Acquisition of ETEC resistance to polymyxin B is reduced by co-incubation with high concentrations of OMVs. At hourly time-points for 0-7 h of co-incubation, equivalent volumes of the samples described below were streaked on each plate in a pattern indicated by the template diagram. Top row: ETEC co-incubated with (A) nothing, (B, D) a high concentration of ETEC OMV (2 μg/mL) and polymyxin B (3.5 μg/ml), or (C) polymyxin B alone (3.5 μg/mL). Samples were streaked either on LB agar (A-C), or LB containing 5 μg/ml polymyxin B (D-E). (E) ETEC co-incubated with ETEC OMV (3 μg/mL) and polymyxin B (3.5 μg/mL) for 5 h, then an additional 5 μg/mL polymyxin B was added, and plated on LB containing 5 μg/mL polymyxin B. Resistance was seen by hour 7 without decreasing cell population significantly. Bottom row: ETEC co-incubated with (F) nothing, or (G, I) 1.4 μg/mL ETEC OMV and 3.5 μg/ml polymyxin B, and (H, J) polymyxin B alone (3.5 μg/mL), streaked on LB (F-H) or LB containing 5 μg/mL polymyxin B (I-J). (n = 9 for all experiments).