OMVs alter antibiotic resistance phenotype in ETEC Adaptive (longer-term) bacterial resistance to polymyxin is typically based

on the upregulation of genes which lead to the modification of LPS [27, 33]. We wondered whether OMV-mediated defense would affect the onset of adaptive resistance of ETEC to polymyxin #ITF2357 randurls[1|1|,|CHEM1|]# B. A mid-log liquid culture of ETEC was treated with polymyxin B (3.5 μg/mL) and concurrently supplemented with either a relatively high concentration of ETEC OMVs (2 μg/mL) or buffer. Samples were taken hourly for up to 7 h post treatment, spread on LB agar and LB agar containing polymyxin B, and the plates inspected after 12 h incubation at 37°C (Figure 4). As expected from the results described earlier, ETEC cultures supplemented

with OMVs survived better compared to cultures that did not contain added OMVs (Figure 4B, C). However, we further observed that these bacteria were not able to grow on plates containing polymyxin B (Figure 4D). This suggests that the bacteria survived to a greater extent but did not become adapted to resist polymyxin. Figure 4 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 Caspase inhibitor 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 C1GALT1 (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). To test if the bacteria in the OMV-supplemented culture were simply incapable of becoming adaptively resistant, an additional 5 μg/ml polymyxin B was added at hour 5 after the OMV-polymyxin B co-incubation and the culture was then plated on polymyxin B-containing agar. Resistant ETEC were observed without a detectable decrease in cell number after 7 h (Figure 4E). This result demonstrated that the OMV-protected ETEC had the capacity to adapt to high levels of antibiotic and achieve resistance if the polymyxin dose was increased beyond the amount the OMVs could protect. This reasoning was confirmed in further experiments in which we used a lower OMV concentration (0.7 μg/ml) with the same concentration of polymyxin B.