Background  In recent years, it has been found that bacteria can secrete bacterial extracellular vesicles (BEVs) to play important biological functions. Engineering modified BEVs for drug delivery carrier construction and new vaccine development have become new research hotspots. However, there is currently a lack of comparative studies on the relevant properties of BEVs from different sources.

  Objective  To investigate the physicochemical properties and biological characteristics of BEVs derived from G + bacteria represented by Staphylococcus aureus and G- bacteria represented by Escherichia coli.

  Methods  In this study, a tangential flow filtration system (TFF) and ultracentrifugation were used to construct a method for the extraction of BEVs. The BEVs of Staphylococcus aureus and Escherichia coli were compared by electron microscope morphology, particle size, potential, protein production, and immune cell activation.

  Results  BEVs with double-membrane structure in cup-shaped and saucer-packed structures could be seen under transmission electron microscope, and the size was about 20-300 nm. Most of the BEVs of Staphylococcus aureus were 50.75 ± 0.00 nm in diameter, and the particle size range was 37.84 - 396.1 nm; Most of the E. coli outer vesicles were 22.15 ± 1.92 nm in diameter, and the particle size range was 13.54-78.82 nm; The BEVs of Staphylococcus aureus and Escherichia coli were negatively charged at (-19.7 ± 0.17) mV and (24.1 ± 2.55) mV, respectively; The protein yields of BEVs of Staphylococcus aureus and E. coli were (0.94 ± 0.04) mg and (1.71 ± 0.19) mg, respectively; The number of particles was 4.00 × 10¹¹( ± 1 × 10¹⁰)/mL, 9.47 × 10¹¹( ± 2.52 × 10¹⁰)/mL; The ratio of particle number/protein amount was 5.59 × 10¹¹( ± 1.6 × 10¹⁰), 4.24 × 10¹¹( ± 6.43 × 10¹⁰), respectively; After adding S. aureus vesicles, the proportion of CD86-positive cells in the macrophage cell line RAW264.7 was 20.23%, which was 45.9 times higher than that of the control group (PBS group) (P＜0.001); The proportion of CD206-positive cells was 4.5%, which was 34.6 times higher than that in the control group (P＜0.001). After adding E. coli-derived BEVs, the proportion of CD86-positive cells in RAW264.7 was 11.16%, which was 25.4 times higher than that in the control group (P＜0.001); The proportion of CD206-positive cells was 2.09%, which was 16.1 times higher than that in the control group (P＜0.05).

  Conclusion  In this study, BEVs from Staphylococcus aureus and Escherichia coli are successfully extracted by the tangential flow filtration system and ultracentrifugation. And the diameter of BEVs from S. aureus is slightly larger than that of BEVs from E. coli, and the zeta potential is slightly higher than that of E. coli. Under the same condition, the output of BEVs from Staphylococcus aureus is lower than that of Escherichia coli, but it has a stronger ability to activate immune cells.