Abstract
Multidrug-resistant Acinetobacter baumannii is a top-priority pathogen globally and polymyxins are a last-line therapy. Polymyxin dependence in A. baumannii (i.e., nonculturable on agar without polymyxins) is a unique and highly-resistant phenotype with a significant potential to cause treatment failure in patients. The present study discovers that a polymyxin-dependent A. baumannii strain possesses mutations in both lpxC (lipopolysaccharide biosynthesis) and katG (reactive oxygen species scavenging) genes. Correlative multiomics analyses show a significantly remodeled cell envelope and remarkably abundant phosphatidylglycerol in the outer membrane (OM). Molecular dynamics simulations and quantitative membrane lipidomics reveal that polymyxin-dependent growth emerges only when the lipopolysaccharide-deficient OM distinctively remodels with ≥ 35% phosphatidylglycerol, and with "patch" binding on the OM by the rigid polymyxin molecules containing strong intramolecular hydrogen bonding. Rather than damaging the OM, polymyxins bind to the phosphatidylglycerol-rich OM and strengthen the membrane integrity, thereby protecting bacteria from external reactive oxygen species. Dependent growth is observed exclusively with polymyxin analogues, indicating a critical role of the specific amino acid sequence of polymyxins in forming unique structures for patch-binding to bacterial OM. Polymyxin dependence is a novel antibiotic resistance mechanism and the current findings highlight the risk of 'invisible' polymyxin-dependent isolates in the evolution of resistance.