Unanticipated Lipid Redistribution Mechanism of Action by Conjugated Oligoelectrolyte Antibiotics

Antimicrobial resistance (AMR) is one of the most pressing global health threats, urgently requiring new classes of antibiotics with differentiated mechanisms of action (MOA). Conjugated oligoelectrolytes (COEs) represent a molecular platform for designing antimicrobial agents structurally distinct from commercially available drugs. However, questions remain regarding their MOA. Herein, we show that COE treatment causes distinct phenotypes from well-established membrane-active antibiotics, with differences arising from structural variations, such as pendant group hydrophobicity. This was revealed through bacterial cytological profiling approaches, single-cell quantitative morphological analysis, and dye localization following treatment against Gram-negative (<i>Escherichia coli</i>) and Gram-positive (<i>Bacillus subtilis</i>) bacteria. <i>E. coli</i> treatment with <b>PNH2</b> and <b>1B</b> resulted in micrometer-sized membrane vesicles, which are absent in <b>2-2H</b>-treated cells. COE-treated <i>B. subtilis</i> featured overproduction of regions of increased fluidity (RIFs), relative to untreated cells. In contrast to the originally postulated membrane pinching mechanism, these findings support a MOA for COEs that relies predominantly on membrane restructuring, thereby providing new guidelines for further COE-based antibiotic design.