Leveraging the Attributes of Mucor hiemalis-Derived Silver Nanoparticles for a Synergistic Broad-Spectrum Antimicrobial Platform

Abstract

Driven by the need to engineer robust surface coatings for medical devices to prevent infection and sepsis, incorporation of nanoparticles has surfaced as a promising avenue to enhance non-fouling efficacy. Microbial synthesis of such nanoscale metallic structures is of substantive interest as this can offer an eco-friendly, cost-effective, and sustainable route for further development. Here we present a Mucor hiemalis-derived fungal route for synthesis of silver nanoparticles, which display significant antimicrobial properties when tested against six pathological bacterial strains (Klebsiella pneumoniae, Pseudomonas brassicacearum, Aeromonas hydrophila, Escherichia coli, Bacillus cereus, and Staphylococcus aureus) and three pathological fungal strains (Candida albicans, Fusarium oxysporum, and Aspergillus flavus). These antimicrobial attributes were comparable to those of established antibiotics (streptomycin, tetracycline, kanamycin, and rifampicin) and fungicides (amphotericin B, fluconazole, and ketoconazole), respectively. Importantly, these nanoparticles show significant synergistic characteristics when combined with the antibiotics and fungicides to offer substantially greater resistance to microbial growth. The blend of antibacterial and antifungal properties, coupled with their intrinsic "green" and facile synthesis, makes these biogenic nanoparticles particularly attractive for future applications in nanomedicine ranging from topical ointments and bandages for wound healing to coated stents.

Keywords: Mucor hiemalis; antibacterial activity; antifungal activity; green synthesis; silver nanoparticles.

FIGURE 1

(A) Colony morphology; (B) Mass culture of Mucor hiemalis; (C) Microscopic view of M. hiemalis processed with lactophenol cotton blue stain; (D) Optical images of biosynthesized AgNP showing a range of colors from colorless (start of biosynthesis) to reddish-brown after 24 h; (E) UV-Vis spectra of M. hiemalis-derived AgNPs at various reaction times; (F) Reaction saturation curve indicating the evolution of the absorption band as a function of time.

FIGURE 2

Morphological and chemical characterization of M. hiemalis-derived AgNP. (A) SEM image of biogenic AgNPs (scale bar indicates 4 μm); (B) EDX spectrum of biogenic AgNPs; (C) TEM image of the biogenic AgNPs (scale bar indicates 100 nm); (D) histogram of the size distribution of biogenic AgNPs.

FIGURE 3

XRD spectrum showing the crystalline nature of the M. hiemalis-derived AgNPs. Further characterization details are included in Section “Results.”

FIGURE 4

FTIR spectra of (A) cell extract and (B) M. hiemalis-derived AgNPs.

FIGURE 5

Comparative analysis of antibacterial efficacy of M. hiemalis-derived AgNP. (A) Zone of inhibition against four Gram-negative bacterial pathogens, Klebsiella pneumoniae (Kp), Pseudomonas brassicacearum (Pb), Escherichia coli (Ec), and Aeromonas hydrophila (Ah), and two Gram-positive bacterial pathogens, Bacillus cereus (Bc) and Staphylococcus aureus (Sa); (B) Measurements of the antibacterial activity of AgNPs (NP) at 5, 10, 20, and 30 μg/ml concentration against the six bacterial pathogens; and (C) Effect of biologically synthesized AgNPs (NP) and antibiotics [streptomycin (S), tetracycline (T), kanamycin (K), and rifampicin (R)] used individually against the bacteria and in combination. The potential synergistic effect of biologically synthesized AgNPs with antibiotics was investigated at a uniform 10 μg/ml concentration for all the agents. Experiments were performed in triplicates; mean ± SD are shown, the asterisk (^∗) above the error bar represents statistically significant differences between the action of the biogenic NPs and the control group (p-value < 0.05).

FIGURE 6

Comparative analysis of antifungal efficacy of M. hiemalis-derived AgNP. (A) Zone of inhibition measured against three fungal pathogens: Candida albicans (Ca), Fusarium oxysporum (Fo), Aspergillus flavus (Af); (B) Measurements of the antifungal activity of AgNPs (NP) at 15, 30, 45, and 60 μg/ml concentration against the three pathogens; and (C) Effect of biologically synthesized AgNPs and fungicides [amphotericin B (A), fluconazole (F), and ketoconazole (K)] used singly and in combination against the pathogenic fungi. All measurements were performed for a constant AgNP concentration of 30 μg/ml for commercial fungicides. DMSO (dimethyl sulfoxide) (D) and fungal extract (FE) were employed as negative controls. Experiments were performed in triplicates; mean ± SD are shown, the asterisk (^∗) above the error bar represents statistically significant differences between the action of the biogenic NPs and the control group (p-value < 0.05).