Comparative Study of Antimicrobial Activity of Silver, Gold, and Silver/Gold Bimetallic Nanoparticles Synthesized by Green Approach

Nanotechnology is one of the most recent technologies. It is uncertain whether the production of small-size nanoparticles (NPs) can be achieved through a simple, straightforward, and medicinally active phytochemical route. The present study aimed to develop an easy and justifiable method for the synthesis of Ag, Au, and their Ag/Au bimetallic NPs (BNPs) by using Hippeastrum hybridum (HH) extract, and then to investigate the effects of Ag, Au, and their Ag/Au BNPs as antimicrobial and phytotoxic agents. Ag, Au, and their Ag/Au BNPs were characterized by UV-visible spectroscopy, FT-IR spectroscopy, XRD, EDX, and SEM analysis. XRD analysis conferring to the face of face-centered cubic crystal structure with an average size of 13.3, 10.72, and 8.34 nm of Ag, Au, and Ag/Au BNPs, respectively. SEM showed that Ag, Au, and Ag/Au BNPs had spherical morphologies, with calculated nano measurements of 40, 30, and 20 nm, respectively. The EDX analysis confirmed the composition of elemental Ag signal of the HH-AgNPs with 22.75%, Au signal of the HH-AuNPs with 48.08%, Ag signal with 12%, and Au signal with 38.26% of the Ag/Au BNPs. The Ag/Au BNPs showed an excellent antimicrobial efficacy against Gram-positive Staphylococcus aureus, Actinomycetes meriye, Bacillus cereus, Streptococcus pyogenes, Methicillin-resistant Staphylococcus aureus, Micrococcus luteus, Streptococcus pneumonia, and Gram-negative Klebsiella pneumonia, Escherichia coli, and Serratia marcescens bacterial strains, as well as against three fungal strains (Aspergillus niger, Aspergillus fumigatus, and Aspergillus flavus) compared to HH extract, HH-AgNPs, and HH-AuNPs. However, further investigations are recommended to be able to minimize potential risks of application.


Introduction
The green synthesis (phytosynthesis) of nanoparticles (NPs) is one of the emergent fields in nanotechnology and nanoscience. Different properties such as electrical, physical, optical, and chemical significantly affect the size and shapes of NPs. Nowadays, NPs, mostly silver (Ag) and gold (Au), are extensively used in biomedicine [1], drug delivery [2], agriculture [3,4], antioxidants [5], as anticancers [6], antibacterials [7], antifungals [8], and ant biofilms agents [9]. For centuries, silver nanoparticles (AgNPs) have been the most widely used antibacterial agents in the healing of wounds, bandages, and medication. [10]. Currently, due to the characteristic antimicrobial potential, AgNPs are also used in other

Factors Effecting Synthesis of NPs
The plant extract, induced HH-AgNPs, HH-AuNPs, and Ag/Au BNPs solutions were exposed to UV-vis spectroscopy during the reduction of ions by HH extract to study the mechanism of Ag, Au, and Ag/Au BNPs formation. The recorded spectra at different pH, such as 4, 5, 6, 7, 8, 9, 10, 11, and 12, are shown in Figure S1A-C. The spectra recorded during the reduction of ions at various pH showed the maximum absorbance with a sharp peak of 417 nm at pH 12, 576 nm at pH 4, and 542 nm at pH 6, which corresponds to the Ag, Au, and Ag/Au BNPs, respectively. The spectra recorded at different concentrations of HH plant, such as 0.5, 1, 1.5, and 2 mL, are shown in Figure S2A-C. The recorded spectra at different HH plant concentrations showed the maximum absorbance with a sharp peak of 417 nm, 576 nm, and 542 nm at 1 mL concentration of plant, which corresponds to the Ag, Au, and Ag/Au BNPs. The UV-vis spectra of Ag, Au, and Ag/Au bimetallic NPs in an aqueous medium at different AgNO3/HAuCl4·3H2O concentrations (0.25, 0.5, 1, and 1.5 mM) were noted at the range of 200 to 800 nm wavelength, which indicated the sharp peaks of 417 nm, 576 nm, and 542 nm at 1 mM salt concentration, which corresponds to the Ag, Au, and Ag/Au BNPs ( Figure S3A-C). In the present research work, NPs were studied at different temperature ranges (20 to 100 °C). Broadband appeared at a lower temperature of 20 °C for Ag, Au, and Ag/Au BNPs, while sharp bands of 417 nm, 576 nm, and 542 nm, which correspond to the Ag, Au, and Ag/Au BNPs, appeared at 40 °C; however, with further increase in temperature beyond 40 °C, the band becomes broad and final broadband with lower absorbance appeared at 100 °C ( Figure S4A-C). Figure S5A-C indicated the reaction time effect on HH-reduced Ag, Au, and Ag/Au BNPs. A broad peak with lower absorption appeared after 1, 2, and 3 h of the stirring. Due to the continuous reduction of Ag and Au ions by HH extract, the absorp-

Factors Effecting Synthesis of NPs
The plant extract, induced HH-AgNPs, HH-AuNPs, and Ag/Au BNPs solutions were exposed to UV-vis spectroscopy during the reduction of ions by HH extract to study the mechanism of Ag, Au, and Ag/Au BNPs formation. The recorded spectra at different pH, such as 4, 5, 6, 7, 8, 9, 10, 11, and 12, are shown in Figure S1A-C. The spectra recorded during the reduction of ions at various pH showed the maximum absorbance with a sharp peak of 417 nm at pH 12, 576 nm at pH 4, and 542 nm at pH 6, which corresponds to the Ag, Au, and Ag/Au BNPs, respectively. The spectra recorded at different concentrations of HH plant, such as 0.5, 1, 1.5, and 2 mL, are shown in Figure S2A-C. The recorded spectra at different HH plant concentrations showed the maximum absorbance with a sharp peak of 417 nm, 576 nm, and 542 nm at 1 mL concentration of plant, which corresponds to the Ag, Au, and Ag/Au BNPs. The UV-vis spectra of Ag, Au, and Ag/Au bimetallic NPs in an aqueous medium at different AgNO 3 /HAuCl 4 ·3H 2 O concentrations (0.25, 0.5, 1, and 1.5 mM) were noted at the range of 200 to 800 nm wavelength, which indicated the sharp peaks of 417 nm, 576 nm, and 542 nm at 1 mM salt concentration, which corresponds to the Ag, Au, and Ag/Au BNPs ( Figure S3A-C). In the present research work, NPs were studied at different temperature ranges (20 to 100 • C). Broadband appeared at a lower temperature of 20 • C for Ag, Au, and Ag/Au BNPs, while sharp bands of 417 nm, 576 nm, and 542 nm, which correspond to the Ag, Au, and Ag/Au BNPs, appeared at 40 • C; however, with further increase in temperature beyond 40 • C, the band becomes broad and final broadband with lower absorbance appeared at 100 • C ( Figure S4A-C). Figure S5A-C indicated the reaction time effect on HH-reduced Ag, Au, and Ag/Au BNPs. A broad peak with lower absorption appeared after 1, 2, and 3 h of the stirring. Due to the continuous reduction of Ag and Au ions by HH extract, the absorption peak increased with time. A final, clear, sharp peak of 417 nm, 576 nm, and 542 nm corresponds to the Ag, Au, and Ag/Au BNPs observed after 24 h of the HH and Ag/Au ions' reaction. The stability of the HH-reduced Ag, Au, and Ag/Au BNPs was studied at different periods (1 day, 3 months, and 6 months). Sharp peaks with maximum absorbance are reported after 1 day and 3 months of NPs formation; however, after 6 months these peaks became broad and also showed low absorbance for all three types of NPs ( Figure S6A-C).
Molecules 2022, 27, x FOR PEER REVIEW 4 of 25 tion peak increased with time. A final, clear, sharp peak of 417 nm, 576 nm, and 542 nm corresponds to the Ag, Au, and Ag/Au BNPs observed after 24 h of the HH and Ag/Au ions' reaction. The stability of the HH-reduced Ag, Au, and Ag/Au BNPs was studied at different periods (1 day, 3 months, and 6 months). Sharp peaks with maximum absorbance are reported after 1 day and 3 months of NPs formation; however, after 6 months these peaks became broad and also showed low absorbance for all three types of NPs ( Figure S6A-C).

XRD Analysis of NPs
XRD is a technique that is used for determining the size and crystalline nature of the sample. In the present study, the Ag, Au, and Ag/Au BNPs were analyzed by XRD.

XRD Analysis of NPs
XRD is a technique that is used for determining the size and crystalline nature of the sample. In the present study, the Ag, Au, and Ag/Au BNPs were analyzed by XRD.   Table 2.        The average crystalline size of HH-induced NPs is calculated by using Debye-Sherrer's Formula (3); "D" is the average crystalline size, k is a geometric factor (0.9), λ is the wavelength of the X-ray radiation source, and β is the angular full width at half maximum (FWHM) of the XRD peak at the diffraction angle θ. For the four major peaks, the average crystalline size for each of the samples Ag, Au, and Ag/Au BNPs was found to be 13.3, 10.72, and 8.34 nm, respectively.

EDX Analysis of NPs
The elemental constituents and relative abundance of the biosynthesized Ag, Au, and Ag/Au BNPs were obtained from EDX analysis, as shown in Figure 6A-C. EDX analysis established the existence of elemental Ag signal of the AgNPs with 22.75%, Au signal of the AuNPs with 48.08%, and Ag signal with 12%/Au signal with 38.26% of the Ag/Au BNPs. The vertical axis displays the number of X-ray counts, while the horizontal axis displays energy in KeV. Identification lines for the major emission energies for Ag and Au are displayed, and these correspond with peaks in the spectrum, thus giving confidence that Ag and Au have been correctly identified in Ag, Au, and Ag/Au BNPs. Thus, the EDX analysis unveils the complete chemical alignment and purity of HH-induced Ag, Au, and (C)

Materials
The Hippeastrum hybridum plant was collected in April from Bannu Khyber Pakhtunkhwa (city), and was identified by a taxonomist, Dr. Tahir Iqbal, at the department of Botany, University of Science and Technology, Bannu. Ten clinically isolated bacterial strains, seven Gram-positive Actinomycetes meriye ATCC 35568, Streptococcus pyogenes ATCC 700294, Bacillus cereus ATCC 14579, Staphylococcus aureus ATCC 29213, Methicillin-resistant Staphylococcus aureus ATCC 43300, Micrococcus luteus ATCC 4698, Streptococcus pneumonia ATCC 6303, and three Gram-negative Klebsiella pneumonia ATCC 700603, Escherichia coli ATCC BAA-2471, and Serratia marcescens ATCC BAA-3111 were used. Three fungus strains, Aspergillus fumigatus, Aspergillus niger, and Aspergillus flavus, were obtained from the microbiology lab of Biotechnology. Brine shrimps, sea salts, nutrient agar, and Sabourad Dextrose Agar (SDA) were obtained from Sigma. All other reagents used were of analytical grade.

Plant's Extraction
After identification, the Hippeastrum hybridum plant was washed using water, shade dried, and ground to a fine powder. About 250 g of the fine plant powder was mixed with 70% methanol in 1:3, and then retained for extraction at room temperature for 7 days. After that, it was filtered by using Whatman No 1 filter paper, after which the methanol was vaporized at 37 • C to obtain a pure crude methanolic extract of sample, which was then kept in the refrigerator at 4 • C for more studies [30].

Synthesis of Ag, Au, and Ag/Au BNPs
For the synthesis of Ag, Au, and Ag-Au BNPs from the HH extract, the standard protocol of [31] was followed with minor modification. Solutions of about 10 mM (0.01 M) of AgNO 3 and HAuCl 4 ·3H 2 O were prepared in 50 mL deionized water. The 10 mM AgNO 3 and HAuCl 4 ·3H 2 O solutions were further diluted 10 times to obtain 1 mM AgNO 3 and HAuCl 4 ·3H 2 O solutions. 0.1 M NaOH, ≥98%, and 0.1 M HCl were used for pH regulation. The HH plant extract was prepared in deionized water (1 gm/100 mL) and agitated quietly for about 1 h on a magnetic stirrer for complete dissolution. The solution was then centrifuged at 6000 rpm for 30 min to obtain the desired product. The supernatant was reserved for further use in NPs synthesis. About 50 mL of the plant supernatant was mixed with 500 mL of: 1 mM AgNO 3 (pH 12), 1 mM HAuCl 4 ·3H 2 O (pH 4), and AgNO 3 /HAuCl 4 ·3H 2 O (1:1) solution (pH 6). After 4 h, the reduction of Ag, Au, and Ag/Au BNPs was detected visually using color change. For the complete settlement and stabilization of NPs, the resultant solution was stored for 24 h at room temperature. After that, the NPs were monitored using a UV-Visible spectrophotometer. The colloidal suspension was centrifuged at −4 • C at 10,000 rpm by cold centrifuge for about 10 min; the supernatants were discarded and the pellet (containing NPs) was lyophilized to obtain the powder form. The powder was further characterized and tested for different biological activities.

Factors Affecting Synthesis Rate, Size, and Shape of NPs
Ag, Au, and Ag/Au BNPs synthesis was determined by using different factors such as pH, AgNO 3 /HAuCl 4 ·3H 2 O concentration, HH plant extract concentration, temperature, time, and stability time. To study the effect of basic and acidic conditions, pH of the reaction mixture ranged from 4 to 12 by using NaOH and HCl (0.1 M) solution. To study the effect of AgNO 3 and HAuCl 4 ·3H 2 O salt concentration, its concentration varied to 0.25, 0.5, 1, and 1.5 mM. To study the effect of HH extract concentration on NPs synthesis, its concentrations varied from 0.5, 1, 1.5, and 2 mL. To study the temperature effect, the NPs synthesis was carried out at different reaction temperature ranges (20,40,60,80, and 100 • C). To study the rate of completion of the reaction, NPs were synthesized at different time intervals (1, 2, 3, and 24 h). The synthesized NPs' stability was studied after 1 day, 3 months, and 6 months.

Characterization
In the aqueous solution, Ag, Au, and Ag/Au BNPs' concentrations were confirmed by using a SHIMADZU UV SPECTROPHOTOMETER (UV-1800). The purified Ag, Au, Ag/Au BNPs, and HH plants extract were examined for the presence of different phytochemicals by using a Fourier Transform-Infrared (FT-IR) Shimadzu (IR Prestige-21) spectrometer (Japan). The crystalline nature of the Ag, Au, and Ag/Au BNPs was determined by using the JDX-3532 (JEOL JAPAN) X-ray diffractometer (XRD) with λ-1.54 A • wavelength. The size and shape of Ag, Au, and Ag/Au BNPs were determined by using JEOL Scanning Electron Microscope (SEM) Model JSM-5910 (Japan). The presence of elements in synthesized Ag, Au, and Ag/Au BNPs was determined by using electron diffraction X-ray spectroscopy (EDX).

Antibacterial Activity (Agar Diffusion Method)
Antimicrobial activities of AgNO 3 salt, HAuCl 4 ·3H 2 O salt, HH extracts, HH-AgNPs, HH-AuNPs, and BNPs were determined according to the procedure [32]. Bacterial suspensions were prepared by caring 1 CFU of bacterial strain from the maintained slants in 0.9% NaCl solution, followed by incubation at 37 • C for 24 h. Nutrient agar (2.8 g/100 mL dH 2 O), autoclaved at 121 • C for 15 min. About 25 mL media (cooled to room temperature) were added to each Petri plate and then kept back for solidification. By swab, a good amount of each bacterial suspension was taken and streaked on the Petri plates. Per plate, 5 wells (3-6 mm) were dug with a sterilized cork borer, with 1 central hole surrounded by 4 holes. All of the wells were properly labeled. DMSO (20 µL, as well as AgNO 3 salt, HAuCl 4 ·3H 2 O salt, HH extracts, HH-AgNPs, HH-AuNPs, and BNPs (concentrations of 30, 60, and 90 µg/mL), were added to the surrounding holes. Levofloxacin (final concentration of 1 mg/mL in DMSO) was added to the central holes. DMSO served as a negative control. Zones of inhibition were measured after a day with a graduated ruler. A clear zone of bacterial inhibition was observed around the holes. The diameter of the clear zone was measured in mm. Inhibition by active ingredients of the extract was determined by measuring linear growth (mm) in Petri plates concerning levofloxacin (mm), a standard antibiotic (positive control).

Antifungal Bio-Assay
SDA media (6.5 g/100 mL dH 2 O) were autoclaved at 121 • C for 15 min and kept at room temperature for cooling (40-50 • C). To each test tube, 7 mL media, along with 67 µL AgNO 3 salt, HAuCl 4 ·3H 2 O salt, HH extracts, HH-AgNPs, HH-AuNPs, and BNPs from the concentrations 20, 40, 60, 80, and 100 µg/mL were added, and kept in slant position for solidifying inside the laminar flow hood. DMSO served as negative control and terbinafine as a positive control. Then, the particular fungal strains (A. niger, A. flavus, and A. fumigatus) were homogeneously spotted in each test tube. The test tubes were then sealed with cotton plugs. The entire setup was placed in an incubator with open water at 30 • C. Growth inhibition was determined by measuring linear growth [32].

Statistical Analysis
All the data were expressed as mean ± SE. Normal data distribution was confirmed.

Discussion
In nanoscience, the design and development of nanomaterials with unusual optoelectronic and physicochemical features act as a cornerstone. The present study discloses the bioreduction properties of Ag and Au ions into Au, Ag, and Ag/Au BNPs using ethnopharmacologically important HH plant extract. In the present investigation, Ag and Au NPs were synthesized from AgNO 3 and HAuCl 4 ·3H 2 O, while Ag/Au BNPs were manufactured by treating the HH extract with a mixture of both AgNO 3 /HAuCl 4 ·3H 2 O (1:1). The synthesis of NPs was monitored visually with a change in the color of reaction mixtures under optimum conditions. After 24 h, the NPs became stable and no more synthesis was detected, which was monitored with UV-vis spectroscopy. These results are supported by the [33,34] report of bimetallic alloys.
To accomplish maximum production of NPs, different intrinsic factors, such as pH, salt concentrations, plant concentration, time, temperature, and stability period were studied. The present results justify the earlier report [35] and confirm the effect of different intrinsic factors on the maximum synthesis of NPs. An ideal pH is required for the synthesis of controlled shape/size NPs [36,37]. As a result of extensive screening with different pH, syntheses of Ag, Au, and Ag/Au BNPs were successful with pH 12, 4, and 6, respectively. These results showed that AgNPs' synthesis is supported by basic conditions and suppressed by acidic conditions; AuNPs' is supported by highly acidic conditions, and Ag/Au BNPs' synthesis is supported by nearly neutral conditions [23,38]. In the current study, NPs' reduction and capping were achieved with the different AgNO 3 , HAuCl 4 ·3H 2 O salt, and HH plant concentrations. Screening with various concentrations of AgNO 3 , HAuCl 4 ·3H 2 O salt, and plant synthesis of Ag, Au, and Ag/Au BNPs was successful, with 1 mM salt and 1 mL plant concentration. Medicinal plants are a rich source of phytochemicals that act as reducing, capping, and mediators for NPs. However, the composition of these active secondary metabolites varies from plant to plant, dependent on the nature, part, type of plant, and method followed for the extraction of these metabolites [39]. Thus, a slight increase in the HH extract and slat concentration beyond 1 mL and 1 mM, respectively, increases the wavelength. Thus, this slight variation in the absorbance values is because of the change in the particle size. The results of the present research are also supported by [23], who reported an absorbance at 414 nm for the 1 mL Calligonum polygonoides and 1 mM salt with high absorbance of 2.375. The results of the present research work are also supported by [31], who reported the synthesis of NPs by using Gloriosa superba leaf extract. The Ag/Au BNPs showed an absorption peak at 542 nm, which is in good agreement with the previous results of the BNPs (Ag:Au) 1:1 ratio [30]. The temperature of the reaction medium determines the nature of NPs [40]. In this study, NPs were synthesized at varying temperature ranges (20 to 100 • C). At a lower temperature of 20 • C, a broad peak with low absorbance was obtained, but with an increase in temperature up to 40 • C, a sharp peak was recorded. With a further increase in temperature, the peak become broader and broader and final broad peaks were revealed for Ag, Au, and Ag/Au BNPs at the highest temperature of 100 • C. Thus, from these changes in peaks with temperature, it is revealed that NPs synthesis required an optimal temperature for its stability [23,41,42]. The quality, size, shape, and type of NPs are greatly influenced by incubated reaction time [43]. After mixing the HH extract with 1 mM salt, no color change was observed after 1 h, 2 h, and 3 h, but after 24 h of incubation an absorption spectrum of 417 nm, 576 nm, and 542 nm were reported, which corresponds to the Ag, Au, and Ag/Au BNPs. From the time study, it is revealed that an optimum time is required for stable NPs synthesis [23,44]. The reactivity and potency of the NPs greatly depends on their stability. The NPs have an optimum stability time, after which their potency is decreased or completely lost. In the present research, the stability of the NPs was checked at different periods of 1 day, 3 months, and 6 months. Sharp peaks at 17 nm, 576 nm, and 542 nm, which correspond to the Ag, Au, and Ag/Au BNPs, appeared after incubation in the dark for 1 day to 3 months. However, after 6 months the absorption band became broad and showed low absorbance. The present research study indicated that NPs remained stable for about 3 months, but after 6 months they lost their stability. The results are best supported by [45], who used Kiwifruit juice at room temperature for NPs' synthesis and revealed NPs in the range of 5 to 25 nm which remain stable for more than one month. Mean particle size and stability of NPs increased at 72 h of stirring time [44]. Thus, the variations in the reaction time may occur due to reasons such as: particle aggregation due to long time storage; particles shrinking or growing during long storage; the shelf life of particles, and so forth. All of these affect the reactivity and potential of the particles [46].
The phyto-components present in aqueous plant extract that are responsible for stabilizing and mediating the NPs were illustrated using FTIR analysis. The scientific studies reported that FTIR is ideal for forecasting functional moieties. In the present study, various vibrational stretches occurred at different peaks, which correspond to polyhydroxy, phenol, carboxyl, proteins, lipids, amide, alkynes, alkene, etc. The FTIR analysis delineated the different functional groups, such as hydroxyl, ketones, carboxyl, amino, alkaloids, amide, etc., that are responsible for the induction of metal ions to NPs [47,48]. The present result of FTIR analysis is justified by the prior findings of [49]. Stimulatingly, in plant-induced NPs, the phytochemicals of the plant played a significant role in the stabilization of NPs, which is critical for transcription and its applicative properties. These results are also supported by the earlier reports of [50,51].
The XRD peaks of Ag, Au, and Ag/Au BNPs exhibited four brag reflections which agree with the face-centric cube crystalline nature of Ag, Au, and Ag/Au BNPs. The well-resolved and strong pattern of the XRD showed that both Ag and Au NPs that are formed due to the reduction of Ag+ and Au+ ions are crystal-like. The Ag/Au BNPs that are formed by the combined reduction of Ag and Au ions are not different from the Ag and Au monometallic NPs, which can be similarly described as a crystal lattice structure. The mean calculated sizes were found to be 13.3, 10.72, and 8.34 nm for Ag, Au, and Ag/Au BNPs, respectively. The obtained results are justified by the earlier reports of [31,52].
The EDX analysis was used for the confirmation of elemental composition, purity, and relative abundance of NPs [53,54]. The percentages of Ag, Au, and Ag/Au metal in the present study were found to be appreciable, such as the Ag signal of the AgNPs with 22.75%, Au signal of the AuNPs with 48.08%, and Ag signal with 12%, and Au signal with 38.26% of the Ag/Au BNPs. The other elements which are shown by EXD analysis served as capping organic agents which are bounded on the NPs surface [55,56].
The surface morphology of NPs was studied by SEM analysis. In the present study, the SEM analysis showed an image of high density, spherical, and monodispersed NPs. The white individual spots represent the NPs, while the longer spots are the collection of NPs in the SEM photograph. The NPs from the SEM photograph have been observed with a diameter of 40, 30, and 20 nm of Ag, Au, and Ag/Au BNPs, respectively. The capping agent of the plant extract indicates the stabilization of the NPs because they were not in direct contact even in the aggregated form. The larger NPs during SEM measurements may be due to the combination of the smaller NPs. The obtained results are justified by the earlier reports of [57,58].
Different pathogenic bacteria cause universal infections: S. aureus causes skin infections, pneumonia, osteomyelitis, and endocarditis; K. pneumonia causes bloodstream infections, wound pneumonia, infections at surgical site, and meningitis; A. meriye can result in abscesses, pain, and inflammation; S. pyogenes causes pneumonia, necrotizing fasciitis, myonecrosis, scarlet fever, and bacteremia; E. coli causes cholangitis, cholecystitis, infection in urinary tract, bacteremia, and traveler's diarrhea; S. marcescens causes severe infections, bacteremia, pneumonia, infection in urinary tract, infection in biliary tract, meningitis, wound infection, and endocarditis, a foodborne pathogen; B. cereus can produce toxins that result in two types of gastrointestinal infection: the emetic (vomiting) and the diarrhea syndrome; MRSA causes severe skin infections; M. luteus is an adaptable pathogen that causes endocarditis, meningitis, septic arthritis, in HIV positive patients causes chronic cutaneous infections and catheter infections; and S. pneumoniae causes various infections such as pneumonia, sinusitis, osteomyelitis, otitis media, septic arthritis, bacteremia, and meningitis [59][60][61]. Due to the resistance of microbes to drugs, researchers are looking at the development of innovative agents against bacteria [62]. The NPs synthesized by the green approach are applied extensively in different biomedical applications [63]. The NPs kill microorganisms due to their interaction with the bacterial cell membranes. The NPs, such as the Ag, Au, and Ag/Au BNPs, exhibited the lowest noxiousness and thermal and pH resistance, and thus proved to be an outstanding antibacterial agent that could be useful in biomedical applications [64]. The green-synthesized Ag, Au, and Ag-Au NPs and HH plant extract displayed promising antibacterial activity against A. meriye, S. pyogenes, B. cereus, S. aureus, MRSA, M. luteus, S. pneumonia, K. pneumonia, E. coli, and S. marcescens in a dose-dependent manner. In this study, the appreciable antibacterial potential of Ag/Au BNPs compared to MNPs depends on morphology, surface area, particle size and shape, and surface polarity [65].
Like bacteria, different pathogenic fungi also cause systemic illness of the mouth, skin, lungs, blood, and liver, as well as causing hypersensitive reactions, tinea cruris, and athlete's foot [66][67][68][69]. For the therapeutic management of invasive systemic fungal infections, only about 10 antifungal drugs are permitted in the United States of America by the Food and Drug Administration (FDA) authority [66,67]. From the present observation, the highest fungicidal activity was observed with Ag/Au BNPs when compared with HH plant, Ag, and Au NPs at a similar concentration. The significant antifungal effect of Ag/Au BNPs compared to single metal Ag/Au NPs is due to their small size; as confirmed by investigators, the smaller the size of the particle, the greater the inhibition [70,71]. In comparison, [72] has analyzed the antifungal effect of NPs against species of aspergillus, revealing potential to control fungal growth. Moreover, [73] could be able to gain an antifungal effect against Aspergillus species at 80 µg/mL concentration of NPs. This outcome is supported by the aforementioned research, which was accompanied by [74], who reported that the NPs' interaction with lysed cells' intracellular substances caused their coagulation and the particles were thrown out of the liquid system. The Au and Ag ions mechanism of inhibition action on micro-organisms demonstrates that the microbe treatment by metal ions prompts loss of DNA, its capability replication and translation, as well as other cellular enzymes and proteins that are necessary for the production of ATP (coenzyme adenosine triphosphate), resulting in living cell inactivation of [75]. It has also been assumed that metal NPs mainly affect the function of membrane-bound enzymes in the chain of respiration [76].

Conclusions
In this study, medicinally active phytochemical-induced HH-AgNPs, HH-AuNPs, and Ag/Au BNPs were synthesized from HH extract, an indigenous plant found in abundance in Pakistan. These NPs were characterized by the following techniques: UV-vis spectroscopy, FTIR, XRD, EDX, and SEM. For antimicrobial activity, Ag and Au have always been excellent choices. Finally, this investigation revealed that Ag/Au BNPs have better antimicrobial activity than HH plant extract, HH-AgNPs, and HH-AuNPs. This work assimilates microbiology and nanotechnology, thus leading to possible advances in antimicrobial agents' formulation. However, future studies on the antimicrobial effect of these NPs on microbes are essential to completely assess its potential use as a novel biocidal material.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/molecules27227895/s1, Figure S1: UV-vis spectrum of NPs biologically synthesized by using the HH plant extract at different pH (Both acidic and basic).