Isolation and Characterization of CRKP Outer Membrane Vesicles

OMVs were extracted by an electrophoresis and dialysis-based method (ELD) with a 300 kDa cut-off dialysis bag [17 (link)]. Specially, 200 ml of DMEM (Gibco, Shanghai, China) inoculated with the single colony was incubated at 37 ℃ with shaking at 200 rpm for 20–24 h. The culture solution was centrifuged at 10,000×g for 10 min to collect the culture supernatant, which was then filtered through a 0.22 μm filter (Beyotime, Shanghai, China) to remove bacteria and bacterial debris. Next, the OMVs of CRKP was isolated from filtered supernatant as previously described [17 (link)].
The OMVs of CRKP were photographed by transmission electron microscopy (TEM). Five µL of OMV suspension fixed with anhydrous ethanol was dropped onto a 200-mesh copper wire at room temperature for 1 min. Then, the sample was drained with filter paper and negatively dyed with 2% uranium acetate for 1 min. Afterwards, the excess dye was drained with filter paper. Then, the OMVs were observed by a Tecnai G2 Spirit Bio transmission electron microscope (FEI, America). NanoSight NS500 (Thermo Scientific, America) was used for determination of OMVs, and the particle size and concentration of OMVs were analyzed by NTA software.