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.IMPORTANCEMultidrug-resistant Acinetobacter baumannii is designated a World Health Organization "critical priority" pathogen, and polymyxins remain the few effective treatment options, particularly in low- and middle-income countries. However, polymyxin heteroresistance poses a major global clinical challenge, and its mechanistic basis remains poorly defined. Most antimicrobial studies rely on population-level measurements, obscuring the biological consequences of phenotypic heterogeneity. Here, we demonstrate that a subset of polymyxin-treated, propidium iodide-positive (PI⁺) A. baumannii cells, typically classified as non-viable, retain the capacity to regrow. By isolating PI⁺ and PI-negative (PI^-) subpopulations, we uncover distinct metabolic adaptations under polymyxin exposure: PI⁺ cells exhibit elevated phosphatidylethanolamine levels, whereas the PI^- cells activate their arginine metabolism. These findings reveal an unrecognized layer of metabolic heterogeneity underlying polymyxin exposure. Our work challenges conventional interpretations of viability assays and highlights the importance of subpopulation-resolved analyses for identifying metabolic vulnerabilities to enhance polymyxin efficacy while limiting resistance emergence.