Bleach does not remove the EVs or their activity, but EDTA does. Each polystyrene slide in (a) and (b) was pretreated with pEVs (12.5 μg/ml) just prior to the experiment. a) Confocal image of the polystyrene surface after treatment for one hour with HEPES buffer, a 10% bleach solution or 0.1M EDTA. Rhodamine BR-18-labeled EVs were still evident on both the HEPES- and bleach-treated surfaces but were predominantly removed from the surfaces treated with EDTA. b) The bleach-treatment did not significantly reduce the inhibitory activity of the EVs. A. baumannii biofilm formation in the bleach-treated EV-coated wells was similar with the Control wells, with 44% and 56% inhibition, respectively. In contrast, treatment with EDTA completely removed the inhibitory activity (p<0.001). c) Treatment of the EVs with EDTA in liquid samples completely abolished their ability to block A. baumannii biofilm development. The EDTA sample was compared with the control (i.e., no pEV pretreatment) (p <0.001).