Flow Cytometry Analysis of Perturbations in the Bacterial Cell Envelope Enabled by Monitoring Generalized Polarization of the Solvatochromic Peptide UNR-1

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