High-resolution analysis of synergistic effects between membrane active peptides and classical antibiotics on bacterial membranes

The increasing prevalence of antimicrobial resistance highlights the need for a deeper understanding of how membrane-active compounds interact with bacterial membranes. Model membrane systems provide a powerful platform for studying these interactions under controlled conditions. In this work, giant unilamellar vesicles (GUVs) mimicking Gram-negative bacterial membranes were investigated using advanced fluorescence microscopy approaches. Membrane organization and biophysical changes induced by antimicrobial treatment were characterized using the environment-sensitive probe NR12A in combination with spectral imaging on a Zeiss LSM 980 microscope. Quantitative analysis of generalized polarization (GP) enabled the detection of subtle alterations in membrane order and lipid packing at different treatment conditions. Complementary fluorescence microscopy approaches were employed to visualize membrane responses at high spatial resolution and to assess heterogeneity within individual vesicles. Our results demonstrate that advanced optical microscopy techniques can reveal nanoscale-relevant membrane remodeling processes that are not accessible through conventional bulk measurements. These findings highlight the potential of modern fluorescence imaging and quantitative membrane analysis for elucidating the mechanisms of membrane-targeting antimicrobials and for supporting the development of novel anti-infective strategies.