Assay of Antibacterial and Antifungal Activity of Silver Nanoparticles Accompanying Different Impurities

No doubt that antimicrobial compounds such as antibiotics are the basic tools in clinical medicine. Increase in antimicrobial resistance may cause several human diseases. The potential of drugs and other agents can be improved by using nanoparticles. Wide use of antimicrobial compounds resulted in formation of resistance in microbes. There is demand to look for novel agents for therapy. Novel silver nanoparticles accompanied different impurities were assayed against Streptococcus pyogenes and Staphylococcus aureus (Gram-positive bacteria), Pseudomonas aeruginosa and Proteus mirabilis (Gram-negative bacteria). In addition to Aspergillus niger, A. terrus, Penicillium cluclauni and Rhizopus stolonofer fungi. Four Candida spp. were also assayed: C. krusei, C. famata, C. parapsilosis and C. utiliz. The method which applied in assaying these microorganisms was diffusion method. Dilution method was obtained for the tested nanoparticles to nd the minimum inhibitory concentration MIC. The annealing temperature of the tested nanoparticles affected on antifungal and antibacterial activities. It was found that the preparation of nanoparticles at room temperature gave higher activities against tested antimicrobial than that at high temperatures.


Introduction
Pathogenic bacteria that are antibiotic resistant cause great challenges in health worldwide (Mantravadi et al. 2019). Bacteria can resist old and newly discovered antibiotics through inherent and acquired mechanisms (Abusaiba and AL-Harmoosh 2020). Fluconazole was the primary therapeutic agent for treatment of fungal infections. Now it is only a fungistatic agent against Candida spp. where both inherent and acquired resistance has been reported (Berkow and Lockhart 2017). Resistance includes alteration in the target of the drug, increase in e us of the drug, and presence of different pathways for target sterol production (Jarvis 1995). The spread of drug-resistant microorganisms represents huge challenges in healthcare worldwide (Sharafutdinov et al. 2020). It was reported that Aspergillus sp had the lowest antifungal activity in comparison to caspofungin and triazoles (Lalitha et al. 2007). Generally, the preparation of nanoparticles has a great effect on the antimicrobial study. There are two methods to prepare different nanoparticles materials, solid state and wet methods. In the solid-state method, bulk nanoparticles are prepared; while in the wet method, nanoparticles are prepared (Nadaroglu et  The novelty of this work is the assaying of silver nanoparticles accompanying different impurities against different opportunistic fungal and bacterial spp. It is reported from previous studies of silver nanoparticles accompanying different impurities showed fascinating physical properties that can be used as alternative drug nanomaterials (El-Bassuony and Abdelsalam 2020 (2, 3)). Thus, the aim of the present work is based on the study of antimicrobial effect on a novel synthesized silver nanoparticle accompanied different impurities against opportunistic bacterial and fungal species.
All materials were mixed in distilled water; urea was added drowsily. The mixture was heated to 250 o C till and the samples were collected to be ground for 0.5 h. Finally, some samples were annealed at 400 o C and were ground also for half an hour. Thus, the prepared samples were Ag nanoparticles accompanied iron oxide impurities α, γ-Fe 2 O 3 (Ag-Fe), Ag nanoparticles accompanied iron and nickel oxide impurities α, γ-Fe 2 O 3 , NiO (Ag-Fe-Ni), Ag nanoparticles accompanied delafossite and chromite (Ag-D-C), Ag nanoparticles accompanied delafossite (Ag-D) as-prepared and at 400 o C.

Materials
Bacterial and fungal species were supplied via Microanalytical Center, Faculty of Science, Cairo University.

Identi cation and growth of micro-organisms
Fungi had grown on potato dextrose agar (PDA) media and incubated for seven days at 25 °C (Adwic, El-Nasr chemical Co. Egypt). The fungal species were then identi ed based on some characters such as: shape of conidia/spores, colony shape, colony pigmentation and presence of septa (Moubasher 1993;Maadon et al. 2018;Raper and Fennell 1965). Bacteria had grown on nutrient agar and blood agar plates. The colonies had been recognized biochemically and morphologically (Holt et al. 1994).

Antimicrobial study of silver nanoparticles accompanied different impurities
The antimicrobial activity of silver nanoparticles accompanied different impurities were tested against Aspergillus niger, A. terrus, Penicillium cluclauni and Rhizopus stolonofer fungal species. Also, the nanoparticles were tested against Candia spp.: C. famata, C. parapsilosis, C. krusei and C. utiliz, fungal species. The tested fungi were added to fresh PDA media before solidi cation at concentration 10 6 colony forming unit/ml (CFU/ml). Then, 100 mg/ml of the investigated nanoparticles was added into the surface of agar plates which were incubated for 5 days at 27 °C. The standard antifungal drug which used in the present work is 100 mg/ml of Fluconazole however 100 mg/ml of Ampicillin was used as a control (Saadabi et al. 2012).

The tested bacteria Staphylococcus aureus and Streptococcus pyogenes (Gram positive bacteria), Proteus mirabilis and
Pseudomonas aeruginosa (Gram-negative bacteria), were added at concentration 10 6 (CFU/ml) before solidi cation. Then, 100 mg/ml of silver nanoparticles accompanied different impurities was added into the surface of the agar plates which were incubated for 2 days at 35 °C.
Estimation of minimum inhibitory concentration MIC (minimum inhibitory concentration) was determined by Pershin method (Pershin 1971). The inhibitory effect of minimum concentration of silver nanoparticles accompanied different impurities on bacterial and fungal growth was obtained by Serial Dilution method using bifold dilution concentrations from 6.25, 12.5, 25, 50, and 100 mg/ml. The dimethyl sulfoxide (DMSO) solvent was used to dissolve the tested nanoparticles. The culture media for bacteria is Mueller-Hinton broth liquid medium however that for fungi is and Sabouraud liquid media. For bacteria the test inoculums are 5 × 10 4 CFU/ml bacteria per ml however for fungi the test inoculums are 10 3 CFU/ml fungal spores per ml. Then, the tested compounds were added onto agar surface after inoculation with the test organism with incubation of plates at 30 °C. Finally, the lowest concentration that showed no growth (MIC) was obtained after incubation o 2-7 days.

Statistical analysis
Statistical analysis was obtained using SPSS (Statistical Package of the Social Sciences) version 22. The homogeneity of different treatments was examined using Duncan's test. The signi cant results were at P < 0.05, whereas the insigni cant results were at P > 0.05. The reported data is (mean ± standard deviation (SD)) ( Tahir

Results
Assay of synthesized silver nanoparticles accompanied different impurities against fungal species Tables 1 & 2 showed the highest antifungal effect against tested fungal species while Tables 4 &5 showed  Assay of synthesized silver nanoparticles accompanied different impurities against bacterial species One can obtain from the results of antibacterial activity, that the sensitivity of Gram-positive bacteria to the investigated samples was greater than that of Gram-negative bacteria. Table 3 showed that Ag-Fe, followed by Ag-D and Ag-D-C, showed the highest antibacterial effect against the tested bacterial species and the lowest MIC values with respect to the remaining tested nanosamples that showed lesser antibacterial activity and high MIC values as shown in Table 6.

Structural properties
It is reported from previous studies in details the physical and magnetic properties of silver nanoparticles accompanied by different impurities (El-Bassuony AAH, Abdelsalam HK 2020(2, 3)). Table 7 showed the crystallite size and the particle size estimated from X-ray diffraction pattern (XRD), eld emission scanning electron microscopy (FESEM) and atomic force microscopy (AFM). These analyses emphasize the nanosize of the investigated samples. Moreover, the crystallite size increased by increasing the annealing temperature as shown from Table (7).

Discussion
The great demand from many researchers to nd novel drugs to overcome the microbial resistance problem that caused Nanoparticles can be prepared by many methods however ash technique gave very ne nanoparticles and low cost method which was chosen in preparation of the investigated samples. The annealing temperature is an important parameter that affect the particle size of the nanosamples. Thus, the more e cient annealing temperature is low temperature that gives ne nanoparticles which could be able to penetrate the membrane of the microbial cell and destroy the internal molecules causing There is another publication to study cytotoxicity assay with concentrations 6.25, 12.5, 25, 50, and 100 mg/ml. Furthermore, it is required to study the investigated samples in vivo using animal models to study the treatment of fungal and bacterial infections.
To summarize, in the present study, novel silver nanoparticles accompanied different impurities were synthesized and showed that Ag-D and Ag-D-C had high in vitro antifungal effect, while Ag-Fe was the most powerful compound against bacteria. The best activities was shown for compounds that synthesized at 30°C. Finally, Ag-D, Ag-D-C and Ag-Fe are promising compounds that could be used as an alternative drug in the future.

Declarations
Authors' contributions MAS and AAH conceived and designed research. MAS and HAK conducted experiments. AAH contributed new reagents. AAH and HAK analyzed data. MAS and AAH wrote the manuscript. All authors read and approved the manuscript.

Compliance with ethical standards
This article does not contain any studies with human participants or animals performed by the authors.

Con ict of interest
The authors declare that they have no con ict of interest.

Compliance with Ethical Standards
This article does not contain any studies with human participants or animals performed by any of the authors.

Data availability
The datasets generated during and/or analysed during the current study are available from the corresponding author on  Data is represented as mean ± standard deviation.
In the same raw, the values marked with the same superscript letter are similar (P>0.05), whereas those marked with different ones are signi cantly different (P<0.05). Data is represented as mean ± standard deviation.
In the same raw, the values marked with the same superscript letter are similar (P > 0.05), whereas those marked with different ones are signi cantly different (P < 0.05). Data is represented as mean ± standard deviation.
In the same raw, the values marked with the same superscript letter are similar (P > 0.05), whereas those marked with different ones are signi cantly different (P < 0.05).    Table 7 Values of Crystallite size from X-ray analysis (XRD), particle size estimated from eld emission scanning electron microscopy (FESEM) and particle size estimated from atomic force microscopy (AFM) of the synthesized silver nanoparticles accompanied different impurities  Figure 1 Representative photos showing the antimicrobial activity of the tested nanoparticles against opportunistic microorganisms