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 at membrane-level. Kinetic data regarding antibiotic binding and lytic action were 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.

This work was supported by project PTDC/QUIBIQ/112929/2009 from Fundação para a Ciência e a Tecnologia– Ministério da Educação e Ciência (FCT-MEC, Portugal). JMF and DGalso acknowledge FCT-MEC for fellowships SFRH/BD/70423/2010 and SFRH/BPD/73500/2010, respectively, and ASV for funding within the FCT Investigator Programme (IF/00803/2012). Work at Pompeu Fabra University was supported by grants from the Spanish Ministry of Economy and Competitiveness (SAF2011-24899) and from Generalitat de Catalunya (SGR2009-0492).