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ABSTRACT: Hemolysin (HlyA) produced by some stains of Escherichia coli is considered to be an important virulence factor of those bacteria. On the other hand, reactive oxygen species (ROS) have been reported to be involved in the pathogenesis of different diseases via oxidative stress generation. The purpose of this study was to analyze the capacity of HlyA to induce oxidative stress in whole blood cultures (WBCs). To this end, ROS production, the damage induced in lipids and proteins, and the antioxidant defense system was evaluated in blood cultures exposed to low concentrations of HlyA. We found that HlyA increased the level of free radicals detected by chemiluminescence assay. Moreover, lipid peroxidation and protein damage was significantly increased in cultures treated with HlyA in comparation with those found in control cultures. On the other hand, a decrease in total antioxidant capacity of plasma and in the activity of superoxide dismutase (SOD) was observed in plasma from blood treated with HlyA. Collectively, our data demonstrate that low concentrations of E. coli hemolysin induced oxidative stress in WBCs with the induction of different oxidative damage biomarkers.
Toxicon 04/2013; · 2.51 Impact Factor
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ABSTRACT: This study investigates new aspects of the possible role of antioxidant defenses in the mechanisms of resistance to ciprofloxacin in Proteus mirabilis. Four ciprofloxacin-resistant variants (CRVs), selected in vitro by repeated cultures in a sub-minimum inhibitory concentration (MIC) concentration of ciprofloxacin, attained different levels of antibiotic resistance and high Ferric reducing antioxidant power, with 10(-6) frequencies. However, no mutations occurred in positions 83 or 87 of gyrA, 464 or 466 of gyrB, or 78, 80 or 84 of parC, suggesting that resistance took place without these typical mutations in DNA gyrase or topoisomerase IV. Assays with ciprofloxacin and the pump inhibitor carbonyl cyanide m-chlorophenylhydrazone showed that in addition to the antioxidant mechanisms, the influx/efflux mechanism also contributed to the increase in the resistance to ciprofloxacin in one CRV. Moreover, lipid oxidation to malondialdehyde and protein oxidation to carbonyls and advanced oxidation protein products were higher in sensitive than in the resistant strains, as a new factor involved in the mechanisms of resistance in P. mirabilis. The oxidative stress cross-resistance to telluride in CRVs enhanced the role of the antioxidants in the ciprofloxacin resistance of P. mirabilis, which was reinforced during the assays of reduction of susceptibility to ciprofloxacin by glutathione and ascorbic acid.
FEMS Microbiology Letters 11/2011; 327(1):25-32. · 2.04 Impact Factor
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ABSTRACT: 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.
Cell biochemistry and biophysics 07/2011; 61(3):467-72. · 3.34 Impact Factor
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ABSTRACT: Diverse chemical and physical agents can alter cellular functions associated with oxidative metabolism, thus stimulating the production of reactive oxygen species (ROS) and reactive nitrogen intermediates (RNI) in planktonic bacterial physiology. However, more research is necessary to determine the precise role of cellular stress in biofilm. The present study was designed to address the issues of Staphylococcus aureus biofilm formation with respect to the generation of oxidative and nitrosative stress. We studied three pathogenic S. aureus clinical strains and an ATCC strain exposed to a different range of culture conditions (time, temperature, pH, reduction and atmospheric conditions) using quantitative methods of biofilm detection. We observed that cellular stress could be produced inside biofilms, thereby affecting their growth, resulting in an increase of ROS and RNI production, and a decrease of the extracellular matrix under unfavorable conditions. These radical oxidizers could then accumulate in an extracellular medium and thus affect the matrix. These results contribute to a better understanding of the processes that enable adherent biofilms to grow on inert surfaces and lead to an improved knowledge of ROS and RNI regulation, which may help to clarify the relevance of biofilm formation in medical devices.
FEMS Microbiology Letters 02/2011; 315(1):23-9. · 2.04 Impact Factor
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ABSTRACT: Shiga toxin-producing Escherichia coli are important food-borne pathogens. The main factor conferring virulence on this bacterium is its capacity to secrete Shiga toxins (Stxs), which have been reported to induce apoptosis in several cell types. However, the mechanisms of this apoptosis have not yet been fully elucidated. In addition, Stxs have been shown to stimulate macrophages to produce nitric oxide (NO), a well-known apoptosis inductor.The aim of this study was to investigate the participation of NO in apoptosis of rat peritoneal macrophages induced by culture supernatants or Stx2 from E. coli. Peritoneal macrophages incubated in the presence of E. coli supernatants showed an increase in the amounts of apoptosis and NO production. Furthermore, inhibition of NO synthesis induced by addition of aminoguanidine (AG) was correlated with a reduction in the percentage of apoptotic cells, indicating participation of this metabolite in the apoptotic process. Similarly, treatment of cells with Stx2 induced an increase in NO production and amount of apoptosis, these changes being reversed by addition of AG. In summary, these data show that treatment with E. coli supernatants or Stx2 induces NO-mediated apoptosis of macrophages.
Microbiology and Immunology 01/2011; 55(4):231-8. · 1.30 Impact Factor
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ABSTRACT: 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.
International journal for parasitology 11/2010; 40(13):1531-8. · 3.39 Impact Factor
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ABSTRACT: 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.
Fundamental and Clinical Pharmacology 04/2010; 24(6):771-6. · 1.80 Impact Factor
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ABSTRACT: Antibiotic resistance and antioxidant defense were induced by ciprofloxacin in planktonic Proteus mirabilis and compared with the natural antibiotic resistance of biofilm. Resistant variants (1X and 1Y) were obtained from cultures of the sensitive wild type "wt" strain 1 in the presence of the antibiotic. Planktonic strain 1 exhibited oxidative stress with increases in the reactive oxygen species (ROS) and consumption of NO in the presence of ciprofloxacin, whereas 1X and 1Y suffered non-significant rises in ROS generation, but produced and consumed more NO than sensitive strain 1. The two resistant variants were more resistant to telluride than wt and showed increased levels of intracellular superoxide dismutase (SOD) and glutathione (GSH). However, ciprofloxacin did not stimulate oxidative stress in biofilm. The production of ROS and NO with or without ciprofloxacin was less significant in biofilms than in an equivalent number of planktonic bacteria; sensitive and resistant strains did not present differences. On the other hand, SOD and GSH were more elevated in the biofilm than in planktonic bacteria. In summary, these results indicate that ciprofloxacin can induce resistance by the enhancement of antioxidant defense in planktonic bacteria, similar to the natural resistance occurring in biofilm. This feature may be added to the factors that regulate the susceptibility to this antibiotic.
Biochemical and Biophysical Research Communications 02/2010; 393(1):84-8. · 2.48 Impact Factor
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ABSTRACT: 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.
FEMS Microbiology Letters 02/2010; 303(1):101-5. · 2.04 Impact Factor
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ABSTRACT: The chemiluminescence of luminol, a measure of oxidative stress, increased immediately as a consequence of reactive oxygen species (ROS) stimulated by this antibiotic. The effect of Ch was dose dependent with maximum stimulus at 8 mg/ml (Vmax); above this concentration the cells began to reduce the production of ROS. The oxidative injury of Ch was counteracted by water extracts of Berberis buxifolia lam, Zizyphus mistol Griseb and Prosopis alba, indigenous fruits from Argentina. The relatively light units (RLU) emitted decreased immediately as a consequence of a protective effect exerted by the extracts of these fruit extracts on blood cells. The three indigenous fruit extracts reduced to a different extent the oxidative injury caused by Ch. B.buxifolia lam exhibited the highest antioxidant capacity followed by Z.mistol Griseb. Water extracts of both fruit extracts were the most effective against the oxidative stress, while P.alba presented better antioxidant capacity in the ethanolic fraction obtained. Hexane extracts showed low protective action on blood cells, with little reduction of area under curve (AUC) of RLU plotted versus time. Leukocytes remained viable in blood samples incubated for 3h with Ch and water extracts of B. buxifolia lam or Z. mistol Griseb (97.1% and 92.5% viability by Trypan blue exclusion, respectively); whereas with Ch only the cells were stressed and viability decreased to 30%. The three fruit extracts protected the viability of leukocytes in parallel with the decrease of ROS. Erythrocytes were not lysed in the presence of Ch.
Medicina 01/2010; 70(1):65-70. · 0.47 Impact Factor
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ABSTRACT: 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.
Basic & Clinical Pharmacology & Toxicology 08/2008; 103(4):349-53. · 2.18 Impact Factor
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ABSTRACT: Chloramphenicol is a toxic antibiotic used for certain infections, though aplastic anaemia is one of its side-effects. The results of our experiments showed that blood cells suffered oxidative stress in the presence of chloramphenicol, with a significant increase in reactive oxygen species (ROS) detected by luminol-chemiluminescence (CL). The extract of fruits of Eriobotrya japonica markedly decreased ROS in leukocytes and erythrocytes, the oxidative stress caused by this antibiotic. Nitro Blue Tetrazolium (NBT) assay with purified leukocytes demonstrated that the antioxidant action of E. japonica caused an intracellular reduction in ROS, and that the extracts decreased these promoters of oxidative stress to normal levels in the cytoplasm. Determinations of nitric oxide (NO) generation indicated that E. japonica extracts also inhibited the stimuli of NO provoked by chloramphenicol. This study showed that the immediate antioxidant effect of E. japonica could be associated with the action of vitamin A. The protective action of this fruit was seen on mature leukocytes and erythrocytes, beneficial effect on blood cells suggest that its extract could be used as an antioxidant agent complementing the administration of chloramphenicol, as a modern-day extension to its traditional use in Chinese medicine.
The American Journal of Chinese Medicine 02/2007; 35(5):875-85. · 1.98 Impact Factor
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ABSTRACT: 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.
Molecular and Cellular Biochemistry 05/2006; 285(1-2):29-34. · 2.06 Impact Factor
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ABSTRACT: We investigated an Enterobacter cloacae strain exhibiting high hemolytic and leukotoxic activity. Monomeric and polymeric forms of the toxin showed similar effects on blood cells, although the polymer was more active than the monomer. Fluorescence microscopy revealed that both forms of the FITC-labeled toxin interacted with leukocytes, principally with neutrophils. Prelytic concentrations of polymeric and monomeric toxin significantly increased the production of reactive oxygen species (ROS) in neutrophils. Conversely, lytic concentrations of both toxin forms showed an increase followed by a decrease of ROS due to neutrophil damage. Monocytes did not show oxidative stress at all the toxin concentrations assayed. The toxin-neutrophil interaction at prelytic concentrations of toxin-stimulated ROS production and led to oxidative stress with subsequent cell death by apoptosis. However, high concentrations of E. cloacae toxin damaged leukocytes, producing lysis before the trigger of apoptosis, which suggests that the toxic effect is concentration dependent. The inhibition of oxidative stress observed with genistein and chloroquine suggests a potential involvement of the tyrosine kinase and nitric oxide synthesis pathways in E. cloacae toxin-mediated elevation of ROS.
International Journal of Medical Microbiology 07/2005; 295(2):109-16. · 4.17 Impact Factor
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ABSTRACT: The photosensitizing properties of six anthraquinones (AQs): soranjidiol (1), soranjidiol-1-methyl ether (2), rubiadin (3), rubiadin-1-methyl ether (4), damnacanthal (5) and damnacanthol (6), isolated from leaves and stems of Heterophyllaea pustulata Hook. f. (Rubiaceae) were studied. By means of photobiological and photophysical methods in vitro, the type of photosensitization that these metabolites are capable of producing was determined. Whereas the photosensitized generation of superoxide anion radical (O(2)(-)) (Type I) was evaluated in leukocyte suspensions, singlet molecular oxygen ((1)O(2)) production (Type II) was examined in organic solution. In addition, the quantum yield of (1)O(2) (Phi) in chloroform was measured for those AQs that generate it. It was established that 4 behaves exclusively as a Type I photosensitizer. By contrast, the others AQs act by both types of mechanisms, among which 5 showed the largest Phi of (1)O(2).
Journal of Photochemistry and Photobiology B Biology 02/2005; 78(1):77-83. · 2.81 Impact Factor
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ABSTRACT: A new toxin of Enterobacter cloacae was purified and studied by SDS-PAGE electrophoresis with the purpose of investigating its ability to generate polymers and their molecular mass. Monomer of 13.3 kDa and structures of multimeric mass were detected. The toxin of 66 kDa was the most abundant form of toxin. This polymer and the monomer were selected to examine blood cells damage. Membrane pores caused by both toxin forms seemed to be of similar dimension (estimated in 3.6 nm by experiments with osmotic protectors) and were able to lyse erythrocytes and leukocytes. The results obtained indicate that polymerization and pore formation are involved in the molecular events that participate in the cytotoxic effects of E. cloacae toxin. Immunization of rabbits with 13.3kDa toxin generated antibody response capable of inhibiting oxidative stress as well as hemolytic and leukotoxic effects. Immunoblotting indicated that monomer and polymer reacted with antihemolysin serum. The importance of E. cloacae toxin "in vivo" was studied in animals by means of assays performed in peritoneum of rats, inoculated with the hemolytic strain (C1) and a non-hemolytic variant (C4). Both strains stimulated infiltration of leukocytes in peritoneum, but C1 caused cell death and lysis wheras assays with C4 maintained the viability of leukocytes even within 5 h after extraction of samples.
Microbiological Research 02/2005; 160(2):203-11. · 2.31 Impact Factor
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ABSTRACT: Oxygen consumption by Staphylococcus aureus ATCC 29213 sensitive to ciprofloxacin was determined with an oxygen selective electrode. Increase in the O(2) consumption was observed with 0.45 micromL(-1) ciprofloxacin while higher concentrations gave rise to a reduction of O(2) consumption. Resistant S. aureus strain did not show increase of O(2) consumption in presence of ciprofloxacin. Nitro Blue Tetrazolium assay showed that production of reactive oxygen species (ROS) increased intracellularly in sensitive bacteria incubated with this antibiotic. The exposition to UV light (360 nm) augmented the intracellular oxidative stress of S. aureus and provoked increment of ROS in extracellular media. Generation of singlet oxygen O(2) ((1)Delta(g)) in S. aureus was measured by means of oxidation of methionine. The absorbance of methionine was monitored at 215 nm and a clear decrease was detected when sensitive S. aureus was stressed with ciprofloxacin. Sodium azide and 2,5-dimethylfuran were used to reinforce the evidence of O(2) ((1)Delta(g)) generation during oxidative stress. Assays with methionine and 2,5-dimethylfuran demonstrated that resistant S. aureus did not increase the production of O(2) ((1)Delta(g)) in the presence of antibiotic. DNA oxidation was investigated in presence of O(2) ((1)Delta(g)) generated by laser excitation of perinaphthenone and subsequent energy transfer. Deactivation of O(2) ((1)Delta(g)) by reaction with DNA of sensitive and resistant bacteria was observed. According to the results obtained, the effect of ciprofloxacin in S. aureus led to an increment of O(2) ((1)Delta(g)) generating oxidative stress in the bacteria.
Journal of Photochemistry and Photobiology B Biology 11/2004; 76(1-3):13-8. · 2.81 Impact Factor
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ABSTRACT: MutS is part of the bacterial mismatch repair system that corrects point mutations and small insertions/deletions that fail to be proof-read by DNA polymerase activity. In this work it is shown that the disruption of the P. aeruginosa mutS gene generates the emergence of diverse colony morphologies in contrast with its parental wild-type strain that displayed monomorphic colonies. Interestingly, two of the mutS morphotypes emerged at a high frequency and in a reproducible way and were selected for subsequent characterization. One of them displayed a nearly wild-type morphology while the other notably showed, compared with the wild-type strain, increased production of pyocyanin and pyoverdin, lower excretion of LasB protease and novel motility characteristics, mainly related to swarming. Furthermore, it was reproducibly observed that, after prolonged incubation in liquid culture, the pigmented variant consistently emerged from the mutS wild-type-like variant displaying a reproducible event. It is also shown that these P. aeruginosa mutS morphotypes not only displayed an increase in the frequency of antibiotic-resistant mutants, as described for clinical P. aeruginosa mutator isolates, but also generated mutants whose antibiotic-resistant levels were higher than those measured from spontaneous resistant mutants derived from wild-type cells. It was also found that both morphotypes showed a decreased cytotoxic capacity compared to the wild-type strain, leading to the emergence of invasive variants. By using mutated versions of a tetracycline resistance gene, the mutS mutant showed a 70-fold increase in the reversion frequency of a +1 frameshift mutation with respect to its parental wild-type strain, allowing the suggestion that the phenotypical diversity generated in the mutS population could be produced in part by frameshift mutations. Finally, since morphotypical diversification has also been described in clinical isolates, the possibility that this mutS diversification was related to the high frequency hypermutability observed in P. aeruginosa CF isolates is discussed.
Microbiology 06/2004; 150(Pt 5):1327-38. · 3.06 Impact Factor
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ABSTRACT: Staphylococcus aureus and Escherichia coli sensitive to chloramphenicol incubated with this antibiotic suffered oxidative stress with increase of anion superoxide (O2-). This reactive species of oxygen was detected by chemiluminescence with lucigenin. S. aureus, E. coli, and Enterococcus faecalis sensitive to ciprofloxacin exhibited oxidative stress when they were incubated with this antibiotic while resistant strains did not show stimuli of O2-. Other bacteria investigated was Pseudomonas aeruginosa, strains sensitive to ceftazidime and piperacillin presented oxidative stress in presence of these antibiotics while resistant strains were not stressed. Higher antibiotic concentration was necessary to augment O2- in P. aeruginosa biofilm than in suspension, moreover old biofilms were resistant to oxidative stress caused by antibiotics. A ceftazidime-sensitive mutant of P. aeruginosa, coming from a resistant strain, exhibited higher production of O2- than wild type in presence of this antibiotic. There was relation between antibiotic susceptibility and production of oxidative stress.
Biochemical and Biophysical Research Communications 05/2004; 317(2):605-9. · 2.48 Impact Factor
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ABSTRACT: Enterobacter cloacae toxin was purified in the form of monomer and polymer. Both forms stimulated the generation of reactive oxygen species (ROS) at sublytic concentration; the oxidative stress produced was studied by using chemiluminescence (CL). The alteration generated caused death of leukocytes, especially at high toxin concentration. Polymeric toxin produced more oxidative stress than the monomeric one. Cytometry allowed the detection of more toxin binding to neutrophils rather than to monocytes or lymphocytes. There was binding at 4 degrees C, and the amount of toxin in the cells increased at 37 degrees C. The interaction of toxin with leukocytes was evident even after 100 degrees C treatment of toxin during 5 min. The incubation with 2-mercaptoethanol was not necessary for toxin binding.
Current Microbiology 10/2002; 45(3):171-4. · 1.82 Impact Factor
