Antimicrobial and Anti-Biofilm Activity of Dichlorophen-Functionalized Gold Nanoparticles Against Carbapenem-Resistant Enterobacteriaceae

Panjie Hu,1,2 Huale Chen,1,2 Changrui Qian,1,2 Qingxia Fu,1,2 Shihang Zhang,1,2 Zeyu Huang,1,2 Haifeng Liu,1,2 Cui Zhou,1,2 Mo Shen,1,2 Tieli Zhou1,2 1Department of Clinical Laboratory, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang, People’s Republic of China; 2Key Laboratory of Clinical Laboratory Diagnosis and Translational Research of Zhejiang Province, Wenzhou, Zhejiang, People’s Republic of ChinaCorrespondence: Mo Shen, Department of Clinical Laboratory, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang, People’s Republic of China, Email [email protected] Tieli Zhou, Department of Clinical Laboratory, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang, People’s Republic of China, Email [email protected]: The global emergence and spread of carbapenem-resistant Enterobacteriaceae (CRE) represent a major threat to effective clinical antimicrobial therapy, highlighting the urgent demand for alternative treatment strategies. This study aims to develop dichlorophen-functionalized gold nanoparticles (DDM_Au NPs) as a novel approach to combat CRE and their associated biofilms.Methods: Two structurally related antiparasitic compounds, bithionol and dichlorophen, were functionalized with Au NPs using a one-pot synthesis technique and thoroughly characterized. Their antibacterial activity was assessed through standard antimicrobial susceptibility testing and bacterial growth curve analysis. Antibiofilm properties were evaluated using crystal violet staining, scanning electron microscopy, and confocal laser scanning microscopy. The underlying mechanism of action was investigated by measuring reactive oxygen species production and assessing bacterial membrane permeability. Biocompatibility was evaluated via hemolysis assays, in vivo murine studies, and Galleria mellonella infection models. A urinary catheter model contaminated with biofilms, along with murine models of abdominal and pulmonary infection, was employed to assess device-associated applicability and therapeutic efficacy in vivo.Results: DDM_Au NPs demonstrated potent antibacterial activity against CRE, with minimum inhibitory concentrations ranging from 4 to 16 μg/mL. These nanoparticles effectively inhibited biofilm formation and promoted the disruption of mature biofilms, resulting in bacterial load reductions of 2– 6  log10 CFU/mL on infected urinary catheters. Mechanistic studies revealed that their antimicrobial activity was primarily driven by disruption of bacterial membrane integrity and induction of intracellular oxidative stress through elevated reactive oxygen species production. Notably, DDM_Au NPs exhibited favorable biocompatibility and significantly reduced bacterial burdens at infection sites by 4– 5 log10 CFU/mL, while also alleviating inflammatory responses and limiting tissue damage across multiple animal infection models.Conclusion: This study introduces a streamlined and effective strategy for achieving both antibacterial and antibiofilm effects using antiparasitic drug-functionalized Au NPs. DDM_Au NPs show strong promise as innovative antimicrobial agents for treating clinical CRE infections and reducing environmental contamination in healthcare environments. Keywords: anti-biofilm, antiparasitic drug, carbapenem-resistant Enterobacteriaceae, dichlorophen, gold nanoparticle