Raman spectroscopy reveals growth phase-dependent molecular differences in bacterial membrane vesicles

Bacterial membrane vesicles (BMVs) have attracted significant attention as highly efficient transport vehicles for molecules crossing biological barriers and as key mediators in infection processes. Based on this increasing interest, the need for standardized isolation protocols and comprehensive analytical approaches becomes apparent. Here, we evaluated BMVs from the human pathogen <i>Pseudomonas aeruginosa</i>, isolated at six distinct growth phases, using physicochemical assays, functional characterization, and Raman spectroscopy. Conventional analyses revealed growth phase-dependent differences in protein content, surface charge, and immunogenicity. Raman spectroscopy provided detailed molecular fingerprints, identifying shifts in protein-to-lipid ratios, increased lipid saturation, and alterations in protein secondary structure during later growth phases. Importantly, the absence of nucleic acid-specific spectral markers confirmed the outer membrane origin of the vesicles. Together, these findings demonstrate that the timing of BMV isolation critically determines their molecular composition and functional properties and establish Raman spectroscopy as a powerful label-free tool for semi-quantitative profiling of BMVs.IMPORTANCE<i>Pseudomonas aeruginosa</i> is an opportunistic gram-negative pathogen and a leading cause of severe nosocomial infections. Its secreted bacterial membrane vesicles (BMVs) are increasingly recognized as mediators of pathogenicity and as potential therapeutic delivery systems. However, the lack of standardized and sensitive analytical techniques has hindered systematic characterization. Our study highlights the profound impact of the bacterial growth phase on BMV composition and immunogenicity. It introduces Raman spectroscopy as a chemically selective, label-free method for detecting subtle yet biologically relevant molecular changes. These insights provide a framework for improved standardization in BMV research and underscore the potential of Raman-based approaches in advancing both fundamental microbiology and translational applications.