MACROMOLECULAR OXIDATION BY REACTIVE OXYGEN SPECIES IN Pseudomonas aeruginosa EXPOSED TO DIFFERENT ANTIBIOTICS
P¨¢ez, Paulina Laura; Becerra, Mar¨ªa Cecilia and Albesa, In¨¦s.
Dpto. Farmacia. Facultad de Ciencias Qu¨ªmicas. Universidad Nacional de C¨®rdoba. Haya de
e-mail: plpaez@fcq.unc.edu.ar
Oxidative stress results from an imbalance between the production of reactive species and antioxidant defense, with proteins being a major target for oxidants as a result of their abundance in biological systems. Oxidized proteins are better substrates for proteolytic digestion, and the proteolytic pathway can provide a valuable line of ¡°secondary antioxidant defense¡±. Furthermore, free radicals can attack directly polyunsaturated fatty acids in membranes and initiate lipid peroxidation. We have previously demonstrated that ciprofloxacin, ceftazidime and piperacillin were able to generate an increase of superoxide anion in bacterial species. In the present work, we have compared the macromolecule oxidative damage occurring in Pseudomonas aeruginosa incubated with antibiotics with the aim of obtaining a better comprehension of the effects of oxidative stress involved in the mechanism of action of different classes of antibiotics. The minimum inhibitory concentration of P. aeruginosa to ceftazidime, piperacilin and ciprofloxacin was determined according to the norms of the Clinical and Laboratory Standards Institute. The oxidation of lipids was evaluated by means of formation of malondialdehyde (MDA). This strain was incubated with ciprofloxacin 5 ¦Ìg/mL, ceftazidime 4 ¦Ìg/mL, piperacilin 128 ¦Ìg/mL or phosphate saline buffer (control). MDA levels were expressed per mg of proteins (nmol/mg P). The carbonyl residues were determined by spectrophotometry, bacterial suspensions were incubated with ciprofloxacin (sub-MIC, MIC and supra-MIC) for 0, 2 and 4 h, before samples were incubated with 2,4-dinitro-phenylhydrazine solution. To determine the Advanced Oxidation Protein Products (AOPP), bacterial suspensions were incubated with ciprofloxacin (sub-MIC, MIC and supra-MIC) or phosphate saline buffer for 24 h, followed by the addition of IK and acetic acid. At 24 h of incubation, the three antibiotics assayed showed increases of MDA. Carbonyl residues levels also increased after 2 h of incubation with respect to the basal, while at 4 h these levels were reduced to the basal level. AOPP increased to a maximum of 450 and 700 meq chloramine T/mg proteins at 4 h with ciprofloxacin with respect to the basal level. In summary, we analyzed the extent of the alteration of macromolecules such as lipids and proteins as a result of ROS generation, which compromises the cell viability. Oxidative injury caused by the three antibiotics studied induced an increase in the concentration of MDA, which was time-dependent. The resulting damage was irreversible and resulted in the degradation of proteins, leading to the formation of carbonyl residues and AOPP. Based on these results, injury by oxidation of macromolecules should be added to the mechanisms of action previously described for these antibiotics.
