Despite the intensive study of antibiotic-induced bacterial permeabilization, its kinetics and molecular mechanism
remain largely elusive. A new methodology that extends the concept of the live–dead assay in flow cytometry
to real time-resolved detection was used to overcome these limitations. The antimicrobial activity of pepR
was monitored in time-resolved flow cytometry for three bacterial strains: Escherichia coli (ATCC 25922),
E. coli K-12 (CGSC Strain 4401) and E. coli JW3596-1 (CGSC Strain 11805). The latter strain has truncated lipopolysaccharides
(LPS) in the outer membrane. This new methodology provided information on the efficacy of the antibiotics
and sheds light on their mode of action atmembrane-level. Kinetic data regarding antibiotic binding and
lytic actionwere retrieved.Membrane interaction and permeabilization events differ significantly among strains.
The truncation of LPS moieties does not hamper AMP binding but compromises membrane disruption and bacterial
killing. We demonstrated the usefulness of time-resolved flow cytometry to study antimicrobial-induced
permeabilization by collecting kinetic data that contribute to characterize the action of antibiotics directly on
bacteria.