Antibacterial activity and mechanism of action of zinc oxide nanoparticles against Campylobacter jejuni

Abstract

The antibacterial effect of zinc oxide (ZnO) nanoparticles on Campylobacter jejuni was investigated for inhibition and inactivation of cell growth. The results showed that C. jejuni was extremely sensitive to treatment with ZnO nanoparticles. The MIC of ZnO nanoparticles for C. jejuni was determined to be 0.05 to 0.025 mg/ml, which is 8- to 16-fold lower than that for Salmonella enterica serovar Enteritidis and Escherichia coli O157:H7 (0.4 mg/ml). The action of ZnO nanoparticles against C. jejuni was determined to be bactericidal, not bacteriostatic. Scanning electron microscopy examination revealed that the majority of the cells transformed from spiral shapes into coccoid forms after exposure to 0.5 mg/ml of ZnO nanoparticles for 16 h, which is consistent with the morphological changes of C. jejuni under other stress conditions. These coccoid cells were found by ethidium monoazide-quantitative PCR (EMA-qPCR) to have a certain level of membrane leakage. To address the molecular basis of ZnO nanoparticle action, a large set of genes involved in cell stress response, motility, pathogenesis, and toxin production were selected for a gene expression study. Reverse transcription-quantitative PCR (RT-qPCR) showed that in response to treatment with ZnO nanoparticles, the expression levels of two oxidative stress genes (katA and ahpC) and a general stress response gene (dnaK) were increased 52-, 7-, and 17-fold, respectively. These results suggest that the antibacterial mechanism of ZnO nanoparticles is most likely due to disruption of the cell membrane and oxidative stress in Campylobacter.

FIG. 1.

Antibacterial activities of ZnO nanoparticles against C. jejuni, S. enterica serovar Enteritidis, and E. coli O157:H7. Freshly grown bacterial cultures (10⁸ to 10⁹ CFU/ml) were treated with a range of concentrations of ZnO nanoparticles. Culturable cell numbers were determined at the time intervals after treatment shown on the figure. The values for CFU/ml are the means of 12 replicates. Error bars indicate standard deviations of the means.

FIG. 2.

Scanning electron microscopic images of C. jejuni. (A) C. jejuni cells in the mid-log phase of growth were treated with 0.5 mg/ml of ZnO nanoparticles for 12 h under microaerobic conditions. (B) Untreated cells from the same growth conditions were used as a control.

FIG. 3.

EMA-qPCR of C. jejuni membrane integrity. Mid-log-phase cells exposed to different concentrations of ZnO nanoparticles were briefly treated with (black bars) and without (white bars) EMA. Inhibition of DNA amplification was quantified by real-time PCR of the hipO gene. Reduced DNA amplification in cells exposed to 0.3 and 0.5 mg/ml of ZnO nanoparticles indicates a certain degree of membrane leakage in the treated cells.

FIG. 4.

Relative gene expression levels of ZnO nanoparticle-treated and untreated C. jejuni. C. jejuni cells in the late log phase of growth were treated with 0 or 0.1 mg/ml of ZnO nanoparticles for 30 min. Transcripts of the selected genes were quantified by RT-qPCR, and data were analyzed using the comparative critical threshold (ΔΔCT) method. The relative expression ratio for each gene is presented as a log₂ value in the histogram. A ratio greater than zero (>0) indicates upregulation of gene expression and a ratio below zero (<0) indicates downregulation. Error bars indicate standard deviations for three replicates.