Phytosynthesized of silver nanoparticles; antimicrobial, antibiofilm activities against E.coli pathogenic isolated from Urinary Tract Infection

The most common cause of urinary tract infections (UTI) is uropathogenic Escherichia coli , which is often resistant to antibiotics. E. coli can form biofilms on urinary catheters. The biofilm protects E. coli against various factors. In this study, silver nanoparticles (AgNPS) were synthesized using Trifolium pratense L. extract as a reducing agent. The AgNPs were characterized by visible UV spectroscopy, a diffraction pattern (XRD), transmission electron microscopy (SEM), and energy dispersion spectroscopy (EDX). The AgNPs had a spherical shape with an average particle of 19 nm. Biological properties were also evaluated using biofilm inhibition, anticancer, antimicrobial activity. The brine shrimp lethality assay was applied to evaluate the anticancer activity of the nanoparticles. The silver nanoparticles with LC 50 (1.3 μg/ml) had the highest cytotoxicity activity. The antimicrobial activity of nanoparticles was evaluated by the agar diffusion method, minimum inhibitory concentration, and minimum bactericidal concentration in the range of 1.00 to 0.0312 and 2.00 to 0.0312, respectively. The nanoparticles exhibited a high antimicrobial effect against human E.coli pathogenic strains isolated from Urinary Tract Infection. The effect of biofilm inhibition on antibiotic resistant clinical strains by silver nanoparticles showed that silver nanoparticles inhibited biofilm between 21.12 to 97.10%.

Catalytic, electronic, magnetic, optical, and antimicrobial properties of metal nanoparticles lead them to be used in various fields, including chemistry, energy, and medicine (Anandalakshmi, Venugobal, & Ramasamy, 2016).
The properties of nanoparticles depend on their size, morphology and distribution. Nanoparticles such as silver, gold, zinc oxide, platinum have medical and pharmaceutical applications. These nanoparticles can also be used in products such as toothpaste and cosmetics (Hashoosh, Fadhil, & Al-Ani, 2014).
Physical and chemical methods are utilized to synthesize and stabilize nanoparticles (Sharma, Yngard, & Lin, 2009). These methods include solution reduction, photochemical reactions in reverse micelles, electrochemical reduction, heat evaporation, and radiation assisting techniques. Physical and chemical methods have generally been applied successfully in synthesizing nanomaterials in large quantities over a short period of time (Allafchian, Mirahmadi-Zare, Jalali, Hashemi, & Vahabi, 2016).
However, these methods successfully produce nanoparticles; they are harmful to human health and the environment due to hazardous and toxic chemicals (Dhand et al., 2016). An environmentally friendly and costeffective method is used to produce nanoparticles (Behravan et al., 2019). The antimicrobial activity of silver nanoparticles is confirmed against a wide range of microorganisms. Also, recent studies showed that silver nanoparticles are antimicrobial agents. However, Sondi (Dixon, 2004).
The objective of this study is to synthesize AgNPs using T. pratense. Also, the biological activity of the nanoparticles was performed using anticancer, antibiofilm, and antimicrobial activity against uropathogenic Escherichia coli isolated from urinary tract infections. Furthermore, the silver nanoparticles were characterized by UV-Visible, XRD, FE-SEM, and EDX methods.

Collection, identification, and extraction
Trifolium pratense flowers were collected in spring near Yasuj, Kohgiluyeh, and BoyerAhmad Province, Iran. The flowers are thoroughly washed with two litres of distilled water and stored at room temperature for 2 days, then split into small pieces. To prepare the aqueous extract, 10 g T. pratense was placed in 100 ml sterile distilled water (ratio: 1:10) for 20 min at 50 °C. The extract was isolated with Whitman filter paper and kept for further processing at 4 °C.

Characteristics of silver nanoparticles
The plant extract was added to 1mM of AgNO 3 solution and incubated at 60 °C. Also, AgNO 3 solution was incubated as a control. The synthesis of silver nanoparticles was confirmed using colour change to brown. To evaluate the synthesis of Ag nanoparticles, the absorbance was measured using a Schimadzu (Model No. UV 1800) spectrophotometer in the range of 300 to 800 nm at different times.
Moreover, the Fourier-transform infrared spectroscopy (FT-IR) spectra of silver nanoparticles were measured using FTIR spectrometer (Brucker, Germany) with a KBr bullet in the range of 4000-400 cm 1 . The crystalline silver nanoparticles were determined by X-ray diffraction (XRD) (Panalytical, Netherlands). Furthermore, the size of the nanoparticles was evaluated by the Debye-Scherrer formula (Ajitha, B., Reddy, Y. A. K., & Reddy, P. S. (2014)). Also, the energy dispersive Xray (EDX) and scanning electron microscope (SEM) (Tescan, Czech) was performed to investigate the morphology and the chemical composition of the nanoparticales.

Antibacterial activity
The antimicrobial effect of the nanoparticles was measured according to the protocol of Clinical and Laboratory Standard Institute (Clinical and Laboratory Standards Institute, (2020)). The antibacterial effect of the AgNps was determined against positive and negative bacteria by the agar diffusion method.
Afterwards, 10 µl of the nanoparticle solution (30 mg/ml) was inserted into the well. The plates were incubated at 37 °C for 24 h. The antibacterial activity was evaluated by measuring the diameter of inhibition zones.

Determination of minimum inhibition concentration
In this method, the minimum inhibition concentration (MIC) was calculated for the susceptible microorganisms to the nanoparticles. The MIC value was evaluated using the microdilution method. For this purpose, 95 µl of TSB medium, 5µl bacterial suspension adjusted 0.5 McFarland, and 100 µl of different concentrations silver nanoparticles (0.03125, 0.0625, 0.025, 0.25, 0.5). (0, 1 and 2 mg/ml) were added to each well. Furthermore, 195 µl of culture medium and 5 µl of the suspension were used as controls. The microplate was then incubated at 37 °C for 24 h. Microbial growth was characterized by the presence of turbidity at the bottom of the well.
To measure lethality, after 24 h, 5μl of each clean-well was inoculated on nutrient agar medium and incubated at 37 °C for 24 h. The concentration that did not grow after 24 h resulted in the killing of the bacteria, and the lowest concentration was considered the minimum bactericidal concentration.

Biofilm inhibition assay
Sixteen human pathogenic E.coli strains were isolated and collected from suspected infected Urinary tract infection (UTI) patients. According to microplate biofilm assay, the antibiofilm effect of the silver nanoparticles was evaluated against E.coli strains.
At first, 100 μl of the nanoparticles (2mg/ml) and 100 µl of each of the diluted strains were added to each well. Moreover, 200 µl of TSB and the bacteria suspensions were negative and positive control, respectively. The microplate was incubated for 24 h at 37 °C. The 1% crystal violet was then added to each well, and after 10 min, the crystal violet was washed with distilled water and allowed to dry. Finally, 200μl of acetic acid was added to each well, and after 15 min, the optical density (OD) was measured using ELISA at 570 nm. The following formula calculated the percentage of biofilm inhibition of the samples (Stepanović et al., 2007).
In this equation, C is equal to the mean biofilm absorbance, the mean absorbance of well containing the sample affected. B is the mean absorbance of the wells containing the medium (control). It was also repeated three times to ensure the test.

Evaluation of anticancer activity
The cytotoxicity activity of biosynthesized AgNPs was investigated using Brine Shrimp Lethality Assay (BSLA).
Artemia salina eggs were grown in artificial seawater (pH 9) for 48 h. The different concentrations (0, 10, 100, 300, 500, 700, and 1000 µg) of biosynthesized Ag NPs were added to the vial, including 5 ml seawater and ten brine shrimp larvae, and incubated at 25 °C for 24h. Brine shrimp death was observed at regular intervals. Vincristine sulfate (VS) was applied as a positive control. These tests were repeated three times. The lethality percentage was recorded according to formula (

Characterization of nanoparticles
The formation of extracellular was observed with a change color from yellowish to deep brown due to the excitation of the localized surface plasmon vibrations of the nanoparticles.
The changing color from yellowish to brown confirmed the synthesis of the nanoparticles.

FTIR
Comparing the spectral pattern of silver nanoparticles and the extract showed that nanoparticles contain compounds in the extract. They are usually formed as a layer around the nanoparticles and can play a role in the stability of nanoparticles. The results showed that peaks in regions 3404 cm-1 determined OH bond in phenols and alcohols. Moreover, the absorption band indicated in region 2925 cm -1 corresponds to CH stretching vibration in alkyls (methylene group), and 1607 cm -1 corresponding to NH bonds in first amines. Furthermore, the band at 1404 cm -1 corresponding OH stretching indicates that phenols. Whereas the absorption band determined in region 1073 cm -1 correspond to the CO stretching in the first alcohols. Phenolic compounds present in the plant are among the major contributors to reducing silver ions and the synthesis of nanoparticles.

SEM analysis
The surface morphology and structure of the synthesized silver nanoparticles were characterized using SEM. Figure 4 exhibited that the silver nanoparticle was an almost spherical shape with a range between 19to 46 nm and an average size of 34.42 nm. The results of the size distribution analysis of silver nanoparticles are shown in (Figure. 3).

EDX
The EDX result confirmed the present silver as a major element. Moreover, the present O, C, and N were determined in the EDX spectrum (Figure3). The presence of biomaterial is one of the advantages of nanoparticles synthesized using plant extracts compared to chemical methods.

XRD
X-ray diffraction is used to study the structure of crystalline materials. The spectral region results can obtain information on the structure, material, and quantities of the elements (Cullity & Stock, 2001). X-ray diffraction analysis was used to investigate and study the synthesized silver nanoparticles (Figure3). Where β is the width of the peaks at half the maximum height, λ is the X-ray wavelength equal to 54.1 nm. Moreover, ϴ is the angle between the reflected beam and the radiation, and D is the crystal size.

Antimicrobial activity
The antimicrobial effect of the silver nanoparticles, the extract and composite of extract and silver nanoparticles were measured by the present or absent zone of the inhibition, and the results are shown in Table1. Table1. Antimicrobial activity of silver nanoparticles and the plant extract.  The results showed that the nanoparticles had an antimicrobial effect. In contrast, the extract shows no antimicrobial activity.
The nanoparticles showed a zone of inhibition against B. subtilis, S. epidermidis and P. aeruginosa (9 mm). Moreover, S.paratyphi A serotype and exhibited the inhibition zone (10 mm), and the zone of S. aureus was 7 mm.
The composite of extract and silver nanoparticles has no antimicrobial effect except for two strains: B. subtilis (9 mm) and S. epidermidis (7 mm).

Biofilm Inhibition
The antibacterial effect of the silver nanoparticles was evaluated against sixteen E.coli strains isolated from patients with urinary tract infections. The results show that the MIC and MBC of silver nanoparticles range from 1.00 to 0.0312 and 2.00 to 0.0312, respectively ( Table 2).
The biofilm formation ability was measured for each assay as a control. The percentage of biofilm inhibition of the silver nanoparticles was between 21.12 to 97.10%. The nanoparticles exhibited high ability antibiofilm against E. coli 579, E. coli 4701, E. coli 228, E. coli 726, E. coli 4828, E. coli 885, and E. coli 3059. Furthermore, the lowest ability antibiofilm was against E. coli 5149.

Cytotoxicity
The anticancer activity of the silver nanoparticles and the percentage of dead larvae were determined at different concentrations (μg/ml).
The previous study determined that LC 50 (0.5-1 mg/ml) was weak the toxicity against A. salina. Moreover, The LC 50 between 0 and 0.1 mg/ml exhibited high toxicity and 0.1-0.5 mg/ml moderate cytotoxicity. Additionally, LC 50 above 1 mg/ml indicated the lack of toxicity against A. Our study showed that the silver nanoparticles with LC 50 1.3 μg/ml had high cytotoxicity and anticancer properties. Furthermore, the LC 50 of vincristine sulfate was 0.751 μg/ml as a positive control. Based on these results, T. pratense L. extract showed high cytotoxicity (Figure.

Conclusion
For the first time in the present study, silver nanoparticles were synthesized using the T. pratense. The antimicrobial and antibiofilm activity of the nanoparticles were determined. Additionally, the formation of silver nanoparticles was confirmed using UV-Vis, XRD, SEM, EDX, and FTIR. However, it can be concluded that this study reports a novel, rapid, economical, and environmentally friendly procedure for the production of silver nanoparticles.
Based on results, the nanoparticles have anticancer, antimicrobial, and antibiofilm activity against UTI. Silver nanoparticles may play a role in neutralizing cell adhesives, thus preventing biofilm formation. Bacterial biofilm is highly resistant to antibiotics. Therefore, silver nanoparticles may play a major role in biofilm formation on urinary catheters. Green synthesis of silver nanoparticles using Atrocarpus altilis leaf extract and