Evaluating a Genetic Approach to Expand the Capacity Of Bacterial Cytological Profiling to Delineate Antibiotic Mechanisms Of Action

Abstract

Understanding how antibiotics affect bacterial cells beyond their primary target is essential for developing next-generation therapeutics. Traditional profiling methods primarily focus on identifying a drug’s direct target, often overlooking the downstream cellular effects of antibiotic treatment. This study evaluates a modified Bacterial Cytological Profiling (BCP) approach that uses a panel of Escherichia coli mutants with deletions in transcription-associated genes to characterize antibiotic mechanisms of action beyond their primary target. By integrating GFP-based translational readouts with measurements of DNA content (DAPI), membrane structure (FM4-64FX), and cell size (FSC), analyzed via flow cytometry, we examined cellular responses to two transcription-blocking rifamycins: rifampicin (RIF) and rifabutin (RFB). We found that many mutants exhibited consistent and gene-specific cytological responses to RIF, highlighting the method’s ability to reveal downstream effects of RNAP inhibition. Additionally, drug-specific phenotypes emerged between RIF and RFB, demonstrating that the modified BCP approach can resolve subtle mechanistic differences between closely related antibiotics. Together, these findings validate the modified BCP approach as a tool for uncov- ering antibiotic action with greater depth and resolution, offering valuable insights for future drug development.