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ISSN: 2157-7439
Journal of Nanomedicine & Nanotechnology
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Green Synthesis, Characterization and Antibacterial Activity of Silver Nanoparticles of Endophytic Fungi Aspergillus terreus

Reena Rani, Dushyant Sharma, Monika Chaturvedi and Yadav JP*

Department of Genetics, M. D. University, Rohtak, 124001, Haryana, India

*Corresponding Author:
Jaya Parkash Yadav
Department of Genetics
M.D. University, Rohtak-124001
Haryana, India
Tel: 9109416474640
E-mail: [email protected]

Received date: August 17, 2017; Accepted date: August 24, 2017; Published date: August 31, 2017

Citation: Rani R, Sharma D, Chaturvedi M, Yadav JP (2017) Green Synthesis, Characterization and Antibacterial Activity of Silver Nanoparticles of Endophytic Fungi Aspergillus terreus. J Nanomed Nanotechnol 8: 457. doi: 10.4172/2157-7439.1000457

Copyright: © 2017 Rani R, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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Abstract

In present study, the silver nanoparticles were synthesized using endophytic fungi Aspergillus terreus, isolated from Calotropis procera. Synthesized nanoparticles were characterized using various spectroscopic techniques. The nanoparticles were reported oval to spherical in shape. The average size of silver nanoparticles was 16.54 nm. Gas chromatography-mass spectrometry analysis of Aspergillus terreus showed the presence of 17 compounds. The synthesized silver nanoparticles showed considerable antibacterial activity against tested bacterial strains: 9 American type culture collection reference (ATCC) and 3 multidrug resistance (MDR) strains. Synthesized nanoparticles showed significant antibacterial activity against Salmonella typhi (16.67 ± 0.58 mm), Staphylococcus aureus (15.67 ± 0.58 mm), and Escherichia coli (15.67 ± 0.58 mm). Minimum inhibitory concentration was reported in range of 11.43 μg/ml to 308 μg/ml. Cell leakage analysis reported an increase in protein leakage level and degradation of nucleic acid after treatment with silver nanoparticles. The present study concluded that endophytic fungi Aspergillus terreus isolated from Calotropis procera can be used as a source for synthesis of silver nanoparticles and suggesting as an effective antibacterial agent

Keywords

Aspergillus terreus; Antibacterial activity; Gas chromatography-mass spectrometry (GC-MS); Multidrug resistance (MDR); Silver nanoparticles; Protein leakage analysis

Introduction

Plants in nature accommodate diverse group of symbiotic and nonsymbiotic microorganisms and these microbes play significant role in plant development, growth, and protection. This association of plant and microbes is significant for their survival in stressed environment [1]. Endophytic fungi are among the organisms, which are in symbiotic association with plants, along with bacteria and yeast.

Due to development and dissemination of antibiotic resistance genes, it is difficult or impossible to treat bacterial infections. At present time development of multidrug resistance (MDR) in bacterial strains is one of the most alarming threats. Rapid development of drug resistance among bacteria threatens the extraordinary health benefits that have been achieved after discovery of first antibiotic “Penicillin”. As bacteria have intrinsic ability to develop drug resistance there is continuous need of development of new antibiotics or materials, alternative treatment therapies and novel treatment approaches that can cope with this serious issue [2]. Non-traditional antibacterial agents are thus grabbing more attentions and offers great opportunities to overcome resistance. Biosynthesized silver nanoparticles (AgNPs) emerge as viable alternative for treatment of bacterial infections.

Over last few decades, nanotechnology rapidly emerges as an important field of science dealing with synthesis and manipulation of particle structure on nanoscale (1-100 nm). Due to very small size, nanoparticles acquire new physiochemical properties like catalytic activity, electric and thermal conductivity etc. as compared to their bulk. These physiochemical properties of nanoparticles are responsible for rapidly increase in their application in various fields like medical, textile, drug delivery, catalysis, environmental remediation, biological labeling, electronics, mechanics, chemical industries, and optics [3,4]. Nanoparticles can be synthesized using various methods: physical, chemical, and biological. Physical and chemical methods include synthesis by evaporation-condensation, laser ablation, microwave, chemical reduction, vapours deposition, sol-gel process, laser pyrolysis etc. Biological methods include synthesis using extracts from plants, fungi, algae, bacteria, and agricultural waste. Biological synthesis approach offers various advantages over other physical and chemical methods in terms of rapid synthesis, cost, eco-friendly, and less toxicity. Synthesis of metallic nanoparticles by using biological materials is a bio-redox reaction mainly carried out by secondary metabolites, cellular enzymes, and other cellular constituents. The biological materials for synthesis of nanoparticles include algae, fungi, yeast, bacteria, actinomycetes and plants. Though chemical method of silver nanoparticles resulted in a high yield as comparative to biological method but this technique is not suitable due to involvement of hazardous chemicals and high cost of production. Various factors like pH, temperature, incubation time, and method of synthesis, types of biological material and their cellular content influenced the size, shape and activity of synthesized silver nanoparticles [5]. Among various noble metals, silver is most commonly used for the synthesis of nanoparticles due to its stability, electrical conductivity, catalytic activity, and surface plasmon resonance (SPR) [6].

As endophytic fungi possess large diversity but there is a limited number of reports on synthesis of AgNPs using endophytic fungi extracts are available. They provide several advantages over the bacteria, i.e. easy handling, simple nutrient requirement, secretion of large amount of extracellular protein being eukaryote, possess metal intake and tolerance capability [7,8]. Despite these facts, during extracellular biosynthesis of nanoparticles using fungi, they offer easier downstream processing than bacteria [9]. Therefore, in the present study, our attempt was to synthesized extracellular AgNPs using Aspergillus terreus (A. terreus) isolated from healthy Calotropis procera (C. procera). The synthesized nanoparticles were further subjected for their characterization and antibacterial efficacy against MDR and reference bacterial strains.

Materials and Methods

Isolation and identification of endophytic fungi

Healthy tissues (leaf, stem, and root) of C. procera plant were collected from M. D. University, Rohtak, Haryana, India. Plant identification was confirmed by voucher number MDU 4602. Sterilization of tissues was carried out using Schulz et al. [10] procedure. Plant tissues were washed thoroughly under running tap water and were surface sterilized, first immersed in 70% ethanol for 60 sec, followed by sodium hypochloride (4% of available chlorine) for 3 min and then dipped in 75% of ethanol for 30 sec. Finally, sterilized samples were rinsed with autoclaved distilled water thrice and allowed to dry in laminar air flow. The plant samples were cut into small pieces (of approximately of same size) and placed on the Petri plates containing potato dextrose agar (PDA, Himedia Pvt. Ltd. India) complemented with streptomycin (100 mg/L, Himedia Pvt. Ltd. India) to prevent endophytic bacterial growth. Petri plates were incubated at 28 ± 2°C, observed on regular basis for endophytic fungal growth. Pure culture of endophytic fungi was obtained after continuous transfer of hyphal tips on fresh PDA plates. Identification of isolated fungi was carried by PCR using ITS1 and ITS4 primer pair. All isolated endophytic fungi screened for antibacterial activity and A. terreus showed significant activity against tested bacterial strains. Hence, it was further selected for synthesis of AgNPs.

Extracellular synthesis of silver nanoparticles

A. terreus was inoculated in Potato Dextrose Broth (PDB) and incubated at 28 ± 2°C on an incubator shaker with 150 rpm for 6 days. Fungal biomass was filtered using Whatman filter paper no.1 and washed repeatedly with distilled water to remove the media. 20 g of fungal biomass taken into a flask of 500 ml containing 100 ml of distilled water and boiled for 15-20 min. Biomass was filtered using Whatman filter paper no.1 and filtrate was used further for synthesis of AgNPs. 20 ml of fungal extract was mixed with aqueous solution of silver nitrate (80 ml; 2mM). Mixture was incubated at room temperature.

Phytochemical analysis using GC-MS

GC-MS analysis of A. terreus was carried out using GC-MS analyzer (BRUKER SCION 436-GC SQ, USA). The column used was Rtx®-5 of 30 m length, 0.25 mm column inside diameter with 0.25 μm film coating. Sample was filtered through Whatman™ FILTER DEVICE (0.2 μm). Helium (99.999%) gas was used as carrier with a flow rate of 1 ml/min in split mode. A volume of 1 μL of fungal extract was injected to column with 280°C inlet temperature. The temperature of oven initially set at 70°C for 2 min and then it was elevated at rate of 7°C/min up to 320°C. Temperature of the ion sources was maintained at 250°C. The mass spectrum of compounds present in fungal extract was obtained by electron ionization at 70 eV and detector operates in scan mode 30 to 500 Da atomic units. Total running time was 22.5 min including 3 min solvent delay. The obtained spectrum of the extract was compared with the database of national institute of science and technology (NIST) library.

Characterization of synthesized nanoparticles

Four different techniques were used for characterization of synthesized AgNPs. Initial characterization was performed by UV-Vis spectroscopy using Shimadzu UV-2450 spectrophotometer, Japan. Wavelength range for absorption was 300-525 nm and distilled water was used as blank. Reaction mixture was centrifuged to concentrate synthesized AgNPs at 12000 rpm for 15 min. Finally, unbounded capping material was removed by repeating washing (4 times) with double distilled water. Thereafter, the obtained pellet was lyophilized to obtain synthesized AgNPs in powdered form. Fourier Transform Infra-red Spectroscopic analysis (FTIR) was used for detection of different functional groups involved in stabilization and capping of synthesized nanoparticles. Dried powder form of nanoparticles was analyzed using Alpha FTIR-ATR (Bruker, Germany). Characteristic peaks were recorded in between 400-4000 cm-1 at resolution 4 cm-1. Analysis was performed twice for the confirmation of transmittance spectra. The surface morphology of synthesized nanoparticles was studied using Scanning Electron Microscopy (SEM) technique. Lyophilized AgNPs were coated on stabs and images were obtained scanning electron microscope EVO18 Zeiss (CARL ZEISS, Germany) at 20 kV voltage. The shape and size of mycosynthesized AgNPs were determined by transmission electron microscopy (TEM) analysis. Firstly, lyophilized AgNPs were suspended in methanol. A drop of suspended nanoparticles put on copper grid and allowed to dry at room temperature. Images were obtained with Tecnai, G 20 (FEI) at 200 KV with different magnification. SEM and TEM analysis were carried by availing facility of SAIF at AIIMS, New Delhi.

Antibacterial activity

Tested bacterial strains: Antibacterial activity of AgNPs synthesized from A. terreus extract was screened against total 12 bacterial strains; 9 reference (Pseudomonas aeruginosa ATCC 27853, Serratia marcescens ATCC 27137, Shigella flexneri ATCC 12022, Salmonella typhi ATCC 13311, Escherichia coli ATCC 25922, Proteus mirabilis ATCC 43071, Klebsiella pneumoniae ATCC 700603, Enterococcus faecalis ATCC 29212, and Staphylococcus aureus ATCC 259323) and 3 multidrug resistance (MDR) strains (Escherichia coli MDREC1, Klebsiella pneumoniae MDRKP2, and Pseudomonas aeruginosa MDRPA3). MDR strains were obtained from the PGIMS (Microbiology Department), Rohtak, Haryana, India.

Agar well diffusion assay: Antibacterial activity of AgNPs was determined using agar well diffusion assay [11]. 24 hour old inoculated bacteria (100 μL) were uniformly spread on nutrient agar Petri plates. Wells of 6 mm were made with the help of sterile borer. Stock solutions of nanoparticles at different concentrations (10 mg/ml, 20 mg/ml, 30 mg/ml, and 40 mg/ml) were prepared in dimethyl sulphoxide (DMSO, 1/10th diluted). 20 μl of each concentration was added to well for all bacterial strains tested. Streptomicin (HiMedia laboratories Pvt. Ltd. India, 10 μg/disc) was used as standard, while same volume of fungal filterate and silver nitrate used as control. Zone of inhibition of nanoparticles was measured with the help of a standard transparent scale HiAntibiotic ZoneScaleTMC (HiMedia Laboratories Pvt. Ltd. India).

Minimum inhibitory concentration (MIC): Minimum inhibitory concentration (MIC) is the lowest concentration of an antibacterial agent to inhibit the growth of bacterial growth. MIC values for synthesized AgNPs were determined using micro broth dilution method of Sarker et al. [12]. A volume of 50 μl of each sterile nutrient broth and normal saline was added to each well of microtitre plate. 50 μl of nanoparticle solution dissolved in DMSO (25 mg/ml) was added to the first row of the microtitre plate followed by serial dilution across. 10 μl of each resazurin (indicator) and bacterial inoculums (approximately 1.5 × 108 CFU/ml) were added to each well. Plates were wrapped in cling film to prevent dehydration of bacteria and incubated at 37°C for 24 h. Experiments were performed in triplicate to avoid any error. Change of color from purple to pink or colorless indicated the growth of bacteria. The lowest concentration at which no color change observed was considered as the MIC value AgNPs.

Cell leakage analysis

Protein leakage analysis: Bacterial cultures were treated with AgNPs (10 mg/ml) and incubated at 35 ± 2°C for 8 h. After incubation bacterial culture were centrifuged at 4°C (6000 rpm, 5min) and supernatant were collected. Bradford [13] method was used to determine the intracellular protein leakage in treated and control bacterial supernatant The assay mixture consists of 150 μl of supernatant and 150 μl of Bradford reagent, incubated at room temperature in dark and absorbance was measured at 595 nm using ELISA reader. Bovine serum albumin (BSA) was used as standard. Agarose gel electrophoresis was performed for treated and control supernatant to determine the nucleic acid leakage.

Results

Phytochemical analysis using GC-MS

GC-MS analysis of A. terreus extract indicated the presence of 17 compounds when compared with NIST database (Table 1 and Figure 1). The major compounds identified were (E)-9-octadecenoic acid ethyl ester (21.952%), hexadecanoic acid (16.591%), ethyl ester (16.591%), oleic Acid (11.382%), dodecanoic acid (8.488%), eicosanoic acid, ethyl ester (7.264%), and docosanoic anhydride (7.177%).

S. No. RT Name of compounds % area Common name Class of compound
1 7.956 Dodecanoic acid 8.488 Capric acid Saturated fatty acid
2 8.11 Dodecanoic acid, ethyl ester 4.31 Lauric acid, ethyl ester Lipids
3 9.556 Tetradecanoic acid, ethyl ester 3.259 Ethyl myristate Fatty acid
4 10.232 Eicosanoic acid, 2-ethyl-2-methyl-, methyl ester 1.59 Arachidic acid, esters Lipids
5 10.74 Ethyl 9-hexadecenoate 2.716 Palmitelaidic acid ethyl ester Fatty acid esters
6 10.879 Hexadecanoic acid, ethyl ester 16.591 Palmitic acid Fatty acid
7 11.51 Docosanoic anhydride 2.445 Behenic anhydride Carboxylic acid
8 11.802 Gamolenic Acid 3.373 Gamma linoleic acid Fatty acid
9 11.925 (E)-9-Octadecenoic acid ethyl ester 21.952 Oleic acid ethyl ester Fatty acid
10 12.079 Eicosanoic acid, ethyl ester 7.264 Eicosanoic acid, ethyl ester Carboxylic acid
11 12.679 Docosanoic anhydride 7.177 Behenic anhydride Carboxylic acid
12 13.172 Di(2-ethylhexyl)adipate 1.185 Diethylhexyl adipate Ester
13 13.541 Gamolenic Acid 1.656 Gamma linoleic acid Fatty acid
14 13.679 Oleic Acid 11.382 Oleic acid Fatty acid
15 13.817 Docosanoic anhydride 3.458 Behenic acid anhydride Carboxylic acid
16 16.171 10-Bromodecanoic acid, ethyl ester 1.395 10-Bromodecanoic acid, ethyl ester Carboxylic acid
17 16.309 Squalene 1.759 Squalene  Steroids

Table 1: GC-MS analysis of ethyl acetate extract of endophytic fungi A. terreus.

nanomedicine-nanotechnology-spectrum

Figure 1: GC-MS spectrum of A. terreus.

Characterization of synthesized silver nanoparticles

UV-Vis spectrophotometric technique is one of the most commonly used techniques for initial characterization of synthesized nanoparticles. Change in color was observed from pale yellow to grayish after addition of aqueous extract of endophytic fungi to 2 mM silver nitrate solution. This change in color is due to SPR phenomenon exhibited by metal nanoparticles in the aqueous solution. A strong peak specific for the production of AgNPs was observed at 410-425 nm as shown in Figure 2 [14]. FTIR analysis was generally carried out to identify the functional group involved in reduction of silver ion to metallic AgNPs. The observed spectrum is represented in Figure 3. Peaks at different wavelength correspond to different functional groups. FTIR spectra showed peaks at 529, 558, and 593 cm-1 represented C-Br stretching suggesting the presence of alkyl halides. Peaks at 628, 667 and 697 cm-1 were related to C-Cl stretching, suggested the presence of halogens. The bands at 930 and 951 cm-1 correspond to =C-H bending; they may be confined for presence of alkenes. Peaks at 1406 and 1436 cm-1 showed the O-H bending could be attributed to the presence of carboxylic acids. Peak at 1995 cm-1 suggested the N=C=S stretching may corresponds to peresence of isothiocynate. Peaks at 2818, 2912 and 2996 cm-1 regions arising from C-H stretching of alkanes compounds were observed. The band at 3385 cm-1 corresponds to O-H bending which suggested the presence of phenols.

nanomedicine-nanotechnology-absorption-spectra-nanoparticles

Figure 2: Representing UV-Vis absorption spectra of synthesized silver nanoparticles using A. terreus at range of 300-525 nm.

nanomedicine-nanotechnology-analysis-endophytic-fungus

Figure 3: Representing image of FTIR analysis of AgNPs of endophytic fungus A. terreus.

SEM analysis of AgNPs showed almost spherical structure (Figure 4). Nanoparticles were dispersed; no aggregates indicated stabilization of the synthesized AgNPs [15]. TEM analysis provides detail information about size and surface morphology. TEM analysis of AgNPs predominated with spherical and oval particles represented an average size of 16.45 nm (Figure 5).

nanomedicine-nanotechnology-scanning-electron-microscopy

Figure 4: Representing image of scanning electron microscopy (SEM) of AgNPs of endophytic fungus A. terreus.

nanomedicine-nanotechnology-transmission-electron-microscopy

Figure 5: Representing image of transmission electron microscopy (TEM) of AgNPs of endophytic fungus A. terreus.

Antibacterial activity

Antibacterial activity of synthesized AgNPs was tested at various concentrations i.e. 10 mg/ml, 20 mg/ml, 30 mg/ml, and 40 mg/ml using agar well diffusion method. The zone of inhibition for reference bacterial strains was ranging from 13.67 ± 0.58 to 16.67 ± 0.58 mm (Table 2). Antibacterial activity is directly related with concentration of AgNPs, as it increases with increase in concentration. It was observed that mycosynthesized AgNPs exhibited considerable antibacterial activity with reference as to controls (fungal filterate and silver nitrate). S. typhi strain was found to be most susceptible as showed highest zone of inhibition (16.67 ± 0.58 mm), followed by E. coli, and S. aureus (15.67 ± 0.58 mm), S. marcescens (15.33 ± 0.58 mm). Lowest zone of inhibition was reported for K. pneumoniae (MDR) (13.33 ± 0.58 mm), and K. pneumoniae ATCC strain was reported to be least susceptible as showed minimum zone of inhibition among all tested reference strains (13.67 ± 0.58 mm). The MIC of synthesized AgNPs from A. terreus against different reference bacterial strains was 11.43-308 μg/ml. ATCC strains S. typhi, E. coli, S. aureus, S. flexneri, and S. marcescens showed the MIC values of 11.43 μg/ml. MIC values observed for P. aeruginosa was 32.2 μg/ml, for P. mirabilis and E. faecalis was 102 μg/ml, for K. pneumoniae was 308 μg/ml (Figure 6).

Bacterial strains Synthesized silver nanoparticles  Fungal supernatant Silver nitrate Standard (10 µg/disc)
10 mg/ml 20 mg/ml 30 mg/ml 40 mg/ml
E. coli (MDR) 13.33 ± 0.58 13.67 ± 0.58 14.67 ± 0.58 14.67 ± 0.58 - - 22.33 ± 0.58
K. pneumoniae (MDR) 12.33 ± 0.58 12.67 ± 0.58 13.33 ± 0.58 13.33 ± 0.58 - - 22.67 ± 0.58
P. aeruginosa (MDR) 14.00 ± 1.00 15.00 ± 1.00 15.33 ± 0.58 15.33 ± 0.58 - - 21.00 ± 1.00
P. mirabilis 11.67 ± 0.58 12.67 ± 0.58 13.33 ± 0.58 14.67 ± 0.58 - - 25.00 ± 1.00
P. aeruginosa 12.67 ± 0.58 13.00 ± 1.00 13.67 ± 0.58 14.33 ± 0.58 - - 23.00 ± 1.00
S. typhi 15.33 ± 0.58 15.67 ± 0.58 16.00 ± 1.00 16.67 ± 0.58 - - 21.67 ± 0.58
S. marcescens 12.67 ± 0.58 13.33 ± 0.58 14.67 ± 0.58 15.33 ± 0.58 - - 22.33 ± 0.58
S. flexneri 14.33 ± 0.58 14.33 ± 0.58 14.67 ± 0.58 14.67 ± 0.58 - - 20.33 ± 0.58
E. coli 14.67 ± 0.58 15.67 ± 0.58 15.67 ± 0.58 15.67 ± 0.58 - - 25.33 ± 0.58
E. faecalis 14.00 ± 1.00 15.00 ± 1.00 15.00 ± 1.00 15.00 ± 1.00 - - 22.67 ± 0.58
K. pneumoniae 13.00 ± 0.58 13.33 ± 1.00 13.67 ± 0.58 13.67 ± 0.58 - - 24.67 ± 0.58
S. aureus 14.67 ± 0.58 14.67 ± 0.58 15.33 ± 0.58 15.67 ± 0.58 - - 24.67 ± 0.58

Table 2: Representing antibacterial activity of AgNPs at different concentrations, synthesized by using A. terreus against different reference and MDR bacterial strains.

nanomedicine-nanotechnology-tested-bacterial-strains

Figure 6: MIC of AgNPs against tested bacterial strains.

The zone of inhibition for MDR bacterial strains was ranging from 13.33 ± 0.58 to 15.33 ± 0.58 mm (Table 2). P. aeruginosa (MDR) strain was reported to be most susceptible as showed highest zone of inhibition (15.33 ± 0.58 mm). K. pneumoniae (MDR) strain was found to be least susceptible for synthesized AgNPs. MIC values observed for P. aeruginosa (MDR) was 34.2 μg/ml, for E. coli was 102 μg/ml, for K. pneumoniae was 308 μg/ml (Figure 6).

Cell leakage analysis

The amount of protein present in supernatant after treatment with AgNPs was quantified by using Bradford assay. In treated cells higher protein content was found than control. The protein leakage from the treated cell was reported in higher amount as compared to the control (Figure 7). Highest amount of extracellular protein after treatment with AgNPs was reported for S. typhi. Agarose gel electrophoresis showed the degraded band of nucleic acid after application of nanoparticles.

nanomedicine-nanotechnology-protein-bacterial-strains

Figure 7: Graph representing estimation of protein leakage level in different bacterial strains after treatment with silver nanoparticles of A. terreus.

Discussion

After discovery of Penicillin, fungi were thoroughly investigated for presence of antimicrobial compounds. These compounds (secondary metabolites) are not only serves as antimicrobial agents but also provide a prototype structure for chemical synthesis of new antibacterial agents. There are several examples which indicated fungal metabolites serve as source directly or indirectly for production of drug molecules [16,17]. Many studies have been carried out to evaluate antibacterial activity of endophytic fungi and for isolation new antibacterial compounds [11,18]. But synthesis of AgNPs takes advantage of their small size and bioactivity of phytoconstituents. Endophytic fungi mediated synthesis of AgNPs provides an attractive and environmentally safe substitute to chemically and physically produced nanoparticles. In present study, the selected endophytic fungal strain i.e. A. terreus exhibited ability to synthesize AgNPs extracellularly. After synthesis, characterization of AgNPs is necessary to predict their physicochemical properties that could have a significant impact on their biological activity. Characterization techniques provides informations like size, shape, charge, solubility, aggregation, and function group involved in reduction and capping of nanoparticles [19,20]. Therefore, we used different characterization technique i.e. UV-Vis spectrophotometer, FTIR, SEM, and TEM for the analysis of synthesis of nanoparticles.

GC-MS analysis of A. terreus extract was carried out for identification of detailed phytoconstituents. It mainly showed the presence of carboxylic acids, esters, and steroids. They are reported to involve in the synthesis of nanoparticles [21,22]. It was previously reported that secondary metabolites present in endophytic fungi are responsible for synthesis and stabilization of AgNPs [8]. The present study hypothesizes that these metabolites might be responsible for the reduction of silver ions and formation of stable AgNPs.

First indication of nanoparticles production is the change in color of solution [23]. Generally, appearance of brown color indicates the production of AgNPs due to SPR phenomenon. Similar indications were also reported [22,24]. Many studies reported that spherical AgNPs show maximum absorbance between 400 nm to 450 nm. The intensity and peak position due to SPR are related with size, morphology and dielectric properties of synthesized nanoparticles [25,26]. Slow decrease in absorbance after peak indicated the polydispersed behavior of AgNPs in solution [27,28].

The exact mechanism involved in nanoparticle synthesis may vary with biological extracts due to presence of different secondary metabolites and enzymes. Generally, phytochemicals responsible for synthesis of nanoparticles are terpenoids, flavones, ketones, aldehydes, amides, and carboxylic acids [3]. A. terreus showed the presence of tannins, flavanoids, phenols, diterpenes, alkaloids, glycosides and carbohydrates (data is not published yet). FTIR analysis of synthesized nanoparticles indicated the presence of many functional groups. Functional groups played important role in reduction and capping of silver ion to AgNPs [29,30]. Characteristic peaks showed the presence of alcohols and phenols which have been reported to assist reduction of silver ion to AgNPs [16]. Some studies also suggested the reduction and capping of silver ion is due to presence of NADHdependent reductases in endophytic fungal extracts [31,32]. SEM and TEM analysis were performed to know the morphology and size of synthesized nanoparticles. It was predicted to be round and oval shape of nanoparticles may be due to capping and stabilization of nanoparticles by secondary metabolites present in extract of A. terreus. TEM observations are in line with many earlier reports for AgNPs synthesized using various fungal extracts [33,34]. In present method, synthesis of silver nanoparticles was done extracellularly. Silver nanoparticles can be synthesized intracellularly but extract out the synthesized nanoparticles require cell lysis which is tedious and relatively costly [35].

In present study, we synthesized and characterized AgNPs from A. terreus and evaluate their antibacterial potential. Significant antibacterial activity of AgNPs was reported against all tested bacterial strains. Increase in zone of inhibition was reported with the increase in concentration of AgNPs. AgNPs inhibited the growth of reference and MDR bacterial strains effectively while, no zone of inhibition was reported for fungal filtrate and silver nitrate. So, the antibacterial activity of synthesized AgNPs may be attributed to their small size and high surface area which enable them to penetrate inside the bacterial cells.

There are various suggested mechanisms of action of AgNPs; AgNPs have ability to anchor to the bacterial cell wall, followed by penetration, thus change the permeability of cell membrane and results in bacterial cell death, produce free radicals when come in contact with bacterial cells [36], and they may release silver ion and these ion interact with thiol groups of many vital enzymes and inactivate them [37]. Many previous studies reported the antibacterial potential of endophytic fungi A. terreus [11,18]. Few studies also reported the synthesis of AgNPs from A. terreus and their bioactivity isolated from different host plants [38,39]. Abeer et al. [38] reported antimicrobial activity AgNPs synthesized from Aspergillus terreus KC462061 isolated from date palm against various fungal strains (Candida albicans, Aspergillus flavus, Aspergillus fumigates, and Aspergillus niger), and bacterial strain (Staphylococcus aureus) using the agar well diffusion method. Remarkable inhibitory in vitro antidermatophytic activity was found against Trichophyton rubrum, Epidermophyton floccosum and Trichophyton mentagrophytes of AgNPs synthesized using A. terreus isolated from host plant Rhizophora annamalayanna [39].

From literature it has been found that AgNPs have the capability to penetrate the bacterial cells thereby causing membrane dissociation. Protein and nucleic acid component thus released can be used as a marker to know cell integrity when compared with control cells i.e. without exposure to nanoparticles. Present study showed high amount of leaked protein and nucleic acids from the treated bacterial strains suggesting the irreversible loss of plasma membrane integrity. Increase in protein leakage and nucleic acid degradation analysis support our finding of disruption of bacterial membrane as reported in other studies also [40,41].

Conclusion

The present study indicates the synthesis of AgNPs using endophytic fungi A. terreus. Synthesized nanoparticles were characterized using various spectrophotometric techniques for their shape, size and functional group involved in bioreduction and stabilization of silver ions.

AgNPs showed significant antibacterial activity against reference and MDR strains. GC-MS analysis was carried out for detailed phytochemical analysis. Thus the biosynthesized AgNPs may be used as an alternative treatment therapy to control bacterial infections.

Acknowledgement

We would like to acknowledge AIIMS, New Delhi for providing SEM and TEM analysis facility. This research work was financially supported by a grant from DST [DST-INSPIRE, IF-140413] & UGC-SAP [F.20/2012(SAP-II)].

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