Real-time single-cell imaging reveals accelerating lipid peroxyl radical formation in Escherichia coli triggered by a fluoroquinolone antibiotic

Abstract

Formation of reactive oxygen species (ROS) induced by bactericidal antibiotics has been associated with a common, non-specific, mechanism of cellular death. Herein we report real-time single cell fluorescence studies on E. coli stained with a fluorogenic probe for lipid peroxyl radicals showing generation of this form of ROS when exposed to the minimum inhibitory concentration (MIC) and 10xMIC of the fluoroquinolone antibiotic ciprofloxacin (3 and 30 μM, respectively). Single-cell intensity-time trajectories show an induction period followed by an accelerating phase for cells treated with antibiotic, where initial and maximum intensity achieved following 3.5 h incubation with antibiotic showed dose dependent average values. A large fraction of bacteria remains viable after the studies, indicating ROS formation is occurring a priori of cell death. Punctate structures are observed, consistent with membrane blebbing. Addition of a membrane embedding lipid peroxyl radical scavenger, an α-tocopherol analogue, to the media increased the MIC of ciprofloxacin. Lipid peroxyl radical formation precedes E. coli cell death and may be invoked in a cascade event including membrane disruption and consequent cell wall permeabilization. Altogether, our work illustrates that lipid peroxidation is caused by ciprofloxacin in E. coli, and suppressed by α-tocopherol analogues. Lipid peroxidation may be invoked in a cascade event including membrane disruption and consequent cell wall permeabilization. Our work provides a methodology to assess antibiotic induced membrane peroxidation at the single cell level, this methodology provides opportunities to explore the scope and nature of lipid peroxidation in antibiotic induced cell lethality.