Standardizing Bacterial Extracellular Vesicle Purification: A Call for Consensus.

Bacteria constitutively produce extracellular vesicles (EVs), which play a pivotal role in linking bacterial physiology to therapeutics. Despite significant advances over the past decade, critical challenges persist in isolating EVs due to the absence of standardized purification methods. While the International Society for Extracellular Vesicles provides well-defined minimal guidelines for human EVs, bacterial EV research still faces inconsistencies across laboratories, limiting reproducibility and broader utility. This review provides an in-depth overview of technical aspects of bacterial EV purification and gives future perspectives for achieving high-purity and reproducibility when isolating EVs from diverse bacterial species. It further addresses the necessity of distinguishing bacterial EVs from host-derived vesicles and potential contaminants, particularly in clinical and microbiota contexts. Establishing gold-standard protocols and defining bona fide bacterial EVs in parallel with the rigor seen in human EV studies will advance our understanding of their roles in host-microbe interactions, microbiota dysbiosis, and therapeutic potential.