title: Monitoring antibacterial permeabilization in real time using time-resolved flow cytometry.
abstract: 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.
authors.affiliation: Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Av. Prof. Egas Moniz, Lisbon 1649-028, Portugal.
authors.forename: João Miguel
authors.initials: JM
authors.lastname: Freire
authors.affiliation: Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Av. Prof. Egas Moniz, Lisbon 1649-028, Portugal.
authors.forename: Diana
authors.initials: D
authors.lastname: Gaspar
authors.affiliation: Department of Experimental and Health Sciences, Pompeu Fabra University, Barcelona Biomedical Research Park, Barcelona E-08003, Spain.
authors.forename: Beatriz Garcia
authors.initials: BG
authors.lastname: de la Torre
authors.affiliation: Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Av. Prof. Egas Moniz, Lisbon 1649-028, Portugal.
authors.forename: Ana Salomé
authors.initials: AS
authors.lastname: Veiga
authors.affiliation: Department of Experimental and Health Sciences, Pompeu Fabra University, Barcelona Biomedical Research Park, Barcelona E-08003, Spain.
authors.forename: David
authors.initials: D
authors.lastname: Andreu
authors.affiliation: Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Av. Prof. Egas Moniz, Lisbon 1649-028, Portugal. Electronic address: macastanho@medicina.ulisboa.pt.
authors.forename: Miguel A R B
authors.initials: MA
authors.lastname: Castanho
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D023181:Antimicrobial Cationic Peptides / Q000494:pharmacology*; D001681:Biological Assay; D002462:Cell Membrane / Q000737:chemistry / Q000187:drug effects*; D002463:Cell Membrane Permeability / Q000187:drug effects*; D004926:Escherichia coli / Q000737:chemistry / Q000187:drug effects*; D005434:Flow Cytometry / Q000379:methods; D005456:Fluorescent Dyes; D007700:Kinetics; D008070:Lipopolysaccharides / Q000737:chemistry*; D050296:Microbial Viability / Q000187:drug effects; D009930:Organic Chemicals; D011419:Propidium; D013045:Species Specificity; D013050:Spectrometry, Fluorescence; D013997:Time Factors
chemical_list: D000900:Anti-Bacterial Agents; D023181:Antimicrobial Cationic Peptides; D005456:Fluorescent Dyes; D008070:Lipopolysaccharides; D009930:Organic Chemicals; C103389:SYTO 9; D011419:Propidium
keywords: Antimicrobial action; Antimicrobial peptide; Lipopolysaccharide; Live/dead assay; Peptide–lipid interaction; Time-resolved flow cytometry