Enhanced Cellular Internalization: A Bactericidal Mechanism More

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Enhanced Cellular Internalization: A Bactericidal Mechanism More Relative to Biogenic Nanoparticles than Chemical Counterparts Madhuree Kumari,†,# Shatrunajay Shukla,‡,# Shipra Pandey,† Ved P. Giri,† Anil Bhatia,† Tusha Tripathi,† Poonam Kakkar,‡ Chandra S. Nautiyal,† and Aradhana Mishra*,† †

CSIR-National Botanical Research Institute, Rana Pratap Marg, Lucknow, 226 001, India CSIR-Indian Institute of Toxicology Research, Vishvigyan Bhawan 31, Mahatma Gandhi Marg, Lucknow, 226 001, India

‡

S Supporting Information *

ABSTRACT: Biogenic synthesis of silver nanoparticles for enhanced antimicrobial activity has gained a lot of momentum making it an urgent need to search for a suitable biocandidate which could be utilized for efficient capping and shaping of silver nanoparticles with enhanced bactericidal activity utilizing its secondary metabolites. Current work illustrates the enhancement of antimicrobial efficacy of silver nanoparticles by reducing and modifying their surface with antimicrobial metabolites of cell free filtrate of Trichoderma viride (MTCC 5661) in comparison to citrate stabilized silver nanoparticles. Nanoparticles were characterized by visual observations, UV−visible spectroscopy, zetasizer, and transmission electron microscopy (TEM). Synthesized particles were monodispersed, spherical in shape and 10−20 nm in size. Presence of metabolites on surface of biosynthesized silver nanoparticles was observed by gas chromatography−mass spectroscopy (GC-MS), energy dispersive X-ray analysis (EDAX), X-ray diffraction (XRD), and Fourier transform infrared spectroscopy (FTIR). The antimicrobial activity of both silver nanoparticles was tested against Shigella sonnei, Pseudomonas aeruginosa (Gram-negative) and Staphylococcus aureus (Gram-positive) by growth inhibition curve analysis and colony formation unit assay. Further, it was noted that internalization of biosynthesized nanoparticles inside the bacterial cell was much higher as compared to citrate stabilized particles which in turn lead to higher production of reactive oxygen species. Increase in oxidative stress caused severe damage to bacterial membrane enhancing further uptake of particles and revoking other pathways for bacterial disintegration resulting in complete and rapid death of pathogens as evidenced by fluorescein diacetate/propidium iodide dual staining and TEM. Thus, study reveals that biologically synthesized silver nanoarchitecture coated with antimicrobial metabolites of T. viride was more potent than their chemical counterpart in killing of pathogenic bacteria. KEYWORDS: green synthesis, Trichoderma viride, silver nanoparticles, bactericidal activity, nanoparticles characterization, bacterial disintegration the particles against multidrug resistant microbes,7,8 research has now been focused on to increase the antimicrobial potential and deciphering the mode of action involved.9,10 A number of methods have been employed to enhance the antimicrobial potential of silver nanoparticles including modification in shape, size and change in surface corona by chemical modifications,5,11,12 however biological means also bear equal potential

1. INTRODUCTION Pathogens have always been a cause of suffering to human beings by causing destruction of human health1 resulting in a huge loss on social and economic front.2 After they acquired resistance to multiple drugs, it has become more difficult to fight against the pathogenic agents.3 Silver has been known for its antimicrobial potential and widely used for food and water safety for ages.4 Advancement in nanotechnology has created new possibilities to synthesize silver nanoparticles to enhance the potential of metal against pathogens.3,5,6 Owing to the excellent antimicrobial activities of © 2017 American Chemical Society

Received: December 2, 2016 Accepted: January 4, 2017 Published: January 4, 2017 4519

DOI: 10.1021/acsami.6b15473 ACS Appl. Mater. Interfaces 2017, 9, 4519−4533

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ACS Applied Materials & Interfaces

mL of sterile deionized water for 3 days and filtered again to obtain cell free filtrate containing metabolites. For synthesis of silver nanoparticles, aqueous silver nitrate solution at a final concentration of 1 mM was added to the reaction flasks containing cell-free filtrate and incubated at 28 °C on a rotary shaker (150 rpm) for 16 h at pH 5.5. Flasks containing cell free filtrate without silver nitrate and aqueous solution of silver nitrate without cell free filtrate of T. viride served as control. To separate out the silver nanoparticles from crude cell free filtrate, the filtrate was centrifuged (Sigma 2-16KL, Germany) at 20 000 rpm for 20 min10 and thus obtained supernatant was filtered through 0.22 μ syringe filters (Millipore). The washing step was repeated before every step of characterization to remove unattached metabolites from the surface of biosynthesized particles. 2.4. Estimation of Protein, Phenolics, and Carbohydrate Content in Cell Free Filtrate and BSNP. To know the reducing potential of cell free filtrate and nature of capping agents of BSNP, concentration of proteins, phenolics and carbohydrate was estimated. Protein and phenolics concentrations were measured by Bradford and Folin-Ciocalteu’s method25,26 Carbohydate content was estimated by anthrone reagent method as described by Weiner,27with some modifications. 2.5. Synthesis of Citrate-Stabilized Silver Nanoparticles (CSNP). The citrate stabilized silver nanoparticles was synthesized by following protocol of Ratyakshi and Chauhan (2009)28 with some modifications. Briefly, 100 mL of 1 mM AgNO3 was heated to boiling to which 10 mL of 1% trisodium citrate was added drop by drop and mixed vigorously. The solution was heated until the color change was observed from colorless to pale yellow and was allowed to stand at 28 °C for 16 h before use. 2.6. Instrumentation. The reduction of silver ions to silver nanoparticles (BSNP and CSNP) was monitored through UV−vis spectroscopy29 (Thermo specrtoscan UV 2700) after preliminary observation for color change of cell free filtrate. For observation of hydrodynamic diameter, monodispersity and zeta potential, dynamic light scattering (DLS) was performed (Malvern, nanoseries zetasizer). The hydrodynamic diameter and the polydispersity index (PDI) were calculated from three individual measurements using intensity distribution. Transmission electron microscopy (TEM) (Technai 1321 G2 Spirit TWIN, USA) was carried out to measure the particle size and shape of nanoparticles biosynthesized. Washed samples were filtered through 0.22 μ syringe filters (Millipore) and sonicated for 2 min. A drop of solution was placed immediately on Formvar coated copper grid after sonication and left overnight for drying. Bright field TEM studies were carried out at 80 kV. Energy Dispersive X-ray Analysis (EDAX) spectra of the nanoparticles, cell free filtrate and silver salt were carried out on a scanning electron microscopy (Quanta FEG 450, FEI, Netherlands).The samples were freeze-dried and casted on a glass substrate. The electron backscatter diffraction was used during the analysis, and the EDS aperture was set to 60 μm and operated at 20 kV. The crystalline phase was detected using X-ray diffraction (XRD) analysis. For Fourier-Transform Infra-Red spectroscopy measurements, the nanoparticles were freeze-dried and diluted with potassium bromide in the ratio of 1:100. The FT-IR spectra of samples were recorded on a FT-IR instrument (Agilent Cary 630). All measurements were carried out in the range of 400−4000 cm−1. 2.7. Metabolite Profiling by GC-MS Analysis. To investigate the antimicrobial compounds present in cell free filtrate of T. viride involved in coating of silver nanoparticles, metabolite profiling was done through GC-MS using Thermo Trace GC Ultra coupled with Thermo fisher DSQ II mass spectrometers. Cell free filtrate and washed biomineralized silver nanoparticles (BSNP) were lyophilized and concentrated to obtain their powder form. The synthesized AgNPs was collected by centrifugation at 20 000 rpm for 20 min and thoroughly washed with deionized and double distilled water to remove unreacted or loosely bound Ag+ ions.10 Chromatographic separations of metabolites were carried out on 30 m × 0.25 mm Thermo TR50 column (polysiloxane column coated with 50% methyl and 50% phenyl groups). X-calibur software was used to process the chromatographic and mass spectrometric data. The GC oven

to modulate shape, size and surface of nanoparticles in green and environment friendly manner,13,14 for enhancement of antimicrobial efficacy. Though some reports demonstrate the higher efficacy of biogenic silver nanoparticles over their chemical counterparts,15 but the feasibility of the process is not convincing and the mechanism of antibacterial property exerted remains unknown yet.15 Biological moieties which demonstrate high antimicrobial activity can be utilized to reduce and stabilize metal ions to form nanoparticles capped with antimicrobial compounds, thereby enhancing the antagonistic potential of particles and reducing time and cost at the same time. Trichoderma sp. are soil fungi known for their role in plant growth promotion, pathogen control, and bioremediation.16,17 They are the prolific producer of many secondary metabolites, including antibacterial and antifungal peptides, peptaibols, enzymes, and organic compounds18,19 which may play a vital role in capping and stabilizing the nanoparticles, thus exhibiting enhanced antibacterial activity. The particular isolate used in this study, Trichoderma viride (MTCC 5661) has already proved its potential in rapid biosynthesis of gold nanoparticles and alteration in their shape and size by changing the physicochemical conditions.17,20 Several modes of action of silver nanoparticles have been proposed to elucidate the mechanism for their antibacterial activity including DNA unwinding, membrane disruption and binding with important biological molecules.9,10 Some reports suggest that irreversible damage on bacterial cells and cytoplasmic membrane is cause of antibacterial activity against Staphylococcus aureus and Pseudomonas syringae while some make modification of intracellular ATP levels responsible for bacterial cell death.21 While the root cause of antibacterial activity of silver nanoparticles is yet to be potentially deciphered, a plausible elucidation regarding the enhanced activities of biogenic silver nanoparticles in comparison with their similar chemical counterpart is also needed. In this study, we investigate the potential of T. viride (MTCC 5661) to enhance the antimicrobial activity of biosynthesized silver nanoparticles (BSNP) in comparison to citrate stabilized silver nanoparticles (CSNP) of similar shape and size. An in depth study has also been carried out to elucidate the reasons for higher antimicrobial activities of BSNP in comparison to CSNP of similar morphology.

2. MATERIALS AND METHODS 2.1. Materials. Silver nitrate (AgNO3, 99.99%), sodium citrate, 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA), fluorescein diacetate (FDA), propidium iodide (PI), and MTT [3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide dye] were purchased from Sigma-Aldrich. Media were purchased from Himedia lab, Mumbai, India. All other chemicals and reagents were of highest purity analytical grade. 2.2. Biocontrol Activity. The fungal isolate (T. viride, MTCC 5661) was obtained from lab repository of division of plant microbe interactions, CSIR-NBRI, and checked for siderophore, indole acetic acid production, phosphate solubilization, and amylolytic, proteolytic, cellulolytic, and chitinolytic acivity.22−24 2.3. Fungal Cell Free Filtrate Preparation and Synthesis of Biogenic Silver Nanoparticles (BSNP). Potential biocontrol isolate T. viride (MTCC 5661) was used for biosynthesis of silver nanoparticles. To prepare cell free filtrate, culture was grown in 100 mL of potato dextrose broth (PDB) for 4 days at 28 °C on rotatory shaker/incubator (80 rpm). After 4 days, the biomass obtained was filtered from Whatmann filter paper no. 1 and washed with autoclaved MQ water for 3 times. The washed biomass was resuspended in 100 4520


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ACS Applied Materials & Interfaces temperature was maintained at 70 °C for 5 min, then gradually increased by 5 °C per minute until it reached 290 °C and this temperature was maintained for 5 min. The sample was injected in the split mode at a splitting ratio of 1:16. Helium was used as a carrier gas and set at a constant flow rate of 1 mL min−130 2.8. Comparison of Antibacterial Activity of Biosynthesized Silver Nanoparticles (BSNP) and Sodium-Citrate-Stabilized Silver Nanoparticles (CSNP). To compare antibacterial activity of biosynthesized silver nanoparticles (BSNP) and sodium citrate stabilized silver nanoparticles (CSNP), stock of 100 μg/mL of both biologically synthesized and sodium citrate stabilized silver nanoparticles were prepared. Two Gram-negative human pathogen Shigella sonnei (isolated from wastewater), Pseudomonas aeruginosa (ATCC 15692), and one Gram-positive human pathogen Staphylococcus aureus (ATCC 33591) were selected because of their severe pathogenicity and abundance in soil and water. For broth assay, nutrient broth (NB) were supplemented with 1, 2, and 5 μg/mL of BSNP and CSNP diluted from the stock of 100 μg/mL. 1% of 16 h grown cultures of pathogenic bacteria (109 CFU/mL) were inoculated into the above prepared media and growth was observed at different time intervals of 1, 2, 3, 4, 5, and 6 h in terms of CFU/mL. NB without nanoparticles was used as control. Further, to nullify the possibility that nanoparticles are increasing lag time without inhibiting the bacterial growth, bacterial growth was also observed at 12 and 24 h. For measuring the optical density (OD) of pathogens, freshly grown bacterial cells were taken in NB tubes and different concentration of BSNP and CSNP (1, 2, and 5 μg/mL) were added to bacterial cultures. Optical density was measured spectrophotometrically using a Spectramax PLUS 384 microplate reader (SoftMaxPro version 5.1; Molecular devices, USA) at time intervals of 1, 2, 3, 4, 5,6, 12, and 24 h at 600 nm. To investigate the synergistic effect of cell free filtrate with biogenic silver nanoparticles, antibacterial effect of cell free filtrate was also estimated until 24h. Briefly, 10 and 25% cell free filtrate were mixed with prepared media and bacterial growth was measured in terms of CFU/mL and spectrophotometrically. NB tubes without nanoparticles served as control. 2.9. Immobilization of Silver Nanoparticles on Cotton and Disk Diffusion Studies. Immobilization of silver nanoparticles on cotton and disk diffusion studies were carried out following the method of Mishra et al.17 with some modifications. 100 μL of overnight grown culture of S. aureus was spread uniformly on nutrient agar plates. Pre sterilized cotton of 1 cm2 was placed on the center of the plates and pipetted with 100 μL of 1 μg/mL silver nanoparticles in water and allowed to air-dry. Similar method was followed for another set of disks where the PVP (10% w/v) were added for better immobilization. The plates were incubated at 28 °C for 24 h, after which the zone of inhibition was observed. 2.10. Internalization of CSNP/BSNP. Flow cytometric (FACS) analysis was performed for the assessment of CSNP/BSNP internalization in bacterial cells (S. sonnei, S. aureus, and P. syringae) according to the method of Suzuki et al.31 with some modifications using light scattering principles. The analysis is based on the principle that increase in the intensity of side scattered (SSC) light with constant intensity of forward scattered (FSC) light in cells reveals increased granularity of cells correlated to cellular uptake of NPs. In brief, 1 × 106 bacterial cells/mL were seeded in 6-well culture plates and exposed to 2 μg/mL concentration of CSNP/BSNP for 1 h and 2h. After indicated incubation period, bacterial cells were harvested and resuspended in 500 μL of 1× PBS. Analysis was made using flow cytometer equipped with 488 nm laser (FACS Canto II and FACS Diva software (version 6.1.2) BD Biosciences, San Jose, CA, USA). 2.11. Determination of Oxidative Stress (ROS) Level. Intracellular level of reactive oxygen species and other adducts in vehicle and BSNP/CSNP treated bacterial cells were monitored at different time points (30, 60, and 120 min) using specific ROS sensitive fluorescent probe 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA), (Sigma). After indicated incubation time, DCFH-DA (10 μM) was added to vehicle and CSNP/BSNP (1 μg/mL and 2 μg/ mL) treated bacterial cells. Vehicle and BSNP/CSNP treated samples were further incubated for 30 min in bacterial incubator. Fluorescence

intensity was monitored on a spectrofluorometer (Varioskan Flash, Thermo) at excitation and emission wavelengths of 504 and 529 nm, respectively. Results were shown as fold change increase of ROS level as compared to control. 2.12. Assessment of Live/Dead Population by FDA/PI Staining. Live/dead population quantification in vehicle and NPs treated bacterial cells was done using FDA/PI staining.10 FDA is a nonfluorescent and cell permeable viability probe, which inside the live cells, deesterified by cellular esterases and gets converted into a highly fluorescent compound fluorescein, while propidium iodide is a cell impermeable nucleic acid stain and internalized only when membrane is compromised. In brief, 1 × 105 bacterial cells/mL were seeded in 96well culture plate and exposed to different concentrations of CSNP/ BSNP (1 and 2 μg/mL) for 1 and 2 h. After indicated incubation time, cells were incubated with 10 μM FDA and 50 μg/mL PI for 30 min and fluorescence intensity of FDA and PI was monitored on a spectrofluorometer (Varioskan Flash, Thermo) at excitation and emission wavelengths of 496/515 and 493/636 nm, respectively. Index of live/dead population was shown as ratio of FDA to PI fluorescence and data is presented as fold change of FDA/PI fluorescence as compared to control. 2.13. Morphological Characterization of Bacterial Cell. One percent preinoculum of bacterial cells (16 h old) were added to 2 mL of NB for 4 h and kept at 28 °C under rotary (180 rpm) condition. After 4 h of growth, treatment of BSNP and CSNP (2 μg/mL) were given to pathogens. After indicated time, bacterial cells were harvested, washed with 0.1 M sodium cacodylate buffer and fixed with 2% paraformaldehyde and 2.5% glutaraldehyde mixture for 2 h at 4 °C. Fixed cells were subsequently washed with 0.1 M cacodylate buffer and post fixed with 1% osmium tetra-oxide for 2−3 h. Postfixed cells were carefully centrifuged and dehydrated in ascending series of acetone (15%, 30%, 60%, 90%, and 100%), 2 times with propylene oxide (10 min each), embedded in Araldite and DDSA medium and baked at 65 °C for 48 h.) Sample blocks were cut by an ultramicrotome (Leica EM UC7, Vienna, Austria), mounted on copper grids and doubly stained with uranyl acetate and lead citrate. Stained samples were examined by Transmission Electron Microscope (TECNAI G2 SPIRIT, FEI, USA) equipped with Gatan Orius camera.32 2.14. Tetrazolium Dye Reduction Assay. To check the effect of BSNP and CSNP on normal cell line, tetrazolium dye reduction assay was carried out. Cell viability of vehicle and BSNP/CSNP-treated rat kidney epithelial cells (NRK-52E) was assessed by the tetrazolium dye MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] as described by Mosmann et al. (1983).33 Briefly, cells were plated in 96-well plates (1 × 104 cells/well) and treated with a range of BSNP/ CSNP concentrations (1, 2, and 5 μg/mL) for 3 h. After indicated incubation time, media was replenished; MTT (0.5 mg/mL) was added and incubated for 4 h. Subsequently, media was aspirated and 200 μL of DMSO was added to dissolve the formazan crystals and plate was read at 570 nm using a Spectramax PLUS 384 microplate reader (SoftMaxPro version 5.1; Molecular devices, USA). 2.15. Statistical Analysis. All the experiments were performed at least three times and quantitative variables are represented in terms of means ± SD in histograms. Student’s t test was used for assessing significance of difference between treated and non treated group of experiment. Differences were considered statistically significant when *P < 0.05, **P < 0.01, and ***P < 0.001 were significantly different from the control.

3. RESULTS AND DISCUSSION 3.1. Biocontrol Activity. T. viride showed ability to produce siderophore, indole acetic acid, phosphate solubilization, and demonstrated amylolytic, chitinolytic, and proteolytic activities (Table 1) which play an important role in antagonistic behavior toward other pathogenic microbes21 and may act as reducing and stabilizing agent during nanoparticle biosynthesis. 3.2. Estimation of Protein, Phenolics, and Carbohydrate in Cell Free Filtrate and BSNP. Trichoderma sp. are 4521


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ACS Applied Materials & Interfaces Table 1. Physiological and Biochemical Characteristics of Biocontrol Agent T. viride traits

T. viride

total siderophore indole acetic acid phosphate solubilization proteolytic activity amylolytic activity cellulolytic activity chitinase activity

152.6 ± 5.2 μg/mL 58.36 ± 0.7 μg/mL 13.32 ± 0.6 μg/mL + + − +

known to produce many important secondary metabolites and peptibols in their extracellular secretion.16 T. viride (MTCC 5661) was found to be a prolific producer of phenolics and carbohydrates, which are important constituent of secondary metabolites. It produced 18.975 nmol gallic acid/mL of phenolics and 268.66 μg/mL of carbohydrate in cell free filtrate (Table 2). The protein concentration in cell free filtrate

Figure 1. UV−visible spectra of cell free filtrate of T. viride (a), biosynthesized silver nanoparticles (BSNP) after 16 h of incubation (b), and citrate stabilized silver nanoparticles (CSNP) (c). Inset graph shows the color change of the same.

Table 2. Total Protein, Carbohydrate, and Phenolics Content in Cell-Free Filtrate and BSNP traits

cell free filtrate

BSNP

total protein (μg/mL) total carbohydrate (μg/mL) total phenolics (nmol GAE/mL)

21.6 ± 1.75 268.66 ± 4.40 18.97 ± 0.70

27.5 ± 2.03 387.33 ± 2.02 5.72 ± 0.18

nanoparticle biosynthesis have demonstrated the role of proteins in bioreduction of metal salt to nanoparticles.34,35 3.3.2. Particle Size Determination and Morphological Characterization of Biosynthesized and Chemically Synthesized Nanoparticles. To observe the particle size, monodispersity and zeta potential of CSNP and BSNP, nanoparticles were characterized through dynamic light scattering (DLS). The CSNP showed a hydrodynamic average diameter (hydrodynamic size) of 71.58 ± 0.14 nm and PDI index of 0.249 ± 0.01, while BSNP showed hydrodynamic size of 74.8 ± 0.70 nm and PDI index of 0.239 ± 0.026 indicating the monodispersity of silver nanoparticles (Figure S1a,b). In culture media, i.e. nutrient broth (NB), CSNP and BSNP showed hydrodynamic size of 71.58 ± 0.50 and 71.35 ± 0.82 nm with PDI value of 0.370 ± 0.002 and 0.248 ± 0.002, respectively (Figure S1c, d). Zeta potential of CSNP was −19.9 ± 0.47 mV while BSNP showed potential of −26.36 ± 0.14 mV (Figure S1e, f). Though the size and PDI values of BSNP in NB were as consistent as it was in water, the PDI of CSNP shifted from 0.249 in water to 0.370 in NB indicating increase in polydispersity of the CSNP. To check the stability of BSNP and CSNP, their monodispersity index (PDI) was measured after six months of storage. BSNP showed good monodispersity (PDI-0.265), while PDI of CSNP increased from 0.249 during synthesis to 0.438 after long-term storage (Figure S1h, g). The results demonstrates the capabilities of cell free filtrate to act as a better capping and stabilizing agent than citrate. The DLS instrument measures the shell thickness of capping bio-organic compounds along with the actual size of the metallic core36 resulting in larger hydrodynamic diameter than the actual size of the particles. For getting the actual size of particles and their morphological aspect, TEM studies of BSNP and CSNP were carried out. Most of the particles were spherical in shape (Figure 2a, b), while some were pseudospheres. Particle size histogram showed that particle size was below 40 nm, having 60% of particles in the range of 10−25 nm (Figure 2). Both BSNP and CSNP were well dispersed and not aggregated. Even after six months, particles were monodispersed in case of BSNP while CSNP aggregated after one month of storage at room temperature. It may be due to capping of particles by extracellular secretions of T. viride, which prevented the self-

was found to be 0.0216 mg/mL. Secondary metabolites and proteins, both act as strong reducing and capping agents for nanoparticles.12 Further to know the involvement of cell free filtrate in coating of biogenic nanoparticles, protein, phenolics and carbohydrate was estimated in BSNP. The concentration of protein, phenolics, and carbohydrate in BSNP was 0.0275 mg/ mL, 5.725 nmol gallic acid/mL, and 387.33 μg/mL, respectively (Table 2). 3.3. Characterization of Nanoparticles. 3.3.1. Characterization of the BSNP and CSNP by UV−vis Absorption Spectroscopy. The addition of aqueous silver nitrate solution to cell free filtrate of T. viride and sodium citrate solution resulted in gradual change of color from colorless to brown after 16 h of incubation, indicating the formation of BSNP and CSNP respectively (Figure 1). The pure silver nitrate solution did not show any color change during entire time duration. The change in color of the solution is a characteristic property of the synthesized silver nanoparticles observed due to excitation of surface plasmon resonance (SPR) in the nanoparticles in the visible range of 400−500 nm.16 CSNP showed maximum absorbance at 431 nm, depicting synthesis of silver nanoparticles.16 Absence of any shoulder peaks near the 350 nm absorption peaks in BSNP and CSNP indicated that no bulk silver is generated. Cell free filtrate without addition of silver nitrate did not show any peak absorbance in the particular range, while the absorption peak was recorded at 422 nm after 16 h of addition of silver nitrate to cell free filtrate of T. viride (Figure 1). A peak at 280 nm was observed in cell free filtrate of T. viride which clearly indicated the role of amino acids of protein in synthesis and stabilization of biosynthesized silver nanoparticles.12 BSNPs remained stable and dispersed even after six months of synthesis, at room temperature. It was an indication that proteins present in cell free filtrate were capable enough to coat and stabilize the synthesized particles. Earlier reports of 4522


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Figure 2. TEM micrographs of BSNP (a) and CSNP (b) nanoparticles with their size distribution. Scale bar-50 nm.

secondary metabolites and proteins of cell free filtrate of T. viride in shaping the surface composition of the nanoparticles. 3.4.2. FT-IR Spectroscopy. Characterization of functional groups on the surface of biosynthesized silver nanoparticles (BSNPs) were investigated using a Fourier-transmission infrared spectroscopy. Different peaks at 669, 757, 1155, 1215, 1384, 1737, 2926, 2854, and 3019 cm−1 (Figure S4), obtained in FT-IR spectrum of BSNP, indicated the presence of different biomolecules. The band at 669 and 757 cm−1 denoted C−Br stretch and C−Cl stretch of alkyl halides, while 1155, 1215, and 2926 cm−1 indicated involvement of C−N stretch of aliphatic amines and stretching vibration of amide II respectively. The band at 1384 cm−1 demonstrated stretching vibration of aromatic amines. Nanoparticle biomolecules interactions can occur through several means including the electrostatic attraction of negatively charged groups of organic molecules and involvement of amine residue of enzymes.12 Presence of different bands depicting structures of enzymes and organic compounds in both cell free filtrate and silver nanoparticles clearly demonstrated the role of extracellular secondary metabolites and proteins of T. viride in capping of Ag nanoparticles. 3.4.3. Metabolite Profiling. After confirmation of involvement of secondary metabolites in capping of BSNP, gas chromatography−mass spectrometry (GC-MS) analysis was carried out to investigate the details of antimicrobial metabolites present in water fraction of cell free filtrate of T. viride. A total of 40 metabolites were identified in which 16 were found to be involved in coating of silver nanoparticles. The

aggregation of BSNP. Previous studies on biosynthesis of nanoparticles also supported the role of biomolecules as synthesizing as well as capping agents.10,37 3.4. Presence of Proteins and Secondary Metabolites on the Surface of BSNP. 3.4.1. EDAX Spectroscopy and XRD Analysis. EDAX analyses of cell free filtrate of T. viride, silver salt and biosynthesized silver nanoparticles were carried out separately to know the bioelements involved in coating of BSNP (Figure S2). Strong signals of C, O, Na, Mg, Al, Si, S, Cl, K, and Ca were observed in EDAX spectrum of cell free filtrate indicating the presence of diverse series of bioelements which were absent in spectrum of silver nitrate salt. Further analysis of biosynthesized silver nanoparticles showed C, O, Mg, P, K, and Fe as major component apart from silver, reflected that the particles were well capped with several biomolecules which were present as secondary metabolites and proteins of cell free filtrate of T. viride. Earlier studies have also demonstrated the role of bioelements such as enzymes and secondary metabolites in biosynthesis and capping of silver nanoparticles38 but the exact name of metabolites and their effect on properties of nanoparticles synthesized has not been elucidated yet. XRD analysis of cell free filtrate demonstrated a hump at 2θ value of 20 and several amorphous peaks demonstrating the presence of several organic compounds while the peaks of silver, chlorargyrite and S-benzyl cysteine were observed in biogenic silver nanoparticles (Figure S3). Zhang et al., 2016,39 in their studies, have observed broad humps typical of amorphous BSA by XRD analysis. Presence of several amorphous peaks in both, cell free filtrate and biogenic silver nanoparticles demonstrated the important role of organic compounds derived from 4523


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Table 3. Quantification of Metabolites Present in Coating of Silver Nanoparticles by Cell-Free Filtrate of T. viride Using GC-MS Analysisa relative concentration (%) S. no.

RT (min)

metabolites

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40

6.8 9.34 9.8 11.52 11.88 11.96 12.5 16.03 17.45 17.61 19.7 21.89 22.02 22.35 22.71 23 23.24 23.45 24.36 25.14 25.47 27.37 27.46 28.66 29.67 30.31 30.54 30.74 31.08 31.42 31.7 33.1 34.66 34.83 34.89 36.48 36.94 43.94 45.83 50.49

ethylene glycol propanoic acid glycolic acid 2-furoic acid N-carboxy glycine pyruvic acid butanoic acid glycerol succinic acid glyceric acid 2-furyl glycolic acid L-glutamic acid erythronic acid erythritol 2-hydroxyglutaric acid T-muconic acid threonic acid 4-hydroxyphenylethanol α-hydroxy-α-methylglutaric acid arabinonic acid succinic acid xylitol D-mannose ribonic acid D-ribo-hexitol arabino-hexonic acid D-fructose D-gluconic acid D-glucose D-galactose D-glucitol galactonic acid inositol 2-keto-D-gluconic acid N-acetyl glucosamine glucoson α-D-glucopyranose D-turanose sucrose α-D-glucopyranoside

a

206, 234, 220, 184, 235, 232, 248, 308, 262, 322, 286, 405, 262, 410, 364, 286, 424, 282, 378, 364, 438, 512, 569, 526, 526, 540, 569, 466, 569, 569, 614, 628, 612, 554, 538, 524, 692, 846, 918, 676,

193, 228, 208, 169, 220, 220, 235, 293, 249, 309, 272, 355, 261, 395, 335, 273, 409, 180, 365, 349, 423, 422, 394, 421, 409, 525, 408, 451, 466, 376, 422, 524, 508, 506, 523, 429, 505, 480, 437, 543,

fragmentation pattern (m/z)

T. viride filtrate

BSNP coated with T. viride filtrate

191, 219, 177, 125, 190, 177, 233, 218, 172, 292, 257, 274, 189, 218, 305, 197, 379, 126, 247, 259, 305, 319, 320, 307, 333, 335, 263, 363, 320, 319, 346, 433, 367, 437, 345, 345, 356, 361, 379, 455,

0.4 4.79 0.56 0.31 0.55 1.43 1.57 3.65 0.35 0.78 9.36 0.3 1.09 3.02 0.3 0.98 1.48 0.19 0.4 0.28 7.2 9.19 0.73 0.74 4.11 2.09 0.25 ND 4.17 1.37 ND 0.64 1.07 0.18 0.23 0.74 0.27 1.27 0.49 0.9

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

1.49 ± 0.13 3.14 ± 0.37 0.46 ± 0.04

147, 103, 73 147, 117, 73, 66, 59 147, 133, 73, 66, 59 95, 73 148, 147, 89, 86, 73, 59 147, 116, 75, 73, 66 218, 177, 75, 73, 66 177, 147, 133, 103, 73, 59 147, 83, 75, 73, 55 205, 189, 147, 103, 73 183, 169, 147, 94, 75, 73 244, 184, 142, 111, 75, 73, 55 171, 129, 103, 75, 73, 59 204, 149, 117, 103, 73 248, 203, 157, 129, 75, 73, 53 169, 147, 75, 73, 52 292, 221, 205, 148, 117, 73 103, 73 186, 129, 85, 73, 55 217, 189, 147, 117, 73, 59 221, 189, 147, 74, 73 217, 205, 147, 103, 73 244, 189, 157, 129, 73 218, 189, 147, 103, 73 243, 205, 129, 73, 69, 59 245, 147, 129, 73 217, 173, 147, 103, 73 319, 271, 229, 169, 102, 73 229, 205, 160, 147, 73 217, 205, 160, 147, 103, 73 307, 205, 157, 147, 103, 73 333, 218, 207, 189, 147, 73, 59 305, 265, 191, 129, 73 319, 257, 147, 117, 75, 73, 56 304, 232, 173, 103, 73, 57 217, 174, 103, 73, 56 300, 217, 160, 147, 103, 73 287, 217, 147, 103, 73, 69, 55, 53 361, 289, 217, 147, 73 273, 208, 134, 103, 73, 69

0.04 0.44 0.04 0.04 0.06 0.13 0.14 0.3 0.03 0.06 0.8 0.02 0.1 0.2 0.02 0.01 0.1 0.01 0.05 0.03 0.57 0.75 0.07 0.74 0.3 0.2 0.02

ND ND 0.37 ± 0.04 0.69 ± 0.07 3.76 ± 0.42 ND ND ND ND ND 1.1 ± 0.11 ND ND ND ND ND ND ND ND ND ND 2.4 ± 0.2 ND 5.84 13.41 34.05 8 0.68

± 0.33 ± 0.14 ± ± ± ± ± ± ± ± ±

0.07 0.09 0.02 0.02 0.07 0.03 0.1 0.06 0.08

± ± ± ± ±

0.5 1.14 0.16 0.92 0.06

ND 1.11 ± 0.11 ND ND ND ND 9.98 ± 0.7 1.39 ± 0.18 ND

ND = not detected; ± = SD; the significance level between the metabolites in obtained by two tailed paired t test.

showing potent antimicrobial activity against a wide range of Gram-positive and Gram-negative bacteria. Similarly, other sugar derivative, glucitol is also known for its antimicrobial potential.43 Inositol and trisamine, which are precursors of many antimicrobial agents,44,45 were also present in the coating of biosynthesized silver nanoparticles. A recent study by Kobylarz et al. (2014)46 has shown that derivative of propanoic acid act as precursor of siderophore and several antibiotic agents. Presence of propanoic acid in coating of silver nanoparticles can also contribute toward enhancing the antimicrobial potential. The results clearly indicates the involvement of plethora of antimicrobial compounds in cell free filtrate of T. viride to cap the silver nanoparticles which play a vital role in increasing the antagonistic potential of biosynthesized silver nanoparticles against pathogens.

metabolites identified comprised of organic acids, sugars, intermediates of carbohydrate metabolism, amino acids and their derivatives (Table 3). Among 40 identified compounds, 25 were potent antimicrobial agents or precursors of several antimicrobial metabolites. A number of organic acids and alkaloids of antimicrobial potential40,41 including propanoic acid, glycolic acid, butanoic acid, gluconic acid and erythritol were involved in coating of silver nanoparticles. A significant increase in gluconic acid and glucitol were observed among the compounds involved in coating of BSNP. Higher concentration of gluconic acid might have resulted from oxidation of glucose which can also contribute toward enhancing antimicrobial efficacy of BSNP. Earlier, Nieto-Peñalver et al. (2014)42 reported production of this organic acid from direct oxidation of glucose by a pyrroloquinoline-quinone-linked glucose dehydrogenase in a plant growth-promoting bacterium, 4524


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Figure 3. Growth curve in terms of absorbance of S. sonnei treated with different concentrations of CSNP (a) and BSNP (b). P.aeruginosa treated with different concentrations of CSNP (c) and BSNP (d) and S. aureus treated with different concentrations of CSNP (e) and BSNP (f). Data represent mean ± SD of three different experiments. *P < 0.05, **P < 0.01, ***P < 0.001, significantly different from the control.

3.5. Antimicrobial Activity of BSNP in Comparison to CSNP. Antibacterial property of BSNPs were tested against S. sonnei, S. aureus and P. aeruginosa and compared with CSNP. S. sonnei, causative agent of shigellosis is common water borne pathogen. It has acquired resistance against multiple antibacterial drugs, posing a health problem in developing countries.47 S. aureus is a well-known pathogen associated with skin infection. Similarly, P. aeruginosa has been adjudged one of the top three causes of opportunistic human infection for a very long time.48 The efficacy of cell free filtrate of T. viride as antimicrobial agent was tested at two different concentrations (10 and 25%). The concentration dependent growth inhibition in all the three tested pathogens were observed in terms of CFU/mL and absorbance at 600 nm (Figure S5). The results obtained support the hypothesis that BSNP coated with cell free filtrate having potent antimicrobials may enhance antimicrobial potential of BSNP in comparison to CSNP. With all the three pathogens tested, 1 μg/mL of BSNP was as potent as 5 μg/mL of CSNP in terms of turbidity (Figure 3), however with P. aeruginosa, antibacterial activity of BSNP and CSNP was comparable to each other.

To know the effect of both nanoparticles on growth curve of bacteria, CFU studies were carried out. A concentration dependent decrease in bacterial population was observed with both BSNP and CSNP (Figure 4). With S. sonnei, 50% killing efficiency was achieved after 4 h of treatment with 5 μg/mL of CSNP, while it was achieved after 3 h of 2 μg/mL of BSNP treatment (Figure 4a and b). Similarly, 5 μg/mL of BSNP treatment resulted in 100% growth inhibition of P. aeruginosa and S. aureus after 3 and 4 h of treatment respectively (Figure 4d and f), while CSNP was able to complete inhibition at 6 h in S. aureus but unable to do so even after 6 h treatment in P. aeruginosa (Figure 4e and c). Many antibacterial agents are known to extend the lag phase of microbes without killing them efficiently. To nullify this possibility, bacteria were further treated with different concentration of BSNP and CSNP for 12 and 24h. No further increase in bacterial count and absorbance was obtained even after 24h with both, BSNP and CSNP (Figure S6,7), proving their potency as superior antimicrobial agent. In terms of CFU/ mL, critical concentration of BSNP for P. aeruginosa was 2 μg/ mL, while CSNP was not able to inhibit growth completely even at 5 μg/mL after 24h of treatment (Figure S6). The critical concentration for S. sonnei and S. aureus after treatment 4525


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Figure 4. CFU curve of S. sonnei treated with different concentrations of CSNP (a) and BSNP (b). P. aeruginosa treated with different concentrations of CSNP (c) and BSNP (d) and S. aureus treated with different concentrations of CSNP (e) and BSNP (f). Data represent mean ± SD of three different experiments.

of BSNP was 1 and 2 μg/mL respectively after treatment for 24 h in terms of absorbance, while with CSNP it was 5 and 5 μg/ mL in same experiment conditions (Figure S7). Antimicrobial properties of silver nanoparticles are known to depend upon a number of factors including particle size, shape, and type of coating10,13 which can be manipulated to get a better antimicrobial property. The coating of antimicrobial agents from extracellular metabolites of T. viride containing spectrum of antimicrobial compounds19 makes it an excellent pathogen control agent (Table 1). At this stage, it became necessary to carry out an in depth study to decipher the mode of action of used by BSNP and CSNP while acting as an antibacterial agents. 3.6. Immobilization of Silver Nanoparticles on Cotton and Disk Diffusion Studies. Disc diffusion studies were done with S. aureus as a model pathogen because of its increasing multidrug resistance and susceptibility toward BSNP as described earlier. Figure S8 explains the enhanced antimicrobial properties of BSNP against S. aureus in immobilization with sterile water and PVP. The BSNP exhibited zone of inhibition 2.025 and 2.19 mm as compared to CSNP in water and PVP 0.075 and 0.09 mm, respectively (Figure S8). The cotton immobilized with PVP demonstrated higher antimicrobial activity as compared to the cotton immobilized with sterile water. PVP acts as a coat of polymer preventing agglomeration

of particles and thus maintaining their antimicrobial activity.49 Similar use of polymers has been demonstrated earlier for retaining the silver nanoparticles even after several washings.50 Being a better antimicrobial agent against pathogens such as S. aureus, and BSNP showing much higher antimicrobial activity as compared to CSNP, it can find its application in wound dressing, coating on paints, contact lens and medical devices. Silver nanoparticles synthesized from cell-free filtrate of T. viride has potential to revolutionize the pharmaceutical industry and biomedical technologies in a green and eco-friendly manner. Further, an attempt was made to gain an insight into the mode of action of antibacterial activity of BSNP and CSNP. 3.7. Cellular Internalization of Nanoparticles. Flow cytometric analysis was carried out for the assessment of BSNP/CSNP internalization in pathogenic bacteria according to the method of Suzuki et al.31 using light scattering principles. Earlier reports are available that granulocytes scattered more light in 90° directions because of the presence of granules in cytoplasm. This is hypothesized that the NPs in bacterial cells behave as granules and scattered more light in a dose dependent manner.28 There was a significant increase in SSC mean value from 8574, 4328, and 1727 (vehicle treated) to 36, 321, 5039, and 17 777 in BSNP treated and 35 576, 6323, and 10 301 in CSNP-treated S. sonnei, P. aeruginosa, and S. aureus, 4526


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Figure 5. Particle uptake analysis of CSNP and BSNP in S. sonnei (a), P. aeruginosa (b), and S. aureus (c) when compared to vehicle treated bacteria, respectively after 2 h treatment as assessed by flow cytometry.

mL) of BSNP and CSNP at different time points (30′, 60′, and 120′) was assessed using 2′,7′- dichlorodihydrofluorescein diacetate (DCFH-DA), a ROS specific fluorescent probe. It is a redox sensitive probe which gets oxidized to DCF (dichlorofluorescein) in the presence of ROS.10 BSNP showed a significant concentration and time dependent increases in ROS generation in terms of fold increase in DCF fluorescence. BSNPs were able to produce ROS up to 2.82 fold, (P < 0.05) in S. sonnei, 1.87 fold, (P < 0.01) in P. aeruginosa and 2.28 fold, (P < 0.01) in S. aureus at 2 h as compared to respective vehicle treated bacteria, while CSNPs could produce ROS up to 1.34 fold, (P < 0.01) in S. sonnei, 1.92 fold, (P < 0.01) in P. aeruginosa, and 1.49 fold, (P < 0.05) in S. aureus at 2 h as compared to vehicle treated control group (Figure 6). Results demonstrated that, the ROS generating potential of BSNP and CSNP were comparable until 30 min after addition of nanoparticles to all pathogenic bacteria. After 60 min, BSNP generated ROS increased significantly in all three pathogens, while no such significant increase was observed with CSNP except in the case of P. aeruginosa. ROS generating potential of nanoparticles can directly be correlated to the internalization of respective nanoparticles. The pathogens (S. sonnei and S. aureus) which demonstrated significantly higher internalization of BSNP showed higher production of ROS in comparison to P. aeruginosa. Higher internalization of BSNP showed higher production of ROS in S. sonnei and S. aureus while lesser in P.

respectively, after 2 h of time interval (Figure 5). During initial studies of particle uptake after 1h, similar trend in increase in granularity was found (Figure S9) There was a significant increase in uptake of BSNP and CSNP in S. sonnei and S. aureus as compared to vehicle treated bacteria. Further, results showed that BSNPs tend to get internalized more effectively in pathogenic bacteria as compared to CSNP. However, cellular uptake of BSNP and CSNP in P. aeruginosa has not been observed much as in S. sonnei and S. aureus as compared to vehicle treated bacteria. Though silver has high affinity for sulfur and phosphorus moiety of proteins forming Ag−N and Ag−O bonds,10 initial binding and uptake is highly dependent upon the surface corona of particles.51 CSNP stabilized with citrate, has a weakly bound capping agent (citrate),51 whereas BSNP stabilized with proteins and secondary metabolites are bound with strong capping agents. As a result, CSNP may tend to aggregate more resulting in reduced surface area, whereas BSNP stabilized with secondary metabolites of T. viride possess higher surface area and less aggregation. Aggregation of particles may result in lesser uptake and internalization of particles (Figure 5).52 Moreover, proteolytic and amylolytic activities of the isolate (Table 1) give BSNP a higher potential to damage cell membrane of pathogens resulting in higher uptake of particles. 3.8. Determination of ROS. Generation of intracellular level of ROS in pathogenic bacteria exposed to (1 and 2 μg/ 4527


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Figure 6. Assessment of bacterial ROS generation using DCFH-DA fluorescent probe at different time intervals in pathogenic bacteria. Fold change of ROS level in S. sonnei treated with CSNP (a) and BSNP (b), in P. aeruginosa treated with CSNP (c) and BSNP (d), and in S. aureus treated with CSNP (e) and BSNP (f). Data represent mean ± SD of three different experiments. *P < 0.05, **P < 0.01, ***P < 0.001 significantly different from the control.

aeruginosa. This might be the reason for aggravated bactericidal potential of BSNP as compared to CSNP.53,54 Secondary metabolites and proteins of Trichoderma are also known for their ability to produce ROS in pathogenic microbes.55 Derivatives of different organic acids present in Trichoderma which may play an important role in coating BSNP have been reported to enhance the production of ROS in pathogens.56,57 Combination of both, silver nanoparticles and coating of metabolites provides BSNP an edge over CSNP in generating ROS in pathogens which ultimately lead to ROS mediated bacterial cell death. Mukherjee et al.54 have shown in their study that coating of secondary metabolites of Lantana montevidensis enhanced anticancer efficacy of gold nanoparticles. 3.9. Quantification of Membrane Damage and Live/ Dead Population by FDA/PI Dual Staining. Quantification of membrane damage and subsequent death of pathogens by BSNP and CSNP treatment was done by FDA/PI dual staining. Ratio of FDA to PI was used as a marker of membrane damage and subsequent cell death as FDA is converted to fluorescein (a highly fluorescent compound) by live cell only while PI was

able to stain nucleic material only when bacterial membrane is compromised. BSNP treatment resulted in increased membrane damage and bacterial cell death as compared to CSNP and vehicle treated bacteria. BSNP treatment was able to decrease FDA/PI ratio up to 0.15-fold, P < 0.001 in S. sonnei, 0.10-fold, P < 0.001 in P. aeruginosa and 0.03-fold, P < 0.001 in S. aureus (control taken as 1-fold), respectively, while CSNP treatment could decrease FDA/PI ratio up to 0.70-fold, P < 0.001 in S. sonnei, 0.09-fold, P < 0.001 in P. aeruginosa, and 0.86-fold, P < 0.01 in S. aureus respectively at 2 h when compared to vehicle treated S. sonnei, P. aeruginosa, and S. aureus respectively (Figure 7a−c). The results obtained here are in agreement with the previous results obtained (Figure 6) during ROS generation. Earlier, with S. sonnei and S. aureus, uptake of particles and generation of ROS were much higher in BSNP as compared to CSNP treatment resulting in increased cell death of bacterial population. However, with P. aeruginosa, both uptake of nanoparticles and generation of ROS were comparable with BSNP and CSNP at 2 h, depicting no significant difference in membrane damage and cell death between BSNP and CSNP 4528


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Figure 7. Index of live/dead bacterial population as fold change of FDA/PI fluorescence at different time intervals. Fold change of FDA/PI fluorescence in S. sonnei treated with CSNP (a) and BSNP (b), P. aeruginosa treated with CSNP (c) and BSNP (d), and S. aureus treated with CSNP (e) and BSNP (f). Data represent mean ± SD of three different experiments. *P < 0.05, **P < 0.01, ***P < 0.001 significantly different from the control.

until 2 h of treatment. In general, P. aeruginosa was the most susceptible pathogen in responding to both types of silver nanoparticles, BSNP and CSNP in terms of ROS production and cell death (Figures 5b, and 7d), however, as the time duration of treatment increased, bactericidal action of BSNP proved to be much effective in comparison to the CSNP. 3.10. Effect on Bacterial Morphology. To further confirm the antibacterial properties of BSNP and CSNP and find out ultrastructural changes in bacterial morphology, transmission electron microscopy was carried out. Healthy rod-shaped S. sonnei and P. aeruginosa with smooth cell surface were observed in vehicle treated control, while round bacterial cells with intact membrane were observed in vehicle treated control of S. aureus (Figure 8). After treatment with BSNP and CSNP, cell surface became rough and wavy while distribution of cytoplasm was uneven and scattered showing remarkable difference from the vehicle treated control cells (Figure 8) in all three pathogens. However, more pronounced damage was observed after BSNP treatment where complete loss of cellular integrity, disruption in bacterial cell membrane and complete

scattering of cytoplasmic content was evident with all the pathogens tested. Higher internalization of nanoparticles and ROS generation in case of BSNP as compared to CSNP might be reason for increased membrane damage, which is also advocated by FDA/ PI dual staining and TEM analysis. Earlier reports of Zhou et al.,51 Chen at al.,2 and Wu et al.58 have also demonstrated the effect of surface modification on antimicrobial activities of nanoparticles. 3.11. Mechanism of Action. On the basis of the results obtained, mechanism of enhanced antibacterial activity of BSNP as compared to CSNP can be summarized in following steps: (i) Increased internalization of nanoparticles in pathogens. (ii) Increased ROS generation in pathogens. Increased internalization and less aggregation of BSNP resulted in excessive ROS generation in bacterial cells as compared to CSNP exposed bacterial cells (Figure 6). Metabolites present in cell free filtrate of T. viride may be strongly contributing to increased production of ROS. Increase in the ROS generation resulted in excessive oxidative stress and severe damage to 4529


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Figure 8. TEM micrographs of S. sonnei treated with vehicle (a), CSNP (b), and BSNP (c). P. aeruginosa treated with vehicle (d), CSNP (e), and BSNP (f). S. aureus treated with vehicle (g), CSNP (h), and BSNP (i).

Figure 9. Schematic representation of comparison between BSNP coated with several antimicrobial metabolites and CSNP coated with citrate. The picture also depicts the complete inhibition of S. aureus by synergistic action of antimicrobial metabolites and silver nanoparticles of BSNP as compared to partial inhibition caused by CSNP.

bacterial membrane. (iii) Increased morphological and membrane damage. Higher uptake of nanoparticles and thus generated oxidative stress results in higher damage of cell membrane and disruption of cell structure as evident by micrographic images (Figure 8). (iv) Increased antibacterial activity. Coating of metabolites and proteins of cell free filtrate of Trichoderma spp., enhanced uptake of nanoparticles, higher oxidative stress and complete damage to pathogen cell organelles and cell membrane. BSNP acted as a superior antimicrobial agent as compared with its chemical counterpart (Figure 9). 3.12. Tetrazolium Dye Reduction Assay. Cell viability assay (MTT assay) was also performed on NRK-52E to confirm safety of synthesized nanoparticles and no significant

toxicity was reported in NRK-52E up to 3 h (Figure S10). Earlier reports have also proved that silver nanoparticles were cytotoxic toward cancer cell line without causing any cytotoxicity to normal and healthy cell lines.59,60

4. CONCLUSIONS Our study demonstrates simple, low cost, and green approach to enhance antimicrobial efficacy of silver nanoparticles using cell free filtrate of potent biocontrol agent T. viride. The role of biological molecules involved in coating of nanoparticles has often been overlooked in determining their antimicrobial properties. This study focuses on selection of suitable biological candidate which along with nanoparticles biosynthesis can coat the nanoparticles with its potent antimicrobial secondary 4530


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Surface Corona Towards Enhancing the Antibacterial Performance of Tyrosine-Capped Ag Nanoparticles. Nanoscale 2014, 6 (2), 758−765. (4) Lemire, J. A.; Harrison, J. J.; Turner, R. J. Antimicrobial Activity of Metals: Mechanisms, Molecular Targets and Applications. Nat. Rev. Microbiol. 2013, 11 (6), 371−384. (5) Dong, B.; Belkhair, S.; Zaarour, M.; Fisher, L.; Verran, J.; Tosheva, L.; Retoux, R.; Gilson, J.-P.; Mintova, S. Silver Confined within Zeolite EMT Nanoparticles: Preparation and Antibacterial Properties. Nanoscale 2014, 6 (18), 10859−10864. (6) Hwang, G. B.; Noimark, S.; Page, K.; Sehmi, S.; Macrobert, A. J.; Allan, E.; Parkin, I. P. White Light-Activated Antimicrobial Surfaces: Effect of Nanoparticles Type on Activity. J. Mater. Chem. B 2016, 4 (12), 2199−2207. (7) Rai, M.; Deshmukh, S.; Ingle, A.; Gade, A. Silver Nanoparticles: The Powerful Nanoweapon Against Multidrug-Resistant Bacteria. J. Appl. Microbiol. 2012, 112 (5), 841−852. (8) Kanmani, P.; Lim, S. T. Synthesis and Structural Characterization of Silver Nanoparticles Using Bacterial Exopolysaccharide and its Antimicrobial Activity Against Food and Multidrug Resistant Pathogens. Process Biochem. 2013, 48 (7), 1099−1106. (9) Wang, Y.; Li, P.; Xiang, P.; Lu, J.; Yuan, J.; Shen, J. Electrospun Polyurethane/Keratin/AgNP Biocomposite Mats for Biocompatible and Antibacterial Wound Dressings. J. Mater. Chem. B 2016, 4 (4), 635−648. (10) Ramalingam, B.; Parandhaman, T.; Das, S. K. Antibacterial Effects of Biosynthesized Silver Nanoparticles on Surface Ultrastructure and Nanomechanical Properties of Gram-Negative Bacteria viz. Escherichia coli and Pseudomonas aeruginosa. ACS Appl. Mater. Interfaces 2016, 8 (7), 4963−4976. (11) Das, S. K.; Khan, M. M. R.; Parandhaman, T.; Laffir, F.; Guha, A. K.; Sekaran, G.; Mandal, A. B. Nano-Silica Fabricated with Silver Nanoparticles: Antifouling Adsorbent for Efficient Dye Removal, Effective Water Disinfection and Biofouling Control. Nanoscale 2013, 5 (12), 5549−5560. (12) Jain, N.; Bhargava, A.; Majumdar, S.; Tarafdar, J.; Panwar, J. Extracellular Biosynthesis and Characterization of Silver Nanoparticles using Aspergillus flavus NJP08: A Mechanism Perspective. Nanoscale 2011, 3 (2), 635−641. (13) Makarov, V.; Love, A.; Sinitsyna, O.; Makarova, S.; Yaminsky, I.; Taliansky, M.; Kalinina, N. “Green” Nanotechnologies: Synthesis of Metal Nanoparticles using Plants. Acta Naturae 2014, 6 (1), 35−44. (14) Narayanan, S.; Sathy, B. N.; Mony, U.; Koyakutty, M.; Nair, S. V.; Menon, D. Biocompatible Magnetite/Gold Nanohybrid Contrast Agents via Green Chemistry for MRI and CT Bioimaging. ACS Appl. Mater. Interfaces 2012, 4 (1), 251−260. (15) Aziz, N.; Faraz, M.; Pandey, R.; Shakir, M.; Fatma, T.; Varma, A.; Barman, I.; Prasad, R. Facile Algae-Derived Route to Biogenic Silver Nanoparticles: Synthesis, Antibacterial, and Photocatalytic Properties. Langmuir 2015, 31 (42), 11605−11612. (16) Mahfooz, S.; Singh, S. P.; Rakh, R.; Bhattacharya, A.; Mishra, N.; Singh, P. C.; Chauhan, P. S.; Nautiyal, C. S.; Mishra, A. A Comprehensive Characterization of Simple Sequence Repeats in the Sequenced Trichoderma Genomes Provides Valuable Resources for Marker Development. Front. Microbiol. 2016, 7, 575. (17) Mishra, A.; Kumari, M.; Pandey, S.; Chaudhry, V.; Gupta, K.; Nautiyal, C. Biocatalytic and Antimicrobial Activities of Gold Nanoparticles Synthesized by Trichoderma sp. Bioresour. Technol. 2014, 166, 235−242. (18) Kumari, M.; Pandey, S.; Giri, V. P.; Bhattacharya, A.; Shukla, R.; Mishra, A.; Nautiyal, C. S. Tailoring Shape and Size of Biogenic Silver Nanoparticles to Enhance Antimicrobial Efficacy Against MDR Bacteria. Microb. Pathog. 2016, DOI: 10.1016/j.micpath.2016.11.012. (19) Mukherjee, P. K.; Horwitz, B. A.; Kenerley, C. M. Secondary Metabolism in Trichoderma−A Genomic Perspective. Microbiology 2012, 158 (1), 35−45. (20) Kumari, M.; Mishra, A.; Pandey, S.; Singh, S. P.; Chaudhry, V.; Mudiam, M. K. R.; Shukla, S.; Kakkar, P.; Nautiyal, C. S. PhysicoChemical Condition Optimization During Biosynthesis Lead to

metabolites and proteins. Majority of the biosynthesized particles were spherical in shape and ranged from 10 to 20 nm in size. Coating of secondary metabolites and proteins on silver nanoparticles was confirmed by EDAX, XRD FT-IR and GC-MS. An attempt was made to get an insight into the mechanism of enhanced antibacterial activity. Membrane rupturing and loss of cellular integrity of pathogens by BSNP treatment itself demonstrates its high potential of antimicrobial activity in comparison with chemically synthesized silver nanoparticles against Gram-positive and Gram-negative pathogens. BSNP was able to penetrate bacterial membrane more easily as compared to CSNP. Enhanced internalization of BSNP in to the pathogenic bacteria led to increased ROS generation. Increased ROS generation stimulated many pathways related to membrane leakage and morphological alteration of bacterial cells. The intense damage caused by BSNP ultimately results in disruption of bacterial structure leading to their cell death. Synergistic effects of biosynthesized silver nanoparticles stabilized with the antimicrobial metabolites of T. viride certainly opens up new opportunities for pharmaceuticals and agriculture pharmaceuticals industries in an eco-friendly manner.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.6b15473. Further information regarding zeta size and potential of BSNP and CSNP, EDAX, XRD, and FT-IR of BSNP, growth inhibition of pathogens by cell free filtrate, BSNP, and CSNP (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: 91 522 2297987. Fax: +91 522 2205839. ORCID

Aradhana Mishra: 0000-0002-3288-2736 Author Contributions #

M.K. and S.S. contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Authors thank CSIR−Indian Institute of Toxicology Research for providing TEM and EDAX facility. This study was partially funded by network project of Council of Scientific and Industrial Research (CSIR) “Root SF BSC0204” and “BSC0112 NanoSHE”. MK thanks CSIR for awarding her Senior Research Fellowship (SRF).

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REFERENCES

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