Biogenic Gold Nanoparticles as Potent Antibacterial and Antibiofilm Nano-Antibiotics against Pseudomonas aeruginosa

Abstract

Abstract: Plant-based synthesis of eco-friendly nanoparticles has widespread applications in many fields, including medicine. Biofilm-a shield for pathogenic microorganisms-once formed, is difficult to destroy with antibiotics, making the pathogen resistant. Here, we synthesized gold nanoparticles (AuNPs) using the stem of an Ayurvedic medicinal plant, Tinospora cordifolia, and studied the action of AuNPs against Pseudomonas aeruginosa PAO1 biofilm. The synthesized AuNPs were characterized by techniques such as ultraviolet-visible spectroscopy, Fourier-transform infrared (FTIR) spectroscopy, energy-dispersive X-ray diffraction, X-ray diffraction, scanning electron microscopy (SEM), and transmission electron microscopy. The AuNPs were spherically shaped with an average size of 16.1 nm. Further, the subminimum inhibitory concentrations (MICs) of AuNPs (50, 100, and 150 µg/mL) greatly affected the biofilm-forming ability of P. aeruginosa, as observed by crystal violet assay and SEM, which showed a decrease in the number of biofilm-forming cells with increasing AuNP concentration. This was further justified by confocal laser scanning microscopy (CLSM), which showed irregularities in the structure of the biofilm at the sub-MIC of AuNPs. Further, the interaction of AuNPs with PAO1 at the highest sub-MIC (150 µg/mL) showed the internalization of the nanoparticles, probably affecting the tendency of PAO1 to colonize on the surface of the nanoparticles. This study suggests that green-synthesized AuNPs can be used as effective nano-antibiotics against biofilm-related infections caused by P. aeruginosa.

Keywords: CLSM; Con A-FITC; Pseudomonas aeruginosa; Tinospora cordifolia; biofilm; gold nanoparticles; nano-antibiotics.

Figure 1

Characterization of synthesized gold nanoparticles. (A) Ultraviolet-visible (UV-Vis) and (B) Fourier-transform infrared (FTIR) spectra of green-synthesized gold nanoparticles (AuNPs). (C) Scanning electron microscopy (SEM) analysis showing different nanoparticles scattered without agglomeration.

Figure 2

Energy-dispersive X-ray (EDX) spectra of green-synthesized AuNPs.

Figure 3

X-ray diffraction (XRD) analysis of green-synthesized AuNPs.

Figure 4

Electron microscopic analysis of green-synthesized gold nanoparticles (AuNPs). (A) Transmission electron microscopy (TEM) image representing different sizes of green-synthesized AuNPs. (B) Histogram showing the average particle size as calculated by ImageJ. (C) Individual AuNP at greater magnification, indicating the spherical nature of the particle.

Figure 5

Scanning electron microscopic analysis of biofilm structure. SEM images of the biofilms formed on the glass coverslips after 24 h of incubation. (A) Control; (B), (C), and (D) treated with 50, 100, and 150 µg/mL of Tinospora cordifolia (TC)-AuNPs, respectively.

Figure 6

Confocal laser scanning microscopy (CLSM) images of the biofilm formed by P. aeruginosa PAO1 on the glass coverslips after 24 h of incubation. (A) Control; (B), (C), and (D) treated with 50, 100, and 150 µg/mL of TC-AuNPs, respectively.

Figure 7

Ultrastructural changes caused by gold nanoparticles as analyzed by TEM. (A) Images of the control setup of P. aeruginosa PAO1 without treatment with nanoparticles. (B) Images of the experimental setup of PAO1 treated with 150 µg/mL of AuNPs. Blue arrows indicate nanoparticles outside the cell, whereas white arrows indicate internalized nanoparticles.