ANTIBACTERIAL ACTIVITY OF CIPROFLOXACIN MEDIATED BY OXIDATIVE STRESS IN A CO2 ATMOSPHERE CONTROLLED

CANO ARISTIZÁBAL V; PARAJE MG; ALBESA I; DUKAN S; PÁEZ PL

Córdoba

Congreso; XI CONGRESO ARGENTINO DE MICROBIOLOGÍA GENERAL SAMIGE; 2015

Sociedad Argentina de Microbiología General

Oxidative stress is caused by exposure to reactive oxygen intermediates, such as superoxide anion (O2?-), hydrogen peroxide (H2O2), and hydroxyl radical (OH?), which can damage proteins, nucleic acids, and cell membranes. In cells CO2 is a main by-product of metabolism. It also constitutes the main physiological pH-buffering system in higher eukaryotic organisms and is required for the growth of many microorganisms. CO2 levels have become a major point of focus of the global community, because of their contribution to the greenhouse effect. The best known effect of increasing CO2 concentration is global warming, but large increases in CO2 concentration (to 1% or 10%) are also known to affect cellular biochemical reactions, leading to an increase in intracellular oxidative stress in human neutrophils, pulmonary inflammation in mouse and increased virulence or bactericidal activities of various pathogenic bacteria. However, current and predicted concentrations are not of this order of magnitude; hence, the probable, direct effects of CO2 on living organisms at the predicted concentrations remain unclear. E. coli ATCC 25922 was grown aerobically in liquid Luria?Bertani (LB) broth, at 37 °C, with shaking at 160 rpm. When the OD600 reached 0.5, cells were exposed to various concentrations of CO2 (50 ppm or 5 %). LB agar plates with and without ciprofloxacin (CIP) 0, 2.56 y 256 µg/mL in the medium were allowed to equilibrate for 20 h at the CO2 level to be tested. Serial dilutions of cell suspensions in phosphate buffer (0.05 M, pH 7.4) were prepared and Escherichia coli cells were spread on LB agar plates and incubated in the presence of either 50 ppm or 5% of CO2. Colonies were counted after incubation at the CO2 concentration tested for 16 h at 37 °C. When the antimicrobial activity of CIP was evaluated against E. coli in the presence of CO2, it was observed that the antibiotic activity decreased as the concentration of CO2 is increased. At concentrations of 5% CO2, CIP concentration necessary for bactericidal activity is 256 µg/mL. This value is well above the minimum bactericidal concentration of CIP against E. coli ATCC 25922. To evaluate the ROS participation in the mechanism of action of CIP, the procedure described previous was done in the presence of an inhibitor of the formation of OH?, 2,2 dipyridyl, which is an iron chelator and therefore an inhibitor of the Fenton reaction which is a major mechanisms of formation of OH?. The effect of CO2 on oxygen toxicity disappeared in these conditions, providing further evidence for the hypothesis that CO2 directly exacerbates CIP toxicity mediated by OH?. We show here that atmospheric CO2 modify death rates due to oxidative stress mediated by CIP in E. coli in a dose-specific manner. This effect is correlated with a reduction in OH? generated by ciprofloxacin in a high concentration of CO2. Thus, CO2 modify ROS toxicity mediated by ciprofloxacin.