EXTRACELLULAR BIOSYNTHESIS, OPTIMIZATION, CHARACTERIZATION AND ANTIMICROBIAL POTENTIAL OF ESCHERICHIA COLI D8 SILVER NANOPARTICLES

This study highlights the optimization of extracellular biosynthesis and antimicrobial efficiency of silver nanoparticles (AgNPs) using the crude metabolite of Escherichia coli D8 (MF06257) strain. The bacterial strain had been isolated from a sewage water stream located in Damietta City, Egypt. The optimum conditions for AgNPs production were at temperature 35°C, pH 7 and 1.5mM silver nitrate. The AgNPs biosynthesis was detected in culture filtrate within 1-2 minutes at room temperature (25±2°C) and sunlight. The characterization of AgNPs was studied by UV-Vis spectroscopy (maximum absorbance at 429 nm), X-ray diffraction (XRD) pattern (crystal planes were 110, 111, 200, 211, 220, and 311), transmission electron microscopy (TEM) (AgNPs were spherical in shape ranging from 6 to 17 nm), Fourier transforminfrared (FTIR) spectroscopy (the bands of symmetric and asymmetric amines were assigned at 3421.1 and 2962.13 cm, the stretching vibration band of aromatic and aliphatic (C-N) exist at 1392.35 and 1122.37 cm bands), Zeta potential analyser (AgNPs had a negative charge value; -33.6 mV) and size distribution by volume (the presence of capping agent enveloping the AgNPs with a mean size of 136.0294.3 nm). Nitrate reductase (NR) was assayed as an important partner in the optimized production (the rate of NR reached to 2.18 U/ml). The study demonstrated that AgNPs are potent inhibitors of Staphylococcus aureus, E. coli, Pseudomonas aeruginosa, Alternaria alternata, Fusarium oxysporum and Aspergillus flavus. The antimicrobial activity of AgNPs was studied by TEM. TEM micrographs showed an inhibition of S. aureus cell multiplication. In case of F. oxysporum, a reduction in the size of treated cells, formation of a mucilage matrix connecting the hyphal cells together, the appearance of a big vacuole, lipid droplets an a severe leakage of cytoplasmic contents were detected. AgNPs exhibited MIC values of 6.25μg/ml and 50 μg/ml against S. aureus and Candida albicans, respectively. In addition, AgNPs showed synergy effects by their combination with fluconazole that increased fold areas especially against A. niger, A. flavus and F. oxysporum.

Experimental procedure

Extracellular biosynthesis of silver nanoparticles
Escherichia coli D8 MF06257 was grown in nutrient broth (NB) medium and incubated at 37°C for 48 hours at 150 rpm. After an incubation period, the bacterial crude metabolites were collected throughout centrifuging at 5000 rpm for 20 minutes aseptically. Two hundred µL of bacterial crude metabolites were added to 20 ml of an autoclaved aqueous solution of 1mM silver nitrate (1% v/v) in triplicates. The negative control was prepared by adding 200 µL of the NB medium into 20 ml of the silver nitrate solution. All samples were incubated at 150 rpm for 5 days at 37°C in dark. After incubation, the appearance of brown colour was observed and measured spectrophotometrically as an indication of the production of AgNPs (Shahverdi et al., 2007). The reaction mixtures were measured in the range of 370 to 750 nm at a resolution of 1 nm using a UV-Vis spectrophotometer (Beckman DU-40) against control test tube as the blank (silver nitrate solution and nutrient broth medium) (Krishnaraj et al., 2012).

Characterization of biosynthesized silver nanoparticles
The X-ray diffraction (XRD) pattern of the AgNPs was recorded at 2θ values between 10° and 80° using a Cu X-ray tube at 40 kV and 30 mA with the X-ray diffractometer (model LabX XRD-6000, Shimadzu, Japan) at Nanotechnology Center, Kafrelsheikh University, Egypt).
The following characterizations of AgNPs were performed at TEM Unit at Mansoura University, Egypt. The shape and size of the optimized AgNPs were examined using TEM, a drop coating of nanocolloidal solution into carboncoated copper grid (Type G 200, 3.05 µM diameter, TAAP, USA) was prepared and kept overnight under vacuum desiccation before loading them onto a specimen holder. TEM micrographs of samples were taken using TEM instrument operated at an accelerating voltage of 200 kv using TEM (JEOL, JEM-2100, Japan). Size distribution by volume and charge of AgNPs were measured by Zeta Potential Analyser (Malvern Zetasizer Nano-ZS90, Malvern, UK). A colloidal solution was used in this instrument by withdrawing 1 ml of solution into the instrumental cuvette for measuring (Ruud et al., 1976;Hanaor et al., 2012). The AgNPs capping agents were analysed by Fourier Transform Infrared Spectroscopy (FTIR) spectrum. It was done for the freeze-dried powder of AgNPs using FT/IR-4100 type A in the diffuse reflectance mode at a resolution of 16 cm -1 at the range of 400-4000 cm -1 (Siddique et al., 2013).

Nitrate reductase assay
The assay of nitrate reductase (NR) was performed according to Harley (1993) depending on the reduction of nitrate into nitrite by nitrate reductase (NR). The NR activity was calculated pertain the amount of the produced nitrite during 60 minutes using 10 ml of sample. Production of one μmol nitrite/h/ml was defined as one unit of NR activity (U/ml).

Minimal inhibitory concentration (MIC)
The MIC values for S. aureus ATCC25923 and C. albicans ATCC10231 were measured using broth microdilution method according to the guidelines of the National Committee for Clinical Laboratory Standards (NCCLS) (Clinical Laboratory Standards, 2008;2017). A 0.5 McFarland standard of S. aureus ATCC25923 and C. albicans ATCC10231 were grown on Mueller-Hinton broth (MHB) and RPMI broth medium, respectively. Serial solutions of AgNPs, Penicillin G and Fluconazole (6.25-125 μg/ml in water) were tested. Mixtures were incubated at 37 °C and 35 °C for S. aureus ATCC25923 and C. albicans ATCC10231, respectively. After 48 hr, the growth turbidity was measured using a spectrophotometer against the growth control at 630 nm wavelength to determine n-values for each antimicrobial agent.

Transmission Electron Microscopy (TEM) of nanosilver treated microorganisms
The exponential-phase cultures of S. aureus ATCC25923 and F. oxysporum f. sp. lycopersici Fol4287 were subjected to silver nanocolloids (6.25, 50, 100 and 150 μg/ml and 50, 100 and 150 μg/ml, respectively) for 2 hours at 37°C and 30°C, respectively. Normal bacteria and fungi were included as controls. The cell cultures were centrifuged at 5000 rpm for 20 minutes, and then washed 3 times with distilled water. Fixative solution (2.5% glutaraldehyde in 0.1 M cacodylate buffer at pH 7) was added and left for 20 minutes at room temperature. The fixative was removed and then 0.1 M buffer was added for washing and postfixed with osmium tetroxide (2%, in the same buffer) for 90 minutes. The fixed cells were dehydrated using graded series of ethanol. The dehydrated cells were embedded in Epon-Araldite (1:1) mixture for 1 hour that polymerized at 65°C for 24 hours. The cells were cross section using an ultra-microtome (50 μm), doublestained with uranyl acetate and lead citrate and exposed to observation on carboncoated copper grids (Type G 200, 3.05 µM diameter, TAAP, U.S.A.) using TEM (JEOL JEM-2100, Japan).

Statistical analysis
The data were statistically analyzed using software system SPSS version 18. All values in the experiments were expressed as the mean ± standard deviation (SD) and were analyzed with one-way Analysis of Variance (ANOVA). The significant level was set at p<0.05.

Optimization of biosynthesized AgNPs
Escherichia coli D8 MF06257 biosynthesized AgNPs within 72 hours in dark conditions. The production of AgNPs was demonstrated by the peak at 429 nm in the UV-Vis spectra. Using 1% of bacterial supernatants and 1.5 mM concentration greatly enabled AgNPs synthesis ( Figure 1a). According to pH value, the brown colour appeared at pH (5-6) and its intensity was increased with the increase in pH value ( Figure 1b). Stable and monodispersed AgNPs were synthesized at pH7. It was found that 35oC was the optimal temperature for AgNPs synthesis (Figure 1c). The brown colour appeared within 72 hours ( Figure 1d) during incubation in dark conditions while biosynthesis occurred throughout a minute in case of the presence of solar irradiation ( Figure 1e).

Nitrate reductase activity
The activity of NR was measured and calculated in the E. coli D8  (Table 3) and the diameter of inhibition zone. Table 2 Antibacterial potential of AgNPs in comparison with benzylpenicillin (Penicillin G) as a standard drug in addition to the synergy action.
The less inhibition effects of Fluconazole (15, 17 and 18 mm) were against the pathogenic fungi F. oxysporum f. sp. lycopersici Fol4287, A. flavus Link ex Fries group and A. niger van Tiegh, respectively. However, the biosynthesized AgNPs revealed significant synergistic effects when companied with Fluconazole in addition to its antifungal activities showing higher fold areas (Table 4).

Minimal inhibitory concentration
The biocidal action of C. albicans ATCC10231 growth was significantly higher at the concentrations of AgNPs 50,100 and 125 μg/ml than lower concentrations. Fluconazole inhibited C. albicans ATCC10231 at 125 μg/ml (Figure 6a). Both AgNPs and Penicillin G showed the same MIC values (6.25 μg/ml) against S. aureus ATCC25923 in addition to complete inhibition at 25 μg/ml (Figure 6b).

TEM of nanosilver treated microorganisms
Antimicrobial activities of AgNPs against S. aureus ATCC25923 and F. oxysporum f. sp. lycopersici Fol4287 were easily demonstrated by TEM analysis as shown in Figures 7 and 8. TEM micrographs showed the morphological changes of the treated S. aureus ATCC25923 cells and inhibition of cell multiplication. The treated F. oxysporum f. sp. lycopersici Fol4287. TEM micrographs showed many changes, including reduced size of treated cells, the formation of a mucilage matrix connecting the hyphal cells together, the appearance of big vacuole and lipid droplets with severe leakage of cytoplasmic contents.

DISCUSSION
Bacteria are considered as an excellent source for the extracellular biosynthesis of nanomaterials. There is a bigwig whack to discover novel bacterial strains having motivated biological potential (Galvez et al., 2019). The crude metabolite of E. coli D8 MF06257 was used as a reducing agent, solvent typology and capping agent in the NPs extracellular biosynthesis. This type of synthesis is safe, renewable, simple, eco-friendly and cost-effective (Saifuddin et al., 2009). This biosynthesis was performed within 1-2 minutes at room temperature and sun light. The colour alteration into brown was due to the excitation of surface plasmon vibrations in the AgNPs (Baalousha et al., 2006). The reduction of silver ions may be resulting from the NADH dependent enzymes activity present in the crude metabolite and/or some redox agents such as sulfur-containing proteins (Krishnaraj et al., 2012). The present study reported the ability of E. coli D8 to produce NR (NADH dependent enzymes) with enzyme activity 2.18 µmol/hr/ml while it was about 0.152 µmol/ hr/ mL for B. subtilis as reported by Saifuddin et al. (2009). The deactivated NR of E. coli D8 metabolite (by heating) did not exhibit any synthesis of AgNPs in the dark condition, while it produced AgNPs in sunlight after 1-2 minutes indicating NR is not the only factor in the silver ion reduction. The aggregation of NPs is considered as a common problem which decreases their biological potential. The outer capping agents determine the size and morphology of NPs by preventing their aggregation (Duan et al., 2015). The biosynthesized AgNPs from E. coli D8 crude metabolite were uniform and monodispersed in size as well as stable for more than 6 months without aggregation at room temperature compared to Trichoderma longibrachiatum AgNPs that produced by Elamawi et al. (2018). The FTIR spectrum confirmed the presence of proteins associated with E. coli D8 AgNPs, which might act as a capping and stabilizing agent. Moreover, the negative charge of AgNPs might increase the repulsion force between particles which minimize their aggregation (Siddique et al., 2013). The biosynthesized AgNPs by the crude metabolite of E. coli D8 exhibited some potent inhibitory activities against all the pathogenic strains. The highest antibacterial activity of AgNPs by E. coli D8 crude metabolite was recorded against S. aureus ATCC25923 followed by E. coli ATCC25922, multi-drug resistant P. aeruginosa ATCC27853, K. pneumoniae ATCC33495 and B. cereus ATCC6633. Generally, the previous antimicrobial activities were more competitive to AgNPs produced by B. licheniformis (Gomaa, 2017) and matched with the antimicrobial activity of S. viridodiastaticus AgNPs (Mohamedin et al.,  2015). The biosynthesized AgNPs by the crude metabolite of E. coli D8 exhibited some potent inhibitory activities against all the pathogenic strains. Furthermore, the antifungal activity of AgNPs exhibited a great interest as E. coli D8 AgNPs showed a significant antifungal activity against C. albicans ATCC10231. Balakumaran et al. (2016) AgNPs produced by A. terreus inhibited the growth of C. albicans with a proximate activity. In addition, E. coli D8 AgNPs possessed a superior potent toxic effect against A. niger followed by A. fumigatus, A. flavus, A. alternata and F. oxysporum f. sp. lycopersici. Similarly, the biosynthesized AgNPs by Streptomyces sp. VITSTK7 showed anti-Aspergillus activity against A. niger, A. flavus and A. fumigatus with antifungal index in the range of 62-75% (Thenmozhi et al., 2013). The AgNPs of E. coli D8 showed a significant antifungal activity against F. oxysporum f. sp. lycopersici that matched with AgNPs, produced by Cryphonectria sp. (Dar et al., 2013). In contrast, the mycelial growth of plant pathogenic A. alternata was less inhibited by E. coli D8 AgNPs than AgNPs produced by A. solani F10 (Abdel-Hafez et al., 2016). E. coli D8 AgNPs increased the antifungal activity of the Fluconazole and increased diameters of ZOI. This synergistic effect was revealed increases in the fold areas especially against A. niger, A. flavus and F. oxysporum f. sp. lycopersici. Gajbhiye et al. (2009) reported that the combination between Fluconazole and AgNPs increased ZOI and fold areas against Phoma glomerata, P. herbarum, F. semitectum, Trichoderma sp. and C. albicans. It was thought that the synergistic effect may be due to formation of AgNP-Fluconazole complex by chelating that lead to more serious damage to microbe's cells (Fayaz et al., 2010). Antimicrobial potential depended on the dose and it increased by increasing the concentration. The MIC values for S. aureus ATCC25923 and C. albicans ATCC10231 were 6.25 μg/ml and 50 μg/ml, respectively.  al., 2013). Both S. aureus ATCC25923 and F. oxysporum f. sp. lycopersici Fol4287 changed in response to their treatment with AgNPs which were easily trapped and absorbed through cell membranes. The changes included inhibition of S. aureus ATCC25923 multiplication in addition to having a low amount of DNA. The untreated hyphal cells of F. oxysporum f. sp. lycopersici Fol4287 showed a normal cell wall, compact cytoplasm, cell membrane and small vacuole. On the other hand, many changes were observed after the treatment by AgNPs such as the formation of a mucilage matrix connecting the hyphal cells together, the appearance of big vacuole and lipid droplets. The accumulating of AgNPs in cytoplasmic membrane and cytoplasm may be the main factor in the major morphological changes in addition to the accumulation in the cell nucleus (Abdel-Hafez et al.,  2016). This accumulation may be indicated to the interaction of AgNPs with DNA (Vahdati and Sadeghi, 2013). In addition, the smallest AgNPs can penetrate the cell membranes and interact with their proteins (including thiol groups in enzymes) leading to blocking, inactivation and cell death (Radzig et al., 2013).

CONCLUSIONS
Escherichia coli D8 (MF062579) crude metabolite was able to synthesize AgNPs within 1-2 minutes in a green and cost-effective method. The presence of protein was confirmed and could be acted as stabilizing and capping agents. This method provides AgNPs possessing competitive size, shape and antimicrobial action beside to synergy potential with Fluconazole against A. niger van Tiegh, A. flavus Link ex Fries group and F. oxysporum f. sp. lycopersici Fol4287. Thusly, E. coli could be developed as a nano-biofactory against pathogenic microbes having severe damage effects on their DNA structure.