Extracellular Vesicles of Drug-Resistant Staphylococcus aureus

Drug-resistant S. aureus BW15 and BWMR26 28 (link) and drug-sensitive S. aureus S29213 were cultured on Luria broth (LB) agar overnight at 37 °C and then a single colony was inoculated into LB medium on a rotary shaker. Then, a 1:100 dilution of the bacteria were cultured at 37 °C in LB medium until they reached late-logarithmic-phase. The bacterial culture was centrifuged at 6000 × g for 20 min to remove the bacteria, followed by filtering the medium through a 0.45 μm vacuum filter. The medium was then centrifuged at 150,000 × g for 2 h at 4 °C (Beckman Coulter, California, USA). The precipitates were considered the EV pellets 24 (link). EV13, EV15, and EV26 were from S. aureus S29213, S. aureus BW15 and S. aureus BWMR26, respectively.
The EV particle morphology was monitored by transmission electron microscopy (TEM; Tecnai 12, Philips, Holland). The hydrodynamic size and potential were measured by Zeta Plus (Malvern Instruments, Worcestershire, UK). The production of EVs from each S. aureus strain was determined using a BCA protein assay and was shown as the total protein (mg) in EVs derived from 1 L of late-logarithmic-phase cultures. The total proteins in the EVs derived from different S. aureus strains were analyzed by SDS-PAGE and the protein abundance of the EV was analyzed by ImageJ 29 (link).