Combination of 2- <i>tert</i> -Butyl-1,4-Benzoquinone (TBQ) and ZnO Nanoparticles, a New Strategy To Inhibit Biofilm Formation and Virulence Factors of Chromobacterium violaceum

Drug-resistant bacteria have been raising serious social problems. Bacterial biofilms and different virulence factors are the main reasons for persistent infections. As a conditioned pathogen, Chromobacterium violaceum has evolved a vast network of regulatory mechanisms to modify and fine-tune biofilm development, contributing to multidrug resistance. However, there are few therapies to combat drug-resistant bacteria. Quorum sensing (QS) inhibitors (QSIs) are a promising strategy to solve antibiotic resistance. Our previous work suggested that 2-<i>tert</i>-butyl-1,4-benzoquinone (TBQ) is a potent QSI. In this study, the combination of zinc oxide nanoparticles (ZnO-NPs) and TBQ (ZnO-TBQ) was investigated for the treatment of Chromobacterium violaceum ATCC 12472 infection. ZnO-NPs attach to cell walls or biofilms, and the local dissolution of ZnO-NPs can lead to increased Zn²⁺ concentrations, which could destroy metal homeostasis, corresponding to disturbances in amino acid metabolism and nucleic acid metabolism. ZnO-NPs significantly improved the efficiency of TBQ in inhibiting the QS-related virulence factors and biofilm formation of C. violaceum ATCC 12472. ZnO-TBQ effectively reduces the expression of genes related to QS, which is conducive to limiting the infectivity of C. violaceum ATCC 12472. Caenorhabditis elegans nematodes treated with ZnO-TBQ presented a significant improvement in the survival rate by 46.7%. Overall, the combination of ZnO-NPs and TBQ offers a new strategy to attenuate virulence factors and biofilm formation synergistically in some drug-resistant bacteria. <b>IMPORTANCE</b> The combination of ZnO-NPs and TBQ (ZnO-TBQ) can compete with the inducer <i>N</i>-decanoyl-homoserine lactone (C₁₀-HSL) by binding to CviR and downregulate genes related to the CviI/CviR system to interrupt the QS system of C. violaceum ATCC 12472. The downstream genes responding to <i>cviR</i> were also downregulated so that virulence factors and biofilm formation were inhibited. Furthermore, ZnO-TBQ presents multiple metabolic disturbances in C. violaceum ATCC 12472, which results in the reduced multidrug resistance and pathogenicity of C. violaceum ATCC 12472. In an <i>in vivo</i> assay, C. elegans nematodes treated with ZnO-TBQ presented a significant improvement in the survival rate by 46.7% by limiting the infectivity of C. violaceum ATCC 12472. In addition, ZnO-TBQ inhibited the generation of virulence factors and biofilm formation 2-fold compared to either ZnO-NPs or TBQ alone. The combination of ZnO-NPs with TBQ offers a potent synergistic strategy to reduce multidrug resistance and pathogenicity.