Chronic CuO Nanoparticles Exposure Enhances Bacterial Antibiotic Sensitivity and Attenuates Bacterial Pathogenicity

Copper oxide nanoparticles (CuO NPs) are widely applied in antimicrobial technologies and consumer products, yet the long-term microbiological consequences of chronic sublethal exposure remain poorly understood. In this study, we demonstrate that Gram-negative bacteria (Escherichia coli ATCC 25922, ATCC 35128, BAA 2452, and Pseudomonas aeruginosa CICC 21636) exposed to CuO NPs for 180 generations developed resistance to the nanoparticle and exhibited increased susceptibility to multiple antibiotics, with inhibition rates rising by up to 29.4% at MIC₅₀. Mechanistic investigations in E. coli ATCC 25922 revealed a multifaceted adaptation response involving (1) a 2.1-fold increase in superoxide dismutase activity to counteract oxidative stress, (2) activation of the Cpx envelope stress response, resulting in more than 2-fold higher extracellular protease activity, and (3) suppression of flagellar biosynthesis (52% fewer flagella) and motility (43% reduction in migration diameter) as an energy-conservation strategy. Although downregulation of outer membrane porins and energy metabolism pathways typically promotes antibiotic resistance, impaired biofilm formation (32.7% reduction in biofilm biomass), closely associated with flagellar dysfunction, has emerged as the dominant factor driving enhanced antibiotic susceptibility. Moreover, reduced host cell damage and attenuated inflammatory responses suggested a concurrent decline in bacterial virulence. These phenotypic changes were largely transcriptionally regulated and attributed mainly to the nanospecific effects of CuO NPs rather than released Cu(II) ions. Collectively, our findings reveal a previously unrecognized trade-off in bacterial adaptation to engineered nanomaterials, offering insights into the dual role of CuO NPs as antimicrobial agents and potential antibiotic sensitizers.