Ehab B. El Domany1*, Tamer M. Essam2, Amr E. Ahmed1 and Ahmed A.Farghli3

1Biotechnology and life Science Department, Faculty of Postgraduate Studies for Advanced Science, Beni-Suef University, Egypt.
2Microbiology and Immunology Department, Faculty of Pharmacy, Cairo University, Kasr Al-Aini Street, Cairo 11562, Egypt.
3Material Science and Nanotechnology Department, Faculty of Postgraduate Studies for Advanced Science, Beni-Suef University, Egypt.

Abstract

Biosynthesis of silver nanoparticles was achieved using cell filtrate from submerged fermentation of Fusariumoxysporum. It was found that AgNO3 reduced to Ag nanoparticles when exposed to the cell filtrate and the colour of solution was dark brown with absorbance peak at 430 nm wavelength. TEM micrograph showed  spherical AgNPs with range 10-25 nm in dimension and was well dispersed. AgNPs show high stability in solution due to biological stabilizing and capping agents released from fungus, and have negative charge -25mv.Biosynthesized AgNPs have high potential antibacterial and antifungal activity, highest inhibitory zone was (27) mm against Candida albicans. The synergistic effect of AgNPs gave highest fold increase(10) against E. coli, followed by (5)fold againstStaphylococcus auras using Azithromycin and levofloxacin as standard antibiotics respectively.

Keywords: Biosynthesis, silver nanoparticles, antibacterial,synergic.

Introduction

Microbial synthesis of nanomaterial is grown fast due to their chemical, optical, electrochemical and electronicproperties.[1] Fabrication of metal nanoparticles using fungi is reliable, ecofriendly and low cost. The green synthesis of nanoparticles can be achieved via the selection of an ecofriendly solvent with environmentally accepted reducing and stabilizing agents[2].Biosynthesis of AgNPs has more  economic advantages than physico-chemical methods which need complex and hi-tech instrumentation facilities, harsh chemicals also nanoparticles for biomedical application should be characterized by lower toxicity and higher their safe usage and this is available in biosynthesis with biocompatible chemicals [3].Several researches have successfully synthesize AgNPs using fungi as reducing agentsfungus ‘‘Fusariumsemitectum’’ used for the extracellular synthesis of silver nanoparticles from silver nitrate solution[4].Silver nanoparticles (AgNPs) have receivedgreatattention due to their interesting and significant antimicrobial properties. Unlike commercial antibiotics, AgNPsreveal their effects via  more inhibitor mechanismnot in a single specific way. Combinations of mechanisms such as damage and change of cellular morphology, disturbingvital cellular enzymes and proteins, depressing the activity of respiratory chain enzymes and finally leading to cell apoptosis[5][6]

The fabricatedAgNPs werecharacterized by UV, TEM, FTIR and Zeta sizer&potential. Finally, the fabricated AgNPs were applied in the field of antibacterial and synergistic studies in comparison with standard antibiotics.

Material and methods

Chemicals and cultures
In the present study the chemicals used are Silver nitrate, nutrient broth , potato dextrose agar  purchased from Himedia (P) Ltd., Mumbai, as starting materials without further purification. Sterile milliQ water was used throughout the experiment. Microoganism used in the experiment are Fusariumoxysporumwas kindly provided by The Regional Center For Mycology and Biotechnology, Azhar University, CairoEgypt. All chemicals and media used were of analytical grade.

Synthesis of nanoparticles
For the synthesis of silver nanoparticles, the biomass of fungus F. oxprosum was prepared by growing the fungus aerobically in a liquid medium MYPG contain 0.3 gram (g.) malt extract, 0.3 g. yeast extract, 0.5 g. peptone and 1 g. glucose in 100 ml deionized water The flasks were inoculated and then incubated on orbital shaker at 25 ± 2 Co and agitated at 120 rpm for 96 h. cell filtrate was obtained by passing it through Whattman filter paper No.1. 150 ml AgNo3 1mM was added to 20 ml free cell filtrate of F. oxprosum and incubated for 48 hours at 30c with agitation 120 rpm.Separatelythe cell filtrate and AgNO3 solution were kept under the same conditions figure (fig.) 1.

Fig. 1. A- cell filtrate B- Cell filtrate with AgNO3 C- AgNO3 only

Table 1. Zone of Inhibition (mm) of AgNPs synthesized by F.oxysporumagainst selected bacterial pathogens

Bacterial pathogen Zone of inhibition (mm)
Mean ± SD
B. subtilis 11.48 ± 0.51
Staph. Aureus 14.04±0.48
E. coli 18.46 ± 0.42
Candida albicans 27.02±0.80

 Characterization of silver nanoparticles
The formation of AgNPs was observed by change in color from paleyellow to brown confirmed by using Uv- visible Spectrophotometer (Shimadzu) operated with 1 nm resolution. TEM images were obtained by JEOL-JEM 2100 (Japan) with an acceleration voltage of 200 KV ,analysis were prepared by coating aqueous AgNPs drops on carbon coated copper grid. Size and potential of AgNPs were measured using Zetasizer Nano-ZS90 (Malvern, UK) by applied diluted sample of AgNPs.Furthercharacterization involved Fourier Transform Infrared Spectroscopy (FTIR) (Perkin–Elmer) analysis ofdrop of silver nanoparticles by scanning the spectrum in the range 450–4,000 cm-1 at a resolution of 4 cm-1.

Antibacterial activity of AgNPs against  pathogenic bacteria
Antibacterial assay
The antibacterial activity of synthesized AgNPs was evaluated using agarwell diffusion method[7]. Pure cultures of selected human pathogenicbacteria were subcultured individually in nutrient broth for 12hrs at 37oC. A 20ml volume ofsterile Mueller Hinton Agar medium was poured into each petriplate and each bacterial strainwas swabbed uniformly into plates using sterile cotton swabs. Wells of 5 mm diameter weremade onto each bacterium inoculated agar plate using sterile gel puncture. 100μl of AgNPssuspension was introduced into the corresponding wells. The bactericidal activity wasdetermined by a clear inhibition zone around the sample loaded well after incubation of platesovernight at 37oC.

Table 2. Synergistic effect of antibiotics in combination with or without AgNPs against selected human bacterial pathogens

Increased zone size (mm) Zone of inhibition (mm)+ Ag Zone of inhibition (mm) disk only Antibiotics (μg/disc) Pathogens
2 30 28 Levofloxacin (5 µg) Bacillus subtilis
1 28 27 Azithromycin(15 µg)
3 29 26 Ciprofloxacin(5 µg)
2 20 18 Amoxicillin(25 µg)
5 30 25 Levofloxacin(5 µg) Staph. aurus
2 28 26 Azithromycin(15 µg)
4 29 25 Ciprofloxacin(5 µg)
3 27 24 Amoxicillin(25 µg)
2 37 35 Levofloxacin(5 µg) E.coli
10 35 25 Azithromycin(15 µg)
1 39 38 Ciprofloxacin(5 µg)
1 18 17 Amoxicillin(25 µg)
19 19 0 Levofloxacin(5 µg) Candida albican
18 18 0 Azithromycin(15 µg)
20 20 0 Ciprofloxacin(5 µg)
13 13 0 Amoxicillin(25 µg)

 

Synergistic effect of AgNPs
The synergistic effect of AgNPs was carried out by disc diffusionmethod. To determine the synergistic effect, four standard antibiotic discssuch as Amoxicillin, Ciprofloxacin, Azithromycinand levofloxacinwere impregnated individuallywith 100μl each of freshly prepared AgNPs and were placed onto the MuellerHilton Agarmedium inoculated with individual test organisms. Standard antibiotic discs alone were used as positive controls. These plates were incubated overnight at 37oC. Afterincubation, the result wasrecorded by measuring the inhibitory zone diameter (mm).

Result and discussion

 The color change of AgNO3 solution from pale yellow to dark brownish yellow indicated theformation of AgNPs. The color change is due to the excitation of surface plasmon vibration inthe NPs [8]. The activemolecules (proteins and Enzymes) present in the Fusariumoxysporum filtrate reduced thesilvermetal ions into AgNPs. The formation of AgNPs was confirmed by intense absorption peak at wavelength435 nm, which are typical absorption bands of spherical AgNPs due to their surface plasmon resonance (Fig. 2). Results were compatible withFusariumoxysporumcell filtrate produce AgNPs solution yielded a maximum absorbance at 436 nm[9].Similar absorption peaks wereobserved in AgNPs formationusing Fusariumspeciescell filtrate with a maximum absorption band at 420 nm[4,8].TEM technique was employed to visualize the size and shape of the silver nanoparticles formed. Fig. 3 shows single spherical silver nanoparticles in shape with range 10-25 nm in size and almost polydispersed. AgNPs showed moderate  stability with negatively charged -25mv fig. 4 due to proteins moiety attached to AgNPsas bio coating agents, also fig. 4shows good size distribution intensity of particles using zeta seizer measurements. Previous reports confirmed that the prepared nanoparticles with zeta potentialvalue greater than +25 mV or less than – 25 mV typically have high degree of stability[11].zeta potential value -12.02 mV for green AgNPs and -10.4 mV for chemical AgNPs[3].

Fig. 2. Absorbance of Biosynthesized AgNPs

Fig. 3. TEM micrograph showed different size of AgNPs with spherical shape

Fig. 4. Zeta sizer and zeta potential

Further FTIR show band at 1637 which can be attributed to carbonyl stretch of amides and thereby could be related to proteins that may be encapsulate[11] fig. 5.

Fig. 5. FTIR of biosynthesized silver nanoparticles

Antibacterial activity
The antibacterial activity of AgNPS was investigated against the human bacterial pathogens such asB.subtilis,S. aureus,E.coliand candida albicanand the result on the inhibitory zone (mm) is represented in Table 1. AgNPS gave thehighest zone of inhibition (27.02mm) against Candida albican, whereas the lowest zone of inhibition (11.48mm) was recordedagainst Bacillus subtilis fig 6. Similarly, aneffectiveantimicrobial activity against higher antibacterial activity against S. typhi than thanB. subtilis a using AgNPs[12] Antibacterial activity against  E. coli  and S. aureus showed good results showing maximum zone of inhibition of 17mm and 16 mm, respectively[13].

Fig. 6. A) Candida albicans (1 AgNPs, 2 cell filtrate) ; B)  B. subtilis (1 AgNPs , 2 Cell filtrate)

The result on synergistic effect of  biosynthesizedAgNPs  is given inTable 2. It revealed that the distinct difference was observed between the inhibitory zones by antibiotics with and without AgNPs,similarly Gold nanoparticles showed synergistic effect against different pathogenic bacteria [2].The enhanced zone of inhibition was observed and it was increased from 25 to 35 mm when the AgNPs were incorporated with Azithromycin antibiotics against E.coli.figure 7.Incontrast, inhibition zone was zero with all tested antibiotics against candida albicanthen show increase of inhibition zone with combination with AgNPs but still lower than AgNPs only Fig. 8.Ciprofloxacin was subsequently shown to be greater against Staph and bacillus than against E.coli.

Fig. 7. 1 Azithromycin with AgNPs 2. Azithromycin only against E.coli

Fig. 8. Shows 1- Antibiotic with AgNPs 2- Antibiotic only.

Conclusion

Biosynthesis of AgNPs has many advantages such as economic viability and easy scale up. Applications of nanoparticles in medical and other fields make this method potentially use for the large-scale synthesis of other inorganic nanomaterials.  Narrow size distribution and small nanosizeAgNPs also offer advantages for self-assembled monolayer formation and enhanced surface area. Silver colloidal solution is biologically well suited and has the potential Antibacterial activity also can be used with other pharmaceutical compound to enhance their activity.

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