Bacterial cytological profiling for mode-of-action discovery of natural product-derived antibiotics: progress, challenges, and future directions.

Covering: 2013 up to 2026The rapid escalation of antimicrobial resistance has outpaced the discovery of natural product (NP)-derived antibiotics, underscoring the need for new strategies to identify and characterize antibacterial agents. NPs have historically dominated antibiotic development due to their structural and mechanistic diversity, yet their chemical complexity, low abundance, laborious dereplication, and challenging mode of action (MoA) identification continue to limit discovery efficiency. Bacterial Cytological Profiling (BCP), an image-based phenotypic screening, has emerged as a powerful approach capable of capturing rich, single-cell-resolved responses, making it particularly well suited for investigating complex NP-derived antibiotic discovery. This review focuses on how BCP, a prokaryotic image-based strategy central to antibiotic MoA elucidation, addresses key challenges in NP-derived antibacterial discovery, including dereplication, deconvolution of multiple MoAs, and identification of novel mechanisms. We also discuss current biological and methodological limitations of the approach and provide practical perspectives for NP researchers seeking to implement BCP in discovery programs. Finally, we examine how lessons from the widely adopted eukaryotic image-based profiling method, the Cell Painting Assay, could inform future BCP development and propose a "BCP v1.0" framework to facilitate broader adoption and ultimately accelerate NP-derived antibiotic discovery.