Green Synthesis of Silver Nanoparticles Using Pseudoduganella eburnea MAHUQ-39 and Their Antimicrobial Mechanisms Investigation against Drug Resistant Human Pathogens

Silver nanoparticles (AgNPs) have shown great promise in biomedical applications. The exact mechanism and mode of action of AgNPs regarding antimicrobial activity are still not well known. Moreover, synthesis of nanoparticles by physical and chemical methods is expensive and not ecofriendly. This study highlights the green, rapid, facile, cost-effective and ecofriendly synthesis of AgNPs using Pseudoduganella eburnea MAHUQ-39 and also investigates their antibacterial mechanisms. The transmission electron microscopy (TEM) image revealed a spherical shape of the AgNPs. The size of the synthesized AgNPs was 8 to 24 nm. The elemental mapping and selected area electron diffraction (SAED) and X-ray diffraction (XRD) patterns revealed the crystalline structure of AgNPs. Fourier-transform infrared spectroscopy (FTIR) analysis identified the functional groups that are involved in the reduction of silver ion to AgNPs. The green synthesized AgNPs exhibited strong antimicrobial activity against multidrug-resistant pathogenic microbes. Minimal inhibitory concentrations (MICs) of Staphylococcus aureus and Pseudomonas aeruginosa were 100 μg/mL and 6.25 μg/mL, respectively, and the minimum bactericidal concentrations (MBCs) of S. aureus and P. aeruginosa were 200 μg/mL and 50 μg/mL, respectively. Our data demonstrated that synthesized AgNPs created structural changes of cells and destroyed the membrane integrity of strains S. aureus and P. aeruginosa. Therefore, AgNPs synthesized by strain MAHUQ-39 can be used as a powerful antimicrobial agent for various therapeutic applications.


Introduction
Nanoparticles (NPs) are a wide class of materials that attract many researchers because of their small size (1-100 nm), unique properties, large surface area, increased reactivity, ability to easily enter the human body and different applications in various fields of modern science, including agricultural and medical sciences. Different kinds of nanoparticles have been reported, including zinc, gold, silver, copper, alginate, titanium and magnesium [1][2][3]. Silver nanoparticles (AgNPs) are broadly used nanoparticles because of their potential applications in various fields of science, including optoelectronics, optics, pharmaceutical and medical sciences for the development of drug delivery systems, therapeutic agents, biosensors, gene therapy, etc. [4][5][6][7]. AgNPs also have been examined for remarkable applications such as on patients for bone cement, trauma, prostheses, dental implants, protective clothing [8,9], etc.
It is reported that AgNPs have strong antimicrobial, antibiofilm, anti-inflammatory and anticancer activities, out of which the antimicrobial properties of AgNPs protect against pathogenic microorganisms, including viruses, bacteria and eukaryotic micro-organisms, have been exploited [3,10].

Characterization of AgNPs Producing Bacterial Strain
The 16S rRNA gene sequence of strain MAHUQ-39 was 1412 bp and the sequence was submitted to NCBI (accession number MK680115). On the basis of the 16S rRNA gene sequence analysis, strain MAHUQ-39 showed the highest sequence similarity with Pseudoduganella eburnea 10R5-21 T (99.5%). The phylogenetic analysis using a neighbor-joining tree also indicated that strain MAHUQ-39 was affiliated with the genus Pseudoduganella ( Figure 1). The biochemical characterization of isolated strain MAHUQ-39 was carried out using API 20 NE and API ZYM; the results are shown in Table 1.

Green Synthesis of AgNPs Using Strain MAHUQ-39
Green synthesis of AgNPs using Pseudoduganella eburnea MAHUQ-39 was confirmed by observing the color of the culture supernatant. The color of MAHUQ-39 culture supernatant changed to deep brown from watery yellow within 24 h of incubation ( Figure 2B), which reveals the synthesis of AgNPs, as the biosynthesized silver nanoparticles cause surface plasmon resonance [3]. Control (without bacteria) did not show any change in color when incubated for the same conditions and period ( Figure 2A). The optimum temperature (30 • C) and AgNO 3 concentration (1 mM final concentration) for the stable synthesis of AgNPs were determined on the basis of ultraviolet-visible (UV-vis) spectral analysis ( Supplementary Figures S1 and S2). The accurate mechanism of synthesis of AgNPs using bacteria is still at an initial stage. Previous reports suggest that various enzymes secreted by bacteria are an important part of the green synthesis of nanoparticles. First, the metal ions are trapped on the surface or inside of bacterial cells and then the trapped metal ions are converted to nanoparticles in the presence of bacterial enzymes [20,21]. Two methods are available for the biosynthesis of silver nanoparticles using microorganisms which can be classified as intracellular or extracellular, depending on the place of nanoparticle synthesis. Extracellular synthesis of nanoparticles is more convenient, facile and cost-effective as the synthesized nanoparticles can be easily and quickly purified, in contrast to intracellular synthesis, which needs difficult and complex purification steps. In the present study, the extracellular methodology was used to green synthesize AgNPs using Pseudoduganella eburnea MAHUQ-39, which is simple, facile, cost-effective and ecofriendly.

Green Synthesis of AgNPs Using Strain MAHUQ-39
Green synthesis of AgNPs using Pseudoduganella eburnea MAHUQ-39 was confirmed by observing the color of the culture supernatant. The color of MAHUQ-39 culture supernatant changed to deep brown from watery yellow within 24 h of incubation ( Figure 2B), which reveals the synthesis of AgNPs, as the biosynthesized silver nanoparticles cause surface plasmon resonance [3]. Control (without bacteria) did not show any change in color when incubated for the same conditions and period ( Figure 2A). The optimum temperature (30 °C) and AgNO3 concentration (1 mM final concentration) for the stable synthesis of AgNPs were determined on the basis of ultraviolet-visible (UV-vis) spectral analysis (Supplementary Figures S1 and S2). The accurate mechanism of synthesis of AgNPs using bacteria is still at an initial stage. Previous reports suggest that various enzymes secreted by bacteria are an important part of the green synthesis of nanoparticles. First, the metal ions are trapped on the surface or inside of bacterial cells and then the trapped metal ions are converted to nanoparticles in the presence of bacterial enzymes [20,21]. Two methods are available for the biosynthesis of silver nanoparticles using microorganisms which can be classified as intracellular or extracellular, depending on the place of nanoparticle synthesis. Extracellular synthesis of nanoparticles is more convenient, facile and cost-effective as the synthesized nanoparticles can be easily and quickly purified, in contrast to intracellular synthesis, which needs difficult and complex purification steps. In the present study, the extracellular methodology was used to green synthesize AgNPs using Pseudoduganella eburnea MAHUQ-39, which is simple, facile, cost-effective and ecofriendly.

Characterization of Green Synthesized AgNPs
The biosynthesis of AgNPs was assured by UV-vis spectral scanning in a range of 300-800 nm; a strong peak at 448 nm was observed ( Figure 2C). It is reported that the absorption peak at about 400-450 nm is due to surface plasmon resonance of AgNPs, which confirmed the formation of AgNPs [22]. Field emission-transmission electron microscopy (FE-TEM) images showed clear morphology of green synthesized AgNPs. FE-TEM analysis disclosed a spherical shape of synthesized AgNPs with a size range of 8 to 24 nm ( Figure 2D,E). Previous studies also showed a similar size and shape of biosynthesized AgNPs [15]. The composition and purity of green synthesized AgNPs were analyzed using an EDX spectrometer. The EDX spectrum exhibited elemental signals of silver atoms in biosynthesized AgNPs at around 3 keV and revealed homogenous distribution of AgNPs ( Figure 3A). Previous reports also suggest that the EDX pattern of AgNPs commonly exhibits an absorption peak nearly at 3 keV [18]. The EDX spectrum also showed other signals for copper and carbon due to the use of a copper grid in the FE-TEM. The highest distribution of silver elements in the sample was found through the elemental mapping of green synthesized AgNPs ( Figure 3B,C). The XRD spectrum of the synthesized AgNPs revealed the diffraction peaks at 2θ values of 38.13 • , 44.31 • , 64.44 • and 77.42 • , matching the corresponding 111, 200, 220 and 311 lattice planes of AgNPs. These diffraction peaks may have resulted from the capping agents that are responsible for the stabilization of nanoparticles. The absence of other peaks reveals the purity of green synthesized AgNPs ( Figure 4A). Similar XRD results were found in previous studies that showed the biosynthesis of silver nanoparticles using microorganisms [3,15]. The elemental mapping and selected area diffraction (SAED) pattern exhibited sharp rings that indicate the crystalline structure of green synthesized AgNPs and correspond to the reflections at 111, 200, 220 and 311 ( Figure 4B). The crystalline structure of biosynthesized AgNPs was confirmed from both the SAED pattern and XRD spectrum.

Characterization of Green Synthesized AgNPs
The biosynthesis of AgNPs was assured by UV-vis spectral scanning in a range of 300-800 nm; a strong peak at 448 nm was observed ( Figure 2C). It is reported that the absorption peak at about 400-450 nm is due to surface plasmon resonance of AgNPs, which confirmed the formation of AgNPs [22]. Field emission-transmission electron microscopy (FE-TEM) images showed clear morphology of green synthesized AgNPs. FE-TEM analysis disclosed a spherical shape of synthesized AgNPs with a size range of 8 to 24 nm ( Figure 2D,E). Previous studies also showed a similar size and shape of biosynthesized AgNPs [15]. The composition and purity of green synthesized AgNPs were analyzed using an EDX spectrometer. The EDX spectrum exhibited elemental signals of silver atoms in biosynthesized AgNPs at around 3 keV and revealed homogenous distribution of AgNPs ( Figure  3A). Previous reports also suggest that the EDX pattern of AgNPs commonly exhibits an absorption peak nearly at 3 keV [18]. The EDX spectrum also showed other signals for copper and carbon due to the use of a copper grid in the FE-TEM. The highest distribution of silver elements in the sample was found through the elemental mapping of green synthesized AgNPs ( Figure 3B,C). The XRD spectrum of the synthesized AgNPs revealed the diffraction peaks at 2θ values of 38.13°, 44.31°, 64.44° and 77.42°, matching the corresponding 111, 200, 220 and 311 lattice planes of AgNPs. These diffraction peaks may have resulted from the capping agents that are responsible for the stabilization of nanoparticles. The absence of other peaks reveals the purity of green synthesized AgNPs ( Figure 4A). Similar XRD results were found in previous studies that showed the biosynthesis of silver nanoparticles using microorganisms [3,15]. The elemental mapping and selected area diffraction (SAED) pattern exhibited sharp rings that indicate the crystalline structure of green synthesized AgNPs and correspond to the reflections at 111, 200, 220 and 311 ( Figure 4B). The crystalline structure of biosynthesized AgNPs was confirmed from both the SAED pattern and XRD spectrum.   The FTIR data indicate that the biological molecules could possibly be involve for both synthesis and stabilization of AgNPs. The present data were supported by the previous report, which showed the microbial synthesis of nanoparticles [23]. The particle size of green synthesized AgNPs was calculated by dynamic light scattering (DLS) analysis. Figure 6 shows the particle size distribution of biosynthesized AgNPs on the basis of volume, number and intensity. The average particle size of synthesized AgNPs was about 141.2 nm with a polydispersity value of 0.369. The polydispersity value indicates the good quality of synthesized AgNPs. Previous reports also showed similar nanoparticle size when synthesized by microorganisms [3,7]. The FTIR data indicate that the biological molecules could possibly be involve for both synthesis and stabilization of AgNPs. The present data were supported by the previous report, which showed the microbial synthesis of nanoparticles [23]. The particle size of green synthesized AgNPs was calculated by dynamic light scattering (DLS) analysis. Figure 6 shows the particle size distribution of biosynthesized AgNPs on the basis of volume, number and intensity. The average particle size of synthesized AgNPs was about 141.2 nm with a polydispersity value of 0.369. The polydispersity value indicates the good quality of synthesized AgNPs. Previous reports also showed similar nanoparticle size when synthesized by microorganisms [3,7].

Antimicrobial Activity of Green Synthesized AgNPs
Drug-resistant pathogenic bacteria have become a major concern for human health, as it is difficult to control these pathogens by using currently available antibiotics. Green synthesized AgNPs can be used to overcome the drawbacks of commercial antibiotics. Biosynthesized AgNPs are well known for their antimicrobial potency against various pathogenic microorganisms [3,15,17,24]. In the current study, AgNPs were synthesized using a biological process and their antibacterial potency was investigated against drug-resistant pathogenic microbes (Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa). Results showed that synthesized AgNPs have significant antimicrobial activity against all tested human pathogens such as S. aureus, P. aeruginosa and E. coli (Figure 7). Figure 7 reveals the clear zone of inhibition around the discs saturated with synthesized AgNPs (0.03 mL/disc), at both 500 μg/mL and 1000 μg/mL. The antibacterial efficacy against pathogenic microorganisms was measured by calculating the diameter of the zone of inhibition ( Table 2). The synthesized AgNPs exhibited the highest antibacterial activity against drug resistant P. aeruginosa followed by E. coli and S. aureus. The results of this study propose that the green synthesized AgNPs were able to control the multidrug resistant pathogenic bacteria and could be used in the medical field. Commercial antibiotics such as vancomycin, erythromycin and penicillin G were also used to check their antibacterial efficacy against human pathogens such as P. aeruginosa, E. coli and S. aureus. All three human pathogens were resistant to the commercial antibiotics vancomycin, erythromycin and penicillin G, and did not show any inhibition zone around the antibiotic discs (Figure 8, Table 3).

Antimicrobial Activity of Green Synthesized AgNPs
Drug-resistant pathogenic bacteria have become a major concern for human health, as it is difficult to control these pathogens by using currently available antibiotics. Green synthesized AgNPs can be used to overcome the drawbacks of commercial antibiotics. Biosynthesized AgNPs are well known for their antimicrobial potency against various pathogenic microorganisms [3,15,17,24]. In the current study, AgNPs were synthesized using a biological process and their antibacterial potency was investigated against drug-resistant pathogenic microbes (Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa). Results showed that synthesized AgNPs have significant antimicrobial activity against all tested human pathogens such as S. aureus, P. aeruginosa and E. coli (Figure 7). Figure 7 reveals the clear zone of inhibition around the discs saturated with synthesized AgNPs (0.03 mL/disc), at both 500 μg/mL and 1000 μg/mL. The antibacterial efficacy against pathogenic microorganisms was measured by calculating the diameter of the zone of inhibition ( Table 2). The synthesized AgNPs exhibited the highest antibacterial activity against drug resistant P. aeruginosa followed by E. coli and S. aureus. The results of this study propose that the green synthesized AgNPs were able to control the multidrug resistant pathogenic bacteria and could be used in the medical field. Commercial antibiotics such as vancomycin, erythromycin and penicillin G were also used to check their antibacterial efficacy against human pathogens such as P. aeruginosa, E. coli and S. aureus. All three human pathogens were resistant to the commercial antibiotics vancomycin, erythromycin and penicillin G, and did not show any inhibition zone around the antibiotic discs (Figure 8, Table 3).

Antimicrobial Activity of Green Synthesized AgNPs
Drug-resistant pathogenic bacteria have become a major concern for human health, as it is difficult to control these pathogens by using currently available antibiotics. Green synthesized AgNPs can be used to overcome the drawbacks of commercial antibiotics. Biosynthesized AgNPs are well known for their antimicrobial potency against various pathogenic microorganisms [3,15,17,24]. In the current study, AgNPs were synthesized using a biological process and their antibacterial potency was investigated against drug-resistant pathogenic microbes (Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa). Results showed that synthesized AgNPs have significant antimicrobial activity against all tested human pathogens such as S. aureus, P. aeruginosa and E. coli (Figure 7). Figure 7 reveals the clear zone of inhibition around the discs saturated with synthesized AgNPs (0.03 mL/disc), at both 500 µg/mL and 1000 µg/mL. The antibacterial efficacy against pathogenic microorganisms was measured by calculating the diameter of the zone of inhibition ( Table 2). The synthesized AgNPs exhibited the highest antibacterial activity against drug resistant P. aeruginosa followed by E. coli and S. aureus. The results of this study propose that the green synthesized AgNPs were able to control the multidrug resistant pathogenic bacteria and could be used in the medical field. Commercial antibiotics such as vancomycin, erythromycin and penicillin G were also used to check their antibacterial efficacy against human pathogens such as P. aeruginosa, E. coli and S. aureus. All three human pathogens were resistant to the commercial antibiotics vancomycin, erythromycin and penicillin G, and did not show any inhibition zone around the antibiotic discs ( Figure 8, Table 3). 8 of 15

Minimum Inhibitory Concentration and Minimum Bactericidal Concentration
In the current study, the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of AgNPs against P. aeruginosa and S. aureus were determined by a standard broth dilution assay. Bacterial growth curves exhibiting the MICs of green synthesized AgNPs were 6.25 µg/mL and 100 µg/mL for P. aeruginosa and S. aureus, respectively, indicating that the synthesized AgNPs strongly suppressed the proliferation of these two pathogens (Figure 9). The MICs of the AgNPs synthesized by strain MAHUQ-39 were much lower than other antimicrobial agents, including biosynthesized ZnO NPs, which were 8 mg/mL and 4 mg/mL against E. coli and S. aureus, respectively [25]. The MBCs of biosynthesized AgNPs for P. aeruginosa and S. aureus were 50 µg/mL and 200 µg/mL, respectively (Figure 10), which were also much lower than those of other antimicrobial agents [25]. The MIC and MBC of green synthesized AgNPs were higher for S. aureus than those for P. aeruginosa. The current and previous data displayed a higher AgNPs susceptibility of Gram-negative bacteria compared to that of Gram-positive bacteria. This might due to distinctions in the composition of their cell wall [26].

Minimum Inhibitory Concentration and Minimum Bactericidal Concentration
In the current study, the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of AgNPs against P. aeruginosa and S. aureus were determined by a standard broth dilution assay. Bacterial growth curves exhibiting the MICs of green synthesized AgNPs were 6.25 μg/mL and 100 μg/mL for P. aeruginosa and S. aureus, respectively, indicating that the synthesized AgNPs strongly suppressed the proliferation of these two pathogens (Figure 9). The MICs of the AgNPs synthesized by strain MAHUQ-39 were much lower than other antimicrobial agents, including biosynthesized ZnO NPs, which were 8 mg/mL and 4 mg/mL against E. coli and S. aureus, respectively [25]. The MBCs of biosynthesized AgNPs for P. aeruginosa and S. aureus were 50 μg/mL and 200 μg/mL, respectively (Figure 10), which were also much lower than those of other antimicrobial agents [25]. The MIC and MBC of green synthesized AgNPs were higher for S. aureus than those for P. aeruginosa. The current and previous data displayed a higher AgNPs susceptibility of Gram-negative bacteria compared to that of Gram-positive bacteria. This might due to distinctions in the composition of their cell wall [26].

Study of Morphogenesis of Treated Cells by FE-SEM
Morphological and ultrastructural changes of P. aeruginosa and S. aureus cells treated with green synthesized AgNPs were directly observed by FE-SEM ( Figure 11). By examining the FE-SEM, the untreated P. aeruginosa cells were seen as typically normal rod-shaped and intact cell surfaces with no damage ( Figure 11A). However, P. aeruginosa cells treated with 1 × MBC of synthesized AgNPs displayed irregularly wrinkled, damaged, deformed and cracked outer surfaces with collapsed cell membranes ( Figure 11B). Similarly, untreated S. aureus cells were also seen as regular plump and sphere-shaped, with intact and smooth cell surfaces ( Figure 11C), while the cells treated with 1 × MBC of biosynthesized AgNPs were deformed and damaged in the middle of the cell. The normal shape of S. aureus cells was changed and appeared as distorted and damaged ( Figure 11D). The morphological changes and damages of bacterial cell walls revealed that the AgNPs might interfere with the normal function of cells. The images revealed a higher sensitivity of P. aeruginosa than S. aureus, which might due to the Gram-negative bacteria and Gram-positive bacteria, as they have a cell wall with different structures and components. Similar results were shown by Chatterjee et al., who investigated the cell damage mechanism of silver nanoparticles against E. coli and S. aureus [26]. There are some mechanisms for the antimicrobial activities of AgNPs that have been proposed and reported. The widely accepted mechanism of antimicrobial activity of AgNPs is that the positively charged silver ions present in AgNPs can interfere with different negatively charged phosphorus or sulfur-bearing bio-macromolecules such as proteins and nucleic acid. This causes structural alterations in bacterial cells, deformations and damages of the cell wall and cell membrane that leads to interruption of metabolic processes, and finally cell death [27]. Reports also suggest that different free radicals that are derived from the surface of nanoparticles are responsible for the damage to the cell membrane by enhancing membrane permeability, which leads to cell death [28].

Study of Morphogenesis of Treated Cells by FE-SEM
Morphological and ultrastructural changes of P. aeruginosa and S. aureus cells treated with green synthesized AgNPs were directly observed by FE-SEM ( Figure 11). By examining the FE-SEM, the untreated P. aeruginosa cells were seen as typically normal rod-shaped and intact cell surfaces with no damage ( Figure 11A). However, P. aeruginosa cells treated with 1 × MBC of synthesized AgNPs displayed irregularly wrinkled, damaged, deformed and cracked outer surfaces with collapsed cell membranes ( Figure 11B). Similarly, untreated S. aureus cells were also seen as regular plump and sphere-shaped, with intact and smooth cell surfaces ( Figure 11C), while the cells treated with 1 × MBC of biosynthesized AgNPs were deformed and damaged in the middle of the cell. The normal shape of S. aureus cells was changed and appeared as distorted and damaged ( Figure 11D). The morphological changes and damages of bacterial cell walls revealed that the AgNPs might interfere with the normal function of cells. The images revealed a higher sensitivity of P. aeruginosa than S. aureus, which might due to the Gram-negative bacteria and Gram-positive bacteria, as they have a cell wall with different structures and components. Similar results were shown by Chatterjee et al., who investigated the cell damage mechanism of silver nanoparticles against E. coli and S. aureus [26]. There are some mechanisms for the antimicrobial activities of AgNPs that have been proposed and reported. The widely accepted mechanism of antimicrobial activity of AgNPs is that the positively charged silver ions present in AgNPs can interfere with different negatively charged phosphorus or sulfur-bearing bio-macromolecules such as proteins and nucleic acid. This causes structural alterations in bacterial cells, deformations and damages of the cell wall and cell membrane that leads to interruption of metabolic processes, and finally cell death [27]. Reports also suggest that different free radicals that are derived from the surface of nanoparticles are responsible for the damage to the cell membrane by enhancing membrane permeability, which leads to cell death [28].

Isolation and Identification of Strain MAHUQ-39
The bacterial strain Pseudoduganella eburnea MAHUQ-39 was isolated from a soil sample collected from a pear orchard, located in Anseong, Republic of Korea. One gm of soil sample was dissolved in 10 mL 0.85% NaCl sterile NaCl solution, serially diluted and spread on R2A agar (Reasoner's 2A agar) plates containing 0.5 gm proteose peptone No. 3, 0.5 gm yeast extract, 0.5 gm casamino acids, 0.5 gm soluble starch, 0.5 gm dextrose, 0.3 gm dipotassium phosphate, 0.3 gm sodium pyruvate, 0.05 gm magnesium sulfate and 15 gm agar in 1000 mL distill water. To check the AgNPs synthesis ability, the isolated strains were streaked on an R2A agar plate supplemented with 1 mM filter-sterilized AgNO 3 solution. The plates were incubated in an incubator for 48 h at 30 • C and then investigated for bacterial growth. The positive strain was preserved at −80 • C as a suspension in R2A broth with 25% glycerol (v/v). Strain MAHUQ-39 has been deposited in the Korean Agricultural Culture Collection (KACC number 21243). Genomic DNA of isolated strain MAHUQ-39 was extracted using bacterial a Genomic DNA extraction kit (Solgent, Korea). The 16S rRNA gene was amplified using bacterial universal primers (27F and 1492R) and sequenced by Solgent Co., Ltd. (Daejeon, Korea). The 16S rRNA gene sequences of related type strains were collected from the EzTaxon-e server and GenBank database [29]. To discover the phylogenetic location of strain MAHUQ-39, its phylogenetic tree was constructed using the neighbor-joining method [30] and the MEGA6 program package [31]. Enzyme activities and biochemical characteristics of strain MAHUQ-39 were examined using API 20NE and API ZYM kits (bioMe'rieux, France) according to the manufacturer's instructions.

Green Synthesis of AgNPs Using Strain MAHUQ-39
For the green synthesis of AgNPs, strain Pseudoduganella eburnea MAHUQ-39 was cultured in 100 mL of R2A broth and incubated in a shaking incubator with 140 rpm for two days at 30 • C. After two days of incubation, the culture supernatant was collected using a centrifuge at 9000 rpm for 15 min. A 0.1 mL (1 M concentration) filter-sterilized AgNO 3 solution was added to 100 mL of supernatant and again incubated in an orbital shaker for 24 h at 140 rpm and 30 • C. Synthesis of AgNPs was confirmed by visually observing the changing color of the culture medium. Finally, the biosynthesized AgNPs were collected by high speed centrifugation at 14,000 rpm for 20 min. The pellets of green synthesized AgNPs were washed with distilled water and then air-dried and finally used for characterization and antimicrobial studies.

Characterization of Green Synthesized AgNPs
The green synthesis of AgNPs was monitored and recorded using a UV-vis (ultraviolet-visible) spectrophotometer in the range of 300-800 nm. The morphology, elemental compositions and purity of synthesized AgNPs were investigated by FE-TEM (field emission-transmission electron microscopy), energy dispersive X-ray (EDX) spectroscopy and elemental mapping and selected area diffraction (SAED) pattern. A drop of biosynthesized AgNPs solution (dissolved in distilled water) was kept on a carbon-coated copper grid, then dried at room temperature and, finally, transferred to the microscope for analysis. The XRD (X-ray diffraction) analysis was carried out using an X-ray diffractometer (D8 Advance, Bruker, Germany) over the 2θ range of 30-90 • , operated with CuKα radiation, at 40 kv, 40 mA and 6 • /min scanning rate. Air-dried samples were used for XRD analysis. The FTIR (Fourier-transform-infrared) spectrum shows the biomolecules that are involved for the biosynthesis and stabilization of AgNPs. FTIR analysis was accomplished by using a PerkinElmer Fourier-transform infrared spectrometer with resolution 4 cm −1 in the range of 400-4000 cm −1 . For FTIR analysis, air-dried samples in powder form were used. Particle size of green synthesized AgNPs were determined by dynamic light scattering (DLS) using a Malvern Zetasizer Nano ZS90 at 25 • C and a 12 • angle. Pure water was used as a dispersive medium with dielectric constant 78.3, viscosity 0.8878 cP and refractive index 1.3328.

Antimicrobial Activity of Green Synthesized AgNPs
The disc diffusion method was used to investigate the antimicrobial efficacy of green synthesized AgNPs against human pathogens. The tested pathogenic microbes (Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli) were grown overnight in MHB Mueller-Hinton (MH) broth. A 0.1 mL sample of overnight growth bacterial culture of each tested pathogen was spread on a Mueller-Hinton (MH) agar plate. The sterile paper discs (8 mm in diameter) containing 0.03 mL of synthesized AgNPs (both 500 µg/mL and 1000 µg/mL, dissolved in distilled water) were placed on the surface of the inoculated MH agar plates. Then, the plates were incubated in an incubator for 24 h at 37 • C. Similarly, the antibacterial activity of commercial antibiotics such as penicillin G (6 µg/disc), erythromycin (15 µg/disc) and vancomycin (30 µg/disc) was tested against S. aureus, P. aeruginosa and E. coli. The inhibition zones were measured after 24 h of incubation. This test was performed in triplets.

Determination of MIC and MBC
S. aureus and P. aeruginosa were selected for this study as Gram-positive and Gram-negative model strains, respectively. Both strains were grown in MH broth at 37 • C for overnight and then the turbidity was adjusted to nearly 10 6 CFUs/mL. MICs of the green synthesized AgNPs were calculated using a broth microdilution assay. Control experiments were carried out without AgNPs. Briefly, serial dilutions of green synthesized AgNPs were prepared in sterile R2A broth with different concentrations ranging from 200 µg/mL to 3.12 µg/mL. A 0.1 mL sample of each AgNPs dilution was kept in a 96-well plate, and inoculated with 0.1 mL of the test organism in R2A broth to a final concentration of 5 × 10 5 CFU/mL, and further incubated in a shaking incubator at 37 • C for 24 h. The MIC was measured as the lowest concentration of AgNPs at which the growth of the bacterium was entirely inhibited after 24 h. The optical density at 600 nm was recorded every 3 h during incubation using an ultraviolet-visible spectrophotometer. MBC was determined by streaking 0.01 mL of each set on an MH agar plate and further incubating at 37 • C for 24 h. The culture plates were watched by the naked eye to determine the lowest concentration (MBC) that blocked bacterial growth [32].

Investigation of Antibacterial Mechanisms via Study of Morphogenesis of Treated Cells
The morphological changes of strains S. aureus and P. aeruginosa were examined by SEM. Logarithmic growth phase cells in a concentration of 1 × 10 7 CFU/mL were treated with the green synthesized AgNPs (dissolved in 0.85% NaCl) at a concentration of 1 × MBC for 6 h. In control, bacterial cultures were treated with 0.85% NaCl solution. Phosphate-buffered saline (PBS) was used to wash the treated cells. A 2.5% glutaraldehyde solution was used to fix the cells for 4 h and then washed with PBS several times. Again, the cells were fixed using 1% osmium tetroxide and then washed with PBS buffer. Fixed cells were dehydrated by different concentrations of ethanol (30%, 50%, 70%, 80%, 95% and 100% for 10 min) at room temperature. The resulted samples were dried using a desiccator. Finally, the cells were placed on SEM metallic stubs and coated with gold. The morphological and structural alterations of the cells were imaged by FE-SEM (JSM-7100F, JEOL, Japan) [32].

Conclusions
The current study reports the green, facile and rapid synthesis of AgNPs using a culture supernatant of Pseudoduganella eburnea MAHUQ-39 and also displays their antibacterial efficacy and mechanisms against drug-resistant human pathogens. The method used in this study for green synthesis of AgNPs is rapid, facile, straightforward, convenient, economical and ecofriendly. The strain Pseudoduganella eburnea MAHUQ-39 is able to synthesis AgNPs within 24 h of incubation. Moreover, an extracellular method was used to synthesize AgNPs that makes the process easy, rapid and convenient, which could be used for high production. Additionally, the green synthesized AgNPs show significant antimicrobial activity against antibiotic-resistant pathogenic strains of E. coli, S. aureus and P. aeruginosa. MICs of P. aeruginosa and S. aureus were 6.25 µg/mL and 100 µg/mL, respectively, and the MBCs of P. aeruginosa and S. aureus were 50 µg/mL and 200 µg/mL, respectively. Synthesized AgNPs can destroy the membrane integrity and cause morphological changes in strains of P. aeruginosa and S. aureus, leading to cell death. This study may help to describe the antibacterial mechanism of green synthesized AgNPs for both Gram-negative and Gram-positive pathogenic bacteria. This is the first report for the green synthesis of AgNPs using Pseudoduganella eburnea MAHUQ-39. The strain Pseudoduganella eburnea MAHUQ-39 could be useful for the massive production of AgNPs, and biosynthesized AgNPs can be applied for various applications in medical and nonmedical fields.
Supplementary Materials: The following are available online at http://www.mdpi.com/1422-0067/21/4/1510/s1, Figure S1: Effect of temperature on the biosynthesis of AgNPs using strain Pseudoduganella eburnea MAHUQ-39 was checked on the basis of UV-vis spectral analysis after 24 h of incubation with 1 mM concentration of AgNO 3 , Figure S2: Effect of salt concentration (AgNO 3 ) on the biosynthesis of AgNPs using strain Pseudoduganella eburnea MAHUQ-39 was checked on the basis of UV-vis spectral analysis after 24 h of incubation at 30 • C.

Conflicts of Interest:
The author declares no conflict of interest.