Decoding bacterial extracellular vesicles: A review on isolation and characterization techniques

Bacterial extracellular vesicles (bEVs) are membrane-bound phospholipid bilayered vesicles in the size range of 20-400 nm. These primarily include outer membrane vesicles (OMV) released by Gram negative bacteria and membrane vesicles (MV) released by Gram-positive bacteria. These vesicles are typically enriched in several molecules including nucleic acids, virulence proteins, toxins, and several others that aid in their crucial roles of transport of biomolecules, cell-cell communication, and microbial pathogenesis. An understanding of bEVs can help researchers develop anti-infective therapies targeting microbes unresponsive to antibiotics. The cornerstone of bEV research lies in effective isolation and characterization. Classical workflows include isolation with ultracentrifugation, size exclusion chromatography, and characterization with nanoparticle tracking analysis. Newer techniques for isolation and characterization include acoustic trapping and resistive pulse sensing respectively. Current review discusses recent developments in extracellular vesicle (EV) research, modifications to improve bEV yield, and provides a brief description of bEV cargo, whose understanding can aid in choosing the appropriate isolation and characterization strategy.