broth was used for optimization studies, which induced high levels of L-dopa under submerged fermentation. A. rutilum gave the maximum L-dopa production (0.89 mg/ml) and tyrosinase activity (1095 U/mg) under the optimized parameters, that is, a temperature of 25 degrees C, pH 5.5, an inoculum size of 2.5 ml, and an incubation time of 72-120 h, with L-tyrosine (5 mg/ml) as substrate. Five resolved bands, with R(f) values of 0.73, 0.60, 0.54, 0.37, and 0.26, were observed, which confirmed the presence of L-dopa. This study involves the elevated profile of L-dopa production. Such study is needed, as L-dopa has the ability to control Parkinson's disease.