ETEC, not ETEC-R, OMVs are protective and induced by polymyxin B. (A) A mid-log culture of ETEC was treated with polymyxin B (4 μg/mL, final concentration) simultaneous with no addition (PMB), 10 μg of WT (K12) OMVs (PMB + OMV), 10 μg of ETEC-derived OMVs (PMB + ETEC OMV), or 10 μg ETEC-R-derived OMVs (PMB + R-ETEC-OMV). Relative growth (% Survival) was determined compared to cultures without antibiotic (Untreated). (n = 9) (B) To titrate OMV-mediated protection for ETEC, ETEC OMVs (final concentrations indicated) were added simultaneously with polymyxin B (5 μg/mL, final concentration) to a mid-log phase ETEC culture and co-incubated 2 h at 37°C. Relative growth (% Survival) was determined compared to cultures without antibiotic. (n = 6) OMV yield was quantitated for mid-log phase cultures of ETEC (C) or ETEC-R (D) treated for 14 h with 3 μg/ml polymyxin B. (n = 6 for both C and D) OMV production was normalized to the CFU/mL of each culture at the time of vesicle harvest, and relative fold-differences compared to untreated cultures are shown.