Macromolecular oxidation by reactive oxygen species in Pseudomonas aeruginosa exposed to different antibiotics

PÁEZ PAULINA L; BECERRA MC; ALBESA I

Villa Carlos Paz

Workshop; II Argentine Workshop on Current Topics in Pseudomonas and Burkholderia Research; 2009

Organizado por la Sociedad Argentina de Microbiología General

Oxidative stress results from an imbalance between the production of reactive species and antioxidant defense, withproteins 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 anions in bacterial species. In the present work, we have compared the macromolecule oxidative damage occurring in Pseudomonas aeruginosa when this strain was incubated with antibiotics, with the aim of obtaining a better understanding of the effects of the oxidative stress generated by antimicrobial of different groups. 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, with the oxidation of lipids  being 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 protein (nmol/mg P), with the carbonyl content being determined by spectrophotometry at 364 nm. Bacterial suspensions were incubated with ciprofloxacin (0.125, 0.4 y 32 ìg/mL) 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 (0.125, 0.4 and 32 ìg/mL) or phosphate saline buffer for 24 h, followed by the addition of IK and acetic acid . The final product was read at 340 nm. After 2 h of incubation with the antibiotics, there was an increase in MDA of 158 and 394% with ceftazidime and piperacilin, respectively. At 24 h of incubation, the three antibiotics assayed showed  increases of MDA. Carbonyl residue levels also rose after 2 h of incubation with respect to the basal, while at 4 h of incubation these levels were reduced. After this time,  a decrease in the values was observed until the the basal level was reached. The AOPP increased to a maximum of 450 and 700 meq chloramine T/mg protein at 4 h of incubation 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 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.