Real-Time Single-Cell Imaging Reveals Accelerating Lipid Peroxyl Radical Formation in Escherichia coli Triggered by a Fluoroquinolone Antibiotic

Abstract

The formation of reactive oxygen species (ROS) induced by bactericidal antibiotics has been associated with a common, nonspecific mechanism of cellular death. Herein, we report real-time single-cell fluorescence studies on Escherichia coli stained with a fluorogenic probe for lipid peroxyl radicals showing the generation of this form of ROS when exposed to the minimum inhibitory concentration (MIC) and 10× MIC 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 of 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. The 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.

Cytoplasmic condensation induced by membrane damage is associated with antibiotic lethality

“…We speculate that α-tocopherol may be less potent in E. coli than in eukaryotes due to potentially limited scavenging potential and uptake. Intriguingly, a recent study developed a lipophilic antioxidant different from α-tocopherol and showed that it prevented lipid peroxidation and fluoroquinolone-induced cell death in E. coli 40 ; together with our results, this observation suggests a varied landscape of chemical compounds that could attenuate lipid peroxidation and, in doing so, mitigate antibiotic lethality.…”

Biodegradable Peptide Polymers as Alternatives to Antibiotics Used in Aquaculture

“…This result indicated that ROS played an important role in the antimicrobial process of K18S4 against S. aureus. Among the possible ROS related antibacterial mechanisms, the increase of intracellular ROS level after the peptide polymer entered the bacteria could cause the damage of bacterial membrane due to membrane lipid peroxidation 57,58 . Characterization on the morphological changes of bacteria by transmission electron microscope (TEM) and scanning electron microscope (SEM) showed membrane lysis and cytoplasm outflow of S. aureus, E. coli and V. anguillurm after treatment with K18S4 (Fig 5i , 5j).…”

Brief survey on organometalated antibacterial drugs and metal-based materials with antibacterial activity

“…44 Of note is also the fact that the analysis of the mechanisms of action for well-established antibacterial drugs revealed their ability to generate oxidative stress in bacteria, which increases/complements their target-specific modes of action. [84][85][86] Applications of gold in therapy have long roots in mankind's history. 87 However, it was not until Robert Koch's observation of the bacteriostatic activity of K[Au(CN) 2 ] followed by the introduction of salvarsan by Paul Ehrlich that metals entered into the antibacterial field following the scientific method.…”

“… 44 Of note is also the fact that the analysis of the mechanisms of action for well-established antibacterial drugs revealed their ability to generate oxidative stress in bacteria, which increases/complements their target-specific modes of action. 84–86 …”