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.氧化铜纳米颗粒（Copper oxide nanoparticles, CuO NPs）已被广泛应用于抗菌技术及消费品中，但慢性亚致死暴露所引发的微生物学远期效应仍未得到充分阐释。本研究显示，经CuO NPs暴露180代的革兰氏阴性菌（Gram-negative bacteria）——包括大肠杆菌（Escherichia coli）ATCC 25922、ATCC 35128、BAA 2452以及铜绿假单胞菌（Pseudomonas aeruginosa）CICC 21636——不仅对该纳米颗粒产生了耐药性，同时对多种抗生素的敏感性显著升高，在半数抑菌浓度（MIC50）下的抑菌率最高可提升29.4%。以大肠杆菌ATCC 25922为模型开展的机制研究揭示了多维度的适应性应答，具体包括：（1）超氧化物歧化酶（superoxide dismutase）活性上调2.1倍，以对抗氧化应激；（2）激活Cpx包膜应激反应（Cpx envelope stress response），使胞外蛋白酶（extracellular protease）活性提升2倍以上；（3）抑制鞭毛生物合成（flagellar biosynthesis），使鞭毛数量减少52%，同时降低运动能力，迁移直径降低43%，以此作为能量节约策略。尽管外膜孔蛋白（outer membrane porins）与能量代谢通路（energy metabolism pathways）的下调通常会促进抗生素耐药性，但与鞭毛功能异常密切相关的生物膜形成受损，其生物膜生物量（biofilm biomass）降低32.7%，已被证实是驱动抗生素敏感性增强的核心因素。此外，宿主细胞损伤（host cell damage）减轻与炎症反应（inflammatory responses）减弱提示细菌毒力同步下降。上述表型变化主要受转录调控（transcriptionally regulated），且其核心诱因是CuO NPs的纳米特异性效应（nanospecific effects），而非其释放的二价铜离子（Cu(II) ions）。综上，本研究结果揭示了细菌在适配工程纳米材料过程中此前未被认知的权衡效应（trade-off），为理解CuO NPs兼具抗菌剂与潜在抗生素增敏剂的双重作用提供了全新视角。