Nova Biotechnol Chim (2022) 21(1): e1023 DOI: 10.36547/nbc.1023 1 Nova Biotechnologica et Chimica Ag/SiO2 nanocomposite mediated by Escherichia coli D8 and their antimicrobial potential Mohamed M. El-Zahed, M. I. Abou-Dobara, Ahmed K.A. El-Sayed, Zakaria A. M. Baka Department of Botany and Microbiology, Faculty of Science, Damietta University, New Damietta, Egypt  Corresponding author: mohamed.marzouq91@du.edu.eg Article info Article history: Received: 20th June 2021 Accepted: 8th November 2021 Keywords: Antimicrobial Biosynthesis Escherichia coli Nanocomposite Silica Silver Abstract Silica (SiO2) has a fundamental role in the recuperation of plants in response to environmental stresses, besides the induction of resistance against plant diseases. Silver nanoparticles (AgNPs) have a superior antimicrobial activity. The combination between SiO2 and AgNPs is a promising approach due to their antimicrobial activity, biological activity, low toxicity, and high stability of the produced nanocomposite. The current study postulated a green method for silver/silica nanocomposite (Ag/SiO2NC) synthesis at room temperature using the crude metabolites of Escherichia coli D8 (MF062579) strain in the presence of sunlight. UV-Vis spectrophotometry, X-ray diffraction (XRD), Fourier transform- infrared spectroscopy (FTIR), and transmission electron microscopy (TEM) analyses have characterized the biosynthesized nanocomposite. TEM study of Ag/SiO2NC showed an average particle size of ~32 – 48 nm whereas AgNPs showed a mean size of 18 – 24 nm. The negative charged Ag/SiO2NC (-31.0 mV) showed potent antimicrobial activity against Bacillus cereus ATCC6633, Klebsiella pneumoniae ATCC33495, Staphylococcus aureus (ATCC25923), E. coli (ATCC25922), Candida albicans (ATCC10231), and Botrytis cinerea (Pers: Fr.). The minimum inhibitory concentration (MIC) test showed a dose-dependent manner of Ag/SiO2NC antimicrobial action. MIC values of Ag/SiO2NC against the tested pathogens exhibited 125 and 6.25 μg.mL-1 as antibacterial and antifungal agents, respectively. TEM micrographs showed changes in the pathogens treated with Ag/SiO2NC including wrinkling, damage, and rupture of the bacterial cell membrane. In addition, the formation of a mucilage matrix connecting the hyphal cells, the appearance of big vacuoles and lipid droplets with severe leakage of cytoplasmic contents of the treated B. cinerea were also recorded. Introduction Nowadays, there is a tendency to use materials after converting them into their nano-form, because of the new and promising advantages and unique properties gained in the new nano-form such as antimicrobial activity, chemical, magnetic, electronic, or mechanical properties because of the change of quantum and surface boundary effects compared with their bulk materials (Chhipa and Joshi 2016; Musere et al. 2021). Nanomaterials can be described as materials with a size of 1 – 100 nm, known as the nano-scale range. Synthesis of nanometals can be done using different methods such as chemical, physical or biological techniques. Conventional hypothesis of chemical synthesis of nanoparticles (NPs) might possess several serious problems due to using expensive toxic chemicals (Zhang et al. 2021). mailto:mohamed.marzouq91@du.edu.eg Nova Biotechnol Chim (2022) 21(1): e1023 2 Physical methods include high radiation and stabilizing agents that might be dangerous for human health and to the environment (Awwad et al. 2020). Accordingly, the green synthesis of NPs by plant extracts or microbial metabolites plays an important role in the reduction of metal ions into NPs and capping them in supporting their stability (Parveen et al. 2016). Biological NPs have different characters compared with chemical or physical NPs, with superior stability and suitable dimensions due to the one-step technique (Narayanan and Sakthivel 2011). Silver nanoparticles (AgNPs) were used as a potent antimicrobial agent during the last decades (Hamad et al. 2020). It showed antimicrobial activity against pathogens such as Escherichia coli (Yang et al. 2021), Staphylococcus aureus (Enan et al. 2021), Klebsiella pneumoniae (Pareek et al. 2021), Candida albicans (Takamiya et al. 2021), and Botrytis cinerea (Ouda 2014). The aggregation of AgNPs is a common problem that decreases their biological activity (El-Dein et al. 2021). One way to enhance the metal NPs stability is by stabilizing them by embedding them inside a polymer, which prevents their aggregation, even at high-volume fractions (Baheiraei et al. 2012). Several previous studies have focused on the synthesis of stable monodisperse silica-coated with nanometals, mainly Ag and gold (Au) (Chen et al. 2017; Si et al. 2019; Li et al. 2021). Silica (SiO2) can act as the platform for developing NPs moreover having antimicrobial properties owing to their large surface area, positive surface charge, and monodispersity. Gankhuyag et al. (2021) reported that SiO2 might increase the stability of the nanometals and prevent their aggregation. In addition, the net positive charge of SiO2 facilitates a greater number of AgNPs to interact with the negatively charged surface of bacteria, resulting in highly efficient antimicrobial activity (Jayasuriya 2017). Egger et al. (2009) and Sohrabnezhad et al. (2020) demonstrated the antibacterial activity of silver/silica nanocomposite (Ag/SiO2NC) against both E. coli and S. aureus. Ag/SiO2NC revealed marked changes in the bacterial cell contents, including the cell wall integrity, metabolism, and genetic stability of Pseudomonas aeruginosa (Anas et al. 2013). Also, Xu et al. (2009) reported the antibacterial effects of Ag/SiO2 core-shell particles against E. coli and S. aureus. Ag/SiO2NC had antifungal potential against B. cinerea as reported by Oh et al. (2006). Youssef and Roberto (2021) demonstrated the antifungal activity of chitosan/silica nanocomposite against B. cinerea. Ag/SiO2NC showed fungicidal activity against the pathogenic fungi in the soybean plants (Fusarium oxysporum and Rhizoctonia solani) as reported by Nguyen et al. (2016) and Aspergillus flavus as reported by Diagne et al. (2020). Hence, new distinctive structures of Ag/SiO2NC could present a new prospect for its antimicrobial activity. The present study aimed to evaluate the ability of the crude metabolite of E. coli D8 (MF062579) for reducing the silver nitrate (AgNO3) into AgNPs extracellularly and also their binding with SiO2 in a new one-step green approach. The antimicrobial potential of Ag/SiO2NC was studied against some pathogenic strains, comparing their activity to the standard commercial antibiotics. Experimental Microbial cultures E. coli D8 (AC: MF062579) and the pathogenic bacterial and fungal strains were obtained from the culture collection of Botany and Microbiology Department, Faculty of Science, Damietta University, Egypt. Chemicals The chemicals included silver nitrate (Panreac Quimica S.L.U, Barcelona, Spain), silica (Silicon dioxide nanoparticles, particle size 190 – 250 nm, mesoporous, pore size 4 nm, Sigma-Aldrich), culture media, and other chemicals (Sigma Aldrich Chemical Pvt. Ltd., India). Penicillin G potassium (Buffered Pfizerpen) and fluconazole (Diflucan) were purchased from Pfizer Inc., New York, NY. Biosynthesis of silver nanoparticles and silver/silica nanocomposite Silver nanoparticles were prepared according to El- Zahed et al. (2021). In brief, E. coli D8 agar slants were sub-cultured on nutrient agar plates (37 °C, 24 h). Then the grown colonies were inoculated into a nutrient broth medium with 0.5 McFarland standard (1 – 2 × 108 CFU.mL-1) and incubated at Nova Biotechnol Chim (2022) 21(1): e1023 3 37 °C/150 rpm for 48 h. Later, the cell-free metabolites of E. coli D8 were collected by centrifugation (3H24RI intelligent high-speed refrigerated centrifuge, Herexi Instrument, and Equipment Co., Ltd) at 8,000 rpm for 20 min and filtration through a sterile 0.22 µm syringe filter (Millex GV, Millipore). For the synthesis of AgNPs, 1.5 mM of AgNO3 solution was mixed with cell-free metabolites (1 % v/v) at room temperature and sunlight. For the synthesis of Ag/SiO2NC, 0.5g of AgNO3 was dissolved into 50 mL of distilled water and then added to another beaker that included 100 g of SiO2. At room temperature, the previous solution was mixed well with 20 mL of E. coli D8 cell-free metabolites in the presence of sunlight. The first indicator for the AgNPs and nanocomposite (NC) formation was the color change from colorless (AgNO3) or white (SiO2) into brown. After 20 min, the AgNPs and Ag/SiO2NC were collected separately by centrifugation at 10,000 rpm for 15 min several times and then dried in an oven at 50 °C for 24 h. Then, the NPs and NC were dried at 185 °C for 5 h (Sadeghi et al. 2013). Characterization of silver/silica nanocomposite Silver/silica nanocomposite spectra were scanned by UV/VIS/NIR Spectrophotometer (V-630, JASCO Corporation, Japan). The X-ray diffraction (XRD) pattern of the Ag/SiO2NC was performed at 2θ values (l = 1.54 °A in the range 10 – 80 °) using a Cu X-ray tube at 40 kV and 30 mA with the X- ray diffractometer (model LabX XRD-6000, Shimadzu, Japan). Fourier transform infrared spectroscopy (FTIR) spectrum of the Ag/SiO2NC was recorded by JASCO FT/IR-4100typeA in the 400 – 4,000 cm-1 range. The size and morphology of AgNPs and Ag/SiO2NC were investigated by TEM (JEOL, JEM-2100, Japan) at an accelerating voltage of 200 kV and using a carbon-coated copper grid (Type G 200, 3.05 μM diameter, TAAP, USA). Charge of AgNPs and size distribution by volume were recorded by Zeta Potential Analyzer (Malvern Zetasizer Nano-ZS90, Malvern, UK). Antimicrobial potential The antimicrobial potential of Ag/SiO2NC was tested against Gram-positive bacteria (S. aureus ATCC25923 and Bacillus cereus ATCC6633), Gram-negative bacteria (E. coli ATCC25922 and K. pneumoniae ATCC33495), yeast (C. albicans ATCC10231), and the phytopathogenic fungus (B. cinerea Pers: Fr.) by agar well diffusion and broth dilution methods. The bacterial, yeast and fungal strains were grown and tested using Mueller Hinton agar (MHA), bacto-casitone agar, and potato dextrose agar (PDA) plates, respectively. 200 μL of 0.5 McFarland standard (1 – 2 × 108 CFU.mL-1) of microbial suspension was used as an initial inoculum for each test. Agar well diffusion method Agar well diffusion assay was performed in vitro against the microbial strains according to the guidelines of the Clinical and Laboratory Standards Institute (Clinical and Laboratory Standards 2006). About 200 μL of 150 μg.mL-1 of SiO2, Ag/SiO2NC, AgNO3, penicillin G potassium (antibacterial), and fluconazole (antifungal) were prepared and added separately into small wells (5 mm diameter of size) that were made into the solidified agar plates. Plates were incubated at 37 °C for 48 h, 30 °C for 48 h, or 28 °C for 5 days, for bacteria, yeast, and fungi, respectively. After the incubation period, inhibition zones were measured in millimeters (mm). Broth dilution method Mueller Hinton, bacto-casitone and potato dextrose broth media test tubes were prepared, autoclaved, and inoculated by 100 μL of microbial suspensions (0.5 McFarland standard (1 – 2 × 108 CFU.mL-1)) in three sets of test tubes containing different dosages of Ag/SiO2NC and Penicillin G potassium (antibacterial) or fluconazole (antifungal) concentrations (6.25 – 125 μg.mL-1). Then, the tubes were incubated at 37 °C/120 rpm for 24 h, 30 °C/120 rpm for 24 h, or 28 °C/120 rpm for 5 days, for bacteria, yeast, and fungi, respectively. The minimal inhibition concentration (MIC) for the tested pathogenic strains was determined by measuring their growth spectrophotometrically at Nova Biotechnol Chim (2022) 21(1): e1023 2 600 nm against negative controls (exclusive of Ag/SiO2NC). The growth inhibition percentage was calculated using the following formula (Eq. 1): % Growth inhibition = (1) where the negative control (broth media exclusive of Ag/SiO2NC) optical density; ODc and the Ag/SiO2NC-treated tested sample optical density; ODt (Clinical and Laboratory Standards 2008; 2017). Ultrastructural study The ultrastructure of Ag/SiO2NC treated E. coli and B. cinerea was studied with TEM (JEOL, JEM- 2100, Japan, 200kV) according to Bozzola (2007). The tested strains were subjected to Ag/SiO2NC (MIC, 6.25 μg.mL-1) for 2 h and compared with untreated bacteria and fungi as controls. The samples were fixed in 2.5 % glutaraldehyde in 0.1M cacodylate buffer at pH 7.0 and then post- fixed in 1 % osmium tetroxide, dehydrated with a graded series of ethanol, embedded in a plastic resin, and sectioned on an ultramicrotome. Ultrathin sections were double‐stained with uranyl acetate and lead citrate and then loaded on carbon- coated copper grids (Type G 200, 3.05 μM diameter, TAAP, U.S.A.). Statistical analysis SPSS software version 18 was used for all the statistical analysis. All values in the experiments were expressed as the mean ± standard deviation (SD) and were analyzed with a one-way Analysis of Variance (ANOVA) with a significant level set at P < 0.05. Results and Discussion Synthesis and characterization of Ag/SiO2NC Synthesis and characterization of Ag/SiO2NC have attracted the attention of the materials community because of their promising properties (Zaferani 2018). The green synthesis approach of those NCs with controllable size and properties has applications in miniaturized catalysts, photonics, optical devices, medical applications moreover it could be used as a potential nanomicrobicide and nanoscale growth regulator in agriculture (Das et al. 2019). Endless progression of microbial antibiotic-resistant mechanisms claims continuous searching for alternative approaches to deal with their risk to humans and plants (Rai et al. 2012). The present study provided a green approach for the synthesis of antimicrobial Ag/SiO2NC mediated by the cell-free metabolites of E. coli D8. FTIR spectrum confirmed the presence of proteins during the bio-reduction process. These proteins, in the E. coli D8 metabolite, might be including the reducing enzymes and/or some redox agents such as sulfur- containing proteins resulting in the bio-reduction of silver ions (Ag+) into AgNPs (Krishnaraj et al. 2012). Also, quinones (menaquinone, demethylmenaquinone, and ubiquinone) found in the E. coli D8 metabolite act as an electron shuttle compound and reduced Ag+ into AgNPs in the presence of sunlight as reported by Duan et al. (2015) and Sharma et al. (2012). The biosynthesis of AgNPs was confirmed through visual observation of the color change of the mixture into brown color producing an obvious absorption peak at 430 nm (Fig. 1). The brown color is because of the excitation of the AgNPs surface plasmon resonance (El-Dein et al. 2021). Granbohm et al. (2018) found the UV-Vis spectra of Ag/SiO2NC powders showed the silver SPR peak at 410 nm. Fig. 1. The UV-Vis spectra of SiO2 and Ag/SiO2NC. The XRD patterns of SiO2 were examined and showed an amorphous SiO2 characteristic diffraction peak at 22.4 °. Ag/SiO2NC XRD pattern revealed peaks at 2θ angles of 32.25 °, 38.25 ° and 44.4 ° corresponding to the reflections of (110), (111) and (200) crystalline planes of the face- 4 Nova Biotechnol Chim (2022) 21(1): e1023 3 centered cubic (FCC) structure of AgNPs (Fig. 2). Also, we had found no other diffraction peaks for silver oxide in the Ag/SiO2NC XRD pattern which showed the coverage of the NC with pure AgNPs (Xu et al. 2015). Fig. 2. The XRD patterns of SiO2 and Ag/SiO2NC. The FTIR spectra of Ag/SiO2NC were analyzed in the region of 400 – 4,000cm-1 (Fig. 3). The vibration bands around 3,431 and 1,613 cm-1 are attributed to the OH and carbonyl group (C=O), respectively. These signals clearly confirm the presence of bacterial compounds bounded on the surface of Ag/SiO2NC that affect protection and stability of the NC. The intense peaks around 3,431 and 2,914 cm-1 are attributed to the primary and secondary amines vibrations bands, respectively. The stretch C-N vibration of aliphatic amines existed at 1,078 cm-1 bands. These signals confirmed the presence of proteins in the Ag/SiO2NC synthesis. Water bands were appeared at around 1,613 cm-1 corresponding to bending vibrations indicating the hygroscopic character of the powdered samples (Singh and Ahmed 2012). Si–O–Si and Si–OH absorptions bands have been observed at 1,078; 783; and 462 cm-1. The Si–O–Ag linkages stretching were also seen at around 691 cm-1. The band appears in the Ag/SiO2NC suggesting bonding between the AgNPs and the oxygen bonded to SiO2. The peaks 450 – 800 cm-1 are probably related to the pseudo- lattice vibrations (Mathur et al. 2006). Fig. 3. The FTIR spectra of SiO2 and Ag/SiO2NC. The Ag/SiO2NC was examined by the TEM (Fig. 4) to investigate the morphology and size of the AgNPs (Fig. 4B). AgNPs are embedded within the matrix and on the surface of the SiO2. TEM image showed small spherical shaped AgNPs having a diameter between 18 – 24 nm. Gu et al. (2011) reported that the AgNPs average particle size on the surfaces of SiO2 had a little increase from 10 to 25 nm as reaction temperature increased. This should be attributed to the higher reduction rate of Ag+ at the elevated reaction temperature. SiO2 has a net positive charge, while Ag/SiO2NC may have a positive or negative surface charge depending on the surface functional group and solution pH (Jana et al. 2007; Jayasuriya 2017). The synthesis of Ag/SiO2NC included binding primary amines as confirmed by the FTIR analysis. The primary amines were deprotonated during the bio-reduction process, were leading to a gradual decrease in the surface positive charge of SiO2 and might approach zero (Jana et al. 2007). On the other hand, the binding between the AgNPs which are capped with highly negative proteins (El-Dein et al. 2021), and SiO2 to give negatively charged Ag/SiO2NC. The biosynthesized Ag/SiO2NC had a negative charge, -31.0 mV (Fig. 5), which matched with Shanthil et al. (2012) results, -33 ± 2 mV and was better than Zhao et al. (2016) and El- Sheshtawy et al. (2020) results (-16.10 mV and -15 mV, respectively). Different studies (Verma and Stellacci 2010; Anas et al. 2013) have studied the interaction of charged nanomaterials with cells showing that positively charged nanomaterials have 5 Nova Biotechnol Chim (2022) 21(1): e1023 2 the greatest efficacy in penetrating the cell membrane. Other studies (Fuller et al. 2008; Martin et al. 2008) have examined the cellular uptake of negatively charged nanomaterials and proposed that negatively charged nanomaterials generate reactive oxygen species (ROS) contributes towards potent bacterial toxicity (Ivask et al. 2010; Agnihotri et al. 2013). A further study of the synthesized Ag/SiO2NC should be taken into account to improve the antimicrobial efficacy of Ag/SiO2NC to have a positive surface charge. The positive charge of the nanomaterials increases the efficient electrostatic interaction with the negative charges of the microbial cell wall (Li et al. 2011). Fig. 4. (a) TEM micrograph of SiO2. (b) TEM micrograph of Ag/SiO2NC. Fig. 5. Zeta potential measurement analysis of Ag/SiO2NC (-31.0 mV). Antimicrobial potential of Ag/SiO2NC The pathogenic bacteria, yeast, and fungi appeared to be more tolerant to SiO2 than Ag/SiO2NC. In this study, Ag/SiO2NC was investigated to determine its antimicrobial action (Fig. 6 and Table 1). The NC revealed very good antimicrobial potential against a wide range of microorganisms such as K. pneumoniae, S. aureus, and B. cinerea. The inhibition of microbial growth due to surface contact with the SiO2 nanocomposite containing AgNPs demonstrated that NC functionalized with 6 Nova Biotechnol Chim (2022) 21(1): e1023 2 the AgNPs has better antimicrobial action than bulk SiO2. The antibacterial potential results of Ag/SiO2NC in He et al. (2012) study revealed that Ag/SiO2NC were sensitive to S. aureus and E. coli and with the inhibition zone diameter 15.3 mm and 10.4 mm, respectively. Lu et al. (2017) studied the combination between chlorhexidine and Ag/SiO2NC and recorded that combination might produce synergistic bactericidal and candidacidal effects and improve the microbicidal efficiency. In addition, Ag/SiO2NC showed antifungal potential against B. cinerea besides the antibacterial and anticandidal actions. Rodríguez-Cutiño et al. (2018) confirmed the antimicrobial properties of Ag/SiO2NC against bacteria such as E. coli, B. cereus, S. typhimurium, and S. aureus in addition to the green squash fungi: B. cinerea and R. solani. Fig. 6. Antimicrobial activity of SiO2, AgNPs, and Ag/SiO2NC; (a) B. cereus, (b) E. coli, (c) K. pneumoniae, (d) S. aureus, (e) C. albicans, and (f) B. cinerea. Table 1. Antimicrobial activity of SiO2, AgNPs and Ag/SiO2NC against the pathogenic microbial strains (Highly significant = *P < 0.05; n = 3). Antibacterial activity (Inhibition zone, mm ± SD) Substance B. cereus E. coli K. pneumoniae S. aureus AgNO3 24 ± 0.06* 26 ± 0* 34 ± 0* 29 ± 0* SiO2 -ve -ve -ve -ve AgNPs 30 ± 0.14* 30 ± 0.14* 38 ± 0* 37 ± 0* Ag/SiO2NC 20 ± 0.06* 22 ± 0.14* 36 ± 0* 34 ± 0* Penicillin G potassium 29 ± 0* 30 ± 0* -ve 26 ± 0.06* Antifungal activity (Inhibition zone, mm ± SD) Substance C. albicans B. cinerea AgNO3 13 ± 0.06* 13 ± 0.14* SiO2 -ve -ve AgNPs 16 ± 0.06* 21 ± 0.14* Ag/SiO2NC 14 ± 0.06* 24 ± 0.14* Fluconazole 15 ± 0* 16 ± 0.14* AgNPs and Penicillin G potassium showed a similar manner of MIC values (6.25 μg.mL-1) against S. aureus, B. cereus, and E. coli compared to Ag/SiO2NC (MIC value, 125 μg. mL -1). The 7 Nova Biotechnol Chim (2022) 21(1): e1023 2 MIC values against B. cinerea were 6.25 and 25 μg.mL-1 for Ag/SiO2NC and fluconazole, respectively. The better growth inhibition percentage of Ag/SiO2NC was against B. cinerea (50.2 %) followed by B. cereus (31.1 %), S. aureus (30.7 %), E. coli (26.6 %), and C. albicans (6.6 %) showing a dose-dependent manner of Ag/SiO2NC antimicrobial action (Fig. 7). The minimum antibacterial concentration of the Ag/SiO2NC is 0.2 and 0.3 μg.mL-1 for Bacillus sp. and E. coli, respectively (Huang 2008). Qasim et al. (2015) suggested Ag/SiO2NC to be a potential antifungal agent for C. albicans 077 showing that this tested human pathogenic fungus was sensitive to Ag/SiO2NC with MIC∼6 μg.mL -1 of Ag/SiO2NC. Vladkova et al. (2020) presented that TiO2/SiO2/Ag nanocomposite totally inhibited the E. coli growth within 30 min to 2 h. The growth of B. cinerea was almost completely inhibited (98.4 %) by Ag/SiO2NC (6.4 µg.mL -1) treatment compared with AgNPs alone (72.43 %, 6.4 µg.mL-1 as reported by Kim (2011). Fig. 7. Growth inhibition percentage of Ag/SiO2NC, AgNPs, and antibiotics at MIC values against S. aureus, B. cereus, E. coli, C. albicans, and B. cinerea. It is known that both, Ag+ and AgNPs are effective antimicrobial agents even though their antimicrobial mechanism is not fully understood (Kędziora et al. 2018). Several studies (Feng et al. 2000; Lara et al. 2011) reported the different mechanism of the antimicrobial action of nanomaterials such as penetrating the cell wall and plasma membrane, ending with damaging DNA molecules. Others suggested that nanomaterials might interact with thiol groups in proteins, which induces the inactivation of microbial proteins. In the presented study, AgNPs bonded on the surface of SiO2 have an opposite charge with Gram- positive bacteria, in that way killing them more easily than Gram-negative bacteria due to the electrostatic attraction. The antimicrobial activities of Ag/SiO2NC are investigated using E. coli and B. cinerea as two model microorganisms. As shown in Fig. 8, untreated E. coli was typically rod-shaped with smooth and intact cell walls. After being treated with Ag/SiO2NC, cell walls became wrinkled and damaged. The separation between the bacterial cell wall and cell membrane was also noted. Fig. 8. The antibacterial action of Ag/SiO2NC on the ultrastructure of E. coli. (a) A negative control (without Ag/SiO2NC). (b) A treated sample (150 μg.mL -1), there are irregular rods (arrows) with lysed cell walls (Ly) and complete cell lysis (Cl). Also, note the separation that occurs between the bacterial cell wall and cell membrane. Ag/SiO2NC 8 Nova Biotechnol Chim (2022) 21(1): e1023 3 With treated B. cinerea (Fig. 9), TEM micrographs showed many changes, including the 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 in comparing to the control. The separation between the fungal cell wall and plasma membrane was also detected in the treated cells. The observed damages of the E. coli and B. cinerea cells after the treatment by Ag/SiO2NC could be because of cellular interactions with the AgNPs. The combined action of adhesion and penetration of AgNPs might illustrate the biocidal action of the NC, plasma membrane being the target of rapid antimicrobial action of AgNPs in E. coli and B. cinerea (Rai et al. 2012). Eckhardt et al. (2013) reported that the binding of AgNPs with microbial proteins might inactivate the electron transport chain, in that way suppressing the respiration and growth of the microbial cells. To establish that the advantages of silver nanocomposites (AgNCs) and the possible mechanisms of their antimicrobial action outweigh the possible risks, the toxicity of AgNPs and AgNCs must be investigated. W Cy V PM V L (a) (b) Fig. 9. The antifungal activity of Ag/SiO2NC on the ultrastructure of B. cinerea. (a) negative control (without Ag/SiO2NC). Note normal cell wall (W), plasma membrane (PM), Vacuole (V), and compact cytoplasm (Cy). (b) The treated sample, note, the big vacuole (V) and lipid droplets (L). Also, note the separation that occurs between the fungal cell wall and plasma membrane (arrow). Conclusion The embedded AgNPs in Ag/SiO2NC mediated by E. coli D8 were characterized as negative- charged (-31.0 mV) and spherical with an average size ranging between 18 and 24 nm. 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