Establishment and application of a vesicle extraction method for clinical strains of Pseudomonas aeruginosa

: Pseudomonas aeruginosa is a versatile pathogen capable of causing illnesses that range from mild infections to life-threatening conditions. Its virulence is driven by a wide array of factors, among which extracellular vesicles (EVs) have gained recognition as important contributors to pathogenicity. Despite this, the full scope of their roles remains unclear. A major barrier to EV characterization is the difficulty of vesicle isolation-procedures are often lengthy, yield is low, and specialized equipment is required. In this study, we assessed the effectiveness of a rapid vesicle extraction method from clinical strains of P. aeruginosa. To that end, we first selected and characterized six phenotypically diverse clinical strains of P. aeruginosa (two reference strains and four clinical isolates, including one strain from a cystic fibrosis patient) and used them to evaluate the vesicle extraction method. The results obtained through SDS-PAGE analysis, Western blot, protein quantification, TEM, dynamic light scattering, and mass spectrometry indicated the presence of vesicles in all samples; however, it was also possible to observe a large number of contaminants in some of them (mainly strains LS07 and Z37). Subsequent enzymatic treatments (DNase and/or alginate lyase) allowed for the elimination of these contaminants, as observed by electron microscopy. Our results suggest that this method is suitable for vesicle extraction from clinical isolates of P. aeruginosa, where traditional methods, such as ultracentrifugation, are not available. However, the phenotypic complexity of these strains presents challenges that current rapid purification methods are ill-equipped to handle, highlighting the need for improved or alternative approaches.IMPORTANCEPseudomonas aeruginosa 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.