A three-dimensional high throughput assay identifies novel antibacterial molecules with activity against intracellular Shigella

Abstract 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 resi...

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Main Authors: Voong Vinh Phat, Andrew Shih Teong Lim, Cristina De Cozar-Gallardo, Maria Isabel Castellote Alvaro, Demetrio Muñoz Alvarez, Elena Fernandez Alvaro, Lluis Ballell-Pages, Sonia Lozano-Arias, Stephen Baker
Format: Article
Language:English
Published: Nature Portfolio 2025-05-01
Series:npj Antimicrobials and Resistance
Online Access:https://doi.org/10.1038/s44259-025-00110-6
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Summary:Abstract 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.
ISSN:2731-8745