Following lipid peroxidation: a single bacteria imaging study

BECERRA M. C; MARTINEZ SR; COSA G; DURANTINI AM

Carlos Paz

Congreso; XIII ELAFOT; 2017

Grupo Argentino de Fotobiología

FOLLOWING LIPID PEROXIDATION: A SINGLE BACTERIAIMAGING STUDYSol R. Martínez a,b, Andrés M. Durantini a, María Cecilia Becerra b,Gonzalo Cosa a.aDepartment of Chemistry and Center for Self-AssembledChemical Structures (CSACS-CRMAA), McGill   University, Montreal, Canada.bInstitutoMultidisciplinario de Biología Vegetal (IMBIV), CONICET, and Facultad deCiencias Químicas, Universidad Nacional de Córdoba, Córdoba, Argentina.e-mail:solmartinez@fcq.unc.edu.arLipid peroxidation(LP) is a crucial step in the pathogenesis of several disease states. This is aself-propagating chain-reaction process initiated by reactive oxygen species(ROS). Oxidation of lipid molecules affects membranes properties jeopardizingcells viability. ROS are not only produced in cells as part of the respiratorychain, different drugs for instance bactericidal antibiotics induces LPaccelerating cellular respiration and consequently enhancing ROS production.The goal of our workwas to assess the LP process caused by Ciprofloxacin in a Gram-negative strain,Escherichia coli ATCC 25922. Weevaluated oxidative damage in real time through a sensitive fluorogenic probe H2B-PMHC. This probe was developed andcharacterized by the group of Prof. Cosa; it presents high specificitytoward lipid peroxyl radicals, the dominant reactive oxygen species encounteredin lipid membranes under oxidative stress. To learn on thephysiological role of antibiotics at the single bacterial level we focused onfluorescence imaging experiments upon excitation with an evanescent beam in aTotal Internal Reflection Fluorescence Microscope (TIRFM).  The sample was incubatedwith increasing antibiotic doses (3, 30, 100 µM) for 12 h monitoring intensityvs. time profiles. Our experiments revealed that LPis a drug concentration-dependent process in E. coli strain. We will also discuss phenotypic heterogeneity inindividual bacterium such as e.g.: the time each bacterium increases ROSproduction (following fluorescence enhancement), the amount of ROS beingproduced and dye distribution. Furthermore, a cell viability assay was assessedusing a death marker (propidium iodine-PI), enabling quantification of deadbacteria from the beginning till the end of the assay. We reasoned that theobserved differences could be due to the metabolic stages of each bacillusindicating that bacteria with a high fluorescent at the beginning of theexperiments were probably in an early dead phase.