Endophytic Bacteria Enterobacter hormaechei Fabricated Silver Nanoparticles and Their Antimicrobial Activity

Antimicrobial resistance (AMR), one of the greatest issues for humankind, draws special attention to the scientists formulating new drugs to prevent it. Great emphasis on the biological synthesis of silver nanoparticles (AgNPs) for utilization in single or combinatorial therapy will open up new avenues to the discovery of new antimicrobial drugs. The purpose of this study was to synthesize AgNPs following a green approach by using an endophytic bacterial strain, Enterobacter hormaechei, and to assess their antimicrobial potential against five pathogenic and four multidrug-resistant (MDR) microbes. UV-Vis spectroscopy, fourier-transform infrared spectroscopy (FTIR), transmission electron microscopy (TEM), scanning electron microscopy-energy dispersive X-ray spectroscopy (SEM-EDX), and zeta potential (ζ) were used to characterize the synthesized AgNPs. Endophytic E. hormaechei-mediated AgNPs (Eh-AgNPs) were represented by a strong UV-Vis absorbance peak at 418 nm within 5 min, forming spherical and polydispersed nanoparticles in the size range of 9.91 nm to 92.54 nm. The Eh-AgNPs were moderately stable with a mean ζ value of −19.73 ± 3.94 mV. The presence of amine, amide, and hydroxyl functional groups was observed from FTIR analysis. In comparison to conventional antibiotics, the Eh-AgNPs were more effective against Bacillus cereus (ATCC 10876) and Candida albicans (ATCC 10231), exhibiting 9.14 ± 0.05 mm and 8.24 ± 0.05 mm zones of inhibition (ZOIs), respectively, while displaying effective inhibitory activity with ZOIs ranging from 10.98 ± 0.08 to 13.20 ± 0.07 mm against the MDR bacteria. Eh-AgNP synthesis was rapid and eco-friendly. The results showed that Eh-AgNPs are promising antimicrobial agents that can be used in the development and formulation of new drugs to curb the menace of antimicrobial resistance in pathogenic and MDR microbes.


Introduction
Antimicrobial resistance (AMR) is a state where microorganisms become resistant to antimicrobial drugs, resulting in ineffective treatment with existing antibiotics. AMR is a global crisis that poses a terrifying challenge to the achievement of universal health coverage. Alarming levels of resistance jeopardize the advancement of many sustainable development goals, including health. The misuse and overuse of persistent antimicrobials have heightened the development and extent of AMR. Increases in AMR are driven by the spread of microbes exposed to antimicrobial compounds and their mechanisms of resistance. AMR is accelerated when antimicrobial compounds are persistent in the environment or in the microbes' hosts [1]. Presently, about 0.7 million deaths occur annually worldwide owing to drug-resistant diseases [2]. Without a sustained effort to control AMR, mortality arising from drug-resistant diseases could rise to 10 million globally per year by 2050 in the worst-case scenario [2]. Nonetheless, AMR is a growing crisis worldwide, Pharmaceutics 2021, 13, 511 3 of 15 as a reliable storehouse of bioactive and chemically novel compounds that may be used in combating numerous pathogenic and drug-resistant microbes affecting humans [18]. Recent reports in the literature suggest that AgNPs fabricated with endophytic bacteria possess effective antimicrobial activities [23,[28][29][30][31]. Taking into account the bio-prospects of endophytic bacteria, the present study aimed to explore the biosynthesis of AgNPs using endophytic E. hormaechei and characterize the synthesized bacteria and their possible applications as antimicrobial agents against pathogenic microbes and multidrug-resistant (MDR) bacteria.

Culture of the Endophyte and Preparation of Cell-Free Extract
Isolates of the endophytic Enterobacter hormaechei (GenBank accession no HQ694367), which had been previously assessed for molecular identification using 16S rRNA [32], were collected from the laboratory of AIMST University, Malaysia. The isolates were grown on nutrient broth (HiMedia Lab. Ltd., Mumbai, India) at 37 • C for 24 h, then taken into 250 mL sterile Luria Bertani broth (HiMedia Lab. Ltd., Mumbai, India) and incubated (37 • C, 24 h, 180 rpm) in a rotary incubator shaker (Innova 40, New Brunswick Scientific Co., New York, NY, USA). The overnight culture was transferred into a 50 mL Beckman tube (Beckman Coulter, Inc., Pasadena, CA, USA) and the bacterial cells were centrifuged at 8000 rpm for 10 min at 4 • C using a centrifuge machine (Avanti J-26 XPI, Beckman Coulter, Inc., Pasadena, CA, USA). The resulting supernatant, termed as cell-free extract (CFE), was used immediately for extracellular synthesis of AgNPs.

Synthesis of Eh-AgNPs
Freshly prepared CFE was added to 100 mL of 0.1 mM silver nitrate (2%, v/v) solution (AgNO 3 , Fisher Scientific, Hampton, NH, USA) and was exposed to bright sunlight (temperature: 32 • C, solar intensity:~72,000 lux) for a period of 60 min. The formation of Eh-AgNPs was monitored at regular time intervals (5,15,30, and 60 min) through observation of the colour change pattern of the reaction mixture. Simultaneously, a similar experiment was carried out under dark conditions (25 • C, 0 lux, 24 h) to explore the role of sunlight in Eh-AgNP synthesis. AgNO 3 solution (0.1 mM) without the presence of CFE was used as the negative control. Pellets of the synthesized nanoparticles in the reaction mixture were centrifuged at 12,000 rpm for 15 min at 4 • C using the centrifuge machine. The pellets were washed (×3) with deionized water and dried in a vacuum dryer (Yamato Scientific Co. Ltd., Tokyo, Japan).

Characterization of Eh-AgNPs
UV-Vis spectroscopy, fourier-transform infrared spectroscopy (FTIR) spectroscopy, TEM, SEM-EDX, and Zeta potential were used to characterize the fabricated Eh-AgNPs [23]. The UV-Vis spectrum of the synthesized Eh-AgNPs was recorded in the range of 200-800 nm using a UV-Vis spectrophotometer (DU-800, Beckman Coulter, Inc., Pasadena, CA, USA). FTIR spectroscopy of the Eh-AgNPs was obtained using an FTIR spectrophotometer (PE 1600, GMI Inc., Ramsey, MN, USA) over the spectrum of 400-4000 cm −1 at a resolution of 4 cm −1 . Morphology, size, and distribution of the Eh-AgNPs were observed using a Philips CM 12 TEM system equipped with Philips Docu Version 3.2 image analysis software (Philips Electron Optics, Eindhoven, The Netherlands) at 120 kV. Further morphology of the Eh-AgNPs was observed using scanning electron microscopy (SEM, Phenom-World B.V., Eindhoven, The Netherlands) running at 15 kV. Energy dispersive X-ray (EDX, Phenom-World B.V., Eindhoven, The Netherlands) spectroscopy was performed using an energy dispersive spectrum to identify elementary compositions of the Eh-AgNPs. Zeta potential of the Eh-AgNPs was calculated usinga zeta potential analyser (Zetasizer, ver. 7.11, Malvern Instruments Ltd., Malvern, UK).
The pathogenic and MDR bacteria were grown on nutrient broth (HiMedia Lab. Ltd., Mumbai, India) at 37 • C for 24 h while pathogenic fungal strains were grown on potato dextrose broth (HiMedia Lab. Ltd., Mumbai, India) at 30 • C for 24 h in sterile screw-cap test tubes. Thereafter, the pathogenic and MDR microbes were cultured on nutrient agar media (HiMedia Lab. Ltd., Mumbai, India) at 37 • C for 24 h while fungal isolates were cultured on potato dextrose agar media (HiMedia Lab. Ltd., Mumbai, India) at 30 • C for 24 h in Petri plates. Colony suspension (~10 6 CFU/mL) for each microorganism was maintained at an equivalent to 0.5 McFarland standard prepared in Mueller-Hinton agar (MHA) (HiMedia Lab. Ltd., Mumbai, India) at pH 7. Microorganisms were swabbed on Petri plates containing MHA to perform the antimicrobial susceptibility test following the disk-diffusion method [33]. Sterile Whatman No. 1 filter paper (Sigma-Aldrich, St. Louis, MO, USA) disks of 6 mm diameter were impregnated with 6 µg and 10 µg of Eh-AgNPs to test the antimicrobial activity against the pathogenic microbes and MDR bacteria, respectively. The Eh-AgNP disks and/or antibiotic disks were placed in the MHA and incubated for 24 h at 37 • C and 30 • C for bacterial and fungal isolates, respectively. A vernier calliper (Mitutoyo, S-530, Mitutoyo Co., Kawasaki, Japan) was used to measure the zone of inhibition (ZOI). Simultaneously, similar experiments were conducted using freshly prepared CFE and AgNO 3 solution as the negative control.
Minimum inhibitory concentration (MIC) was estimated by the broth macrodilution method [36] following CLSI guidelines [37]. For this reason, a trial-and-error method was applied to standardize the working concentrations of Eh-AgNPs [38]. Different concentrations of the Eh-AgNPs in 4 mL nutrient broth were prepared in test tubes following two-fold serial dilution. An aliquot (0.2 mL) of microbial suspension (~10 6 CFU/mL) was added to each test tube. A positive control with microbial suspension (~10 6 CFU/mL) in nutrient broth and negative control with nutrient broth were also maintained. Thereafter, the test tubes with pathogenic and MDR bacteria were incubated at 37 • C for 24 h while those of the pathogenic fungus were incubated at 30 • C for 24 h. The lowest concentration of the Eh-AgNPs at which there was no visible growth of the organisms was considered to be the MIC [36].

Statistical Analysis
The results of the triplicate experiments were expressed as mean ± SD. One-way ANOVA followed by Tukey's honestly significant difference (HSD) test with a significance level of 0.05 were analysed using IBM SPSS for Windows, Version 22.0 (IBM Corp., Armonk, NY, USA).

Synthesis and Characterization of Eh-AgNPs
The reaction mixture of the CFE and AgNO 3 solution turned from whitish to deep yellowish brown (Figure 1) within 60 min. Formation of Eh-AgNPs was confirmed by UV-Vis spectrum of the reaction mixture. A clear surface plasmon resonance (SPR) was formed within 5 min resulting in 418 nm of wavelength in the UV-Vis spectrum ( Figure 2). In contrast, little colour change was observed in the reaction mixture kept in the dark while the negative control mixture revealed no colour change. FTIR analysis was carried out to assess possible functional biomolecules responsible for the reduction of Ag + to Ag 0 . The FTIR spectrum of the Eh-AgNPs showed a number of absorption peaks ( Figure 3). The absorbance peaks at 3190 cm −1 and 2834 cm −1 corresponded to N-H functional groups of primary amine in proteins, while the peak at 1531 cm −1 corresponded to C=N stretching vibration due to secondary amide in proteins [39]. The peaks at 2930, 2917, and 2860 cm −1 were attributed to C-H stretching of aliphatic groups [39]. The peak at 2339 cm −1 corresponded to asymmetric O=C=O stretching of CO 2 , while the weak band at 2110 cm −1 was due to C≡C stretching vibration of the aliphatic groups [40]. The band at 1640 cm −1 attributed to primary amide was due to C=O stretching in proteins and H-O-H deformation of water [39]. The peaks at 1389, 1215, and 1060 cm −1 were assigned to the symmetric deformation of the vibration of O-H groups in alcohols, C-O stretching of aromatic ether, and S=O stretching vibration of sulfoxides, respectively [40]. The peaks at around 808 cm −1 and below were attributed to C-H aromatic vibrations [41]. The Eh-AgNPs were mostly spherical, polydispersed, and inconsistent in size as shown in the TEM image ( Figure 4). Descriptive analysis and a particle-size distribution histogram of 200 arbitrarily selected nanoparticles were obtained from TEM microgram. The particlesize histogram ( Figure 5) revealed the size of the Eh-AgNPs to be in the range from 9.91 nm to 92.54 nm, with a mean of 35.13 ± 14.24 nm. Surface morphology of the Eh-AgNPs as revealed from the SEM image ( Figure 6A) showed the formation of nanoparticles with a higher degree of accumulation without any impurities. The presence of silver was confirmed through EDX. The EDX spectrum ( Figure 6B) indicated a strong signal at 3 keV with different elemental ratios of Ag in different parts. A higher mass percentage of Ag atoms (60.91%) was observed in comparison to atoms of chlorine (11.27%), boron (15.42%), carbon (8.53%), and nitrogen (3.87). The findings of SEM-EDX analysis ( Figure 6A,B) showed the metallic nature of the synthesized AgNPs. Zeta potential was carried out to measure the magnitude of the electrostatic or charge repulsion or attraction among the nanoparticles and to determine the stability of the Eh-AgNPs. The mean of the zeta potential values was found as −19.73 ± 3.94 mV with the range of −20.3 ± 3.53 to −19.3 ± 3.97 mV (Table 1, Figure 7). out to assess possible functional biomolecules responsible for the redu The FTIR spectrum of the Eh-AgNPs showed a number of absorptio The absorbance peaks at 3190 cm −1 and 2834 cm −1 corresponded to N-H of primary amine in proteins, while the peak at 1531 cm −1 correspond ing vibration due to secondary amide in proteins [39]. The peaks at 29 cm −1 were attributed to C-H stretching of aliphatic groups [39]. The corresponded to asymmetric O=C=O stretching of CO2, while the weak was due to C≡C stretching vibration of the aliphatic groups [40]. The attributed to primary amide was due to C=O stretching in proteins mation of water [39]. The peaks at 1389, 1215, and 1060 cm −1 were as metric deformation of the vibration of O-H groups in alcohols, C-O str ether, and S=O stretching vibration of sulfoxides, respectively [40]. Th 808 cm −1 and below were attributed to C-H aromatic vibrations [41]. T mostly spherical, polydispersed, and inconsistent in size as shown ( Figure 4). Descriptive analysis and a particle-size distribution histogr ily selected nanoparticles were obtained from TEM microgram. The gram ( Figure 5) revealed the size of the Eh-AgNPs to be in the rang 92.54 nm, with a mean of 35.13 ± 14.24 nm. Surface morphology of th vealed from the SEM image ( Figure 6A) showed the formation of na higher degree of accumulation without any impurities. The presence firmed through EDX. The EDX spectrum ( Figure 6B

Antimicrobial Activity
The results of the antimicrobial study of the activity of Eh-AgNPs a perimental pathogenic bacteria are presented in Table 2 and Figure 8A−D difference (P < 0.05) was observed among the ZOIs of different pathogenic

Antimicrobial Activity
The results of the antimicrobial study of the activity of Eh-AgNPs against the experimental pathogenic bacteria are presented in Table 2 and Figure 8A-D. A significant difference (p < 0.05) was observed among the ZOIs of different pathogenic microbes. The Eh-AgNPs at 6 µg concentration resulted in the highest ZOI against E. coli (ATCC 10536), of 15.16 ± 0.05 mm with a MIC value of 1.25 µg/mL, while the lowest ZOI was observed against P. aeruginosa (ATCC 10145) as 7.18 ± 0.04 mm with a MIC value of 2.25 µg/mL. The ZOIs of 30.48 ± 0.08 mm and 30.10 ± 0.07 mm produced by the Eh-AgNPs against E. coli (ATCC 10536) and P. aeruginosa (ATCC 10145), respectively, were found to be lower than that of the positive control, ciprofloxacin. By contrast, the ZOI of 11.20 ± 0.07 mm produced by the Eh-AgNPs against S. aureus subsp. aureus (ATCC 11632) was higher than the ZOI of 10.14 ± 0.05 mm produced by the positive control, ampicillin. In contrast, the Eh-AgNPs exhibited better antimicrobial activity against B. cereus (ATCC 10876), producing a ZOI of 9.14 ± 0.05 mm, while that strain was found to be resistant against the positive control, ampicillin. The CFE and AgNO 3 produced no inhibitory activity against the experimental microbes. The fungal strains of C. albicans (ATCC 10231) were found to be resistant against the conventional antibiotic, itraconazole-10 µg, whereas the synthesized Eh-AgNPs effectively inhibited their growth, displaying an 8.24 mm ZOI with MIC of 2.0 µg/mL (Table 2, Figure 8E). The Eh-AgNPs at 10 µg concentration resulted in a significant difference (p < 0.05) in the ZOIs among different MDR bacteria (Table 2, Figure 8F-I). The highest ZOI was observed with MDR E. faecium (ATCC 700221), at 13.20 ± 0.07 mm, with a MIC value of 2.00 µg/mL, while the lowest ZOI was observed with MDR S. pneumoniae (ATCC 700677) at 10.98 ± 0.08 mm, with a MIC value of 6.00 µg/mL. coli (ATCC 10536) and P. aeruginosa (ATCC 10145), respectively, were found to be lower than that of the positive control, ciprofloxacin. By contrast, the ZOI of 11.20 ± 0.07 mm produced by the Eh-AgNPs against S. aureus subsp. aureus (ATCC 11632) was higher than the ZOI of 10.14 ± 0.05 mm produced by the positive control, ampicillin. In contrast, the Eh-AgNPs exhibited better antimicrobial activity against B. cereus (ATCC 10876), producing a ZOI of 9.14 ± 0.05 mm, while that strain was found to be resistant against the positive control, ampicillin. The CFE and AgNO3 produced no inhibitory activity against the experimental microbes. The fungal strains of C. albicans (ATCC 10231) were found to be resistant against the conventional antibiotic, itraconazole-10 μg, whereas the synthesized Eh-AgNPs effectively inhibited their growth, displaying an 8.24 mm ZOI with MIC of 2.0 μg/mL (Table 2, Figure 8E). The Eh-AgNPs at 10 μg concentration resulted in a significant difference (P < 0.05) in the ZOIs among different MDR bacteria (

Synthesis and Characterization of Eh-AgNPs
Synthesis of Eh-AgNPs was rapid (5 min), exhibiting a change in colour of the CFE and AgNO 3 solution due to excitation of strong plasmon resonance resulting from oscillation of silver ions and SPR in the visible region [41], which might correspond to the synthesis of spherical Eh-AgNPs [20,42,43]. The Eh-AgNPs formed at 15, 30, and 60 min time intervals were found to be larger and aggregated in size. The reaction mixture kept in the dark produced some colour change, indicating that both sunlight exposure and CFE are required for the synthesis of Eh-AgNPs. The study clearly demonstrated that the supernatant of the experimental endophytic E. hormaechei possesses reducing and capping agents [15][16][17]. Bacteria-mediated synthesis of AgNPs follows a bottom-up approach. Although the exact mechanism of bacteria-mediated extracellular synthesis of AgNPs is not known, several hypotheses outlining the role of bacterial cell biomolecules involved in the synthesis process have been proposed [9].
The Eh-AgNPs in the FTIR spectrum analysis revealed the presence of proteins and alcohols, among other components. These findings demonstrated the interaction of bacterial cell-secreted functional biomolecules such as amine, amide, and hydroxyl groups with the surfaces of Eh-AgNPs, where these groups acted as capping areas for the stability of the nanoparticles as well as reducing and stabilizing agents that enabled the production of Eh-AgNPs from metal salts [9,12,14,16]. However, the presence of secondary metabolites observed in the FTIR study might be a result of horizontal gene transfer (HGT) to the endophytic E. hormaechei from the host plant [44].
Bacteria-mediated AgNPs may be of variable shapes such as spherical, quasi-spherical, cuboidal, disk-shaped, triangular, hexagonal, rod-shaped, irregular, etc. with the size ranging from 0.5 to 595.00 nm [9,15,16]. The present study corroborated some earlier studies reporting size inconsistency among spherical and polydispersed nanoparticles synthesized using various microbes [28,42,43]. However, the size and shape of extracellularly produced AgNPs depend upon the culture media, reducing agent, and bacterial species used during the synthesis [9].
The strong signal at 3 keV in the EDX spectrum ( Figure 6B) was due to SPR, confirming the presence of silver [28]. Like the present study, different ratios of elemental composition in the synthesized nanoparticles were reported earlier in the literature [28,42]. The presence of other elements such as Cl, B, C, and N ( Figure 6B) observed in the EDX spectrum might be due to the CFE and the carbon grid that were used during sample preparation [28,42].
Zeta potential (ζ) refers to the electro-kinetic potential in colloidal systems, which is related to the short-or long-term stability of emulsions. Emulsions with high negative or positive ζ are electrically stabilized while emulsions with low ζ tend to coagulate or flocculate. The larger the zeta potential, the greater the repulsive force; and the more stable the AgNP, the less the tendency of the suspension system to move towards aggregation [42,45]. The Zeta potential values in the present study (Table 1) indicated that Eh-AgNPs are able to form a moderately stable colloid in aqueous suspension [42,45]. The synthesized nanoparticles were found to be stable after more than 6 months. The present study clearly confirmed the higher stability of nanoparticles compared to those synthesized from Streptomyces xinghaiensis OF1 strain (−15.7 mV) [43], Bacillus sp. MB353 (−18.36 mV) [41], and Bacillus cereus A1-5 (−17.5 mV) [42].
Among human microbiota, C. albicans is the most predominant fungal species that causes a wide range of infections. It is estimated that infections resulting from Candida sp. cause direct medical costs totalling $3 billion in the USA [1]. Recent reports of Candida sp. resistance to antifungal drugs are a serious concern in the healthcare setting [48]. Earlier reports in the literature suggest that bacteria-mediated AgNPs can be an alternative, safe, and effective measure to treat C. albicans [28,43,49,50]. The Eh-AgNPs in the present study displayed more effective antifungal activity than the conventional antibiotic, itraconazole. Similarly, Bacillus methylotrophicus DC3-mediated AgNPs (7-31 nm size) at a concentration of 3 µg were reported to be sensitive antimicrobial agents against C. albicans while it was resistant to the conventional antibiotic, cycloheximide-3 µg [50]. However, higher antifungal efficacy with a 15-mm ZOI was reported with Pseudomonas sp. ef1-mediated AgNPs (20-70 nm size, 25 µL conc.) against C. albicans [28]. Additionally, the effectiveness of Bacillus safensis LAU 13-mediated AgNPs (5-95 nm size) against C. albicans was reported with a MIC of 40 µg/mL [49]. Furthermore, in the present study, the Eh-AgNPs exhibited a lower MIC value than did the Streptomyces xinghaiensis OF1-mediated AgNPs (5-20 nm size) against C. albicans (ATCC 10231), with a MIC of 32 µg/mL [43]. The variations in the MIC values may be attributed to differences in the type of strain used, methods of evaluation [51], and the concentrations of AgNPs used [52]. Although the exact mode of antifungal action of AgNPs against Candida sp. is not yet known, several mechanisms have been elucidated in the literature [53][54][55][56].
The present study revealed that Eh-AgNPs possess antibacterial potential against various MDR pathogens (Table 2, Figure 8F-I). Some previous studies have also reported antimicrobial efficacy of different bacteria-mediated AgNPs against Gram-positive and Gram-negative drug-resistant microbes [23,[57][58][59][60] [57]. Our previous study with endophytic Pantoea ananatis-mediated AgNPs at 10 µg concentration displayed 10.16, 10.20, and 12.16 mm ZOIs against MDR strains of S. aureus subsp. aureus (ATCC 33592), S. pneumoniae (ATCC700677), and E. faecium (ATCC 700221), respectively [23]; these were lower than the present findings shown in Table 2. The antimicrobial activity of Bacillus brevis (NCIM 2533)-mediated AgNPs (41-68 nm size) at different concentrations of 5, 10, 15, and 20 µL was studied against MDR clinical isolates of Gram-positive Staphylococcus aureus and Gram-negative Salmonella typhi [59]. AgNPs were reported to exhibit maximum antimicrobial activity against MDR S. aureus with mean ZOIs of 14, 15, 16, and 19 mm, respectively, while moderate antibacterial activity was reported against MDR S. typhi with mean ZOIs of 0, 7, 7 and 7.5 mm at 5, 10, 15, and 20 µL concentrations, respectively [59]. Another study with Acinetobacter baumannii mediated AgNPs having less stable (zeta potential of −11.7 mV) and a higher particle-size range (37-168 nm) than the AgNPs in the present study were reported to exhibit inhibitory effects against several Gram-negative MDR pathogens, such as Escherichia coli (E3), Pseudomonas aeruginosa (P21), and Klebsiella pneumoniae (K32), with a MIC of 1.53-3.125 µg/mL [60]. AgNPs synthesized using A. baumannii were tested against β-lactams-, aminoglycosides-, and quinolones-resistant E. coli, P. aeruginosa, and K. pneumoniae, and the results showed that the nanoparticles were effective against those microbes with MIC values of 3.1, 1.56, and 3.1 µg/mL, respectively [60]. However, a concrete comparison with other study reports describing different levels of ZOIs and MICs is difficult due to the differences in procedures and concentrations used [29,42,57,59,61] and biological strains used [28,41,42,47,61]. Besides, the shape and size of the synthesized AgNPs may affect their antimicrobial activity against both Gram-positive and Gram-negative bacteria and fungi [62]. The antimicrobial activity of AgNPs against pathogenic microbes may be subject to variation due to differences in microbial structure, molecular dynamics, and sequence [63]. Furthermore, inter-strain differences in bacteria may be attributable to genome versatility resulting from heterogeneity in the bacterial accessory genome, i.e., its lysogen [64], and thus account for the observed antimicrobial variability.
Although the exact mechanism of AgNP antimicrobial activity is yet to be known, several hypotheses have been described to illustrate the antimicrobial mechanisms of AgNPs in the literature [10,13,14]. In brief, (i) adhesion of AgNPs to the microbial cell wall causes disintegration of the cell wall and membrane, leading to leakage of intracellular content and finally disruption of cell integrity and cell death; (ii) intracellular penetration of AgNPs causes degradation and denaturation of bacterial deoxyribonucleic acid (DNA) with ribosomal denaturation, leading to the inhibition of translation and protein synthesis, the inhibition of sugar metabolism resulting from inactivation of phosphomannose isomerase, and the inhibition of protein biosynthesis resulting from inactivation of the enzymatic protein tryptophanase (TNase); (iii) AgNPs produce free radicals and reactive oxygen species (ROS), leading to increased oxidative stress followed by cytotoxic and genotoxic effects; and (iv) AgNPs modulate signal transduction in microbial cells through dephosphorylation of tyrosine, leading to bacterial growth inhibition. However, more research at the molecular level will be necessary to conclusively identify the mechanism of antimicrobial action of AgNPs.

Conclusions
This study provided insights into the emerging role of endophytes towards the synthesis of nanoparticles. To the best of our knowledge, this is the first report on biosynthesis of AgNPs using endophytic E. hormaechei. The synthesis took place within 5 min, which was a rapid process. The Eh-AgNPs were spherical in shape and displayed significant antimicrobial activity against pathogenic B. cereus (ATCC 10876), S. aureus subsp. aureus (ATCC 11632), and C. albicans (ATCC 10231) compared to conventional antibiotics. Additionally, the Eh-AgNPs showed moderate antibacterial activity against MDR strains of S. pneumoniae (ATCC700677), E. faecium (ATCC 700221), S. aureus subsp. aureus (ATCC33592), and E. coli (NCTC 13351). It is evident from the present study that endophytic E. hormaechei could be a novel candidate in facilitating rapid and eco-friendly biosynthesis of AgNPs with potential applications aimed at tackling the antibacterial resistance problem worldwide. Further studies concerning downstream processes, biological and environmental toxicity, as well as mechanisms of antimicrobial action at the molecular level will be required for the application of Eh-AgNPs to the development of independent or synergistic antimicrobial agents for AMR management. However, the present study extends the frontiers of biotechnological application for endophytic bacteria in the field of nanotechnology. The results of the present investigation are promising and feature a growing scientific breakthrough in unexplored roles for endophytic microbes in the formulation of new and alternative antimicrobial agents for the control of drug-resistant pathogens in the near future.

Data Availability Statement:
The data presented in this study are available in this article.