Antimicrobial resistance (AMR) is a growing global health problem, exacerbated by the widespread use of antimicrobials in healthcare, agriculture, and industrial settings. Cationic antimicrobials (CAs) such as the therapeutic antibiotic colistin (COL) and antiseptics cetrimide (CET) and chlorhexidine (CHX) exert their mechanism of action by disrupting bacterial membranes, leading to cell content leakage and death. Tolerance to CAs is rapidly increasing and of the many known AMR mechanisms, the role of outer membrane vesicle (OMV) formation is least understood. In this thesis, we examined OMV isolation methods in a tolA deletion mutant to understand how OMV properties differ when using two common methods (Chapter 3). Using the most robust OMV isolation method, OMV production from a set of CA- tolerant strains were compared to a wild-type (WT) E. coli K-12 BW25113 strain (Chapter 4). OMVs from WT and ∆tolA strains were isolated from culture supernatants by ultradiafiltration and ultracentrifugation, then analyzed with a light scattering based single particle tracking analysis (NTA) to compare OMV isolation differences. We characterized the morphology of OMVs isolated from each strain by cryo- transmission electron microscopy, compared proteomes using liquid chromatography-mass spectrometry, and evaluated susceptibility changes by antimicrobial susceptibility testing (AST). In Chapter 3, we demonstrated that deletion of the IM protein tolA in E. coli, an integral part of the membrane integrity Tol- Pal system, resulted in increased vesicle formation and morphological changes to vesicles, including a new kind of vesicle that we coined “G-OMVs”. In Chapter 4, we found that the CA-adapted strains all had increased OMV formation as compared to WT, where each strain had distinctive morphological alterations; CET-OMVs were encapsulated and aggregated, CHX-OMVs were multilamellar and COL-OMVs were large and amorphous as compared to WT-OMVs. Proteomic analysis highlighted an increase in proteins associated with stress responses, lipid biosynthesis, and protein folding/transport in CA-adapted strains, and identified cytoplasmic and IM protein carryover into vesicles. AST of WT E. coli supplemented with purified CA-adapted OMVs demonstrated that susceptibility could be modulated by the addition of OMVs from CA-adapted strains. Overall, our findings show that CA-adapted E. coli have significant ramifications on OMV production, morphology, and susceptibility, highlighting the need for further research in this field.