Synergistic activity of gold nanoparticles with amphotericin B on persister cells of Candida tropicalis biofilms.

AIM: The antifungal activity was studied on sessile and persister cells (PCs) of Candida tropicalis biofilms of gold nanoparticles (AuNPs) stabilized with cetyltrimethylammonium bromide (CTAB-AuNPs) and those conjugated with cysteine, in combination with Amphotericin B (AmB). MATERIALS/METHODS: The PC model was used and synergistic activity was tested by the checkerboard assay. Biofilms were studied by crystal violet and scanning electron microscopy. RESULTS/CONCLUSIONS: After the combination of both AuNPs and AmB the biofilm biomass was reduced, with significant differences in architecture being observed with a reduced biofilm matrix. In addition, the CTAB-AuNPs-AmB combination significantly reduced PCs. Understanding how these AuNPs aid in the fight against biofilms and the development of new approaches to eradicate PCs has relevance for chronic infection treatment.

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.

The antioxidant activity of a prenyl flavonoid alters its antifungal toxicity on Candida albicans biofilms.

The antioxidant effect of 8PP, a prenylflavonoid from Dalea elegans on Candida albicans biofilms, was investigated. We previously reported that sensitive (SCa) and resistant C. albicans (RCa) biofilms were strongly inhibited by this compound, in a dose-depending manner (50 µM-100 µM), with a prooxidant effect leading to accumulation of endogenous oxidative metabolites and increased antioxidant defenses. In this work, the antifungal activity of high concentrations of 8PP (200-1000 µM), the cellular stress imbalance and the architecture of biofilms were evaluated. Biofilms were studied by crystal violet and confocal scanning laser microscopy (CSLM) with COMSTAT analysis. Superoxide anion radical, the activity of the superoxide dismutase and the total antioxidant capacity were measured. Intracellular ROS were detected by a DCFH-DA and visualized by CSLM; reactive nitrogen intermediates by Griess. An antioxidant effect was detected at 1000 µM and levels of oxidant metabolites remained low, with major changes in the SCa. COMSTAT analysis showed that biofilms treated with higher concentrations exhibited different diffusion distances with altered topographic surface architectures, voids, channels and pores that could change the flow inside the matrix of biofilms. We demonstrate for first time, a concentration-dependent antioxidant action of 8PP, which can alter its antifungal activity on biofilms.

Usnic Acid Activity on Oxidative and Nitrosative Stress of Azole-Resistant Candida albicans Biofilm.

Several studies report that (+)-usnic acid, a lichen secondary metabolite, inhibits growth of different bacteria and fungi; however, the mechanism of its antimicrobial activity remains unknown. In this study, we explored the ability of usnic acid, obtained from Usnea amblyoclada, as an antibiofilm agent against azole-resistant and azole-sensitive Candida albicans strains by studying the cellular stress and antioxidant response in biofilms. The biofilm inhibitory concentration of usnic acid (4 µg/mL) exhibited a significant biofilm inhibition, 71.08 % for azole-resistant and 87.84 % for azole-sensitive C. albicans strains. Confocal scanning laser microscopy showed that the morphology of mature biofilm was altered (reduced the biomass and thickness) in the presence of usnic acid. The antifungal effect was mediated by an oxidative and nitrosative stress, with a significant accumulation of intracellular and extracellular reactive oxygen species detected by confocal scanning laser microscopy and by nitro blue tetrazolium, respectively. In fact, azole-resistant and azole-sensitive C. albicans biofilms treated at the biofilm inhibitory concentration of usnic acid presented 30-fold and 10-fold increased reactive oxygen species measurements compared to basal levels, respectively, and important nitric oxide generation, showing 25-fold and 60-fold increased reactive nitrogen intermediates levels with respect to the controls, respectively. Nonenzymatic and enzymatic antioxidant defenses were increased in both strains compared to biofilm basal levels as response to the increase of oxidant metabolites. The present study shows for the first time that usnic acid can alter the prooxidant-antioxidant balance, which may be the cause of the irreversible cell damage and lead to cell death. Our results suggest that usnic acid could be an alternative for the treatment of Candida infections, which deserves further investigation.

The anthraquinones rubiadin and its 1-methyl ether isolated from Heterophyllaea pustulata reduces Candida tropicalis biofilms formation.

BACKGROUND: Candida tropicalis is increasingly becoming among the most commonly isolated pathogens causing fungal infections with an important biofilm-forming capacity. PURPOSE: This study addresses the antifungal effect of rubiadin (AQ1) and rubiadin 1-methyl ether (AQ2), two photosensitizing anthraquinones (AQs) isolated from Heterophyllaea pustulata, against C. tropicalis biofilms, by studying the cellular stress and antioxidant response in two experimental conditions: darkness and irradiation. The combination with Amphotericin B (AmB) was assayed to evaluate the synergic effect. STUDY DESIGN/METHODS: Biofilms of clinical isolates and reference strain of Candida tropicalis were treated with AQs (AQ1 or AQ2) and/or AmB, and the biofilms depletion was studied by crystal violet and confocal scanning laser microscopy (CSLM). The oxidant metabolites production and the response of antioxidant defense system were also evaluated under dark and irradiation conditions, being the light a trigger for photo-activation of the AQs. The Reactive Oxygen Species (ROS) were detected by the reduction of Nitro Blue Tetrazolium test, and Reactive Nitrogen Intermediates (RNI) by the Griess assay. ROS accumulation was also detected inside biofilms by using 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) probe, which was visualized by CSLM. Superoxide dismutase (SOD) activity and the total antioxidant capacity of biofilms were measured by spectrophotometric methods. The minimun inhibitory concentration for sessile cells (SMIC) was determined for each AQs and AmB. The fractional inhibitory concentration index (FICI) was calculated for the combinations of each AQ with AmB by the checkerboard microdilution method. RESULTS: Biofilm reduction of both strains was more effective with AQ1 than with AQ2. The antifungal effect was mediated by an oxidative and nitrosative stress under irradiation, with a significant accumulation of endogenous ROS detected by CSLM and an increase in the SOD activity. Thus, the prooxidant-antioxidant balance was altered especially by AQ1. The best synergic combination with AmB was also obtained with AQ1 (80.5%) (FICI=0.74). CONCLUSION: Under irradiation, the oxidative stress was the predominant effect, altering the prooxidant-antioxidant balance, which may be the cause of the irreversible cell injury in the biofilm. Our results showed synergism of these natural AQs with AmB. Therefore, the photosensitizing AQ1 could be an alternative for the Candida infections treatment, which deserves further investigation.

Antifungal activity of a prenylated flavonoid from Dalea elegans against Candida albicans biofilms.

BACKGROUND: The continuing emergence of infections with antifungal resistant Candida strains requires a constant search for new antifungal drugs, with the plant kingdom being an important source of chemical structures. PURPOSE: The present study investigated the antifungal effect of 2',4'-dihydroxy-5'-(1''',1'''-dimethylallyl)-8-prenylpinocembrin (8PP, formerly 6PP), a natural prenylflavonoid, on Candida albicans biofilms, and compared this with an azole antifungal (fluconazole) by studying the cellular stress and antioxidant response. STUDY DESIGN/METHODS: The fluconazole sensitive (SCa) and azole-resistant (RCa) C. albicans strains were used, with biofilm formation being studied using crystal violet (CV) and confocal scanning laser microscopy (CSLM). The minimal inhibitory concentration for sessile cells (SMIC) was defined as the concentration of antifungal that caused a 50% (SMIC 50) and 80% (SMIC 80) reduction of treated biofilms. The reactive oxygen species (ROS) were detected by the reduction of nitro blue tetrazolium (NBT), and reactive nitrogen intermediates (RNI) were determined by the Griess assay. The activities of the superoxide dismutase (SOD) and catalase (CAT) antioxidant enzymes and the total antioxidant capacity of the biofilms were measured by spectrophotometric methods. ROS accumulation was also detected inside biofilms by using the fluorogenic dye 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA), which was visualized by CSLM. RESULTS: The SCa and RCa biofilms were strongly inhibited by 8PP at 100 µM (SMIC 80). We observed that cellular stress affected biofilms growth, resulting in an increase of ROS and also of reactive nitrogen intermediates (RNI), with SOD and CAT being increased significantly in the presence of 8PP. The basal level of the biofilm total antioxidant capacity was higher in RCa than SCa. Moreover, in SCa, the total antioxidant capacity rose considerably in the presence of both 8PP and fluconazole. CONCLUSION: Our data suggest that 8PP may be useful for the treatment of biofilm-related Candida infections, through an accumulation of endogenous ROS and RNI that can induce an adaptive response based on a coordinated increase in antioxidant defenses. 8PP may also have a therapeutic potential in C. albicans infections.

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.

Describen el nivel de conocimiento acerca del sistema de farmacovigilancia / Description of levels of knowledge on the pharmacovigilance system

Se demuestra la necesidad de continuar con el fomento de la comprensión y la enseñanza de la farmacovigilancia, así como la formación clínica en la materia y una comunicación eficaz dirigida a los profesionales de la salud y a la opinión pública en general.

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.

Immune neuroendocrine interactions during a fungal infection in immunocompetent or immunosuppressed hosts.

The yeast Candida albicans belongs to the microflora of healthy individuals, although it can infect a variety of tissues ensuing changes in the host's immune status. To evaluate the effect of neuroendocrine input on the early immune response during the fungal infection, we use a 3-day paradigm of chronic varied stress in Wistar rats infected with C. albicans. We find that stress mediators contribute to the spread of the fungus and downregulate critical functions of phagocytic cells at the infection site. Phenotypic and functional alterations of effector cells account for the decreased resistance to candidiasis and condition the development of the adaptive response. Stressed hosts exhibit a higher fungal burden in kidneys and livers associated with hyphal forms. The hepatic inflammatory reaction is compromised with severe steatosis, increment of functional enzymes, marked lipid peroxidation and hepatocyte apoptosis. Moreover, infection-related sickness symptoms are significantly increased by exposure to stress with anorexia, weight loss, lack of leptin and depletion of glycogen depots. Food deprivation exacerbates the liver injury. Stress mediators perturb the complex immune and metabolic program that operates early during fungal spread and promotes severe tissue damage.

Candida albicans-secreted lipase induces injury and steatosis in immune and parenchymal cells.

Virulence depends on opposing reactions between host and pathogen and is intrinsically linked to the host immune status. Virulence factors rely upon microbial attributes that mediate cell damage. While the activity of several Candida albicans hydrolytic enzymes is well characterized, the biological role of lipases is uncertain. In this report, we identified, isolated, and characterized a C. albicans 70 kDa lipase that exhibited maximal activity at physiological pH and temperature. We evaluated the ability of C. albicans lipase to interact with two types of mammalian host cells: macrophages, as crucial immune effector cells involved in fungal control, and hepatocytes, as examples of parenchymal cells compromised during fungal dissemination. Herein, we demonstrate for the first time that an extracellular lipase released by C. albicans directly induced cytotoxicity and promoted the deposition of lipid droplets in the cytoplasm of macrophages and hepatocytes.

An Enterobacter cloacae toxin able to generate oxidative stress and to provoke dose-dependent lysis of leukocytes.

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.

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.

Interaction of bacterial toxin with leukocytes measured by flow cytometry.

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.