Lipid peroxidation in bacteria caused by ciprofloxacin: a single bacteria imaging approach

MARTINEZ SR; COSA G; DURANTINI A; BECERRA M C

Santiago de Chile

Congreso; 25th IAPS Meeting; 2016

Lipid peroxidation in bacteria caused by ciprofloxacin: a single bacteria imaging approachSol Martínez, 1, 2 Andres Durantini, 1 María Cecilia Becerra, 2 and Gonzalo Cosa. 11Department of Chemistry and Center for Self-Assembled Chemical Structures (CSACS-CRMAA), McGill University, 801 Sherbrooke Street West, Montreal, Quebec H3A 0B8, Canada.2 Instituto Multidisciplinario de Biología Vegetal (IMBIV), CONICET, and Facultad de Ciencias Químicas, Universidad Nacional de Córdoba , Córdoba X5000HUA, Argentina.Our research group at Universidad Nacional de Cordoba, Argentina and others have shown that bactericidal antibiotics, such as ciprofloxacin, induce changes in bacteria membrane caused by reactive oxygen species (ROS). ROS, generated as a bacterial response to the antibiotic presence, are responsible for lipid chain peroxidation (LP, also called autoxidation). Here, upon ROS reaction with fatty acids lipid carbon-centered radicals are generated that next trap molecular oxygen under physiological conditions to form lipid peroxyl radicals effective chain carriers in the lipid chain autoxidation, The primary effect of lipid chain autooxidation is a decrease in membrane fluidity, which alters the membrane properties and mainly disrupt membrane-bound proteins. The most widely biomarker used to study LP is malondialdehyde (MDA). MDA reacts with thiobarbituric acid (TBA) to yield a red-colored adduct that can be assayed spectrophotometrically. Nevertheless, the TBA reacting substances test (TBARS) is notoriously nonspecific which has led to substantial controversy over its use for quantification of MDA.The aim of this work was to evaluate the LP caused by Ciprofloxacin in a Gram negative, Escherichia coli ATCC 25922 and a Gram positive model, Bacillus subtilis ATCC 6051. To evaluate oxidative damage a highly sensitive fluorogenic probe H2B-PMHC was used. H2B-PMHC, previously developed and characterized by our collaborators at McGill University, Canada, presents high specificity toward lipid peroxyl radicals, the dominant reactive oxygen species encountered in lipid membranes under oxidative stress. In this work we assessed the damage through ensemble fluorescence experiments and a single bacteria imaging approach. Ensemble experiments revealed that regardless of the antibiotic concentration (30-0.3µM) the E. coli strain experienced the same damage. However B.subtillis only showed oxidative damage at high concentration (Figure A and B). This effect could be related to the differences in both membranes and differences in the antioxidant capacity in the gram positive strain. Imaging our sample upon excitation with an evanescent beam in a Total Internal Reflection Fluorescence Microscope (TIRFM) showed the specific localization of H2B-PMHC and a >15-fold fluorescence intensity enhancement after treating E. coli strain with the antibiotic (Figure C and D). We could detect the LP with a highly sensitive fluorogenic probe, in the Gram negative strain. Our future work will aim at studying the oxidative damage in bacteria with single molecule resolution.