Metal and metal oxide nanoparticles, including copper nanoparticles (CuNPs), display antimicrobial activities and are regarded as promising microorganism inhibitors. Here, we explored the antimicrobial activity of CuNPs in Escherichia coli (E. coli) using two particle sizes (20 and 60 nm) and five concentrations (1, 5, 10, 50 and 100 μg/mL). The result showed a concentration-dependent trend of bactericidal activities for both size groups, with 20 nm particles more effective than 60 nm particles at low concentrations. The membrane disruption caused by CuNPs was confirmed by electron microscopy, PI staining and protein leaking analysis. However, the results of reactive oxygen species generation and genomic DNA damage revealed that the size and concentration of CuNPs were factors affecting the induction of multiple bactericidal mechanisms simultaneously on different scales. Further results of annexin V-PI staining supported this hypothesis by showing the shifting composition of the early-, late- and non-apoptotic dead cells across the CuNP groups. Many CuNP treatment groups were rescued when four mammalian modulators-wortmannin, necrosulfonamide, Z-VAD-FMK, and SBI-0206965-were applied separately. The results suggest the possible existence of bacterial programmed cell death pathways in E. coli which could be triggered by CuNP treatments.
Keywords: apoptosis; bactericide; copper nanoparticles (CuNPs); membrane leakage; programmed cell death; reactive oxygen species (ROS).