Microscopy-Based Multiwell Assay to Characterize Disturbed Bacterial Morphogenesis Upon Antibiotic Action

Abstract

The urgent need of new antimicrobial agents to combat life-threatening bacterial infections demands the identification and characterization of novel compounds that interfere with new and unprecedented target pathways or structures in multiresistant bacteria. Here, bacterial cell division has emerged as a new and promising target pathway for antibiotic intervention. Compounds, which inhibit division, commonly induce a characteristic filamentation phenotype of rod-shaped bacteria, such as Bacillus subtilis. Hence, this filamentation phenotype can be used to identify and characterize novel compounds that primarily target bacterial cell division. Since novel compounds of both synthetic and natural product origin are often available in small amounts only, thereby limiting the number of assays during mode of action studies, we here describe a semiautomated, microscopy-based approach that requires only small volumes of compounds to allow for the real-time observation of their effects on living bacteria, such as filamentation or cell lysis, in high-throughput 96-well-based formats. We provide a detailed workflow for the initial characterization of multiple compounds at once and further tools for the initial, microscopy-based characterization of their antibacterial mode of action.