Phat Voong Vinh, Lim Andrew Shih Teong, De Cozar-Gallardo Cristina, Alvaro Maria Isabel Castellote, Alvarez Demetrio Muñoz, Alvaro Elena Fernandez, Ballell-Pages Lluis, Lozano-Arias Sonia, Baker Stephen
The Hospital for Tropical Diseases, Wellcome Trust Major Overseas Programme, Oxford University Clinical Research Unit, Ho Chi Minh City, Vietnam.
The ALBORADA Drug Discovery Institute, University of Cambridge, Cambridge, United Kingdom.
NPJ Antimicrob Resist. 2025 May 15;3(1):40. doi: 10.1038/s44259-025-00110-6.
The Gram-negative bacterial species Shigella is the second leading cause of diarrhea among children in low and middle-income countries (LMICs) and is a World Health Organization (WHO) priority pathogen. Shigella infections are becoming increasing difficult to treat due to antimicrobial resistance (AMR), leading to an urgent need for new antimicrobial agents with novel modes of action. Shigella pathogenesis is largely intracellular and antibacterial chemicals that preferentially work inside cells may be desirable to limit collateral AMR and block key components of the Shigella infection cycle. Aiming to facilitate the process of identifying antibacterial chemicals that kill intracellular Shigella, we developed a high-throughput screening (HTS) cell-based chemical screening assay. The three-dimensional (3-D) assay, incorporating Shigella invasion into Caco-2 cells on Cytodex 3 beads, was scaled into a 384-well platform for screening chemical compound libraries. Using this assay, we evaluated >500,000 compounds, identifying 12 chemical hits that inhibit Shigella replication inside cells. This simple, efficient and HTS-compatible assays circumvents many of the limitations of traditional screening methods with cell monolayers and may be deployed for antibacterial compound screening for other intracellular pathogens.
革兰氏阴性菌志贺氏菌属是低收入和中等收入国家（LMICs）儿童腹泻的第二大主要病因，也是世界卫生组织（WHO）的重点病原体。由于抗菌药物耐药性（AMR），志贺氏菌感染的治疗变得越来越困难，因此迫切需要具有新作用模式的新型抗菌药物。志贺氏菌的发病机制主要是细胞内感染，优先作用于细胞内的抗菌化学物质可能有助于限制附带的AMR并阻断志贺氏菌感染周期的关键组成部分。为了促进鉴定杀死细胞内志贺氏菌的抗菌化学物质的过程，我们开发了一种基于细胞的高通量筛选（HTS）化学筛选试验。将志贺氏菌侵入Cytodex 3珠上的Caco-2细胞的三维（3-D）试验扩展到384孔平台，用于筛选化合物文库。使用该试验，我们评估了超过500,000种化合物，鉴定出12种抑制细胞内志贺氏菌复制的化学命中物。这种简单、高效且与HTS兼容的试验规避了传统细胞单层筛选方法的许多局限性，可用于其他细胞内病原体的抗菌化合物筛选。
