Probiotic-Mediated Biosynthesis of Silver Nanoparticles and Their Antibacterial Applications against Pathogenic Strains of Escherichia coli O157:H7

The present study aimed to suggest a simple and environmentally friendly biosynthesis method of silver nanoparticles (AgNPs) using the strain Bacillus sonorensis MAHUQ-74 isolated from kimchi. Antibacterial activity and mechanisms of AgNPs against antibiotic-resistant pathogenic strains of Escherichia coli O157:H7 were investigated. The strain MAHUQ-74 had 99.93% relatedness to the B. sonorensis NBRC 101234T strain. The biosynthesized AgNPs had a strong surface plasmon resonance (SPR) peak at 430 nm. The transmission electron microscope (TEM) image shows the spherical shape and size of the synthesized AgNPs is 13 to 50 nm. XRD analysis and SAED pattern revealed the crystal structure of biosynthesized AgNPs. Fourier transform infrared spectroscopy (FTIR) data showed various functional groups associated with the reduction of silver ions to AgNPs. The resultant AgNPs showed strong antibacterial activity against nine E. coli O157:H7 pathogens. Minimum inhibitory concentration (MIC) values of the AgNPs synthesized by strain MAHUQ-74 were 3.12 μg/mL for eight E. coli O157:H7 strains and 12.5 μg/mL for strain E. coli ATCC 25922. Minimum bactericidal concentrations (MBCs) were 25 μg/mL for E. coli O157:H7 ATCC 35150, E. coli O157:H7 ATCC 43895, E. coli O157:H7 ATCC 43890, E. coli O157:H7 ATCC 43889, and E. coli ATCC 25922; and 50 μg/mL for E. coli O157:H7 2257, E. coli O157: NM 3204-92, E. coli O157:H7 8624 and E. coli O157:H7 ATCC 43894. FE-SEM analysis demonstrated that the probiotic-mediated synthesized AgNPs produced structural and morphological changes and destroyed the membrane integrity of pathogenic E. coli O157:H7. Therefore, AgNPs synthesized by strain MAHUQ-74 may be potential antibacterial agents for the control of antibiotic-resistant pathogenic strains of E. coli O157:H7.


Introduction
Silver nanoparticles (AgNPs) refer to nanoparticles with sizes ranging from 1 nm to 100 nm. AgNPs have been widely used in various applications in biomedical science such as in antibiotics, biosensors, drug delivery systems, antimicrobial, anticancer, and anti-inflammatory agents, etc. [1][2][3][4][5][6][7]. Many devices, including textiles, keyboards, and medical devices, now contain AgNPs that continuously release small amounts of silver ions to provide antibacterial protection [8]. AgNPs also have been implemented in the development of various bioactive materials, including polymer composites, because of their high antimicrobial activity. The polymer-based nanoparticles are usually a combination of organic polymers or a specific biomolecule in which an inorganic nanoparticle is embedded. Polymer-based metallic nanoparticles are widely explored due to their versatility, biodegradability, being environmentally friendly, and being biocompatible in nature [9,10]. The successful capping of polymers on AgNPs may prevent the agglomeration of synthesized nanocomposite as well as facilitate the stabilization process [11]. a serious threat to humans. Escherichia coli O157:H7 is a notorious pathogen, and this pathogen is associated with a broad spectrum of illnesses in humans ranging from mild diarrhea and hemorrhagic colitis to the potentially fatal hemolytic uremic syndrome [36]. In this study, we report a newly isolated B. sonorensis MAHUQ-74 from kimchi, which we used for the synthesis of AgNPs using an extracellular method. The culture supernatant of B. sonorensis MAHUQ-74 was used to quickly and easily synthesize AgNPs without adding any reducing agent. Then, resultant AgNPs were characterized, and their antimicrobial activity at the mechanism level was investigated against various pathogenic strains of E. coli O157:H7.

Isolation, Identification, and Characterization of AgNP-Producing Probiotics
Kimchi, a traditional, fermented food product, was collected from the retail market in Anseong, Korea. Briefly, 1 g of kimchi was dissolved in 9 mL of 0.85% (w/v) NaCl solution, serially diluted up to 10 −5 , and 100 µL of each dilution was spread on both nutrient agar (NA) and Reasoner's 2A agar (R 2 A agar) plates [37]. Then, the plates were incubated at 37 • C for 3 days. Single colonies were purified by successive transferring to new NA or R 2 A agar plates. AgNP synthesis ability was screened by culturing all isolates in 5 mL R2A broth media for 48 h at 37 • C. Then, 1 mM AgNO 3 solution was added to the culture supernatant and incubated again for 48 h. Among all of these isolated strains, strain MAHUQ-74 showed strong AgNP synthesis ability. Then, the strain was identified by 16S rRNA gene sequence analysis [38]. The 16S rRNA gene sequence of strain MAHUQ-74 was submitted to GeneBank of NCBI. The 16S rRNA gene sequences of related taxa were obtained from the EzBioCloud server [39]. The phylogenetic tree was created using the MEGA6 program [40] and the neighbor-joining method, to discover the phylogenetic location of isolated strain MAHUQ-74 [41]. The optimum growth conditions of strain MAHUQ-74 including media, temperature and pH, gram-straining reaction, catalase, and oxidase activities were examined according to the previous description [42]. According to the manufacturer's instructions, commercial API ZYM and API 20 NE kits (bioMérieux) were used to further determine enzyme activity and carbon source utilization. The probiotic strain MAHUQ-74 was deposited to the Korean Agricultural Culture Collection (KACC).

Biosynthesis of AgNPs Using Probiotic Strain MAHUQ-74
For the biosynthesis of AgNPs, the B. sonorensis MAHUQ-74 strain was cultured in 100 mL of R 2 A broth and incubated at 37 • C for 3 days in a shaking incubator at 180 rpm. After three days of incubation, the culture supernatant was collected by centrifugation at 10,000 rpm, 4 • C for 10 min in a centrifuge. Then, 0.1 mL (1 M concentration) filter-sterilized AgNO 3 solution was added to 100 mL supernatant and incubated again in an orbital shaker at 180 rpm and 30 • C for 24 h. The synthesis of AgNPs was confirmed by visual observation of the color change. Finally, the biosynthesized AgNPs were collected by centrifugation at 13,000 for 30 min. The precipitate of synthesized AgNPs was washed with distilled water. Then, the air-dried samples were used for characterization and antibacterial studies.

Characterization of Biosynthesized AgNPs
The biosynthesis of AgNPs was monitored by a UV-Vis spectrophotometer in the range of 300-800 nm. By using field-emission transmission electron microscopy (FE-TEM), energy-dispersive X-ray (EDX) spectroscopy, element map, and selective area diffraction (SAED) mode, the morphology, element composition, and purity of the synthesized AgNPs were studied. A drop of AgNP solution was kept on a copper mesh, dried at room temperature, and finally transferred to a microscope for analysis. X-ray diffraction (XRD) analysis was conducted with an X-ray diffractometer (D8 Advance, Bruker, Germany) in the range of 30-90 • over 2θ value, using CuKα radiation, at 40 kV, 40 mA with 6 • /min scanning rate. For XRD analysis, air-dried AgNP samples were used. Fourier transforminfrared (FTIR) spectroscopy showed biomolecules related to the biosynthesis and stability of AgNPs. FTIR analysis was performed by using a PerkinElmer Fourier Transform Infrared Spectrometer (PerkinElmer Inc., Waltham, MA, USA), with a resolution of 4 cm -1 and a range of 400-4000 cm -1 . A Malvern Zetasizer Nano ZS90 (Otsuka Electronics, Osaka, Japan) was used to determine the particle size of the green synthetic AgNPs by dynamic light scattering (DLS) at 25 • C. Pure water was used as a dispersion medium with a dielectric constant of 78.3, a viscosity of 0.8878 cP, and a refractive index of 1.3328.

Antimicrobial Activity of Probiotic-Mediated Synthesized AgNPs
The tested pathogenic microbes (nine different E. coli O157:H7 strains) were grown overnight in LB broth. Briefly, 100 µL of the bacterial culture sample of the tested pathogen was spread on the LB agar plate. Sterile paper discs containing AgNPs 50 µL and 100 µL (1000 µg/mL) were placed on the LB agar plates. Then, the plates were incubated in an incubator at 37 • C for 24 h. Similarly, the antibacterial activity of some commercial antibiotics such as erythromycin (15 µg/disc), vancomycin (30 µg/disc), and penicillin G (10 µg/disc) was tested against nine E. coli O157:H7 strains. The inhibition zones were calculated after 24 h of incubation. The test was performed twice.

Determination of MIC and MBC
The minimum inhibitory concentration (MIC) of probiotic-mediated synthesized AgNPs was measured using the broth microdilution technique. Nine E. coli O157:H7 strains were grown in LB broth overnight at 37 • C, and the turbidity was fixed at around 1 × 10 6 CFUs/mL. Then, 100 mL of test bacterial (1 × 10 6 CFUs/mL) suspension were added to a 96-well plate, after which an equal volume of AgNP solution with various concentrations (3.12-200 µg/mL) was added, and finally, the plates were incubated in a 37 • C incubator for 24 h. Every 3 h of the interval, the absorbance (at 600 nm) was measured in a microplate reader. Minimum bactericidal concentration (MBC) was determined by streaking 8 µL of each set on an agar plate and again incubated for 24 h at 37 • C. The culture plates were observed by direct visualization to determine the MBC that blocked bacterial growth [24].

Morphological Evaluation of Treated Cells Using FE-SEM
The morphological alterations of E. coli O157:H7 (ATCC 35150) were examined using FE-SEM. Logarithmic growth phase cells (1 × 10 7 CFU/mL) were treated with probioticmediated synthesized AgNPs at a concentration of 1 × MBC for 6 h. In the control, the bacterial culture was treated without AgNP solution. The treated cells were washed with phosphate-buffered saline (PBS). The cells were fixed for 4 h using 2.5% glutaraldehyde solution and then, washed several times with PBS. Again, with 1% tetroxide cells were fixed and washed with PBS buffer solution. The fixed cells were dehydrated using different concentrations of ethanol (30 to 100%, every 10% interval) at room temperature for 10 min. Then, the samples were dried with a dryer. Finally, the samples were placed on the SEM metal grid and coated with gold. The morphological and structural changes in the cells were observed using FE-SEM (S-4700, Hitachi, Tokyo, Japan).

Biosynthesis of AgNPs Using Strain MAHUQ-74
Biosynthesis of AgNPs using B. sonorensis MAHUQ-74 was ensured by monitoring the color of the culture supernatant. The color of the MAHUQ-74 culture supernatant turned to deep brown from watery yellow, which indicated the synthesis of AgNPs. The control sample (without bacterial supernatant) did not show any color change when incubated under the same conditions (Figure 2A,B). Optimum temperature (30 • C) and AgNO 3 concentration (final concentration 1 mM) for stable synthesis were determined based on the ultraviolet-visible spectroscopy (UV-Vis) analysis. Two methods are commonly utilized for the biosynthesis of nanoparticles using bacteria-intracellular and extracellular methods. The intracellular method is a more complex and a multi-step process, compared with the extracellular method. In the current study, the extracellular methodology was used to biosynthesize AgNPs, using probiotic bacterial strain MAHUQ-74, which was simple, facile, cost-effective, and ecofriendly.

Characterization of Biosynthesized AgNPs
Probiotic-mediated synthesized AgNPs showed a peak at 430 nm ( Figure 2C), which revealed that AgNPs were fruitfully synthesized. AgNPs are known to exhibit a UV-Visible absorption peak in the range of 400-500 nm [24]. The lower absorption wavelength indicates smaller-sized, spherical NPs [43], which suggested that MAHUQ-74 might synthesize smallsized AgNPs. The results indicated that MAHUQ-74 may be a promising candidate for the biosynthesis of AgNPs. TEM analysis showed that AgNPs were spherical and elliptical, and they dispersed well, without obvious agglomeration. The size of the synthesized AgNPs was 13-50 nm ( Figure 2D-F).
The composition and purity of biosynthesized AgNPs were investigated using an EDX spectrometer. The EDX data revealed elemental signals of silver atoms in probioticmediated synthesized AgNPs at around 3 keV and indicated the homogenous distribution of AgNPs. Some other peaks of copper were also found in the EDX mode due to the use of a copper grid ( Figure 3A). The elemental mapping results showed that the most distributed element in biosynthetic nanoproducts was silver ( Figure 3B-D, Table 2).
AgNO3 concentration (final concentration 1 mM) for stable synthesis were determined based on the ultraviolet-visible spectroscopy (UV-Vis) analysis. Two methods are commonly utilized for the biosynthesis of nanoparticles using bacteria-intracellular and extracellular methods. The intracellular method is a more complex and a multi-step process, compared with the extracellular method. In the current study, the extracellular methodology was used to biosynthesize AgNPs, using probiotic bacterial strain MAHUQ-74, which was simple, facile, cost-effective, and ecofriendly.

Characterization of Biosynthesized AgNPs
Probiotic-mediated synthesized AgNPs showed a peak at 430 nm ( Figure 2C), which revealed that AgNPs were fruitfully synthesized. AgNPs are known to exhibit a UV-Visible absorption peak in the range of 400-500 nm [24]. The lower absorption wavelength indicates smaller-sized, spherical NPs [43], which suggested that MAHUQ-74 might synthesize small-sized AgNPs. The results indicated that MAHUQ-74 may be a promising candidate for the biosynthesis of AgNPs. TEM analysis showed that AgNPs were spherical and elliptical, and they dispersed well, without obvious agglomeration. The size of the synthesized AgNPs was 13-50 nm ( Figure 2D-F).
The composition and purity of biosynthesized AgNPs were investigated using an EDX spectrometer. The EDX data revealed elemental signals of silver atoms in probioticmediated synthesized AgNPs at around 3 keV and indicated the homogenous distribution of AgNPs. Some other peaks of copper were also found in the EDX mode due to the use of a copper grid ( Figure 3A). The elemental mapping results showed that the most distributed element in biosynthetic nanoproducts was silver ( Figure 3B-D, Table 2).   Figure 4A). A recently reported study revealed similar XRD results, in which AgNPs were synthesized using microorganisms [44]. The crystalline structure of probiotic-mediated synthesized AgNPs was confirmed using SAED analysis, which re-   Figure 4A). A recently reported study revealed similar XRD results, in which AgNPs were synthesized using microorganisms [44]. The crystalline structure of probiotic-mediated synthesized AgNPs was confirmed using SAED analysis, which revealed sharp rings corresponding to the reflections of 111, 200, 220, 311, and 222 ( Figure 4B). Both the XRD spectrum and SAED pattern confirmed the crystalline structure of AgNPs.  The FTIR spectrum indicated that the functional molecules including biopolymers such as proteins, enzymes, and amino acids secreted by probiotic bacteria could be involved in both the synthesis and stabilization of AgNPs. The particle size of biosynthesized AgNPs was measured with dynamic light scattering (DLS) analysis. Figure 5B shows the particle size distribution of biosynthesized AgNPs based on intensity, volume, and number. The The FTIR spectrum indicated that the functional molecules including biopolymers such as proteins, enzymes, and amino acids secreted by probiotic bacteria could be involved in both the synthesis and stabilization of AgNPs. The particle size of biosynthesized AgNPs was measured with dynamic light scattering (DLS) analysis. Figure 5B shows the particle size distribution of biosynthesized AgNPs based on intensity, volume, and number. The average particle size of probiotic-mediated synthesized AgNPs was 44.6 nm, and the polydispersity value was 0.406.

Antimicrobial Activity of Probiotic-Mediated Synthesized AgNPs against Different E. coli O157:H7 Strains
The antimicrobial activity of AgNPs is related to their ability to bind microbial DNA, proteins, and enzymes, as well as to alter cell morphology and function [18,45,46]. Small AgNPs have higher antimicrobial activity than large particles, allowing the faster release of Ag+ ions [47]. Many reports have tested the antimicrobial activity of biosynthetic AgNPs against different pathogenic microorganisms, including bacteria, fungi, yeasts, and microbial biofilms. In this study, AgNPs were synthesized using probiotic bacteria B. sonorensis MAHUQ-74, and their antibacterial ability was investigated against nine foodborne pathogenic E. coli O157:H7 strains (Table 3).

Antimicrobial Activity of Probiotic-Mediated Synthesized AgNPs against Different E. coli O157:H7 Strains
The antimicrobial activity of AgNPs is related to their ability to bind microbial DNA, proteins, and enzymes, as well as to alter cell morphology and function [18,45,46]. Small AgNPs have higher antimicrobial activity than large particles, allowing the faster release of Ag+ ions [47]. Many reports have tested the antimicrobial activity of biosynthetic AgNPs against different pathogenic microorganisms, including bacteria, fungi, yeasts, and microbial biofilms. In this study, AgNPs were synthesized using probiotic bacteria B. sonorensis MAHUQ-74, and their antibacterial ability was investigated against nine foodborne pathogenic E. coli O157:H7 strains ( Table 3).
The results showed that the synthesized AgNPs had significant antibacterial activity against all tested foodborne pathogens. The antibacterial efficacy against various pathogenic strains of E. coli O157:H7 was determined by calculating the diameter of the zone of inhibition ( Figure 6, Table 4). It was also found that AgNPs can inhibit the growth of most E. coli O157:H7 strains when treated with a 100 µL AgNP solution. The results of this study revealed that the probiotic bacteria B. sonorensis MAHUQ-74-mediated biosynthesized AgNPs are able to control foodborne pathogenic bacteria, especially pathogenic E. coli O157:H7 strains.  Three antibiotics (erythromycin, vancomycin, and penicillin G) were also tested against the foodborne pathogens E. coli O157:H7. Six of the nine foodborne pathogens were found to be resistant to all three antibiotics included in this study (Figure 7, Table 5).

Determination of MIC and MBC
The MIC and MBC of probiotic-mediated synthesized AgNPs against a total of nine E. coli O157:H7 were determined by a standard broth dilution assay. Bacterial growth curves revealed that the MICs of biosynthesized AgNPs were 3.12 µg/mL for eight E. coli O157:H7 strains ( Figure 8A-H) and 12.5 µg/mL for strain E. coli ATCC 25,922 ( Figure 8I). This result indicated that the probiotic-mediated synthesized AgNPs extremely suppressed the growth of pathogenic strains of E. coli O157:H7.
The MBC of synthesized AgNPs was 25 µg/mL for E. coli O157:H7 ATCC 43895, E. against all tested foodborne pathogens. The antibacterial efficacy against various pathogenic strains of E. coli O157:H7 was determined by calculating the diameter of the zone of inhibition ( Figure 6, Table 4). It was also found that AgNPs can inhibit the growth of most E. coli O157:H7 strains when treated with a 100 µ L AgNP solution. The results of this study revealed that the probiotic bacteria B. sonorensis MAHUQ-74-mediated biosynthesized AgNPs are able to control foodborne pathogenic bacteria, especially pathogenic E. coli O157:H7 strains.

Determination of MIC and MBC
The MIC and MBC of probiotic-mediated synthesized AgNPs against a total of nine E. coli O157:H7 were determined by a standard broth dilution assay. Bacterial growth curves revealed that the MICs of biosynthesized AgNPs were 3.12 μg/mL for eight E. coli

Study of Morphogenesis of E. coli O157:H7-Treated Cells Using FE-SEM
The structural and morphological alterations of E. coli O157:H7 ATCC 35,150 cells treated with probiotic-mediated synthesized AgNPs were directly observed using FE-SEM ( Figure 10). Based on the FE-SEM analysis, it was found that the untreated E. coli O157:H7 cells were intact, normal, and rod-shaped, and the structural integrity of the bacterial cells was good without any damage ( Figure 10A,B). However, after treatment with 1 × MBC of synthesized AgNPs, the shape of E. coli O157:H7 cells became abnormal, irregular, and wrinkled, with the cell membrane entirely collapsed and damaged ( Figure 10C,D).
Although the bactericidal mechanism of AgNPs is not yet fully understood, several hypotheses have been proposed in the literature. The attachment of AgNPs to the bacterial cell membrane results in the formation of a "pit" on the bacterial cell wall, thereby allowing the nanoparticles to enter the bacterial cells in the periplasm [48,49]. As a result, subsequent changes in the DNA of the bacterial cells treated with AgNPs lead to the loss of DNA replication ability, which leads to the inactivation of the expression of proteins and enzymes necessary for ATP production. In the present study, the structural and morphological alterations, damage to bacterial cell walls, and cell membrane indicated that the biosynthesized AgNPs might interfere with the metabolic process and normal cell functions, leading to the death of bacterial cells.   Figure 9A-I).

Study of Morphogenesis of E. coli O157:H7-Treated Cells Using FE-SEM
The structural and morphological alterations of E. coli O157:H7 ATCC 35,150 cells treated with probiotic-mediated synthesized AgNPs were directly observed using FE-SEM ( Figure 10). Based on the FE-SEM analysis, it was found that the untreated E. coli O157:H7 cells were intact, normal, and rod-shaped, and the structural integrity of the bacterial cells was good without any damage ( Figure 10A,B). However, after treatment with 1 × MBC of synthesized AgNPs, the shape of E. coli O157:H7 cells became abnormal, irregular, and wrinkled, with the cell membrane entirely collapsed and damaged ( Figure  10C,D). Although the bactericidal mechanism of AgNPs is not yet fully understood, several hypotheses have been proposed in the literature. The attachment of AgNPs to the bacterial cell membrane results in the formation of a "pit" on the bacterial cell wall, thereby allowing the nanoparticles to enter the bacterial cells in the periplasm [48,49]. As a result, subsequent changes in the DNA of the bacterial cells treated with AgNPs lead to the loss of DNA replication ability, which leads to the inactivation of the expression of proteins and enzymes necessary for ATP production. In the present study, the structural and morphological alterations, damage to bacterial cell walls, and cell membrane indicated that the biosynthesized AgNPs might interfere with the metabolic process and normal cell functions, leading to the death of bacterial cells.

Conclusions
In conclusion, the biological compounds produced by bacteria have high utility value and may play important roles in the production of AgNPs. Biomolecules, including biopolymers such as proteins, enzymes, and amino acids, that are secreted by bacteria are involved in the synthesis process but also may improve the function of synthesized AgNPs. In the present study, we isolated and identified the strain B. sonorensis MAHUQ-74 and used their culture supernatant for the biosynthesis of AgNPs. The AgNPs were analyzed using UV-Vis, FE-TEM, XRD, EDX, FTIR, and DLS. FE-TEM images indicated that the AgNPs were mostly circular, and the size ranged from 13 to 50 nm. The FTIR data indicated that various biomolecules may participate in the synthesis and stabilization of AgNPs. The extracellular method was used for the biosynthesis of AgNPs. Moreover, the biosynthesized AgNPs showed potent antibacterial efficacy against nine pathogenic E. coli O157:H7 strains. The MICs of the AgNPs synthesized by strain MAHUQ-74 were 3.12 to

Conclusions
In conclusion, the biological compounds produced by bacteria have high utility value and may play important roles in the production of AgNPs. Biomolecules, including biopolymers such as proteins, enzymes, and amino acids, that are secreted by bacteria are involved in the synthesis process but also may improve the function of synthesized AgNPs. In the present study, we isolated and identified the strain B. sonorensis MAHUQ-74 and used their culture supernatant for the biosynthesis of AgNPs. The AgNPs were analyzed using UV-Vis, FE-TEM, XRD, EDX, FTIR, and DLS. FE-TEM images indicated that the AgNPs were mostly circular, and the size ranged from 13 to 50 nm. The FTIR data indicated that various biomolecules may participate in the synthesis and stabilization of AgNPs. The extracellular method was used for the biosynthesis of AgNPs. Moreover, the biosynthesized AgNPs showed potent antibacterial efficacy against nine pathogenic E. coli O157:H7 strains. The MICs of the AgNPs synthesized by strain MAHUQ-74 were 3.12 to 12.5 µg/mL for the tested nine E. coli O157:H7 strains. The MBCs of the AgNPs synthesized by strain MAHUQ-74 were 25 to 50 µg/mL for the tested E. coli O157:H7 strains. FE-SEM analysis showed that the biosynthesized AgNPs can cause changes in the morphology and structure of the foodborne pathogenic E. coli O157:H7 strain and destroy the integrity of the membrane, leading to cell death. This is the first study on the use of probiotic B. sonorensis MAHUQ-74 isolated from kimchi for the rapid and facile synthesis of bioactive AgNPs. AgNPs manufactured using B. sonorensis MAHUQ-74 may be potential antimicrobial agents for controlling antibiotic-resistant microorganisms, especially pathogenic strains of E. coli O157:H7.