Evaluation of Antibacterial Activity and Reactive Species Generation of N-Benzenesulfonyl Derivatives of Heterocycles.

Two N-benzenesulfonyl (BS) derivatives of 1,2,3,4-tetrahydroquinoline (THQ) were designed, prepared, and screened for antibacterial activity. This approach was based on combining the two privileged structures, BS and THQ, which are known to be active. The objective of this study was to evaluate the antibacterial activity of BS-THQ and its analogue 4-NH2BS-THQ, and to investigate the roles of reactive oxygen species and reactive nitrogen species in their lethality. Both showed bactericidal activity against Staphylococcus aureus ATCC 29213 and methicillin-resistant S. aureus (MRSA) ATCC 43300, with transmission electron microscopy revealing a disturbed membrane architecture. Furthermore, an increase of reactive oxygen species (ROS) in strains treated with BS-THQ with respect to the control was detected when fluorescent microscopy and spectrophotometric techniques were used. The analogue 4-NH2BS-THQ demonstrated a broader spectrum of activity than BS-THQ, with a minimum inhibitory concentration of 100 µg/mL against reference strains of S. aureus, Escherichia coli and Pseudomonas aeruginosa. The assayed compounds represent promising structures for the development of new synthetic classes of antimicrobials.

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.

In vitro oxidant effects of D-glucosamine reduce adhesión and biofilm formation of Staphylococcus epidermidis.

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.

Nitric oxide-mediated apoptosis in rat macrophages subjected to Shiga toxin 2 from Escherichia coli.

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.

Chemiluminescence determination of antioxidant property of Zizyphus mistol and Prosopis alba during oxidative stress generated in blood by Hemolytic Uremic Syndrome-producing Escherichia coli.

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.

Resistance to ciprofloxacin by enhancement of antioxidant defenses in biofilm and planktonic Proteus mirabilis.

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.

Beneficial effect of Berberis buxifolia Lam, Ziziphus mistol Griseb and Prosopis alba extracts on oxidative stress induced by chloramphenicol.

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.

Eriobotrya japonica counteracts reactive oxygen species and nitric oxide stimulated by chloramphenicol.

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.

Enhanced inhibition of bacterial biofilm formation and reduced leukocyte toxicity by chloramphenicol:ß-cyclodextrin:N-acetylcysteine complex.

The purpose of this study was to improve the physicochemical and biological properties of chloramphenicol (CP) by multicomponent complexation with ß-cyclodextrin (ß-CD) and N-acetylcysteine (NAC). The present work describes the ability of solid multicomponent complex (MC) to decrease biomass and cellular activity of Staphylococcus by crystal violet and XTT assay, and leukocyte toxicity, measuring the increase of reactive oxygen species by chemiluminescence, and using 123-dihydrorhodamine. In addition, MC was prepared by the freeze-drying or physical mixture methods, and then characterized by scanning electron microscopy and powder X-ray diffraction. Nuclear magnetic resonance and phase solubility studies provided information at the molecular level on the structure of the MC and its association binding constants, respectively. The results obtained allowed us to conclude that MC formation is an effective pharmaceutical strategy that can reduce CP toxicity against leukocytes, while enhancing its solubility and antibiofilm activity.

Protective effect of quercetin in gentamicin-induced oxidative stress in vitro and in vivo in blood cells. Effect on gentamicin antimicrobial activity.

We have evaluated the effect of gentamicin and gentamicin plus quercetin on ROS production, endogenous antioxidant defenses (SOD and CAT) and lipid peroxidation in vitro on human leukocytes and in vivo on whole rat blood. Gentamicin generated ROS production in human leukocytes, produced a dual effect on both enzymes dosage-dependent and generated an increase in lipid peroxidation. Quercetin, in leukocytes stimulated by gentamicin, showed more inhibitory capacity in ROS production than the reference inhibitor (vitaminC) in mononuclear cells and a similar protective behavior at this inhibitor in polymorphonuclear cells. Quercetin, in both cellular systems, tend to level SOD and CAT activities, reaching basal values and could prevent lipidic peroxidation induced by gentamicin. The results in Wistar rats confirmed that therapeutic doses of gentamicin can induce oxidative stress in whole blood and that the gentamicin treatment plus quercetin can suppress ROS generation, collaborate with SOD and CAT and diminish lipid peroxidation. Finally, flavonoid and antibiotic association was evaluated on the antimicrobial activity in S. aureus and E. coli, showing that changes were not generated in the antibacterial activity of gentamicin against E. coli strains, while for strains of S. aureus a beneficial effect observes. Therefore, we have demonstrated that gentamicin could induce oxidative stress in human leukocytes and in whole blood of Wistar rats at therapeutic doses and that quercetin may to produce a protective effect on this oxidative stress generated without substantially modifying the antibacterial activity of gentamicin against E. coli strains, and it contributes to this activity against S. aureus strains.

Natural anthraquinones probed as Type I and Type II photosensitizers: singlet oxygen and superoxide anion production.

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).

Pore formation, polymerization, hemolytic and leukotoxic effects of a new Enterobacter cloacae toxin neutralized by antiserum.

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.

Inclusion complexes of chloramphenicol with ß-cyclodextrin and aminoacids as a way to increase drug solubility and modulate ROS production.

The aim of this study was to improve the solubility of chloramphenicol and reduce the production of reactive oxygen species (ROS) in leucocytes induced by this drug, using complexation. Multicomponent complexes were prepared by the addition of ß-cyclodextrin with glycine or cysteine. Nuclear magnetic resonance and phase solubility studies provided information at the molecular level on the structure of the complexes and their association binding constants, respectively. In the solid state, all systems were extensively characterized by Fourier-transform infrared spectroscopy, scanning electron microscopy, thermal analysis and X-ray powder diffraction. Antimicrobial activity of inclusion complexes was investigated by agar diffusion methods. Finally ROS determination by chemiluminescence was used to investigate the effect of complex formation on the potential toxicity in human leucocytes. These studies revealed that multicomponent complexes can increase the aqueous solubility of chloramphenicol as well as reducing the stress by ROS production in leucocytes and maintaining its microbiological activity.

Effect of antibiotics on cellular stress generated in Shiga toxin-producing Escherichia coli O157:H7 and non-O157 biofilms.

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.

Light effect and reactive oxygen species in the action of ciprofloxacin on Staphylococcus aureus.

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.

Macromolecular oxidation in planktonic population and biofilms of Proteus mirabilis exposed to ciprofloxacin.

Diverse chemical and physical agents can alter cellular functions associated with the oxidative metabolism, thus stimulating the production of reactive oxygen species (ROS). Proteins and lipids may be important targets of oxidation, and this may alter their functions in planktonic bacterial physiology. However, more research is necessary to determine the precise role of cellular stress and macromolecular oxidation in biofilms. The present study was designed to evaluate whether ciprofloxacin (CIP) could oxidize the lipids to malondialdehyde (MDA) and the proteins to carbonyl residues and to advanced oxidation protein products (AOPP) in planktonic populations and biofilms of Proteus mirabilis. Incubation with CIP generated an increase of lipid and protein oxidation in planktonic cells, with a greater effect found in sensitive strains than resistant ones. Biofilms showed higher basal levels of oxidized macromolecules than planktonic bacteria, but there was no significant enhancement of MDA, carbonyl, or AOPP with antibiotic. The results described in this article show the high basal levels of MDA, carbonyls, and AOPP, with aging and loss of proliferation of biofilms cells. The low response to the oxidative stress generated by CIP in biofilms helps to clarify the resistance to antibiotics of P. mirabilis when adhered to surfaces.

Impact of ciprofloxacin and chloramphenicol on the lipid bilayer of Staphylococcus aureus: changes in membrane potential.

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.

Hemolysin from Escherichia coli induces oxidative stress in blood.

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.

Nitrosylation: an adverse factor in Uremic Hemolytic Syndrome. Antitoxin effect of Ziziphus mistol Griseb.

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.

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.