The release of outer membrane vesicles (OMVs) from bacteria is an important phenomenon.. OMVs are released spontaneously during the normal growth of Gram-negative bacteria . OMVs have been described in both pathogenic and nonpathogenic Gram-negative bacteria such as Escherichia coli, Shigella spp, Neisseria spp, Porphyromonas spp, Pseudomonas aeruginosa, Helicobacter pylori, Vibrio spp, Salmonella spp, Brucella spp, Actinobacillus spp., Xenorhabdus nematophilus , and Pseudoalteromonas antarctica. OMVs possesses a bilayer membrane and contain components such as lipoproteins, outer membrane proteins (OMP), lipopolysaccharide (LPS), and some periplasmic components. 

Maintain a culture of B. melitensis 16 M (ATCC 23456) and B. melitensis VTRM1 rough mutant derived from B. melitensis 16 M.. Both strains can be cultured on tryptic soy agar (TSA) plates supplemented with 0.7% yeast extract and incubate 36 h at 37°C. Prepare a bacterial suspension  from both strains, adjusted each to 0.5 g of cells per mL of tryptic soy broth, of which 0.5 mL is spread onto each of 100 TSA plates (10 cm diameter) and incubate at 37°C for 48 h.

The OMVs purificationcan be  performed according to the protocol described by Gamazo et al., 1989. Briefly, the bacteria are harvested with a rubber policeman and suspended in 250 mL sterile phosphate-buffered saline (PBS 0.1 M, pH 7.3). The bacterial suspension is centrifuged at 10,000 ×g for 30 min. The supernatant is passed through a 0.22 μm filter (Millipore Corp.), and a sterility test is performed by culturing an aliquot onto a TSA plate followed by incubation for 72 h at 37°C; the filtrate was stored at 4°C during the viability check. The filtered supernatant is centrifuged at 100,000 ×g for 2 h at 4°C. The pellet is washed twice with 25 mL of sterile PBS, and the OMVs were resuspended in 1 mL of sterile PBS. The total protein concentration is determined by using PIERCE-BCA (PIERCE) reagents as per manufacturer’s recommendations. The OMVs samples are divided into 0.5 mL aliquots and stored at −20°C until use.