Jia Zhang,1,2,* Xiaotuan Zhang,2,3,* Zhuocheng Yao,2 Juan Pan,2 Jianzhong Ye,2 Ping Xia,2 Tieli Zhou,2 Jianming Cao1,2 

1School of Laboratory Medicine and Life Science, Wenzhou Medical University, Wenzhou, Zhejiang, People’s Republic of China; 2Department of Clinical Laboratory, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang, People’s Republic of China; 3Department of Clinical Laboratory, The Second Affiliated Hospital, Hengyang Medical School, University of South China, Hengyang, Hunan, People’s Republic of China

*These authors contributed equally to this work

Correspondence: Jianming Cao; Tieli Zhou, Department of Clinical Laboratory, The First Affiliated Hospital of Wenzhou Medical University, Key Laboratory of Clinical Laboratory Diagnosis and Translational Research of Zhejiang Province, Wenzhou, Zhejiang, People’s Republic of China, Tel +86-577-8806-9595; +86-577-8668-9885, Fax +86-577-8806-9595; +86-577-8668-9885, Email wzcjming@163.com; wyztli@163.com

Introduction: Carbapenem-resistant gram-negative bacteria (CR-GNB) pose a significant threat to public health and require immediate attention. The development of novel antibacterial agents against CR-GNB has become an urgent priority, and nanomaterials offer promising solutions due to their unique properties. This study introduces 5-amino-2-mercaptobenzimidazole (5-A-2MBI) functionalized gold nanoparticles (5-A-2MBI_Au NPs) and evaluates their antibacterial activity against CR-GNB.
Methods: The 5-A-2MBI_Au NPs was synthesized using a one-pot method. Its biocompatibility, bactericidal properties, and mechanisms of action were systematically characterized through in vivo and in vitro toxicity tests, antimicrobial susceptibility testing, live/dead staining, membrane permeability and reactive oxygen species (ROS) generation assays, as well as transcriptomic analysis.
Results: The results of this study demonstrate that 5-A-2MBI_Au NPs exhibit excellent antibacterial efficacy against carbapenem-resistant gram-negative bacteria with various resistance mechanisms, with a minimum inhibitory concentration (MIC) of 2 μg/mL. In vivo experiments further confirmed that 5-A-2MBI_Au NPs not only possess effective bactericidal activity but also exhibit satisfactory biocompatibility. Mechanistic studies revealed that 5-A-2MBI_Au NPs enhance bacterial membrane permeability, increase the generation of reactive oxygen species, and disrupt intracellular oxidative stress and succinate synthesis, thereby conferring potent antibacterial activity. This study results demonstrate that 5-A-2MBI_Au NPs exhibit notable antibacterial efficacy against CR-GNB, with a minimum inhibitory concentration of 2 μg/mL. The antibacterial mechanism involves enhanced membrane permeability, increased reactive oxygen species production, and interference with intracellular oxidative stress and succinate synthesis. These mechanisms collectively contribute to the potent antibacterial activity of 5-A-2MBI_Au NPs against CR-GNB.
Discussion: 5-A-2MBI_Au NPs are a novel and highly effective antibacterial agent prepared through a simple process using benzimidazole and HAuCl4• 3H2O. They efficiently eradicate the most challenging multidrug-resistant GNB both in vitro and in vivo while demonstrating excellent biocompatibility. This highlights their potential as a promising antibacterial agent to combat multidrug-resistant GNB.

Keywords: carbapenem-resistant gram-negative bacteria, benzimidazole, gold nanoparticles, biocompatibility, transcriptome analysis