ZnO Nanocrystals Inhibit Escherichia coli Biofilms by Suppressing glgA- and gltB-Dependent EPS Biosynthesis

Xinyuan Tian,1 Chaoyu Zhou,1 Qianyu Bai,1 Yinzhu Chen,1 Runqiu Cai,1 Jiaxin Lai,1 Linyi Ye,1 Wanru Feng,1 Yeru Wang,2 Qingyi Song,3 Tianlong Liu1 1State Key Laboratory of Veterinary Public Health and Safety, China Agricultural University, Beijing, People’s Republic of China; 2Risk Assessment Department, China National Center for Food Safety Risk Assessment, Beijing, People’s Republic of China; 3Anyang CAU Biotechnology Co., Ltd, Anyang, Henan, People’s Republic of ChinaCorrespondence: Tianlong Liu, State Key Laboratory of Veterinary Public Health and Safety, China Agricultural University, Beijing, 100193, People’s Republic of China, Email liutianlong@cau.edu.cn Yeru Wang, Risk Assessment Department, China National Center for Food Safety Risk Assessment, Building 2, No. 37, Guangqu Road, Chaoyang District, Beijing, 100022, People’s Republic of China, Email wangyeru@cfsa.net.cnIntroduction: Bacterial biofilms are a major cause of persistent and device-related infections due to their antibiotic resistance and ability to shelter bacteria. Zinc oxide nanocrystals (ZnO NCs), with their multiple antimicrobial mechanisms, have emerged as efficient antibacterial agents.Methods: In this study, we synthesized rod-shaped ZnO NCs and evaluated their anti-biofilm efficacy against Escherichia coli. Anti-biofilm activity was assessed at sub-minimum inhibitory concentration (MIC) and MIC levels. Transcriptomic analysis and qPCR were employed to examine gene expression changes in E. coli, with further mechanistic validation targeting specific metabolic pathways.Results: At sub-MIC levels, ZnO NCs potently inhibited biofilm formation and eradicated pre-formed E. coli biofilms. These treatments also markedly suppressed the synthesis of key extracellular polymeric substances (EPS) components, while MIC-level treatments effectively degraded existing EPS in mature biofilms. Concurrently, ZnO NCs reduced overall EPS density, loosened biofilm architecture, and increased its structural heterogeneity. Furthermore, bacterial motility (swimming, twitching, and swarming) was strongly impaired across sub-MIC to MIC concentrations. Transcriptomic analysis revealed that ZnO NCs downregulated genes associated with biofilm formation, motility, and amino acid biosynthesis. qPCR indicated that the downregulation of glgA and gltB impaired the synthesis of key EPS components.Conclusion: Mechanistically, we validate that in E. coli, ZnO NCs suppress glgA to disrupt glycogen-derived carbon precursors for polysaccharide synthesis, and downregulate gltB to impair glutamate synthase activity, thereby limiting nitrogen assimilation and amino acid supply for proteinaceous EPS components. These findings elucidate a previously undefined mechanism wherein ZnO NCs dismantle E. coli biofilms by simultaneously targeting two pivotal metabolic nodes (glgA and gltB) that fuel EPS production. This work provides not only a deeper mechanistic insight into anti-biofilm action against E. coli but also supports the potential of ZnO NCs as multi-targeted anti-biofilm agents. This image illustrates the effect of zinc oxide nanocrystals (ZnO NCs) treatment on the biofilm of Escherichia coli. The biofilm contains live and dead Escherichia coli cells, proteins, extracellular DNA, and polysaccharides. The study shows that the use of zinc oxide nanocomposites can affect the structure of the biofilm. Within the Escherichia coli cells, it is shown that ZnO NCs inhibit the expression of the glgA and gltB genes, thereby affecting the synthesis of polysaccharides and proteins. ZnO NCs also affect the assembly of flagella, thereby inhibiting their motility behavior, such as twisting, clustering, and swimming. The arrows in the figure indicate the flow direction of these metabolic processes and their impact on the motility ability of Escherichia coli.ZnO NCs impact E. coli biofilm, altering polysaccharides, proteins and motility.