Biosynthesis of Silver Nanoparticles from Oropharyngeal Candida glabrata Isolates and Their Antimicrobial Activity against Clinical Strains of Bacteria and Fungi

The objective of the present study was one step extracellular biosynthesis of silver nanoparticles (AgNPs) using supernatant of Candida glabrata isolated from oropharyngeal mucosa of human immunodeficiency virus (HIV) patients and evaluation of their antibacterial and antifungal potential against human pathogenic bacteria and fungi. The mycosynthesized AgNPs were characterized by color visualization, ultraviolet-visible (UV) spectroscopy, fourier transform infrared spectroscopy (FTIR), and transmission electron microscopy (TEM). The FTIR spectra revealed the binding and stabilization of nanoparticles with protein. The TEM analysis showed that nanoparticles were well dispersed and predominantly spherical in shape within the size range of 2–15 nm. The antibacterial and antifungal potential of AgNPs were characterized by determining minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC)/ minimum fungicidal concentration (MFC), and well diffusion methods. The MBC and MFC were found in the range of 62.5–250 μg/mL and 125–500 μg/mL, which revealed that bacterial strains were more susceptible to AgNPs than fungal strains. These differences in bactericidal and fungicidal concentrations of the AgNPs were due to the differences in the cell structure and organization of bacteria and yeast cells. The interaction of AgNPs with C. albicans analyzed by TEM showed the penetration of nanoparticles inside the Candida cells, which led the formation of “pits” and “pores” that result from the rupturing of the cell wall and membrane. Further, TEM analysis showed that Candida cells treated with AgNPs were highly deformed and the cells had shrunken to a greater extent because of their interaction with the fungal cell wall and membrane, which disrupted the structure of the cell membrane and inhibited the normal budding process due to the destruction and loss of membrane integrity and formation of pores that may led to the cell death.

performed to confirm the C. glabrata isolates [16]. One to two pure colonies of fresh C. glabrata cultures were again suspended into 500 mL SDB and incubated for 48-72 h at 28 • C. After incubation, the culture was centrifuged at 12,000 rpm for 10 min and the supernatant was collected and stored at 4 • C for the synthesis of AgNPs. All media was purchased from HiMedia, Mumbai, India.

Extracellular Mycosynthesis, Sepration and Purification of AgNPs
For the biosynthesis of AgNPs, 20 mL of collected supernatant was added in 1 mM of silver nitrate and then the solution was kept at room temperature overnight. The reduction of silver nitrate into AgNPs showed a change in color of silver nitrate from colorless to brown, which is an indication of the formation of AgNPs. Further, the synthesized AgNPs were separated and isolated by ultracentrifugation as previously described with slight modification [17,18]. To remove excess, unreduced silver ions and other possible impurities from the supernatant, the reaction mixture containing NPs were centrifuged at 14,000 rpm for 20 min with Milli-Q water. The process of centrifugation was repeated at least four times to ensure a better separation of NPs [17]. The particles separated were then resuspended again in ethanol and then centrifugation was further repeated three times [19]. A dark sediment was formed at the bottom of the centrifugal tube. After that, a dried powder of the AgNPs was obtained by freeze-drying [18], and their characterization was performed.

Characterization of Silver Nanoparticles
The biosynthesized AgNPs were preliminarily characterized by visual observation of the color change of the reaction mixture and then by measuring the absorbance of the colloidal suspension before and after the isolation NPs from the supernatant using UV-Vis spectrophotometer (Perkin-Elmer Lambda 25, Shelton, CT, USA). The preparation of samples for absorbance was accomplished as described in our previous work [20]. Briefly, AgNPs were subjected to mild sonication for 20 min, after which the UV-Vis spectra of AgNPs was monitored in the range of 270-700 nm. Distilled water was used to adjust the baseline [20]. Further, to confirm the presence of protein in the supernatant and their possible role in synthesis and reduction of AgNO 3 was carried out by a simple protein-dye binding assay, i.e., Bradford assay ( Figure A1). The functional group present in the supernatant that was responsible for the synthesis of AgNPs was analyzed by FTIR (SHIMADZU-8400 spectrometer, Tokyo, Japan) in the range of 400 to 4000/cm. Further, the surface morphology, shape, and size of synthesized AgNPs were characterized by transmission electron microscopy (Joel 2100, Tokyo, Japan).

Tested Microorganisms
The clinical isolates of fungal and bacterial species, i.e., C. albicans, C. tropicalis, C. parapsilosis, C. dubliniensis, C. krusei, C. glabrata, S. aureus, E. coli, P. aeruginosa, K. pneomonaie, S. typhimurium, and S. flexneri isolated from the oropharyngeal mucosa of the patients used in this study were obtained from the Department of Microbiology, Jawaharlal Nehru Medical College and Hospital, Aligarh Muslim University, India.

Evaluation of Antimicrobial Activity of Mycosynthesized AgNPs Using a Diffusion Method
The antimicrobial activity of as-synthesized AgNPs against various bacterial isolates, e.g., S. aureus, E. coli, S. typhimurium, S. flexneri, K. pneomonaie, P. aeruginosa, and Candida spp., e.g., C. albicans, C. dubliniensis, C. parapsilosis, C. tropicalis, C. krusei, and C. glabrata were examined using a well diffusion method as previously reported [17]. Briefly, a 6 mm diameter well was made on Nutrient and Sabouraud dextrose agar plates previously inoculated with 100 µL of 1 × 10 6 bacterial and fungal suspension, and then varying concentrations of AgNPs were aseptically filled in the well. The plates were subsequently incubated at 37 • C (Bacteria) and 28 • C (Candida spp.) for 24 h, and the antimicrobial activity was analyzed by measuring the diameter of the inhibition zone (mm) around the well.

Minimal inhibitory concentration (MIC):
The MIC values of mycosynthesized AgNPs against bacterial and Candida isolates were assessed using the method described by Ansari et al. [21].
Minimal bactericidal and fungicidal concentration (MBC/MFC): Further, MBC and MFC values of AgNPs against all tested bacteria and Candida spp. was examined using a method described in References [17,21].

Ultrastructural Morphological Changes Caused by AgNPs in C. albicans: TEM Analysis
The morphological and ultrastructure alteration caused by AgNPs in C. albicans cells were examined by using transmission electron microscope. The sample preparation and analysis procedures were similar to those described in our previous research work [17].

Characterization of Biosynthesized AgNPs
In the present study, C. glabrata supernatant was used for the synthesis of AgNPs. After the addition of C. glabrata supernatant with 1 mM aqueous solution of silver nitrate, a brownish color was observed, which indicated the extracellular biosynthesis of AgNPs ( Figure 1). Previously, it has been reported that the reduction of Ag + into AgNPs can be seen very clearly when the color of the solution changes from colorless to brown due to the surface plasmon resonance [6]. Figure 2 shows the UV-Vis spectroscopy of the as-prepared AgNPs in the range of 270-700 nm and a single strong peak was observed at 460.64 nm, which is the characteristic of AgNPs. Bhat et al. [10] reported that the absorption line of biosynthesized AgNPs was 430 nm when the silver nitrate solution was challenged with C. albicans. The MIC values of mycosynthesized AgNPs against bacterial and Candida isolates were assessed using the method described by Ansari et al. [21].
Minimal bactericidal and fungicidal concentration (MBC/MFC): Further, MBC and MFC values of AgNPs against all tested bacteria and Candida spp. was examined using a method described in References [17,21].

Ultrastructural Morphological Changes Caused by AgNPs in C. albicans: TEM Analysis
The morphological and ultrastructure alteration caused by AgNPs in C. albicans cells were examined by using transmission electron microscope. The sample preparation and analysis procedures were similar to those described in our previous research work [17].

Characterization of Biosynthesized AgNPs
In the present study, C. glabrata supernatant was used for the synthesis of AgNPs. After the addition of C. glabrata supernatant with 1 mM aqueous solution of silver nitrate, a brownish color was observed, which indicated the extracellular biosynthesis of AgNPs ( Figure 1). Previously, it has been reported that the reduction of Ag + into AgNPs can be seen very clearly when the color of the solution changes from colorless to brown due to the surface plasmon resonance [6]. Figure 2 shows the UV-Vis spectroscopy of the as-prepared AgNPs in the range of 270-700 nm and a single strong peak was observed at 460.64 nm, which is the characteristic of AgNPs. Bhat et al. [10] reported that the absorption line of biosynthesized AgNPs was 430 nm when the silver nitrate solution was challenged with C. albicans.  FTIR analysis was performed to identify the potential biomolecules and functional groups responsible for the reduction of silver ions to Ag 0 and stabilization of AgNPs [22]. Figure 3 shows a strong absorption line at 3436.03 cm −1 , which corresponds to the -OH groups that could arise from carbohydrates present in the supernatant, and the peak at 1636.17 cm −1 is due to amides (C-O stretch), i.e., characteristic of the presence of protein and enzymes in the supernatant that confirm the extracellular formation of AgNPs [23]. The peak at 661 cm −1 corresponds to C-H (alkane) and C=H bonding (alkene). In a previous study, Bhat et al. [10] found that the protein biomolecules present FTIR analysis was performed to identify the potential biomolecules and functional groups responsible for the reduction of silver ions to Ag 0 and stabilization of AgNPs [22]. Figure 3 shows a strong absorption line at 3436.03 cm −1 , which corresponds to the -OH groups that could arise from carbohydrates present in the supernatant, and the peak at 1636.17 cm -1 is due to amides (C−O stretch), i.e., characteristic of the presence of protein and enzymes in the supernatant that confirm the extracellular formation of AgNPs [23]. The peak at 661 cm -1 corresponds to C-H (alkane) and C=H bonding (alkene). In a previous study, Bhat et al. [10] found that the protein biomolecules present in the extract of C. albicans bind to the synthesized nanoparticles either by free amino or carboxyl groups, and the released extracellular proteins could possibility stabilize the biosynthesized AgNPs.  FTIR analysis was performed to identify the potential biomolecules and functional groups responsible for the reduction of silver ions to Ag 0 and stabilization of AgNPs [22]. Figure 3 shows a strong absorption line at 3436.03 cm −1 , which corresponds to the -OH groups that could arise from carbohydrates present in the supernatant, and the peak at 1636.17 cm -1 is due to amides (C−O stretch), i.e., characteristic of the presence of protein and enzymes in the supernatant that confirm the extracellular formation of AgNPs [23]. The peak at 661 cm -1 corresponds to C-H (alkane) and C=H bonding (alkene). In a previous study, Bhat et al. [10] found that the protein biomolecules present in the extract of C. albicans bind to the synthesized nanoparticles either by free amino or carboxyl groups, and the released extracellular proteins could possibility stabilize the biosynthesized AgNPs.  The mechanism of biosynthesis of AgNPs using microorganisms are well understood. It was reported that the biomass or supernatant of microorganisms possibly act as bioreductant and capping agents due to the presence of biomolecules such as proteins, amino acids, enzymes, vitamins, and polysaccharides [10,23]. However, the most widely accepted mechanism for the biosynthesis of AgNPs and other nanoparticles using microbes is mainly due to the presence of enzyme Nicotinamide adenine dinucleotide (NADH) and NADH-dependent nitrate reductase [24][25][26]. The role of nitrate reductase in the synthesis of AgNPs has been demonstrated by exploring the purified nitrate reductase in vitro for the synthesis of AgNPs and it was found that the reduction of silver ions happens by means of the transfer of electrons from NADH where enzyme NADH-dependent reductase acts as a carrier [24][25][26].
The TEM micrograph of as-prepared AgNPs exhibited that the synthesized AgNPs were predominantly spherical and oval, well-dispersed, and uniform with a size range of 2-15 nm (Figure 4). Niknejad et al. [8] reported biosynthesis of AgNPs using the yeast S. cerevisiae and they found that AgNPs were mainly spherical and polydispersed within the size of 5-20 nm. The size of AgNPs obtained in our study was much smaller than AgNPs synthesized from Candida utilis, where the size was 20-80 nm [11]. The image shows both individual and aggregated AgNPs (Figure 4). The particles were well-dispersed and not in direct contact due to capping and stabilization of AgNPs by the proteins around the periphery of the nanoparticles (Figure 4), which further confer the involvement of extracellular proteins and enzymes secreted by C. glabrata in the supernatant that acted as reducing and capping agents [22]. predominantly spherical and oval, well-dispersed, and uniform with a size range of 2-15 nm ( Figure  4). Niknejad et al. [8] reported biosynthesis of AgNPs using the yeast S. cerevisiae and they found that AgNPs were mainly spherical and polydispersed within the size of 5-20 nm. The size of AgNPs obtained in our study was much smaller than AgNPs synthesized from Candida utilis, where the size was 20-80 nm [11]. The image shows both individual and aggregated AgNPs (Figure 4). The particles were well-dispersed and not in direct contact due to capping and stabilization of AgNPs by the proteins around the periphery of the nanoparticles (Figure 4), which further confer the involvement of extracellular proteins and enzymes secreted by C. glabrata in the supernatant that acted as reducing and capping agents [22].

Antimicrobial Activity of Biosynthesized AgNPs
Antimicrobial activity of biosynthesized AgNPs against human pathogenic microorganisms, i.e., Candidal spp. and bacterial spp. was evaluated by agar well diffusion and two-fold microdilution methods. A zone of inhibition test of mycosynthesized AgNPs against tested bacteria and Candida spp. at different concentrations are shown in Figures 5 and 6, and it was very clear that AgNPs had an excellent inhibition zone against all strains. The inhibition zone diameter showed that AgNPs were more effective against bacterial strains in comparison to fungi (Figures 5 and 6). No antimicrobial activity has been observed by the C. glabrata supernatant. Further, the antimicrobial properties of AgNPs against various bacterial and fungal strains were examined by determining the

Antimicrobial Activity of Biosynthesized AgNPs
Antimicrobial activity of biosynthesized AgNPs against human pathogenic microorganisms, i.e., Candidal spp. and bacterial spp. was evaluated by agar well diffusion and two-fold microdilution methods. A zone of inhibition test of mycosynthesized AgNPs against tested bacteria and Candida spp. at different concentrations are shown in Figures 5 and 6, and it was very clear that AgNPs had an excellent inhibition zone against all strains. The inhibition zone diameter showed that AgNPs were more effective against bacterial strains in comparison to fungi (Figures 5 and 6). No antimicrobial activity has been observed by the C. glabrata supernatant. Further, the antimicrobial properties of AgNPs against various bacterial and fungal strains were examined by determining the MIC, MBC, and MFC. The MIC and MBC values for all tested bacterial strains were found in the range of 31.25-125 µg/mL and 62.5-250 µg/mL, respectively (Table 1), whereas the MIC and MFC values for fungal strain was in the range of 62.5-250 µg/mL and 125-500 µg/mL, respectively (Table 2). It was found that AgNPs were bactericidal at low concentration (62.5-250 µg/mL) and fungicidal at high concentration (125-500 µg/mL), which revealed that bacterial strains were more susceptible to AgNPs than fungal strains. In general, it was found that gram-negative and gram-positive bacterial strains showed better antimicrobial activity when compared to fungi Candida spp. (Tables 1 and 2). These differences in bactericidal and fungicidal concentrations of the AgNPs were due to the differences in the cell structure and organization of the bacteria and yeast cells. The bacterial cell structure is less complex and they are evolutionarily prokaryotic types, and were therefore unable to fight the toxic effects of AgNPs as effectively as the eukaryotic yeast cells that can resist higher concentrations of AgNPs because of their better cell organization and much more complex structure, and superior detoxification system [27].

Ultrastructural Changes in C. albicans after Exposure to AgNPs: TEM Analysis
The morphological alteration after exposure to biosynthesized AgNPs in C. albicans was investigated using a transmission electron microscope. TEM analysis clearly showed that the untreated C. albicans exhibited a normal and well-conserved cell wall that was mainly composed of an outer layer, an intermediate space, and a thin innermost layer of the cell membrane (Figure 7a). However, the Candida cells after the AgNP treatment showed an aberrant morphological structure and severe damage that was characterized by the formation of "pits" and "pores" that result in the

Ultrastructural Changes in C. albicans after Exposure to AgNPs: TEM Analysis
The morphological alteration after exposure to biosynthesized AgNPs in C. albicans was investigated using a transmission electron microscope. TEM analysis clearly showed that the untreated C. albicans exhibited a normal and well-conserved cell wall that was mainly composed of an outer layer, an intermediate space, and a thin innermost layer of the cell membrane (Figure 7a). However, the Candida cells after the AgNP treatment showed an aberrant morphological structure and severe damage that was characterized by the formation of "pits" and "pores" that result in the rupturing of the cell wall and membrane (Figure 7b,c). Candida cells treated with AgNPs were highly deformed and the cells had shrunken to a great extent (Figure 7b,c). Further, it was observed that the damaged cells exhibited either complete or localized separation of the membrane from the cell wall (Figure 7b,c). Nasrollahi et al. [28] reported that the antifungal activity of AgNPs was due to the formation of "pits" and "pores" on the surfaces of C. albicans and S. cerevisiae cells that led to the cell death. It has been observed that AgNPs not only anchor to cells at several sites (Figure 7b,c; black arrows), but they penetrate inside the cells (Figure 7c; red arrows), which could result in cell lysis. In their study, Vazquez-Muñoz et al. found that AgNPs were non-specifically distributed in different regions of the cytoplasm and cell wall, and the mode of action of AgNPs was due to the releasing of silver ions that induced cell death [29]. In the present study, it was observed that the rupturing, disintegration, and detachment of the cell wall and membrane from the cells led to the death of C. albicans (Figure 7b,c). Kim et al. found that AgNPs exert an antifungal activity because of their interaction with the fungal cell wall and membrane, which disrupts the structure of the cell membrane and inhibits the normal budding process due to the destruction and loss of membrane integrity, and due to the formation of pores that may led to the cell death [30]. Ishida et al. [31] also reported a similar mode of action of AgNPs against yeast Cryptococcus neoformans where they found that the antifungal activity of AgNPs was due to the disruption of the cell wall and cytoplasmic membrane [31]. Hwang et al. [32] found that AgNPs exert an antifungal activity against C. albicans due to the production and accumulation of reactive oxygen species (ROS) and free hydroxyl radicals ( • OH) inside the cells, which regulates and induces the cell death through mitochondrial dysfunctional apoptosis, release of cytochrome c, nuclear fragmentation, DNA damage, and the activation of metacaspases [32]. rupturing of the cell wall and membrane (Figure 7b,c). Candida cells treated with AgNPs were highly deformed and the cells had shrunken to a great extent (Figure 7b,c). Further, it was observed that the damaged cells exhibited either complete or localized separation of the membrane from the cell wall (Figure 7b,c). Nasrollahi et al. [28] reported that the antifungal activity of AgNPs was due to the formation of "pits" and "pores" on the surfaces of C. albicans and S. cerevisiae cells that led to the cell death. It has been observed that AgNPs not only anchor to cells at several sites (Figure 7b,c; black arrows), but they penetrate inside the cells (Figure 7c; red arrows), which could result in cell lysis. In their study, Vazquez-Muñoz et al. found that AgNPs were non-specifically distributed in different regions of the cytoplasm and cell wall, and the mode of action of AgNPs was due to the releasing of silver ions that induced cell death [29]. In the present study, it was observed that the rupturing, disintegration, and detachment of the cell wall and membrane from the cells led to the death of C. albicans (Figure 7b,c). Kim et al. found that AgNPs exert an antifungal activity because of their interaction with the fungal cell wall and membrane, which disrupts the structure of the cell membrane and inhibits the normal budding process due to the destruction and loss of membrane integrity, and due to the formation of pores that may led to the cell death [30]. Ishida et al. [31] also reported a similar mode of action of AgNPs against yeast Cryptococcus neoformans where they found that the antifungal activity of AgNPs was due to the disruption of the cell wall and cytoplasmic membrane [31]. Hwang et al. [32] found that AgNPs exert an antifungal activity against C. albicans due to the production and accumulation of reactive oxygen species (ROS) and free hydroxyl radicals ( • OH) inside the cells, which regulates and induces the cell death through mitochondrial dysfunctional apoptosis, release of cytochrome c, nuclear fragmentation, DNA damage, and the activation of metacaspases [32].

Conclusions
In this present study, a simple, one-step, safe, rapid, pollutant free, cost-effective, and ecofriendly extracellular silver nanoparticles were prepared using the supernatant of yeast C. glabrata as reducing and stabilizing agents for the first time. The synthesized nanoparticles were characterized by UV-visible, TEM, and FTIR spectra. Extracellular synthesis of AgNPs by yeast, i.e., C. glabrate, has advantages over mycelium fungi because separation of the particles is much easier and simpler, takes less time without much sophistication, and therefore, extracellular synthesis of AgNPs has importance from the point of view of large-scale production in an environmentally-friendly approach. The extracellular mycosynthesized AgNPs showed excellent antibacterial and antifungal activity against gram-negative and gram-positive pathogenic bacteria and Candida spp., respectively. Further, the interaction of AgNPs with C. albicans studied by TEM showed the primary attachment and penetration of nanoparticles inside the Candida cell that caused the rupturing, disintegration, and detachment of the cell wall and membrane from the cells that led to the death of C. albicans. Thus, these mycosynthesized AgNPs may lead to the development of appropriate pharmaceuticals and represent an alternative remedy for the treatment of bacterial and fungal infections, but this needs further in vivo cytotoxic studies before being brought into the market.