Antibacterial effect of gold nanoparticles against Corynebacterium pseudotuberculosis

Abstract

Corynebacterium pseudotuberculosis is the etiological agent of chronic caseous lymphadenitis. The bacterium infects goats and sheep causing great economic loss worldwide annually. The present work aims to evaluate the efficiency of gold nanoparticles (AuNPs) and AuNPs - laser combined therapy as antibacterial approaches against C. pseudotuberculosis bacteria in vitro. Gold nanoparticles 25 nm were synthesized by co-precipitation method and characterized by different techniques including; Transmission Electron Microscope (TEM), X-ray Diffraction and Dynamic Light Scattering. Three concentrations of AuNPs (50, 100 and 200 μg/mL) were utilized for estimating the bacterial growth rate and the Minimum Inhibitory Concentration (MIC). The mechanism of interaction between AuNPs and bacteria was evaluated by transmission electron microscopic image analysis. Confocal Laser Scanning Microscopic technique was used to study the cytotoxic action of AuNPs and laser against C. psudotuberculosis. Results revealed that MIC of AuNPs and AuNPs - laser combined therapy were 200 μg/mL and 100 μg/mL respectively. TEM image analysis illustrated that gold nanoparticles penetrated the thick wall of C. psudotuberculosis and accumulated as intracellular agglomerates. Laser light enhanced the antimicrobial activity of gold nanoparticles by at least one fold due to its photo thermal combined effect that might be used as an effective antibacterial approach against C. pseudotuberculosis.

Keywords: Antibacterial agent; C. pseudotuberculosis; Caseous lymphadenitis; Gold nanoparticles; Laser; Sheep and goats.

Graphical abstract

Fig. 1

Characterization of gold nanoparticles (AuNPs). (A): Absorption spectrum of gold nanoparticles. (B): HRTEM image showing spherical shape of prepared gold nanoparticles with average size 25 nm. (C): Particle size distribution of prepared gold nanoparticles showing the average size of 25 nm. (D): XRD pattern analysis indicating the formation of gold nanoparticles with cubic unit crystal.

Fig. 2

Antibacterial activity of AuNPs and Laser induced AuNPs against C. pseudotuberculosis at different gold concentration (0. 50, 100 and 200 µg/ml) and different laser exposure times (5, 10 and 20 min). Plates represent the plate count method describing the number of growth colonies and the growth curves represent the percentage of inhibition rate of all treatments.

Fig. 3

HR-TEM electrograph of intracellular localization of gold nanoparticles within C. pseudotuberculosis bacteria, (A): penetration of AuNPs into the cell wall and accumulation in cytoplasm by HR-TEM. (B): Energy Dispersive X-ray (EDX) image analysis of the intracellular AuNPs aggregates.

Fig. 4

Intracellular mode of action imaged by HR-TEM. (A): untreated bacterial cell. (B): AuNPs induce vacuole formation in cytoplasm. (C): AuNPs-laser induced cellular destruction.

Fig. 5

CLSM images for viable/dead C. pseudotuberculosis bacteria in the AuNPs co-culture and stained with Acridine Orange (AO) green fluorescence and Ethidium Bromide (EB) red fluorescence.. (A) Deionized Water control; (B) 50 μg/mL AuNPs. (C) 100 μg/mL AuNPs; (D) 200 μg/mL AuNPs. Magnification, ×40.

Fig. 6

CLSM images for viable/dead C. pseudotuberculosis bacteria in the AuNPs co-culture and stained with Acridine Orange (AO) green fluorescence and Ethidium bromide (EB) red fluorescence, staining. (A) Laser - deionized Water control; (B) 50 µg/mL AuNPs-Laser. (C) 100 µg/mL AuNPs-Laser; (D) 200 µg/mL AuNPs-Laser. Magnification, ×40.