Open Access

Ehab B. El Domany1 , Tamer M. Essam2, Amr E. Ahmed1 and A.A. Farghali3

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.
J Pure Appl Microbiol. 2017;11(3):1441-1446
https://doi.org/10.22207/JPAM.11.3.27 | © The Author(s). 2017
Received: 02/08/2017 | Accepted: 18/09/2017 | Published: 30/09/2017
Abstract

Biosynthesis of silver nanoparticles was achieved using cell filtrate from submerged fermentation of Fusarium oxysporum. 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 against Staphylococcus 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 electronic properties1. 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 agents2. 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 chemicals3 .Several researches have successfully synthesize AgNPs using fungi as reducing agents fungus ‘‘Fusarium semitectum’’ used for the extracellular synthesis of silver nanoparticles from silver nitrate solution4. Silver nanoparticles (AgNPs) have received great attention due to their interesting and significant antimicrobial properties. Unlike commercial antibiotics, AgNPs reveal their effects via  more inhibitor mechanism not in a single specific way. Combinations of mechanisms such as damage and change of cellular morphology, disturbing  vital cellular enzymes and proteins, depressing the activity of respiratory chain enzymes and finally leading to cell apoptosis5,6

The fabricated AgNPs were characterized 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.

Materials 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 Fusarium oxysporum was kindly provided by The Regional Center For Mycology and Biotechnology, Azhar University, Cairo Egypt. 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, 0.3g. yeast extract, 0.5g. peptone and 1g. glucose in 100 ml deionized water The flasks were inoculated and then incubated on orbital shaker at 25 ± 2°C and agitated at 120rpm for 96h. cell filtrate was obtained by passing it through Whattman filter paper No. 1. 150ml AgNo3 1mM was added to 20ml free cell filtrate of F. oxprosum and incubated for 48 hours at 30°C with agitation 120rpm. Separately the cell filtrate and AgNO3 solution were kept under the same conditions figure (fig.) 1.

Characterization of silver nanoparticles
The formation of AgNPs was observed by change in color from pale yellow 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. Further characterization involved Fourier Transform Infrared Spectroscopy (FTIR) (Perkin–Elmer) analysis of drop 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 agar well diffusion method7. Pure cultures of selected human pathogenic bacteria were subcultured individually in nutrient broth for 12hrs at 37°C. A 20ml volume of sterile Mueller Hinton Agar medium was poured into each petriplate and each bacterial strain was swabbed uniformly into plates using sterile cotton swabs. Wells of 5mm diameter were made onto each bacterium inoculated agar plate using sterile gel puncture. 100µl of AgNPs suspension was introduced into the corresponding wells. The bactericidal activity was determined by a clear inhibition zone around the sample loaded well after incubation of plates over night at 37°C.

Synergistic effect of AgNPs
The synergistic effect of AgNPs was carried out by disc diffusion method. To determine the synergistic effect, four standard antibiotic discs such as Amoxicillin, Ciprofloxacin, Azithromycin and levofloxacin  were impregnated individually with 100µl each of freshly prepared AgNPs and were placed onto the Mueller Hilton Agar medium inoculated with individual test organisms. Standard antibiotic discs alone were used as positive controls. These plates were incubated overnight at 37°C. After incubation, the result was recorded by measuring the inhibitory zone diameter (mm).

RESULTS AND DISCUSSION

The color change of AgNO3 solution from pale yellow to dark brownish yellow indicated the formation of AgNPs. The color change is due to the excitation of surface plasmon vibration in the NPs8. The active molecules (proteins and Enzymes) present in the Fusarium oxysporum filtrate reduced the silver metal ions into AgNPs. The formation of AgNPs was confirmed by intense absorption peak at wavelength 435 nm, which are typical absorption bands of spherical AgNPs due to their surface plasmon resonance (Fig. 2). Results were compatible with  Fusarium oxysporum cell filtrate produce AgNPs solution  yielded a maximum absorbance at 436 nm9. Similar absorption peaks were observed in AgNPs formation using Fusarium species cell filtrate with a maximum absorption band at 420 nm4,8. 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 AgNPs as bio coating agents, also fig. 4 shows good size distribution intensity of particles using zeta seizer measurements. Previous reports confirmed that the prepared nanoparticles with zeta potential value greater than +25 mV or less than – 25 mV typically have high degree of stability11. zeta potential value -12.02 mV for green AgNPs and -10.4 mV for chemical AgNPs3.

Fig. 1. A- cell filtrate B- Cell filtrate with AgNO3 C- AgNO3 only
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 encapsulate11 fig. 5.

Fig. 5. FTIR of biosynthesized silver nanoparticles

Antibacterial activity
The antibacterial activity of AgNPS was investigated against the human bacterial pathogens such as B. subtilis, S. aureus, E.coli and candida albican and the result on the inhibitory zone (mm) is represented in Table 1. AgNPS  gave the highest zone of inhibition (27.02 mm) against Candida albican , whereas the lowest zone of inhibition (11.48mm) was recorded against Bacillus subtilis fig 6. Similarly, an effective antimicrobial activity against higher antibacterial activity against S. typhi than than B. subtilis a using AgNPs12 Antibacterial activity against E. coli and S. aureus showed good results showing maximum zone of inhibition of 17mm and 16 mm, respectively13.

Table (1):
Zone of Inhibition (mm) of AgNPs synthesized by F. oxysporum against 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

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

The result on synergistic effect of  biosynthesized AgNPs  is given in Table 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 bacteria2.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. In contrast, inhibition zone was zero with all tested antibiotics against candida albican then 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.
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)
Fig. 7. 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 nanosize AgNPs 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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© The Author(s) 2017. Open Access. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License which permits unrestricted use, sharing, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.

© The Author(s) 2017. Open Access. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License which permits unrestricted use, sharing, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.