Lightning talk + Poster Australasian Extracellular Vesicles Conference 2020

A new improved method for the isolation of bacterial extracellular vesicles by the depletion of "exosome-like particles " in bacteriological culture medium  (#46)

Lena Le 1 2 , Kate Friesen 3 , Richard L Ferrero 1 2 4
  1. Biomedicine Discovery Institute, Department of Microbiology, Monash Univeristy, Clayton, Victoria, Australia
  2. Centre for Innate Immunity and Infectious Diseases, Hudson Institute of Medical Research, Clayton, Victoria, Australia
  3. Department of Immunology , University of Toronto , Toronto, Canada
  4. Department of Molecular and Translational Sciences , Monash University , Clayton , Victoria, Australia

Bacterial extracellular vesicles (BEVs) are isolated from microorganisms that have been grown in culture media containing animal-derived products. Consequently, these media are likely to contain animal EVs, that may have an effect on downstream analyses. In the present study, we sought to: 1). develop an optimised protocol for BEV purification that would eliminate any animal-derived EVs from bacteriological culture medium; and 2) compare the properties of BEVs isolated using this new protocol with those isolated from bacteria grown in standard medium. For these studies, we used Brain Heart Infusion (BHI), a culture medium commonly used to grow a range of bacterial species. This medium contained particles that were reactive with a lipophilic dye (DiO). Nanoparticle tracking analysis showed that these particles were similar in size to those of BEVs and eukaryotic EVs, suggesting that BHI broth may contain animal-derived EVs. To deplete BHI broth of these “exosome-like particles” (ELPs), we used tangential flow filtration (TFF) with a Vivaflow membrane (50,000 MWCO). The amounts of ELPs were reduced by 1-2 log in the flow-through (FT) fractions compared to the retentate (RET). The ELPs from the two fractions did not react with an epithelial cell EV marker antibody (Epcam), but further experiments with other exosome markers are required. We found that the FT fractions supported bacterial growth better than normal BHI broth. Furthermore, BEVs isolated from bacteria that had been grown in these FT fractions were not only of higher purity and contained a larger number of proteins than those grown in standard BHI medium, but also stimulated higher levels of interleukin-8 production in mammalian cells. This study highlights the impact of the culture medium on the isolation of BEVs, a point that is often overlooked in the field. Overall, we have successfully developed an optimised method for the isolation of BEVs, which reduces the potential effects of “exosome-like particles” in bacteriological culture media.