Fungicidal and antibiofilm activities of gold nanoparticles on Candida tropicalis.

Aim: To investigate the antifungal activity of two different functionalized gold nanoparticles (AuNP), those stabilized with cetyltrimethylammonium bromide and those conjugated with cysteine, and their effects on the architecture of Candida tropicalis biofilms. Materials & methods: Biofilms were studied by crystal violet binding assay and scanning electron microscopy. We investigated the effects of AuNPs on reactive oxygen species, reactive nitrogen intermediates and enzymatic and nonenzymatic antioxidant defenses. Results/Conclusion: The fungicidal activity and cellular stress of both AuNPs affected biofilm growth through accumulation of reactive oxygen species and reactive nitrogen intermediates. However, cetyltrimethylammonium bromide-stabilized AuNPs revealed a higher redox imbalance. We correlated, for the first time, AuNP effects with the redox imbalance and alterations in the architecture of C. tropicalis biofilms.

Biofilms are at least 100­1000-times more resistant to the effects of antimicrobial agents compared with planktonic cells, and nanoparticles have emerged to provide new approaches to improve the safety and efficacy of antimicrobial therapy. The aim of this work was to investigate the antifungal activity with two different functionalized gold nanoparticles. A significant reduction of Candida tropicalis biofilms with alterations in surface topography and architecture was observed, and the oxidative and nitrosative stress affected the biofilms. To the best of our knowledge, this is the first study that attempts to correlate the antibiofilm effects of gold nanoparticles on the redox imbalance against biofilms. These compounds could be an alternative to fungal biofilms infections treatments, applied specifically in biological and medical fields.

Silver bionanoparticles toxicity in trophoblast is mediated by nitric oxide and glutathione pathways.

Silver bionanoparticles (AgNPs) biosynthesized by Pseudomonas aeruginosa culture supernatant have an important antibacterial activity mediated by ROS increase; however their toxicity in human cells is not known. Due to the high susceptibility of the developing tissues to xenobiotics, the aim of this study was to investigate the AgNPs effect on first trimester human trophoblasts. The HTR8/SVneo cell line was treated with AgNPs (0.3-1.5 pM), for 6 and 24 h. Cell viability, reactive nitrogen and oxygen species (RNS and ROS) production, nitric oxide synthase expression, antioxidant defenses and biomolecule damage were evaluated. The exposure to AgNPs produced changes in HTR8/SVneo cell morphology and decreased cell viability. Alterations in redox balance were observed, with an increase in ROS and RNS levels, and NOS2 protein expression. Superoxide dismutase and catalase augmented their activity accompanied with a decreased in glutathione content and glutathione S-transferase activity. Protein oxidation and genotoxic damage were observed at concentrations greater than 0.6 pM. The pre-incubation with l-NMMA, NAC, mannitol and peroxidase demonstrated that AgNPs-induced cytotoxicity was not mediated by HO and H2O2, but nitric oxide and glutathione pathways were implicated in cell death. Since reported AgNPs microbicidal mechanism is mediated by increasing ROS (mainly HO and H2O2) without an increase in RNS, this work indicates an interesting difference in the reactive species and oxidative pathways involved in AgNPs toxicity in eukaryotic and prokaryotic cells. Highlighting the importance of toxicity evaluation to determine the safety of AgNPs with pharmaceutical potential uses.

Flavonoids as protective agents against oxidative stress induced by gentamicin in systemic circulation. Potent protective activity and microbial synergism of luteolin.

The flavonoids effect on gentamicin (GEN)-induced oxidative stress (OS) in systemic circulation was evaluated in terms of reactive oxygen species (ROS) production, enzymatic antioxidant defenses superoxide dismutase (SOD) and catalase (CAT), and lipid peroxidation (LP) in vitro on human leukocytes and in vivo on rat whole blood. The inhibitory activity of ROS was ATS < QTS < isovitexin < vitexin < luteolin. Luteolin, the most active, showed more inhibition in ROS production than vitamin C (reference inhibitor) in mononuclear cells and a slightly lower protective behavior compared to this inhibitor in polymorphonuclear cells. In both cellular systems, luteolin tends to level SOD and CAT activities modified by GEN, reaching basal values and preventing LP. In Wistar rats, GEN plus luteolin can suppress ROS generation, collaborate with SOD and CAT and diminish LP produced by GEN at therapeutic doses. Finally, luteolin and antibiotic association was evaluated on the antimicrobial activity in S. aureus and E. coli showing a synergism between GEN and luteolin on S. aureus ATCC and an additive effect on E. coli ATCC. Therefore, simultaneous administration of luteolin and GEN could represent a potential therapeutic option capable of protecting the host against OS induced by GEN in the systemic circulation while enhancing the antibacterial activity of GEN.

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.

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

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

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

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

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

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

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.