All chemicals used in this study were of analytical grade, procured from Sigma-Aldrich or Merck, India and used as received. All culture media were purchased from HiMedia, India. Double distilled water (ddH₂O) or Milli-Q water was used for all the experiments in this study. For the antimicrobial efficacy study, six bacterial strains namely Klebsiella pneumoniae (KJ938546), Pseudomonas brassicacearum (KJ938545), Aeromonas hydrophila (KM104684), Escherichia coli (MCC 2412), Bacillus cereus (MCC 2243), and Staphylococcus aureus (MCC 2408) were used. The first three strains were procured from the Amity University (Noida, India), while the latter three along with the fungal strain Candida albicans (MCC 1151) were received from the Microbial Culture Collection at National Centre for Cell Science, Pune, India. Additionally, Fusarium oxysporum (NFCCI 245), Aspergillus flavus (NFCCI 384), and Mucor hiemalis (NFCCI 2228) were procured from National Fungal Culture Collection of India, Pune, India.

Mucor hiemalis was grown on potato dextrose broth. Briefly, the pH of the medium was adjusted to 5.1 ± 0.2 and flasks were incubated at 180 rpm at 25°C for 72 h. The pure culture of M. hiemalis was maintained on potato dextrose agar (PDA) slants at 4°C. The mycelia were separated by centrifugation (5000 rpm) for 10 min and filtered using Whatman filter paper No. 1, and washed thrice with deionized water. After washing, 100 ml of deionized water was added to 10 g of wet fungal biomass and incubated for 48 h (180 rpm and 25°C). After incubation, the mycelia were again centrifuged (5000 rpm, 10 min) and filtered. Twenty milliliters of resulting aqueous extract was diluted with 80 ml of water followed by addition of 0.017 g (1 mM) silver nitrate (AgNO₃) in a conical flask and stirred at 25°C in dark condition. Control experiment without AgNO₃ was also carried out under identical conditions. After 24 h, visual observation revealed a color change—from colorless to reddish brown due to reduction of AgNO₃ to AgNPs—of the fungal filtrate. Prior to characterization, 100 ml of the nanoparticle solution was centrifuged at 5000 rpm for 10 min. The supernatant was again centrifuged at 10,000 rpm for 60 min to obtain a pellet. The pellet was re-dispersed into 1 ml of deionized water (Kayalvizhi et al., 2014). Centrifugation and re-dispersion in ddH₂O were repeatedly carried out to ensure better elimination of these extraneous entities followed by lyophilization to obtain powdered nanoparticles. The AgNP yield was determined by weighing the powdered nanoparticles.

To examine the bioreduction of AgNO₃ to AgNPs the absorption spectra were recorded at different temporal points using a double beam UV-Vis spectrophotometer (UV-1800, Shimadzu, Japan). The surface morphology of the biogenic AgNPs was characterized using scanning electron microscope (SEM; Zeiss Evo HD, Jena, Germany) and energy dispersive X-ray analysis (EDX, Oxford Instruments, UK) connected with the SEM. Experiments with acquisition time ranging from 60 to 100 s and an accelerating voltage of 20 kV was performed to confirm the presence of elemental silver inside the biologically synthesized nanoparticles. TEM (Philips, EM-410LS, JEOL, Japan) was also used to observe the morphology of the biosynthesized AgNPs. Acquired images were analyzed using Image J (http://imagej.nih.gov/ij/download.html). For microscopy observations, the sample was prepared by dispersing 5 mg of nanoparticles in 1 ml ethanol and the resulting mixture was sonicated for 30 min. A small drop of the resulting suspension was evenly spread on the grid followed by drying at room temperature and the samples were gold-coated before imaging. Additionally, for vibrational characterization, FTIR measurements (Varian 7000 FTIR, USA) were performed on the KBr pellet prepared with diluted AgNPs. Spectroscopic measurements were carried out in 400–4000 cm^-1 range at a resolution of 4 cm^-1. To evaluate the crystallinity of AgNPs, XRD (Ultima IV, Rigaku, Japan) measurements were also performed and the data was recorded at 2𝜃 range of 20° to 80° having K-beta filter with X-Ray of 1.54056 Å at 40 kV and 30 mA.

Antibacterial and antifungal activities of AgNPs were evaluated using agar well diffusion and disc diffusion method. To determine the antibacterial activities of AgNPs, culture of four Gram-negative bacteria namely Klebsiella pneumoniae (Kp), Pseudomonas brassicacearum (Pb), Aeromonas hydrophila (Ah), Escherichia coli (Ec) and two Gram-positive bacteria, namely Bacillus cereus (Bc) and Staphylococcus aureus (Sa), were prepared by growing a single colony overnight in nutrient broth and adjusting the turbidity to 0.5 McFarland standards. Hundred microliters of bacterial test pathogens were spread onto 25 ml Mueller-Hinton agar medium in the plates. Fifty microliters of different concentrations (5, 10, 20, and 30 μg/ml) of AgNPs and 50 μl of 10 μg/ml antibiotics (streptomycin, tetracycline, kanamycin, and rifampicin) were poured in the wells. Synergistic effect of AgNPs with respective antibiotics was evaluated at 10 μg/ml concentration both for AgNPs and antibiotic. These plates were incubated at 37°C for 24 h and the zones of inhibition were measured. All experiments were simultaneously performed in triplicates.

The antifungal activity was assessed using agar well diffusion method for Candida albicans while the disc diffusion method was used for Fusarium oxysporum, and Aspergillus flavus. The fungal spores were washed with sterile 0.85% saline containing 0.1% tween 80 (v/v). The spore suspension was adjusted with sterile saline to a concentration of 1.0 × 10⁷ spores/ml in a final volume of 100 μl per well. The inocula were stored at 4°C for further use. Dilution of the inocula was cultured on solid PDA to verify the absence of contamination and to check the validity of the inoculum. 15, 30, 45, and 60 μg/ml concentrations of AgNPs and 30 μg/ml of standard commercial fungicides [amphotericin B (A), fluconazole (F), and ketoconazole (K)] were poured into Petri plates and their growth inhibition was observed. Synergistic effect of AgNPs with respective fungicides was measured at 30 μg/ml concentration of the former. All experiments were simultaneously performed in triplicates.

In order to assess the nature of the in vitro interactions between any nanoparticle–drug combinations, the data obtained from the zone of inhibition studies were quantified using the following equation:

Where I is the estimated interaction index, I_(A+B) is zone of inhibition for the combination of AgNP and antibiotic/fungicide, I_A is zone of inhibition of AgNPs alone and I_B is zone of inhibition for antibiotic/fungicide. While the interactions are concentration dependent, since the concentrations of each of these agents was kept constant (equal to 10 μg/ml and 30 μg/ml for the antibacterial and antifungal interaction studies, respectively) the estimated interaction index in Eq. 1 is concentration independent. Importantly, if the 95% confidence intervals of I values of a combination of agents were significantly higher or lower than 1, synergy or antagonism, respectively, was concluded to be present for that particular combination.

Data analysis was carried out using standard statistical software GraphPad Prism. Descriptive statistical measures, especially the mean and standard deviation, were used to summarize the collection of data for each measurement. Two-way analysis of variance was used to evaluate the influence of independent variables as well as possible interactions between them in the antimicrobial efficacy study. Tukey’s procedure was used to determine whether the data show evidence of difference between the various classes of antimicrobial agents.

Mucor hiemalis cultures were first grown in the potato dextrose medium for 3 days (Figures 1A,B). Figure 1C displays the representative microscopic morphology of the culture following staining with lactophenol cotton blue stain. Incubation of the cell extracts with 1 mM AgNO₃ solution resulted in the formation of AgNPs and we observed a steady color change of the solution (Figure 1D). The formation of AgNPs was confirmed using UV-Vis absorption measurements at different time intervals (Figure 1E) with the spectrum obtained at 72 h displaying an absorbance maximum at 420 nm. As seen in Figure 1F, the peak intensity increases over time before plateauing around 72 h. Here, we obtained ca. 45 mg/100 ml of AgNPs from the fungal biosynthesis process and the quantity was consistent across three repeats.

Figure 2A shows a representative SEM image of the biologically synthesized AgNPs. On the region of interest (highlighted by the magenta square in the figure), we performed EDX measurement to determine the elemental composition of the synthesized nanoparticles (Figure 2B). The measured composition was consistent with the expected makeup with Ag accounting for more than 90% of the compositional contributors in the probed region (Supplementary Table S1). The remaining content of carbon, oxygen, and sodium reflect the presence of microbial biomolecules. To further investigate the size distribution, we performed TEM analysis on the M. hiemalis-derived AgNPs (Figure 2C). TEM images depict the spherical morphology of the nanoparticles and the particle size distribution was observed to vary in the range of 5–15 nm with only a very small fraction of the particles having dimensions between 20 and 30 nm (Figure 2D).

As illustrated in the XRD graph of the biologically synthesized AgNPs (Figure 3), four peaks at 2𝜃 values of 38.3°, 46.4°, 63.9°, and 77.0° were observed corresponding to (111), (200), (220), and (311) planes, respectively, of silver that were in agreement with the standard powder diffraction card of Joint Committee on Powder Diffraction, silver file No. 04-0783 (Yang et al., 2011). The XRD results, thus, indicate that the synthesized nanoparticles have a distinctly crystalline nature. We computed the mean size of the ordered (crystalline) domains (D) using the Debye–Scherrer formula:

where λ represents the X-ray wavelength (0.1541 nm), β is the line broadening at half the maximum intensity (equivalent to the full width at half maximum (FWHM)) and 𝜃 is the Bragg’s angle. Based on the different 2𝜃 values, one can estimate a range of average crystalline sizes that is consistent with the previous TEM measurements of Figure 2D.

The FTIR spectra of the cell extract and the biologically synthesized AgNPs are presented in Figure 4. The notable peaks at 3312, 1661, and 1028 cm^-1 in the FTIR spectrum of the extract can be ascribed to the O–H stretching vibration of aromatic compounds (such as phenols), C=C stretching vibration of alkenes and C–N stretching, respectively. The features at 2928 and 2887 cm^-1 can be ascribed C–H stretching vibrations, whereas 1732 and 1553 cm^-1 is likely indicative of C=O stretching vibration of ketones and NH₂ bending vibration of the primary amines, respectively. Furthermore, the bands at 1460, 1252, and 1155 cm^-1 are consistent with peaks representative of CH₂ group bending, asymmetric PO₂^- stretching mode, and C–O stretching mode of the C–OH groups of amino acids. Notably, a gross inspection of Figures 4A,B reveals that the FTIR spectrum of the AgNP solution is considerably less intense and is broadened than that acquired from the cell extract. For instance, the spectral bands at 2928 and 2887 cm^-1 of the cell extract is barely perceptible in the spectrum of the synthesized AgNPs. Notably, reduction in the intensity of the 1028 cm^-1 peak after biosynthesis of AgNPs suggests that one or more cyclic peptides is involved in synthesizing and capping the nanoparticles.

To study the antibacterial efficacy, the biosynthesized AgNPs were tested against six clinically significant pathogenic bacteria, namely Klebsiella pneumoniae, Pseudomonas brassicacearum, Aeromonas hydrophila, Escherichia coli, Bacillus cereus, and Staphylococcus aureus. Fifty microliters of different concentrations of AgNPs were incubated with the bacterial culture and the zone of inhibition was observed for each case (Figures 5A–C). As evidenced from Figures 5A–C, the biogenic nanoparticles display consistent antibacterial action against all the types of bacterial pathogens under consideration—even at a very low concentration of 5 μg/ml. Expectedly, the size of inhibition zone increases with the increase in AgNPs concentration as verified through morphometric analysis of the cultures (Figure 5B). The antibacterial activity of AgNPs was comparable to the commercial antibiotics at the same concentration (Figure 5C), albeit with variations in strength of action of both the AgNP and the antibiotics across the bacterial spectrum.

When AgNPs was mixed with above antibiotics in the same concentration (ca. 10 μg/ml), a substantial increase in the antibacterial activity was observed. To precisely elucidate the nature of synergistic/antagonistic interactions, we computed the interaction index for each combination from the zone of inhibition studies. From Table 1, we observe that the order of the degrees of synergistic interaction between these six species was as follows: Pb > Ah > Ec > Bc > Sa > Kp. The highest degree of synergy was found with the three Gram-negative species, Pb, Ah, and Ec, especially for antibiotics rifampicin (R) and streptomycin (S). Little synergy was observed for Kp, even though it is also a Gram-negative species.

The antifungal characteristic of the synthesized AgNP with three representative fungi, namely Candida albicans (Ca), Fusarium oxysporum (Fo), and Aspergillus flavus (Af) was similarly tested and our observations are shown in Figure 6. Morphometric analysis of the incubated cultures exhibits the pattern of antifungal action with increasing concentration of the nanoparticles (Figure 6B). The antifungal activity of AgNPs was found to produce similar, if slightly smaller zones of inhibition on average, in relation to that obtained with standard fungicides, namely amphotericin B (A), fluconazole (F), and ketoconazole (K), at the same concentration of 30 μg/ml (Figure 6C). Furthermore, when AgNPs was mixed with commercial fungicides, a synergistic antifungal effect was reflected for some combinations. The order of the average degrees of positive interaction between these three species was as follows: Ca > Af > Fo (Table 2).

The antimicrobial properties of silver are well established and, thus, it has been extensively utilized in the medical field (Wright et al., 1999; Fu et al., 2006). In this study, we first sought to test the relative efficacy of the biogenic nanoparticles, derived from M. hiemalis, against a wide spectrum of bacteria and fungi, with respect to the action of commercial antibiotics and fungicides. To study the antibacterial efficacy, the biosynthesized AgNPs were tested against six clinically significant pathogenic bacteria. This cohort was selected to present a challenging and demanding subsets of Gram-positive and Gram-negative bacteria for testing antimicrobial efficacy. This action has been previously explained through a combination of interference with the respiratory chain at the cytochromes (Bragg and Rainnie, 1974) as well as with components of the bacterial electron transport system (Feng et al., 2000), binding DNA and inhibiting its replication (Modak and Fox, 1973).

The antibacterial activity of AgNPs was comparable to the commercial antibiotics at the same concentration, albeit with variations in strength of action of both the AgNPs and the antibiotics across the bacterial spectrum. Additionally, the relative efficacy of the singly used AgNPs was more prominent in the Gram-negative pathogens. Studies by Rhim et al. (2006) and Huang et al. (2011) have spawned similar observations, presumably owing to the thinner cell walls of the Gram-negative strains leading to facile perforation and more rapid absorption. When AgNPs was mixed with above antibiotics in the same concentration (ca. 10 μg/ml), a substantial increase in the antibacterial activity was observed. To precisely elucidate the nature of synergistic/antagonistic interactions, we computed the interaction index for each combination from the zone of inhibition studies. The degree of synergy was lower, on balance, with Gram-positive pathogens reflective of the absence of outer membrane permeability barriers in such bacteria. Collectively, this highlights the presence of synergistic interactions for a large fraction of the AgNPs and antibiotic combinations (rather than simply additive effects) opening the door for combined action against multi-drug resistant strains. This is particularly important in view of the increasing epidemic of drug-resistant pathogens, as several bacteria have developed resistance against conventional antibiotics. It is also notable that biologically synthesized AgNPs have previously shown better synergistic effects as compared to chemically derived AgNPs (Hari et al., 2014). This is attributable to the differential capping of biogenic and chemically synthesized nanoparticles, and is consistent with reports of the direct impact of silver nanomaterial capping on antibiotic sensitive and resistant strains of bacteria (Tiwari et al., 2015).

One plausible explanation for the enhanced antibacterial activity using a combination of AgNP and antibiotics is the bonding between the nanoparticles and antibiotic molecules. The active functional groups of antibiotics such as hydroxyl and amino groups, can be chelated by silver and thereby cover a considerable portion of the surface of AgNPs (Morones-Ramirez et al., 2013). Alternately, the findings can also be driven by the AgNP-induced reactive oxygen species generation that increases the cell permeability, which in turn stimulates the antibiotic action (Fayaz et al., 2010).

In relation to the antifungal activity, the degree of synergy was lowest with Fusarium oxysporum, reflecting the characteristic of the constituents present on the surface of the fungi causing permeability barriers. While the synergy in most combinations was found to be statistically significant, it is worth noting that the degree of synergy was not as strong as that noted in some of the antibacterial studies.