Single-Cell Analysis of Perturbations in the Bacterial Cell Envelope Enabled by Monitoring Generalized Polarization of a Nile Red-Based 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 primordial, there is also a high need for next-generation analytical methods for surveying the physiological state of live bacteria in heterogeneous populations and their response to environmental stress. Here we report a single-cell high-throughput method for monitoring bacterial stress and environmental adaptation based on ratiometric flow cytometry. We used a combination of a sensitive fluorescent molecular tool, the red solvatochromic antimicrobial peptide UNR-1 with defined cellular localization in Gram-positive and Gram-negative bacteria, with a robust protocol of calculating generalized polarization (GP) of fluorescence adapted to flow cytometry. Our methodology enabled rapid detection of perturbations in the bacterial cell envelope caused by heat shock, transfer to a nutrient-poor medium, fixation, and exposure to antibiotics.