PROTEIN OXIDATION BY REACTIVE OXYGEN SPECIES IN STAPHYLOCOCCUS AUREUS AND ESCHERICHIA COLI EXPOSED TO GENTAMICIN

GALERA ILD; ALBESA I; PÁEZ PL

Rosario

Congreso; 2° Reunión Internacional de Ciencias Farmacéuticas; 2012

Introduction Oxidative stress is, in essence, an imbalance between the production of reactive species and antioxidant defenses. It can be defined as a disturbance in the prooxidant-antioxidant balance in favor of the former, thus leading to potential damage [1]. The highly reactive hydroxyl radicals are generated by the presence of hydrogen peroxide and iron (Fenton reaction) or by superoxide anion, hydrogen peroxide, and a metal catalyst (Haber–Weiss reaction). These species are able to oxidize proteins, resulting in the formation of carbonyl groups in some amino acid residues. This study focuses on the effect of gentamicin (GEN) on the generation of oxidants in the cell and the damage they cause to bacterial macromolecules such as proteins. Materials and methods Bacterial Strains and Minimum Inhibitory Concentration (MIC) Assay The MIC was determined according to the norms of Clinical and Laboratory Standards Institute (CLSI) [2] using the standard tube dilution method in Mueller–Hinton medium. The MICs for GEN was determined for the S. aureus (ATCC 29213, 424 and 235) and E. coli (ATCC 25922, 25 and 30) strains. Advanced Oxidation Protein Products (AOPP) 5 ml of bacterial suspensions cultured overnight in TSB at 35 ºC were incubated with 0.5 ml of GEN or phosphate saline buffer for 24 h. 1 ml of the samples was taken at 0, 2, 4, 8, and 24 h of incubation. Then, 50 ll of IK (1.16 M) and 0.1 ml of acetic acid were added. The final product of the reaction was read at 340 nm, with Chloramine-T (50 lM) being used as the standard. The concentrations of AOPP were expressed as chloramine-T equivalents per mg of proteins [3]. Results Staphylococcus aureus ATCC 29213 and 424 exhibited sensitivity to GEN while S. aures 235 was resistant to GEN. The strains ATCC 25922 and 25 of E. coli were sensitive to GEN while E. coli 30 was intermédiate sensitivity. In S. aureus, AOPP was increased for the three concentrations of GEN tested. The maximum production of AOPP was obtained at 1 h of incubation with de antibiotic at concentrations equal to the MIC, below the MIC, or higher than the MIC. The AOPP levels reached the control values at 24 h. In E. coli, AOPP increased to maximum of 1,76 meq chloramine T/mg protein at 1 h of incubation with GEN with respect to the control. Conclusions Oxidative stress is involved in the action of antibiotics, such as aminoglycosides, which induce ROS by means of alteration of membrane proteins. In the present study, we analyzed the extent of the alteration of macromolecules such as proteins as a result of ROS generation, which subsequently affects the cell viability. The damage caused by the antibiotic was irreversible and resulted in the degradation of proteins, leading to the formation of AOPP. Based on these results, injury by oxidation of macromolecules should be added to the mechanisms of action described for the antibiotics. This may include modifications of proteins, which could lead to structural changes by increasing the noxious action described for antibiotics.