Flow Cytometry\nAnalysis of Perturbations in the Bacterial\nCell Envelope Enabled by Monitoring Generalized Polarization of the\nSolvatochromic Peptide UNR‑1

The\nworldwide spread of antibiotic resistance is considered\nto\nbe one of the major health threats to society. While developing new\nantibiotics is crucial, there is also a strong need for next-generation\nanalytical methods for studying the physiological state of live bacteria\nin heterogeneous populations and their response to environmental stress.\nHere we report a single-cell high-throughput method to monitor changes\nin the bacterial cell envelope in response to stress based on ratiometric\nflow cytometry. We used a sensitive fluorescent molecular probe, the\nNile Red-based solvatochromic antimicrobial peptide <b>UNR-1,</b> with defined cellular localization in Gram-positive and Gram-negative\nbacteria. We also developed a robust protocol for calculating the\ngeneralized polarization (GP) of fluorescence adapted to flow cytometry.\nOur methodology enabled rapid detection of perturbations in the bacterial\ncell envelope caused by exposure to antibiotics, heat shock, and other\nfactors.