Establishment and application of a vesicle extraction method for clinical strains of <i>Pseudomonas aeruginosa</i>

is a highly adaptable pathogen, and its extracellular vesicles (EVs) play important yet still not fully understood roles in its virulence. Efficient EV characterization depends on reliable extraction/isolation methods, but current approaches are often slow, low-yielding, and require specialized equipment. This study evaluated a new vesicle extraction method using six phenotypically diverse clinical strains. While vesicles were detected in all samples, some preparations contained contaminants, which were successfully reduced through enzymatic treatments. These findings indicate that the method can be used to extract EVs from clinical isolates, allowing these samples to be used for functional screening. With this approach, only preparations with relevant activities could then be purified using conventional protocols (such as density gradient ultracentrifugation), if required.