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              <p>The gut microbiota is a dynamic ecosystem essential to our health. Gut colonization begins at birth and gradually evolves, increasing its diversity and stability over time. The establishment of the gut microbiota at birth could be crucial for the individual&amp;#39;s future, according to the concept of Developmental Origins of Health and Disease (DOHaD). Several studies have revealed the ability of bacteria to produce extracellular vesicles carrying bioactive molecules. The molecules transported by bacterial extracellular vesicles (BEVs) can be involved in pathogenesis, antibiotic resistance, nutrient absorption, horizontal transfer, and detoxification. Due to differences in the structure of the bacterial envelope, extracellular vesicles of Gram-positive bacteria (MEVs) have long been neglected, and their role in establishing the relationship between the gut microbiota and the newborn has not yet been explored. Our study focused on two primary colonizing Gram-positive bacteria that are prevalent in the newborn gut: Bifidobacterium longum subsp. longum and Lactiplantibacillus plantarum. The aim was to establish a protocol for the extraction and purification of MEVs from these two bacteria. After measuring growth parameters, we implemented a negative staining protocol to observe them using a scanning transmission electron microscope (STEM) to examine the surface of the bacteria. Then, we tested and compared several methods for the extraction, purification, and detection of MEVs from B. longum and L. plantarum. The results obtained by protein equivalent detection and nanoparticle tracking analysis (NTA) showed the presence of MEVs for both strains with a relatively low yield obtained after size exclusion chromatography purification. Optimization of the protocol subsequently led us to significantly improve the yield of purified MEVs. Furthermore, transmission electron microscope (TEM) was used to visualize phenomena potentially related to MEVs production. In parallel, we defined stress conditions close to those encountered in the gut microbiota to understandtheir impact on MEVs production by these bacteria. The results obtained offer new avenues for better understanding the variation in MEVs production and vesicular content during the establishment of postnatal gut microbiota.</p>
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