Synthesis and characterization of silver nanoparticles by "Rhizopus stolonifer" and its activity against multidrug resistant "Escherichia coli" and "Staphylococcus aureus"

Abstract

This study reports the extracellular synthesis of silver nanoparticles by Rhizopus stolonifer and its efficacy against multidrug resistant ( MDR) E.coli and S.aureus isolated from Khwaja Bande Nawas Hospital, Gulbarga, Karnataka. Synthesis of silver nanoparticles (AgNPs) was carried out by using fungal filtrate of R.stolonifer and an aqueous solution of AgNO3. The characterization of AgNPs was made by UV-Visible absorption Spectroscope, Scanning Electron Microscope and Energy Dispersed Spectroscope (SEM-EDS), Transmission Electron Microscope (TEM), Fourier Transform Infrared (FTIR) spectroscopy and Atomic Force Microscope (AFM). TEM micrograph revealed the formation of spherical nanoparticles with size ranging between 3 to 20 nm. Atomic force microscopy gives the three dimensional structure of the particles. The presence of proteins was detected by FTIR spectroscopy. Three dimensional structure of AgNPs was studied by AFM. AgNPs produced by R.stolonifer gave good antibacterial activity against clinical isolates which were multidrug resistant. Here we report the efficacy of mycogenic metal nanosilver against MDR strains which is difficult through conventional chemotherapy.

Keywords: R. stolonifer , silver nanoparticles, MDR-strains.

Nanotechnology is the ability to observe, measure, manipulate and manufacture things at the nanometre scale. A nanometre (nm) is a unit of length 10-9, or a distance of one-billionth of a meter. That’s very small. At this scale you are talking about the size of the atoms and molecules. Recently, scientist has endeavoured the use of microorganisms as possible eco-friendly nano factories for the synthesis of metallic nanoparticles [1]. An important aspect of nanotechnology is the development of toxicity-free synthesis of metal nanoparticles which is a great challenge. Where as chemical synthesis of nanoparticles have adverse effect due to the absorbance of toxic chemicals on the surface. Green synthesis provides advancements over chemical and physical methods as it is environment friendly, cost effective, easily scaled up for large scale synthesis and biological method does not require high pressure, energy and toxic chemicals.

New multidrug resistant strains of bacteria have become a serious problem in public health. The emerging resistances in bacteria and high cost of advanced antimicrobial drugs have encouraged researchers to search for effective and economically viable broadly applicable drugs [2]. It has been known for long time that silver ions are highly toxic to a wide range of bacteria and silver based compounds have been used extensively in bactericidal applications. Silver has one advantage of having broad antimicrobial activities against gram negative and gram positive bacteria. AgNPs are the most effective preparation of silver because of the high surface/volume fraction resulting in a large proportion of silver atoms in direct contact with their environment [2]. It can be expected that the high specific surface area and high fraction of surface atoms of AgNPs will lead to high antimicrobial activity compared to bulk Ag metal [3]. The purpose of this study was to examine the antibacterial activity of silver nanoparticles against multidrug resistant S.aureus and E.coli.

The objective of the work is to synthesize silver nanoparticles from R. stolonifer and to study the antibacterial activity of the biosynthesized silver nanoparticles on gram-positive and gram-negative strains isolated from Khwaja Bande Nawas hospital, Gulbarga. The MDR-strains of E.coli, and S.aureu were selected for antibacterial study with the SNPs produced by R.stolonifer.

2. Materials and Methods

2.1. Biosynthesis of silver nanoparticles

Fungal isolates from soil were inoculated in Malt Glucose Yeast Peptone (MGYP) broth [4] containing yeast extract and malt extract-0.3% each, glucose-1%, peptone-0.5%, at 40oC, in shaking condition (180 rpm) [4]. After incubation of 72h the biomass was filtered and then extensively washed with distilled water to remove the medium components. This biomass was taken into flasks containing 100 ml distilled water and were incubated at the aforesaid condition. After 72h the biomass was filtered again, (Whatman filter paper No.1) the fungal filtrate was used further. Aqueous solution of AgNO3 (1mM AgNO3 of final concentration) was mixed with fungal filtrate and the flasks were agitated at 40oC. periodically, aliquots of only those isolates which showed colour change from yellow to brown were subjected for UV-Visible absorption spectrophotometric study. Control (without silver ions) was also run along with the experimental flasks.

2.2. Characterization of silver nanoparticles

Synthesis of AgNPs was characterized by UV-Visible absorption Spectrophotometer ( T90/T90+ double-beam) with a resolution of 1 nm, which is one of the important technique to verify the formation of metal nanoparticles provided surface plasmon resonance exists for the metal [5] . To detect silver nanoparticle the absorption range is 400 to 450 nm [6]. This surface Plasmon resonance is caused by the coherent oscillation of the free conduction electrons induced by light. The confirmation for the synthesis of elemental silver was made by energy dispersed spectroscope. Transmission electron micrograph pattern were recorded on a carbon-coated copper grid on a Hitachi-H-7500 machine, sample preserved for over 6 months, synthesized by treating silver nitrate solution with cell free filtrate of R.stolonifer . The interaction between protein and AgNPs was analysed by Fourier transform-infrared spectroscope (JASCO FT/IR-3500). Three dimensional structures of biosynthesized silver nanoparticles were observed by atomic force microscopy.

2.3. Source of microorganisms

Two MDR-strains of E.coli and S.aureus from Khwaja Bande Nawas Hospital were used to study the antibacterial efficacy of silver nanoparticles.

2.4 Analysis of the Antibacterial activity of silver nanoparticles

The effect of silver nanoparticles on gram-positive and gram-negative bacteria was investigated by culturing the organisms on Luria-Bertani (LB) agar (106 colony forming units (CFU) of each strain per plate) supplemented with nanoparticles of 0.5, 1, 1.5, 2, 2.5µg/ml. Plates without silver nanoparticles were used as controls. Plates were incubated for 24h at 37oC the number of colonies was counted. The counts on three plates corresponding to a particular sample were averaged.

To examine the MIC of AgNPs and the growth curve of E.coli and S.aureus to AgNPs, different concentration of nanosilver 0.5, 1, 1.5, 2, 2.5 µg/ml was added in LB medium. Each bacterium culture ( S.aureus and E.coli ) was controlled at 105-106 CFU/ml and incubated at 37oC. To establish the antibacterial activity of nanosilver on bacterial growth, the MIC of AgNPs was determined by optical density of the bacterial culture solution containing different concentrations of nanoparticles after 24h.

3. Result

R. stolonifer (Fig.1) was used for the synthesis of silver nanoparticles from aqueous solution of AgNO3. The colour change of the fungal filtrate was noted by visual observation. The appearance of brown colour solution clearly indicates the formation of silver nanoparticles [7, 8]. A series of typical UV-Visible absorption spectra of the reaction solution were recorded at every 24h can be observed in Fig.2. All the spectra exhibit an intense peak at 422 nm corresponding to the surface plasmon resonance frequency of silver nanoparticles. This event clearly indicates that the reduction of the ions occur extracellularly through reducing agents released in to the solution by fungi. Scanning electron microscope clearly shows the biosynthesis of well dispersed nanosilver particles by R.stolonifer. EDS analysis gives the additional evidence for the reduction of silver nanoparticles to elemental silver. The optical absorption peak is seen approximately at 3kev. A representative TEM micrograph shows the AgNPs size ranges between 5 nm to 30 nm. FT-IR spectrum shows the bands at 1633(3) and 1554(4), 1423(5) cm-1. AFM gives clear three dimensional picture of the nanoparticles, the height and width of the particle is measured (5 nm) using the software. Silver nanoparticles at the concentration of 20µg/mL and 25µg/mL were effective against E.coli and S.aureus respectively.

4. Discussion

4.1. UV-Visible absorption Spectroscopy

UV-Visible absorption spectroscopy is one of the most widely used technique for structural characterization of silver nanoparticles. The colour change was caused by the surface plasmon resonance of silver nanoparticles in the visible region [9]. Silver nanoparticles are known to exhibit size and shape dependent surface plasmon resonance bands which are characterized by UV-Visible absorption spectroscopy [10]. Silver nanoparticles showed maximum absorbance at 422 nm, implying that the bio reduction of the silver nitrate has taken place following incubation of the AgNO3 solution in the presence of cell-free extract. Our results are correlating with the reports of Sadowski, and Maliszwaska (2009) with the fungus Penicillium . It is reported that the absorption spectrum of spherical silver nanoparticles presents a maximum between 420 nm and 450 nm [6].

4.2. SEM-EDS

Scanning electron microscope clearly shows the biosynthesis of well dispersed nanosilver particles (Fig. 3) by R.stolonifer . EDS analysis gives the additional evidence for the reduction of silver nanoparticles to elemental silver. The optical absorption peak is seen approximately at 3kev, which is typical for the absorption of metallic silver nanocrystals due to surface plasmon resonance, which confirms the presence of nanocrystalline elemental silver. Spectrum shows strong silver signal along with weak oxygen and carbon peak, which may be originate from the biomolecules that are bound to the surface of nanosilver particles can be seen in Fig.4.

4.3. Transmission electron microscopy

A representative TEM micrograph recorded from drop coated film of a silver nanoparticles sample preserved for over 6 months, this has been deliberately done to study the effect of ageing on the size of the particles. The AgNPs are spherical in shape. All the particles are well separated and no agglomeration was noticed can be observed in Fig.5. Biosynthesized AgNPs size ranges between 5 nm to30 nm. The process of growing silver nanoparticles comprises of two key steps: (a) bioreduction of AgNO3 to produced silver nanoparticles and (b) stabilization and/or encapsulation of the same by suitable capping agents [11]. It is suggest that the biological molecules could possibly perform the function for the stabilization of the AgNPs. Silver nanoparticles synthesized by this route are fairly stable even after prolonged storage. This may be concluded that there is not much agglomeration of the AgNPs even after preserving the colloidal solution for extended periods. Fig.6 clearly represents the stability of AgNPs even after 6 months of storage.

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