Silver Nanoparticles (AgNPs) Biosynthesized by Aspergillus flavus KF946095; their Characterization and Antibacterial Activity

The antimicrobial agents of silver nanoparticles (AgNPs) have been applied a little while back in diverse therapeutic studies. In this analysis, AgNPs were biosynthesized using an ecologically welcomed and cost-effective simple of bio-reduction. An isolate of Aspergillus flavus KF946095 (A. flavus) was found to biosynthesize AgNPs; the size of AgNPs was (56nm) and detected by UV-Vis analysis at (400 nm). The reducing properties for biosynthesis of AgNPs are mainly due to the protein functional surface reactive groups detected by Fourier Transform Infrared spectroscopy (FTIR).Whereas, FTIR for AgNPs showed different peaks at 3994.5, 3201.6, 1801.4, 1643.2 and 1604.7 cm-1 that shared with the biosynthesize and stability of AgNPs as protein capping agents. Transmission Electron Microscope (TEM) confirmed the scattering of biosynthesized AgNPs within a sol with oval and round shapes. The antibiotic susceptibility test was studied for some pathogenic bacteria. Staphylococcus aureus DSM 1104 (S. aureus) appeared to be the more resistant strain; it resisted the action of 6 antibiotics out of 8 ones tested. MIC value of AgNPs was 20μg/mL and antibiotic ciprofloxacin was 30μg/mL. Mixture of MIC values or double MIC values distinctively inhibited the multidrug resistant (MDR) S.aureus.


INTRODUCTION
The existence of antibiotic resistant strains is increased recently and their inhibition by alternative agents are necessary (Goffeau, 2008; Enan et al., 2013;Osman et al., 2016; El-Gazzar., etal.,2020 Abdel-shafi, 2020; . S. aureus is a Gram-positive, coccidshaped, facultative anaerobic and it forms yellow colonies on routine agar medium and forms black colonies with halo zones after its growth on its specific medium Baired Parker agar. S. aureus is non-motile, non-spore former, ferments glucose and produces lactic acid; it shows both aand bblood hemolysis capability and is characterized by positive coagulase reaction (Colombari et al., 2007).
Previous studies (Osman et  From the Elders, silver and its compounds have been considered to be Functional Antimicrobial active factors (Silver, 2003; El-Gazzar and rabie, 2018; Ngozi et al., 2019; El-Gazzar and Enan 2020; El-Gazzar and Ismail, 2020; Samson et al., 2020). Nano-silver has received particular attention as a possible antimicrobial agent, owing in particular to recent advances in metal nanoparticles research (Melaiye et al., 2005). Silver nanoparticles have been widely subjected due to their physicochemical properties (Lok, 2006) and are recently applied as anti-bacterial factors in food storage and health industries. The anti-microbial activity of Ag-Nps has now been well recognized and they are definite to possess anti-inflammatory, anti-viral, anti-platelet and anti-fungal efficiency (Panacek et al., 2009).
The nanoparticles syntheses are characterized by an effective and a potent role in application as novel antimicrobial factors (El-Gazzar and Enan 2020). The newly nanoproducts have already applied against pathogenic bacteria (Kim et al., 2008;. So, the biosynthesis and functionalization of nanoparticles create the most potent antimicrobial products. Thus, this work was aimed to biosynthesize of AgNPs by A.flavus and investigates the antimicrobial effect of AgNps alone or in combination with antibiotics to inhibit MDR S.aureus.

MATERIALS AND METHODS The fungal strain used for biosynthesis of AgNPs
The fungal strain used in this study for biosynthesis of AgNPs was Aspergillus flavus KF946095. It was isolated from soil contaminated with wastes from Photographic Industries (El-Sharkia Governorate, 80 km North Cairo, Egypt) (El-Gazzar, 2015). It was stored in glass beads and subcultred into potato dextrose broth (Oxoid) (Abdel-Salam et al., 2001).

Biosynthesis of AgNPs
A. flavus KF946095 was isolated, identified and characterized to possess the capability to biosynthesize AgNPs by its extracellular enzyme to reduce AgNO 3 solution at 1mM to AgNPs according to El-Gazzar and Rabie, (2018). The color of AgNO 3 at the beginning of the experiment was colorless and the developed AgNPs were visually monitored from the intensity of yellow-brown color. The concentration and physical properties of the developed AgNPs were determined (El-Gazzar et al., 2020).

Characterization of the bio synthesized AgNPs
The prepared post-biotransformation fluid of bio synthesized AgNPs was detected with (T80+UV Flash spectrophotometer PG Instrument LTD), (

Antibacterial assay of AgNPs
Brain Heart infusion agar plates (DifcoTM, Maryland, USA) were prepared and seeded with log phase cells (10 5 CFU/mL) of S. aureus DSM 1104 as it was the most resistant organism to the antibiotics tested, then the sterile singly AgNPs were applied to discs (6mm diameter) which were added onto the above plates compared with controls discs that saturated with sterile distilled water. All samples were incubated at 37°C for 24-48 h. IZDs was determined (Aziz et al., 2014; El-Gazzar and Enan, 2020).

Determination of minimum inhibitory concentration (MIC) of AgNPs
The antibacterial activity of AgNPs was studied by different concentration of AgNPs discs and placed onto Muller Hinton agar (DifcoTM, Maryland, USA) that seeded previously with activity growing cells of S. aureus (10 5 CFU/mL). MIC was visually measured trough the minimum

The efficiency of Ciprofloxacin and AgNPs incorporation against bacteria
The antibiotic Ciprofloxacin that inhibited the S. aureus DSM 1104 was combined with MIC concentration of AgNPs. Sterile filter paper discs were impregnated either by the antibiotic only or by the AgNPs only and also by combinations them and tested for their antimicrobial activity against S. aureus as described above. Mixtures of AgNPs with Ciprofloxacin were prepared as follows: (30 µg/mL Ciprofloxacin + 20 µg/mL AgNPs) and (60 µg/mL Ciprofloxacin + 40 µg/mL AgNPs (Osman et al., 2016)

RESULTS
The preliminary investigation showed that the A.flavus KF946095 changed the color of medium into yellowish brown indicating on conversion of AgNO 3 into AgNPs as revealed from the necessary analysis used in further experiments.
For characterization whether the yellowish brown fluids contain AgNPs or no; this suspension was subjected to spectral scan UVvisible which had maximum absorption at 400 nm corresponding to surface Plasmon resonance indicating the formation of AgNPs (Fig. 1). The diameter of nano-product showed an average particle size at 56 nm that based on intensity distribution (Fig. 2). Moreover, TEM measurements showed well distribution of AgNPs without any agglomeration at 6.49 nm with oval and roundoutline (Fig. 3). The results of FTIR for AgNPs included different peaks at 1643.2 cm -1 , 1604.7cm -1 and 1801.4 cm -1 (Fig. 4). The band at 1643.2 cm -1 and 1604.7 cm -1 classified into the amide bands. Whereas, the band at 1643.2 cm -1 arises due to carbonyl stretch, unsaturated aldhyde, ketone and alkene group. While the band at 1604.7 cm-1 occurs because of N H stretching vibrations in the protein's amide linkages. At1801.4 cm -1 is assigned to c=c stretch alkenyl stretch from the proteins in the solution which is a useful tool for quantifying secondary structure in metal nanoparticles protein interaction. In addition, it shows the presence of 3201.6 and 3994.5 cm −1 bands. The band 3201.6 cm −1 is characteristic of amide II band also alchole / phenol O=H stretch. At 3994.5 cm −1, the band reflected the N=H stretch at the amide.
The results were extended to study the antibiotic sensitivity test of different pathogenic bacteria as given in Materials and Methods. Results are given in Table ( 1). MAR index of the indicator bacteria tested could be arranged in the following descending manner: 75%; 62.5%; 37.5%; 25% for S. aureus; Listeria monocytogenus; Pseudomonas aeruginosa and Escherichia coli, respectively (Table, 1). Since the antibiotic ciprofloxacin was the more effective antibiotic and S.aureus bacterium The experiments were investigated the possibility to inhibit the S.aureus strain that was resistant to 8 antibiotics tested by different concentrations of AgNPs Table (2). AgNPs inhibited the MDR S. aureus strain. The inhibition was dependant on concentration. Inhibitory activity against S. aureus was increased with higher concentrations of AgNPs. Minimum inhibitory concentration (MIC) of AgNPs was 20µg/mL. MIC of ciprofloxacin was 30µg/ disc.      The experiments were further extended to check whether mixtures of both the antibiotic ciprofloxacin (the more effective antibiotic) and AgNPs will give greater inhibitory activity against the MDR S. aureus or no. Mixtures of MIC values of both the antibiotic ciprofloxacin and AgNPs showed obvious and distinctive inhibition of MDR S. aureus more than that obtained by either ciprofloxacin or AgNPs alone. Double amounts of MIC values showed distinctive inhibition of MDR S. aureus also; indicating on synergism between ciprofloxacin and AgNPs (Table,3)

DISCUSSION
The detection of a newly research for suppressing and blocking the various impedance microscopic organisms to antibiotic is urgent (Enan et al., 2013;Enan et al., 2014;Abdel-Shafi et al., 2020). Nanotechnology introduce effectiveness ways in inhibition of pathogenic multidrug resistant bacteria (Gu et al., 2003; El-Gazzar and Ismail 2020).The emergence of nanotechnology in the last decade offers recent biotechnology in development of drugs based nanoparticles (El-Gazzar and Enan, 2020). From the obtained data, the absorbance of AgNPs detected at 400 nm, this is similar to latter published results in this respect (El-Gazzar and Rabie, 2018; El-Gazzar and Enan, 2020).
In addition, the size of such nanoparticles was 56nm. Latter published results showed the ability of fungal strains to biosynthesize nanoparticles (El-Gazzar and Ismail, 2020). The obtained single peak indicated that the quality of the biosynthesized nanoparticles is well suitable (Nakkala et al., 2017).Also, AgNPs was scattered with oval and round shapes. Moreover, the results confirmed the presence of different molecules which are responsible on synthesis with stabilization of AgNPs. This is in confirming with previous results in this respect (El-Gazzar et al., 2020).
FTIR measurements of the prepared nanoparticles were carried out to identify the possible biomolecules presented in nanoparticles solution, for determination the stabilizing mechanism for AgNPs. The FTIR results of AgNPs detected the presence of adverse biomolecules; this is coupled with latter published work (El-Gazzar et al., 2020).Thus, the existence of diverse biomolecules with nanoparticles increases its stability and ability in metal degradation (Dhoble and Kulkarni, 2016; El-Gazzar and Rabie, 2018).
The results employed herein approved that the S. aureus was multidrug resistant bacteria, such strain showed by many authors also to resist the action of different antibiotics authors also to resist the action of different antibiotics . Inhibition of such MDR S. aureus by AgNPs is of interest.
Antibacterial potential of biologically synthesized silver nanoparticles was assessed. AgNPs were found to be extremely potent with inhibition of MSR S. aureus observed at 20 μg/ ml concentration against all test organisms. In addition, to investigate if any synergism existed between the ciprofloxacin antibiotic and AgNPs in their antimicrobial action, mixtures containing MIC values or their doubles of them were tested. These combinations showed distinctive inhibition of MDR S. aureus than that obtained by either Ciprofloxacin or AgNPs.
Silver was characterized by its higher efficiency versa microorganisms. Although the exact underlying mechanisms are yet to be fully elucidated, other literature has indicated that AgNPs interested by the permeability of cell membrane and in turn interferes with the bacterial respiratory chain. In addition, their connection with reactive oxygen species generation (e.g., hydrogen peroxide) and resultant oxidative stress and cell damage (Aziz et al., 2014;Loo et al., 2015).
The findings of the present study have yet again highlighted the significance of the combination between antibiotics and AgNPs as potential antimicrobial agents (Prasad et al., 2016). The extract needs to be analyzed further for isolation and identification of active principles. Biogenic silver nanoparticles are not only extremely effective antimicrobial agents, as deduced from this study, but also economically. Although the AgNPs and antibiotic displayed significant antibacterial activity, a mixture containing both in much lower concentration that have the same efficiency, through a synergistic potency (Abdelshafi et al., 2019). Further work will be necessary to study the toxicity of AgNPs against living tissues and to study the mode of action of AgNPs alone and in combinations with antibiotics.

CONCLUSION
A. flavus KF946095 showed ability to biosynthesize AgNPs that were characterized by possible instrumental analysis herein. One strain of S. aureus DSM 1104 showed to resist the action of many antibiotics; it was inhibited by different AgNPs. The antibiotic ciprofloxacin and AgNPs in combinations showed distinctive inhibition of MDR S. aureus DSM 1104.

ACKNOWLEDGMENTS
Authors would like to thank Prof. Dr Gamal Enan Dean of Faculty of science, Zagazig University, Zagazig, Egypt for his revising the work and the isolates of bacteria.