Metabolic rewiring of bacterial subpopulations governs polymyxin responses in Acinetobacter baumannii

Polymyxins are often last-resort antibiotics against high-priority Gram-negative pathogens, particularly Acinetobacter baumannii . However, most mechanistic studies to date have overlooked the heterogeneity within bacterial populations during polymyxin treatment. Using time-lapse imaging and propidium iodide (PI) staining, we observed that a subset of PI-positive (PI ⁺ ) cells, which are traditionally considered non-viable, were capable of regrowth. This unexpected scenario promoted further investigation into the distinct metabolic responses of PI ⁺ or PI-negative (PI ⁻ ) subpopulations following polymyxin exposure. By combining a synthetic fluorescent polymyxin probe, FADDI-043, with fluorescence-activated cell sorting (FACS), we isolated PI ⁺ and PI ⁻ cells and profiled their metabolic responses. PI ⁺ cells exhibited increased levels of phosphatidylethanolamine, likely to compensate for the severe membrane damage by polymyxins. In contrast, PI ⁻ cells, where polymyxin interacted with bacterial membranes without causing extensive damage, demonstrated broader metabolic adaptations. Notably, arginine metabolism was uniquely upregulated in the PI ⁻ group, and exogenous arginine supplementation conferred protection against polymyxin treatment. Collectively, this is the first study to demonstrate subpopulation-specific metabolic responses to polymyxins, highlighting dynamic and heterogeneous bacterial adaptations. Our findings underscore the importance of single-cell analysis to unravel antibiotic mechanisms and may inform novel metabolic reprogramming strategies to enhance antibiotic efficacy and minimize resistance emergence.