María Cecilia Becerra

Lipids and DNA oxidation in Staphylococcus aureus as a consequence of oxidative stress generated by ciprofloxacin

Ciprofloxacin induced an increment of reactive oxygen species in sensitive strains of Staphylococcus aureus leading to oxidative stress detected by chemiluminescence while resistant strains did not suffer such stress. Oxidation of lipids was performed by employing thiobarbituric acid reaction to detect the formation of the amplified intermediate between reactive species oxygen and cytoplasmic macromolecules, namely malondialdehyde (MDA). The sensitive strain presented higher peroxidation of lipids than the resistant strain. The oxidative consequence for DNA was investigated by means of bacteria incubation with ciprofloxacin and posterior extraction of DNA, which was studied by high performance liquid chromatography (HPLC). Sensitive S. aureus ATCC 29213 showed an increase of 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) respect controls without antibiotic; there was evident increase of the ratio between 8-oxodG and deoxyguanosine (dG) as a consequence of oxidation of dG to 8-oxodG considered the major DNA marker of oxidative stress. The resistant strain showed low oxidation of DNA and the analysis of 8-oxodG/dG ratio indicated lesser formation of 8-oxodG than S. aureus ATCC 29213.

Induction of NADPH oxidase activity and reactive oxygen species production by a single Trypanosoma cruzi antigen.

Trypanosoma cruzi is an intracellular protozoan parasite that predominantly invades mononuclear phagocytes and is able to establish a persistent infection. The production of reactive oxygen species (ROS) by phagocytes is an innate defence mechanism against microorganisms. It has been postulated that ROS such as superoxide anion (O(2)), hydrogen peroxide and peroxynitrite, may play a crucial role in the control of pathogen growth. However, information on parasite molecules able to trigger ROS production is scarce. In this work, we investigated whether cruzipain, an immunogenic glycoprotein from T. cruzi, was able to trigger the oxidative burst by murine cells. By employing chemiluminiscense and flow-cytometric analysis, we demonstrated that cruzipain induced ROS production in splenocytes from non-immune and cruzipain immune C57BL/6 mice and in a Raw 264.7 macrophage cell line. We also identified an O(2)(-) molecule as one of the ROS produced after antigen stimulation. Cruzipain stimulation induced NOX2 (gp91(phox)) and p47(phox) expression, as well as the co-localisation of both NADPH oxidase enzyme subunits. In the current study, we provide evidence that cruzipain not only increased ROS production but also promoted IL-6 and IL-1β cytokine production. Taken together, we believe these results demonstrate for the first time that cruzipain, a single parasite molecule, in the absence of infection, favors oxidative burst in murine cells. This represents an important advance in the knowledge of parasite molecules that interact with the phagocyte defence mechanism.

Chloramphenicol-induced oxidative stress in human neutrophils.

The aim of this study was to evaluate the in vitro effect of chloramphenicol in order to determine its potential toxic effects on human neutrophils, by using assays of reactive oxygen species (ROS) determination, nitrite measurement and antioxidant systems. Chloramphenicol enabled the oxidative stress response of neutrophils and increased the ROS production at 2, 4, 8 and 16 microg/ml, while ROS generation decreased at high concentrations (32 microg/ml). The nitroblue tetrazolium assay shows that neutrophils incubated with chloramphenicol increased the intracellular ROS, with the extracellular production rising with a corresponding increase in antibiotic concentration. Enzymatic activities--superoxide dismutase, catalase and diaphorase enzymes--increased after chloramphenicol treatment, while the glutathione level decreased in neutrophils incubated with antibiotic. The results obtained in the present work suggest that the study of susceptibility to oxidative stress in neutrophils before chloramphenicol treatment could be adequate for in vitro toxicity screening.

Relevance of Biofilms in the Pathogenesis of Shiga-Toxin-Producing Escherichia coli Infection

The present study was designed to determine the relationships among biofilm formation, cellular stress and release of Shiga toxin (Stx) by three different clinical Shiga toxin-producing Escherichia coli (STEC) strains. The biofilm formation was determined using crystal violet stain in tryptic soy broth or thioglycollate medium with the addition of sugars (glucose or mannose) or hydrogen peroxide. The reactive oxygen species (ROSs) were detected by the reduction of nitro blue tetrazolium and reactive nitrogen intermediates (RNI) determined by the Griess assay. In addition, the activities of two antioxidant enzymes, superoxide dismutase (SOD) and catalase (CAT), were studied. For the cytotoxicity studies, Vero cells were cultured with Stx released of STEC biofilms. The addition of sugars in both culture mediums resulted in an increase in biofilm biomass, with a decrease in ROS and RNI production, low levels of SOD and CAT activity, and minimal cytotoxic effects. However, under stressful conditions, an important increase in the antioxidant enzyme activity and high level of Stx production were observed. The disturbance in the prooxidant-antioxidant balance and its effect on the production and release of Stx evaluated under different conditions of biofilm formation may contribute to a better understanding of the relevance of biofilms in the pathogenesis of STEC infection.

Oxidative Stress and Antimicrobial Activity of Chromium(III) and Ruthenium(II) Complexes on Staphylococcus aureus and Escherichia coli

The prevalence of antibiotic resistance has resulted in the need for new approaches to be developed to combat previously easily treatable infections. The main aim of this work was to establish the potential of the synthetic α-diimine chromium(III) and ruthenium(II) complexes (where the α-diimine ligands are bpy = 2,2-bipyridine, phen = 1,10-phenanthroline, and dppz = dipyrido[3,2-a:2′,3′-c]-phenazine) like [Cr(phen)3]3+, [Cr(phen)2(dppz)]3+, [Ru(phen)3]2+, and [Ru(bpy)3]2+ as antibacterial agents by generating oxidative stress. The [Cr(phen)3]3+ and [Cr(phen)2(dppz)]3+ complexes showed activity against Gram positive and Gram negative bacteria with minimum inhibitory concentrations (MICs) ranging from 0.125 μg/mL to 1 μg/mL, while [Ru(phen)3]2+ and [Ru(bpy)3]2+ do not exhibit antimicrobial activity against the two bacterial genera studied at the concentration range used. When ciprofloxacin was combined with [Cr(phen)3]3+ for the inhibition of Staphylococcus aureus and Escherichia coli, an important synergistic effect was observed, FIC 0.066 for S. aureus and FIC 0.064 for E. coli. The work described here shows that chromium(III) complexes are bactericidal for S. aureus and E. coli. Our results indicate that α-diimine chromium(III) complexes may be interesting to open new paths for metallodrug chemotherapy against different bacterial genera since some of these complexes have been found to exhibit remarkable antibacterial activities.

Antioxidative mechanisms protect resistant strains of Staphylococcus aureus against ciprofloxacin oxidative damage

The aim of this investigation was to determine whether the antioxidant defences protect resistant strains of Staphylococcus aureus against ciprofloxacin oxidative damage. Reactive oxygen species (ROS) were determined by chemiluminescence and nitric oxide (NO) was assayed by Griess reaction. The accumulation of ciprofloxacin was examined by fluorometry and oxidation of protein, catalase, ferrous reduction antioxidant potency (FRAP), carbonyls and advanced oxidation protein products (AOPP), studied by spectrophotometry. Ciprofloxacin stimulated higher production of ROS and NO in the susceptible strains than in the resistant ones. There was higher accumulation of antibiotic in sensitive strains than in resistant ones, except for the most resistant strain, which accumulated an elevated amount of antibiotic. The FRAP/ciprofloxacin accumulation ratio of the antibiotic was lower in sensitive than in resistant strains. The most resistant strain exhibited the highest FRAP and presented a high catalase activity. There was oxidation of proteins in the presence of ciprofloxacin, with the carbonyl residues increasing in sensitive and resistant S. aureus. The degradation of carbonyls to AOPP in oxidized proteins was higher in the resistant than in sensitive strains. In conclusion, an increase in antioxidant capacity and a rapid oxidation of carbonyls to AOPP contributed to resistance to ciprofloxacin.

In vitro activity of N-benzenesulfonylbenzotriazole on Trypanosoma cruzi epimastigote and trypomastigote forms.

Chagas disease is still an important health problem in Central and South America. However, the only drugs currently available for specific treatment of this disease may induce toxic side effects in the host. The aim of this work was to determine the activity of N-benzenesulfonylbenzotriazole (BSBZT) against the protozoan parasite Trypanosoma cruzi. The effects of BSBZT and benzotriazole (BZT) were compared to those of benznidazole (BZL) on epimastigote and trypomastigote forms. BSBZT was found to have an in vitro growth inhibitory dose-dependent activity against epimastigotes, with flow cytometry analysis confirming that the treated parasites presented size reduction. BSBZT showed an IC(50) of 21.56 μg/mL (81.07 μM) against epimastigotes at 72 h of incubation, whereas BZT did not affect the growth of this parasite form. Furthermore, the toxic effect of BSBZT, was stronger and appeared earlier (at 24h) in trypomastigotes than in epimastigotes, with the LC(50) of this compound being 28.40 μg/mL (106.79 μM) against trypomastigotes. The concentrations of BSBZT used in this study presented low hemolytic activity and cytotoxicity. Consequently, at concentrations near IC(50) and LC(50) (25μg/mL), BSBZT caused only 2.4% hemolysis and 15% of RAW 264.7 cell cytotoxicity. These results reveal the potential of BSBZT as a prototype in drug design for developing new anti-T. cruzi compounds.

Comparison of Macromolecular Oxidation by Reactive Oxygen Species in Three Bacterial Genera Exposed to Different Antibiotics

Proteins and lipids maybe important targets of oxidation and this may alter their functions. We evaluated whether ceftazidima (CAZ), piperacillin (PIP), chloramphenicol (CMP), and ciprofloxacin (CIP) could oxidize the macromolecules in the three bacterial genera Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa. There was an increase in lipid peroxidation observed in these three species. However, this was lower in the Gram negative bacteria than in S. aureus. A reduction of the carbonyl residue in S. aureus with ciprofloxacin was observed whereas in Gram negative bacteria the antibiotics increased the carbonyl residue with respect to the control. Although the strains suffered a rise in advanced oxidation protein products (AOPP) in the presence of ciprofloxacin, the S. aureus strain had a smaller increase of AOPP than the other strains. The results described in this article provide data about the susceptibility of the three bacterial genera to the oxidative stress induced by the antibiotics studied.

Effect of the association of reduced glutathione and ciprofloxacin on the antimicrobial activity in Staphylococcus aureus.

We report the effect of glutathione and the role of reactive oxygen species (ROS), assayed by a nitro blue tetrazolium reaction, on the antibacterial action of ciprofloxacin, gentamicin and chloramphenicol in Staphylococcus aureus 22 resistant to ciprofloxacin and gentamicin, and in S. aureus ATCC 29213 sensitive to the above three antibiotics. The association of glutathione with ciprofloxacin or gentamicin significantly reduced the value of the minimum inhibitory concentration (MIC) in resistant S. aureus 22, measured using the macrodilution method, with a concomitant increase of intracellular ROS and a decrease of extracellular ROS. However, glutathione did not induce modifications in MIC or ROS generated by chloramphenicol. Furthermore, in the sensitive S. aureus ATCC 29213, the association of glutathione with ciprofloxacin, gentamicin or chloramphenicol did not induce any significant variations of MIC or ROS. There was a correlation between the stimulus of intracellular ROS and the decrease of MIC caused by exogenous glutathione. According to the results obtained, it is possible to modify the sensitivity of resistant strains of S. aureus by the addition of exogenous glutathione.

Linezolid as an eradication agent against assembled methicillin-resistant Staphylococcus aureus biofilms

Methicillin-resistant Staphylococcus aureus (MRSA) infections are a major health problem worldwide. One of the therapeutic options for treating MRSA is linezolid (LZD), which acts by binding to the ribosome bacteria and inhibiting protein synthesis. Bacterial biofilms are assembled communities which are around 10 to 1000 times more resistant to antibiotics than their planktonic counterparts. The aim of this work was to investigate the inhibition profile and the percentage of biofilm eradication in clinical and reference S. aureus strains caused by LZD. The bacterial biomass was assessed by crystal violet staining, and biofilm formation was studied using the XTT assay, with mature biofilm samples being exposed to the antibiotic and the inhibition profile also being measured by XTT. Antibiofilm activity was studied at different times by SEM to evaluate LZD eradication. All the tested strains produced a biofilm matrix, with clinical MRSA forming more biomass. The antibiofilm activity was observed at 10× MIC and revealed that LZD eliminated at least 98% of cell bunch clusters. Our results suggest that LZD is an efficient agent for eradicating MRSA biofilms.