Sphingolipid-mediated vesiculation in multidrug-resistant Sphingobacterium detergens under polymyxin B stress

Among environmentally widespread Sphingobacterium species, the animal feces isolate Sphingobacterium detergens E70 exhibited multidrug antibiotic resistance despite carrying few annotated antibiotic resistance genes (ARGs), prompting investigation of non-canonical mechanisms. This observation aligns with comparative genomic analysis of 62 Sphingobacterium genomes, which revealed only two to four ARGs per strain, typically associated with macrolide, phenicol, or tetracycline resistance. Remarkably, strain E70 lacked canonical ARGs yet displayed high-level resistance across nine antibiotics, including β-lactams and polymyxins. Scanning and transmission electron microscopy identified polymyxin B (PMB) as the most potent envelope-disrupting agent, revealing extensive outer-membrane vesiculation following PMB exposure. Fluorescence-based cytometric and microscopic analyses revealed PMB-induced phenotypic alterations, characterized by enhanced biofilm formation, modified surface charge, and changes in lipid composition. Inhibition of sphingolipid biosynthesis with myriocin markedly reduced vesiculation and impaired growth, indicating that sphingolipids contribute to envelope integrity and stress adaptation under PMB treatment. Confocal imaging with dansyl-labeled PMB showed that myriocin-mediated sphingolipid depletion made the membrane more permeable to PMB and reduced surface fluorescence, reflecting altered membrane environments. Together, these findings uncover a previously unrecognized role of sphingolipids in PMB resistance, that is, by promoting outer membrane vesicle-mediated membrane remodeling, sphingolipids enhance PMB resilience in S. detergens E70 and potentially in other sphingolipid-producing bacteria.

Environmental bacteria often display antibiotic tolerance without carrying canonical resistance genes. We show that Sphingobacterium exploits sphingolipid-dependent outer membrane vesiculation as a structural defense against polymyxin B. Blocking sphingolipid biosynthesis with myriocin suppressed vesiculation and sensitized cells to polymyxin B, indicating that these rare bacterial lipids provide essential sites for drug interaction and membrane remodeling. Our findings reveal a lipid-driven mechanism of vesiculation and highlight sphingolipid metabolism as a potential therapeutic target.