RESUMO
Shiga toxin-producing Escherichia coli (STEC) are important food-borne pathogens, with the main virulence factor of this bacterium being its capacity to secrete Shiga toxins (Stxs). Therefore, the use of certain antibiotics for the treatment of this infection, which induces the liberation of Stxs, is controversial. Reactive oxygen and nitrogen species are also involved in the pathogenesis of different diseases. The purpose of this study was to analyze the effects of antibiotics on biofilms of STEC and the relationships between cellular stress and the release of Stx. To this end, biofilms of reference and clinical strains were treated with antibiotics (ciprofloxacin, fosfomycin and rifaximin) and the production of oxidants, the antioxidant defense system and toxin release were evaluated. Ciprofloxacin altered the prooxidant-antioxidant balance, with a decrease of oxidant metabolites and an increase of superoxide dismutase and catalase activity, being associated with high-levels of Stx production. Furthermore, inhibition of oxidative stress by exogenous antioxidants was correlated with a reduction in the liberation of Stx, indicating the participation of this phenomenon in the release of this toxin. In contrast, fosfomycin and rifaximin produced less alteration with a minimal production of Stx. Our data show that treatment of biofilm-STEC with these antibiotics induces oxidative stress-mediated release of Stx.
Assuntos
Antibacterianos/farmacologia , Toxina Shiga I/metabolismo , Toxina Shiga II/metabolismo , Escherichia coli Shiga Toxigênica/efeitos dos fármacos , Animais , Antioxidantes/farmacologia , Ácido Ascórbico/farmacologia , Biofilmes , Catalase/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Chlorocebus aethiops , Ciprofloxacina/farmacologia , Fosfomicina/farmacologia , Glutationa/farmacologia , Testes de Sensibilidade Microbiana , Óxido Nítrico/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Rifamicinas/farmacologia , Rifaximina , Escherichia coli Shiga Toxigênica/genética , Escherichia coli Shiga Toxigênica/metabolismo , Escherichia coli Shiga Toxigênica/fisiologia , Superóxido Dismutase/metabolismo , Células Vero , Fatores de Virulência/genéticaRESUMO
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.
Assuntos
Biofilmes/crescimento & desenvolvimento , Infecções por Escherichia coli/etiologia , Escherichia coli Shiga Toxigênica/fisiologia , Escherichia coli Shiga Toxigênica/patogenicidade , Animais , Catalase/metabolismo , Chlorocebus aethiops , Meios de Cultura , Infecções por Escherichia coli/metabolismo , Infecções por Escherichia coli/microbiologia , Escherichia coli O157/patogenicidade , Escherichia coli O157/fisiologia , Humanos , Espécies Reativas de Nitrogênio/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Toxinas Shiga/biossíntese , Toxinas Shiga/toxicidade , Superóxido Dismutase/metabolismo , Células VeroRESUMO
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.
Assuntos
Cromo/farmacologia , Escherichia coli/efeitos dos fármacos , Rutênio/farmacologia , Staphylococcus aureus/efeitos dos fármacos , Anti-Infecciosos/farmacologia , Cromo/química , Testes de Sensibilidade Microbiana , Compostos Organometálicos/química , Estresse Oxidativo/efeitos dos fármacos , Fenantrolinas/química , Rutênio/química , Infecções Estafilocócicas/tratamento farmacológico , Infecções Estafilocócicas/microbiologiaRESUMO
The present study was undertaken to explore the interaction of ciprofloxacin and chloramphenicol with bacterial membranes in a sensitive and in a resistant strains of Staphylococcus aureus by using 1-anilino-8-naphthalene sulfonate (ANS). The binding of this probe to the cell membrane depends on the surface potential, which modulates the binding constant to the membrane. We observed that these antibiotics interacted with the bilayer, thus affecting the electrostatic surface potential. Alterations caused by antibiotics on the surface of the bacteria were accompanied by a reduction in the number of binding sites and an increase in the ANS dissociation constant in the sensitive strain, whereas in the ciprofloxacin-resistant strain no significant changes were detected. The changes seen in the electrostatic surface potential generated in the membrane of S. aureus by the antibiotics provide new aspects concerning their action on the bacterial cell.
Assuntos
Cloranfenicol/farmacologia , Ciprofloxacina/farmacologia , Bicamadas Lipídicas/metabolismo , Potenciais da Membrana/efeitos dos fármacos , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/fisiologia , Naftalenossulfonato de Anilina/metabolismo , Testes de Sensibilidade MicrobianaRESUMO
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.
Assuntos
Infecções por Escherichia coli/sangue , Proteínas de Escherichia coli/sangue , Escherichia coli/química , Proteínas Hemolisinas/sangue , Estresse Oxidativo/efeitos dos fármacos , Produtos da Oxidação Avançada de Proteínas/metabolismo , Antioxidantes/metabolismo , Biomarcadores/sangue , Infecções por Escherichia coli/metabolismo , Infecções por Escherichia coli/microbiologia , Humanos , Peroxidação de Lipídeos , Luminescência , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase/metabolismoRESUMO
Toxins of Escherichia coli (STEC) causing Uremic Hemolytic Syndrome (UHS) generate oxidative stress in human blood with more production of nitric oxide (NO) than reactive oxygen species (ROS). Shiga toxin (Stx) together with the hemolysin (Hly) increased lipid oxidation, as evaluated by malondialdehyde MDA and oxidation of proteins. The addition of Ziziphus mistol Griseb extracts decreased NO, ROS, MDA and simultaneously caused an increase in the degradation of oxidized proteins to advanced oxidation protein products (AOPPs) in controls and samples with toxins. Furthermore, the nitrosylated proteins/AOPP ratio was reduced, due to the increase of AOPP. Z. mistol Griseb extracts exhibited a high proportion of polyphenols and flavonoids, with evident correlation with ferrous reduction antioxidant potential (FRAP). The plasma of eight children with UHS showed oxidative stress and NO stimulus, comparable to the effect of toxins during the assays in vitro. UHS children presented high levels of nitrosylated proteins respect to control children of similar age. Although the degradation of oxidized proteins to AOPP rose in UHS children, the nitrosylated proteins/AOPP rate increased as a consequence of the elevated nitrosative stress observed in these patients.
Assuntos
Antioxidantes/farmacologia , Antitoxinas/farmacologia , Síndrome Hemolítico-Urêmica/sangue , Extratos Vegetais/farmacologia , Polifenóis/farmacologia , Ziziphus/química , Produtos da Oxidação Avançada de Proteínas/sangue , Criança , Proteínas Hemolisinas/metabolismo , Humanos , Metabolismo dos Lipídeos/efeitos dos fármacos , Malondialdeído/sangue , Óxido Nítrico/sangue , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/sangue , Toxina Shiga/metabolismo , Toxina Shiga/toxicidade , Escherichia coli Shiga Toxigênica/metabolismoRESUMO
Staphylococcus epidermidis is a common pathogen in medical device-associated infections. Its major pathogenic factor is the ability to form adherent biofilms. In this work, three S. epidermidis strains isolated from infected catheters were chosen with the objective of investigating the effect of D-glucosamine (D-Glu) on reactive oxygen species (ROS) production, adhesion and biofilm formation. The chemiluminescence and nitroblue tetrazolium reduction assays were used to determine ROS production by planktonic S. epidermidis and the microtiter plate assay to quantify in vitro biofilm formation. D-Glu generated a dose-dependent increase in ROS in planktonic cells with maximum stimuli at a concentration of 0.05 mM, and reduced adhesion and biofilm formation. On the other hand, glucose showed an antioxidative stress action and promoted biofilm adhesion and growth. This study suggests a potential application of D-Glu against infections associated with indwelling medical devices, since the oxidative stress caused by this hexosamine in planktonic S. epidermidis contributed to reducing biofilm formation.
Assuntos
Aderência Bacteriana/efeitos dos fármacos , Biofilmes/efeitos dos fármacos , Glucosamina/farmacologia , Oxidantes/farmacologia , Staphylococcus epidermidis/efeitos dos fármacos , Catéteres/microbiologia , Avaliação Pré-Clínica de Medicamentos , Contaminação de Equipamentos , Vidro , Glucose/farmacologia , Técnicas In Vitro , Estresse Oxidativo/efeitos dos fármacos , Poliestirenos , Staphylococcus epidermidis/isolamento & purificação , Staphylococcus epidermidis/fisiologiaRESUMO
Staphylococcus epidermidis is a common pathogen in medical device-associated infections. Its major pathogenic factor is the ability to form adherent biofilms. In this work, three S. epidermidis strains isolated from infected catheters were chosen with the objective of investigating the effect of D-glucosamine (D-Glu) on reactive oxygen species (ROS) production, adhesion and biofilm formation. The chemiluminescence and nitroblue tetrazolium reduction assays were used to determine ROS production by planktonic S. epidermidis and the microtiter plate assay to quantify in vitro biofilm formation. D-Glu generated a dose-dependent increase in ROS in planktonic cells with maximum stimuli at a concentration of 0.05 mM, and reduced adhesion and biofilm formation. On the other hand, glucose showed an antioxidative stress action and promoted biofilm adhesion and growth. This study suggests a potential application of D-Glu against infections associated with indwelling medical devices, since the oxidative stress caused by this hexosamine in planktonic S. epidermidis contributed to reducing biofilm formation.
Staphylococcus epidermidis es un patógeno común en infecciones asociadas a dispositivos médicos. Su factor de patogenicidad más importante es la capacidad para formar biofilms. Se trabajó con tres cepas de S. epidermidis aisladas de catéteres, con las que se efectuaron ensayos de quimioluminiscencia y de reducción de azul de nitrotetrazolio, para determinar la producción de especies reactivas del oxígeno (ERO) en S. epidermidis planctónico, y ensayos dirigidos a cuantificar la formación de biofilm in vitro, empleando placas multipocillos. La D-glucosamina generó un aumento dependiente de la dosis en la producción de ERO en las células planctónicas, con un estímulo máximo a una concentración de 0,05 mM. Este aumento condμlo a la reducción de la adhesión y de la formación de biofilm. La adición de glucosa, en cambio, mostró un efecto anti estrés oxidativo y promovió la adhesión y el crecimiento de biofilm. Este estudio sugiere una posible aplicación de la D-glucosamina contra las infecciones asociadas a dispositivos médicos, ya que el estrés oxidativo provocado por esta hexosamina contribuyó a una menor formación de biofilm.
Assuntos
Aderência Bacteriana/efeitos dos fármacos , Biofilmes/efeitos dos fármacos , Glucosamina/farmacologia , Técnicas In Vitro , Oxidantes/farmacologia , Staphylococcus epidermidis/efeitos dos fármacos , Catéteres/microbiologia , Avaliação Pré-Clínica de Medicamentos , Contaminação de Equipamentos , Vidro , Glucose/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Poliestirenos , Staphylococcus epidermidis/isolamento & purificação , Staphylococcus epidermidis/fisiologiaRESUMO
Staphylococcus epidermidis is a common pathogen in medical device-associated infections. Its major pathogenic factor is the ability to form adherent biofilms. In this work, three S. epidermidis strains isolated from infected catheters were chosen with the objective of investigating the effect of D-glucosamine (D-Glu) on reactive oxygen species (ROS) production, adhesion and biofilm formation. The chemiluminescence and nitroblue tetrazolium reduction assays were used to determine ROS production by planktonic S. epidermidis and the microtiter plate assay to quantify in vitro biofilm formation. D-Glu generated a dose-dependent increase in ROS in planktonic cells with maximum stimuli at a concentration of 0.05 mM, and reduced adhesion and biofilm formation. On the other hand, glucose showed an antioxidative stress action and promoted biofilm adhesion and growth. This study suggests a potential application of D-Glu against infections associated with indwelling medical devices, since the oxidative stress caused by this hexosamine in planktonic S. epidermidis contributed to reducing biofilm formation.(AU)
Staphylococcus epidermidis es un patógeno común en infecciones asociadas a dispositivos médicos. Su factor de patogenicidad más importante es la capacidad para formar biofilms. Se trabajó con tres cepas de S. epidermidis aisladas de catéteres, con las que se efectuaron ensayos de quimioluminiscencia y de reducción de azul de nitrotetrazolio, para determinar la producción de especies reactivas del oxígeno (ERO) en S. epidermidis planctónico, y ensayos dirigidos a cuantificar la formación de biofilm in vitro, empleando placas multipocillos. La D-glucosamina generó un aumento dependiente de la dosis en la producción de ERO en las células planctónicas, con un estímulo máximo a una concentración de 0,05 mM. Este aumento condμlo a la reducción de la adhesión y de la formación de biofilm. La adición de glucosa, en cambio, mostró un efecto anti estrés oxidativo y promovió la adhesión y el crecimiento de biofilm. Este estudio sugiere una posible aplicación de la D-glucosamina contra las infecciones asociadas a dispositivos médicos, ya que el estrés oxidativo provocado por esta hexosamina contribuyó a una menor formación de biofilm.(AU)
Assuntos
Aderência Bacteriana , Biofilmes , Glucosamina/farmacologia , Oxidantes/farmacologia , Staphylococcus epidermidis , Catéteres/microbiologia , Avaliação Pré-Clínica de Medicamentos , Contaminação de Equipamentos , Vidro , Glucose/farmacologia , Estresse Oxidativo , Poliestirenos , Staphylococcus epidermidis/isolamento & purificação , Staphylococcus epidermidis/fisiologiaRESUMO
Staphylococcus epidermidis is a common pathogen in medical device-associated infections. Its major pathogenic factor is the ability to form adherent biofilms. In this work, three S. epidermidis strains isolated from infected catheters were chosen with the objective of investigating the effect of D-glucosamine (D-Glu) on reactive oxygen species (ROS) production, adhesion and biofilm formation. The chemiluminescence and nitroblue tetrazolium reduction assays were used to determine ROS production by planktonic S. epidermidis and the microtiter plate assay to quantify in vitro biofilm formation. D-Glu generated a dose-dependent increase in ROS in planktonic cells with maximum stimuli at a concentration of 0.05 mM, and reduced adhesion and biofilm formation. On the other hand, glucose showed an antioxidative stress action and promoted biofilm adhesion and growth. This study suggests a potential application of D-Glu against infections associated with indwelling medical devices, since the oxidative stress caused by this hexosamine in planktonic S. epidermidis contributed to reducing biofilm formation.(AU)
Staphylococcus epidermidis es un patógeno común en infecciones asociadas a dispositivos médicos. Su factor de patogenicidad más importante es la capacidad para formar biofilms. Se trabajó con tres cepas de S. epidermidis aisladas de catéteres, con las que se efectuaron ensayos de quimioluminiscencia y de reducción de azul de nitrotetrazolio, para determinar la producción de especies reactivas del oxígeno (ERO) en S. epidermidis planctónico, y ensayos dirigidos a cuantificar la formación de biofilm in vitro, empleando placas multipocillos. La D-glucosamina generó un aumento dependiente de la dosis en la producción de ERO en las células planctónicas, con un estímulo máximo a una concentración de 0,05 mM. Este aumento condμlo a la reducción de la adhesión y de la formación de biofilm. La adición de glucosa, en cambio, mostró un efecto anti estrés oxidativo y promovió la adhesión y el crecimiento de biofilm. Este estudio sugiere una posible aplicación de la D-glucosamina contra las infecciones asociadas a dispositivos médicos, ya que el estrés oxidativo provocado por esta hexosamina contribuyó a una menor formación de biofilm.(AU)
Assuntos
Aderência Bacteriana/efeitos dos fármacos , Biofilmes/efeitos dos fármacos , Glucosamina/farmacologia , Oxidantes/farmacologia , Staphylococcus epidermidis/efeitos dos fármacos , Catéteres/microbiologia , Avaliação Pré-Clínica de Medicamentos , Contaminação de Equipamentos , Vidro , Glucose/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Poliestirenos , Staphylococcus epidermidis/isolamento & purificação , Staphylococcus epidermidis/fisiologiaRESUMO
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.
Assuntos
Antioxidantes/metabolismo , Ciprofloxacina/farmacologia , Farmacorresistência Bacteriana , Proteus mirabilis/efeitos dos fármacos , Proteus mirabilis/metabolismo , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , DNA Girase/genética , Peroxidação de Lipídeos , Dados de Sequência Molecular , Estresse Oxidativo , Proteus mirabilis/genéticaRESUMO
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.
Assuntos
Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Bactérias/metabolismo , Substâncias Macromoleculares/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteínas de Bactérias/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Oxirredução/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Carbonilação Proteica/efeitos dos fármacosRESUMO
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.
Assuntos
Apoptose , Infecções por Escherichia coli/microbiologia , Infecções por Escherichia coli/fisiopatologia , Macrófagos Peritoneais/citologia , Óxido Nítrico/imunologia , Toxina Shiga II/imunologia , Escherichia coli Shiga Toxigênica/imunologia , Animais , Células Cultivadas , Infecções por Escherichia coli/imunologia , Feminino , Humanos , Macrófagos Peritoneais/imunologia , Ratos , Ratos WistarRESUMO
This study was undertaken to elucidate the antioxidant effect of Zizyphus mistol and Prosopis alba, with the hypothesis that these fruits can counteract the induction of reactive oxygen species (ROS) caused by toxins produced by Escherichia coli. In the search of nutrients effective against the Hemolytic Uremic Syndrome (HUS), we detected by chemiluminescence a protective role of both plants, due to their natural antioxidants significantly decreasing the levels of ROS induced by toxins from E. coli in blood. The ferric reducing antioxidant power (FRAP) was found to be higher in Z. mistol than in P. alba. The chemical analyses of the phenols and flavonoids present in the fruit extracts indicated that the FRAP correlated with the amount of phenolic compounds, but not with the flavonoids analyzed. Both fruits studied reduce the induction of ROS, and in this way help to prevent the development of complications related to oxidative stress generated in the blood of patients with HUS.
Assuntos
Antioxidantes/farmacologia , Escherichia coli/patogenicidade , Síndrome Hemolítico-Urêmica/sangue , Estresse Oxidativo , Extratos Vegetais/farmacologia , Prosopis/química , Ziziphus/química , Síndrome Hemolítico-Urêmica/microbiologia , Humanos , Luminescência , Espécies Reativas de Oxigênio/metabolismoRESUMO
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.
Assuntos
Biofilmes/crescimento & desenvolvimento , Espécies Reativas de Nitrogênio/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Staphylococcus aureus/fisiologia , Aerobiose/fisiologia , Meios de Cultura/metabolismo , Concentração de Íons de Hidrogênio , Estresse Oxidativo , Plâncton/metabolismo , Staphylococcus aureus/crescimento & desenvolvimento , Staphylococcus aureus/metabolismo , Temperatura , Fatores de TempoRESUMO
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-1ß 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.
Assuntos
Antígenos de Protozoários/imunologia , Cisteína Endopeptidases/imunologia , NADPH Oxidases/biossíntese , Espécies Reativas de Oxigênio/metabolismo , Trypanosoma cruzi/imunologia , Animais , Linhagem Celular , Feminino , Citometria de Fluxo , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Macrófagos/imunologia , Macrófagos/parasitologia , Camundongos , Camundongos Endogâmicos C57BL , Proteínas de Protozoários , Baço/imunologiaRESUMO
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.
Assuntos
Antibacterianos/farmacologia , Antioxidantes/farmacologia , Ciprofloxacina/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Staphylococcus aureus/efeitos dos fármacos , Farmacorresistência Bacteriana , Testes de Sensibilidade Microbiana , Óxido Nítrico/metabolismo , Oxirredução , Espécies Reativas de Oxigênio/metabolismo , Staphylococcus aureus/metabolismoRESUMO
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.
Assuntos
Antibacterianos/farmacologia , Antioxidantes/farmacologia , Cloranfenicol/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Extratos Vegetais/farmacologia , Berberis/química , Citoproteção/efeitos dos fármacos , Humanos , Leucócitos/efeitos dos fármacos , Prosopis/química , Espécies Reativas de Oxigênio/sangue , Ziziphus/químicaRESUMO
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.
Se estudió el efecto antioxidante de tres extractos de frutas autóctonas, Berberis buxifolia lam (michay), Zizyphus mistol Griseb (mistol) and Prosopis alba (algarrobo). Las células sanguíneas humanas sufrieron estrés oxidativo por acción de cloramfenicol (Ch), con un aumento inmediato de especies reactivas del oxígeno (ERO), que fue determinado por quimioluminiscencia con luminol. La respuesta fue dependiente de la dosis, con un máximo a 8 mg/ml. Los extractos de frutas autóctonas de la Argentina fueron capaces de contrarrestar el estrés generado por el antibiótico. El michay y el mistol resultaron más efectivos en la fase acuosa, y el algarrobo fue más antioxidante en extractos etílicos, mientras que las fracciones obtenidas con hexano no fueron activas. La viabilidad de los leucocitos se mantuvo elevada con Ch en presencia de extractos, entre 92.5 y 97.1%, cayendo hasta un 30% con Ch solo. Tanto los eritrocitos como los leucocitos fueron protegidos del efecto estresante por la capacidad antioxidantes de los extractos de las tres frutas investigadas, lo que podría ser importante a considerar en la dieta de niños, y pacientes en general, sometidos a Ch u otras terapias causantes de estrés oxidativo.
Assuntos
Humanos , Antibacterianos/farmacologia , Antioxidantes/farmacologia , Cloranfenicol/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Extratos Vegetais/farmacologia , Berberis/química , Citoproteção/efeitos dos fármacos , Leucócitos/efeitos dos fármacos , Prosopis/química , Espécies Reativas de Oxigênio/sangue , Ziziphus/químicaRESUMO
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.