Oxidative stress generation of silver nanoparticles in three bacterial genera and its relationship with the antimicrobial activity.

Oxidative stress is a condition caused by the high intracellular concentrations of reactive oxygen species (ROS) that includes superoxide anion radicals, hydroxyl radicals and hydrogen peroxide. Nanoparticles could cause rapid generation of free radicals by redox reactions. ROS can react directly with membrane lipids, proteins and DNA and are normally scavenged by antioxidants that are capable of neutralizing; however, elevated concentrations of ROS in bacterial cells can result in oxidative stress. The aim of this work was contribute to the knowledge of action mechanism of silver nanoparticles (Ag-NPs) and their relation to the generation of oxidative stress in bacteria. We demonstrated that Ag-NPs generated oxidative stress in Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa mediated by the increment of ROS and this increase correlated with a better antimicrobial activity. On the other hand, we showed that the oxidative stress caused by the Ag-NPs biosynthesized was associated to a variation in the level of reactive nitrogen intermediates (RNI). Oxidative stress in bacteria can result from disruption of the electronic transport chain due to the high affinity of Ag-NPs for the cell membrane. This imbalance in the oxidative stress was evidentiated by a macromolecular oxidation at level of DNA, lipids and proteins in E. coli exposed to Ag-NPs. The formation of ROS and RNI by Ag-NPs may also be considered to explain the bacterial death.

Inhibition of cytotoxicity of Shiga toxin of Escherichia coli O157:H7 on vero cells by Prosopis alba Griseb (Fabaceae) and Ziziphus mistol Griseb (Rhamnaceae) extracts.

The capacity of Prosopis alba Griseb. and Ziziphus mistol Griseb. fruit extracts to inhibit the toxic action of Shiga toxin (Stx) was investigated. Purification of Stx from Escherichia coli O157:H7 was performed by saline precipitation and affinity chromatography using a column with globotriaosylceramide, while the fruits were subjected to ethanolic or aqueous extractions. The protective action of both fruits was determined by pre-, co-, and postincubation of one 50% cytotoxic dose per ml of Stx with different concentrations of ethanolic and aqueous extracts in confluent monolayers of Vero cells for 72 h at 37°C (5% CO2). The inhibition of the cytotoxic effect of Stx by fruit extracts was determined by the neutral red vital staining technique. The extraction of the polyphenols and flavonoids was effective, and more polyphenols per milligram of dissolved solids were obtained from P. alba than from Z. mistol. However, there were more flavonoids in Z. mistol than in P. alba. Components of both fruits increased the viability of cells treated with Stx when the extracts were preincubated with Stx for 1 h before being applied to the cell cultures, with the ethanolic extract of P. alba showing 95% cell viability at a concentration of 2.45 mg/ml. The extracts were less effective in protecting cells when Stx, extracts, and cells were coincubated together without a previous incubation of Stx; only the concentrations of 19.46 mg/ml for the P. alba aqueous extract and 3.75 mg/ml for the Z. mistol ethanolic extract resulted in the inhibition of cytotoxicity, with 52 and 56% cell viability occurring, respectively. Investigation into this difference in the protection of cells indicated that the protein molecule of Stx suffered degradation to advanced oxidative protein products during preincubation with extracts, principally with P. alba, which exhibited a greater amount of nonflavonoid polyphenols than Z. mistol. The prooxidant action on Stx favored the cells and enhanced the protective action of both fruits.

Relevance of biofilms in pediatric tonsillar disease.

In this investigation, we study the relation between chronic inflammation of the tonsils, clinical features, and the presence of biofilms in the crypts in patients presenting with obstructive hypertrophy and recurrent upper airway pathology. Thirty-six patients who needed to undergo a tonsillectomy for obstructive reasons (aged 1 to 6 years), among which none of them had taken any antibiotics 30 days prior to surgery, were included. Samples were examined with hematoxylin-eosin and Gram staining, fluorescent microscopy, and confocal laser microscopy. The predominance of symptoms were those related to obstructive pathology rather than infection (p < 0.01). All patients had tonsillar hypertrophy (grade III or IV), but an association with adenoids hypertrophy was detected in 66.66% of cases (p < 0.05). 77.28% of tonsils presented biofilms in their crypts, but hypertrophy and tonsillar follicle number were not related to the presence or absence of biofilms. Here, we demonstrated that symptoms like harsh raucous sound, tonsillar and adenoids hypertrophy, apnea, and cervical adenopathies are clearly related to the presence of biofilm in tonsils. Our results allow us to propose that biofilms are involved in the pathogenesis of tonsils and adenoids hypertrophy. The prevention of biofilms formation should be focused in the early stages, attempting to restrain bacterial attachment to the respiratory mucosa.

Evidence of bacterial biofilms in nasal polyposis.

INTRODUCTION: The pathogeny of chronic rhinosinusitis with nasal polyposis (CRS/NP) has not been elucidated. Bacterial exotoxins have been implicated in many inflammatory chronic diseases, such as chronic otitis, chronic tonsillitis, cholesteatomas, and more recently CRS/NP. We propose that the bacteria in CRS/NP are not only present in a planktonic state, but also occur in microbial communities as biofilms. OBJECTIVE: To determine and characterize the presence of biofilms in CRS/NP. METHODS: We performed a prospective study in 12 patients undergoing endoscopic sinus surgery for nasal polyposis. Ten patients without CRS/NP who underwent septoplasty were included as a control group. Tissue samples were obtained from the inferior turbinate mucosae. The bacteria were isolated and typified and the material was examined in vitro using a spectrophotometer, and in vivo using optical microscopy and confocal scanning laser microscopy. RESULTS: Moderate to high in vitro biofilm-forming capacity was detected in 9 out of 12 patients with CRS/NP (mean [SD] optical density values of between 0.284 [0.017] and 3.337 [0.029]). The microorganisms isolated were Staphylococcus (5 patients), Streptococcus viridans, Pseudomonas aeruginosa, Enterococcus faecalis and Streptococcus viridans/Corynebacterium. Biofilms were demonstrated in vivo in 2 patients and no biofilm structures were evident in any of the controls. CONCLUSION: This study demonstrates the presence of bacterial biofilms in patients with CRS/NP. This chronic inflammatory factor might contribute to nasal mucosa damage, increased inflammatory cells in tissue, and the subsequent hyperplasic process.

Lipase of Candida albicans induces activation of NADPH oxidase and L-arginine pathways on resting and activated macrophages.

Candida albicans secretes various hydrolytic enzymes which are considered to be an integral part in the pathogenesis. However, the role of lipases is far from being completely understood and the direct effects of these fungal enzymes during the host-pathogen interaction remain to be established. We recently isolated and characterized an extracellular C. albicans lipase (CaLIP), and demonstrated the ability of this fungal enzyme to interact directly with macrophages (Mvarphi) and hepatocytes and to operate as a virulence factor. Herein, we explored the effects of CaLIP on Mvarphi functions such as oxidative burst and l-arginine metabolism. The study was performed in cells with different activation status: normal-resting Mvarphis and Mvarphis primed in vivo or in vitro with C. albicans. The ability of this fungal factor to modulate the above-mentioned parameters was dependent on cells status, dose, and microenvironment, where the interaction took place. These results constitute a new finding in the biology of candidiasis and could illustrate an additional evolutive advantage for the fungus in the framework of the bidirectional host-pathogen interaction.

Enterobacter cloacae leukotoxin: modulation of reactive oxidant species generated by neutrophils.

A leukotoxin purified from Enterobacter cloacae culture by saline precipitation, gel chromatography and HPLC was studied as a modulator of reactive oxidant species (ROS) produced by human neutrophils. Chemiluminescence showed that stimulation of ROS was achieved at a low leukotoxin concentration, but ROS production decreased when the toxin was applied at concentrations above 30 microg/mL. Also, the addition of 100 microg toxin/mL significantly reduced the activating effect of phorbol myristate acetate (PMA) and low doses of toxin did not produce an opposite effect toward the stimulation produced by PMA. Normal neutrophils showed a linear correlation between the inverse of ROS production and time, but the kinetic reaction changed when toxins were added to the cells and the ROS formation increased directly with time.

Molecular properties and metabolic effect on blood cells produced by a new toxin of Enterobacter cloacae.

A new toxin of Enterobacter cloacae able to lyse erythrocytes and leukocytes was found. Purification of the toxin was performed by salt precipitation, gel filtration, ion exchange and HPLC in C8 column. SDS-PAGE electrophoresis showed more than one bank corresponding to the leukotoxin able to form polymers and aggregate like some pore-forming cytotoxins (RTX). In culture supernatant the toxin showed 1 HU/ml (hemolytic unit) and 1.5 LU/ml (leukotoxic unit); after purification it reached 15 HU/ml and 20 LU/ml. The ratio between HU and percentage red cells affected the lytic capacity. E. cloacae toxin stimulated the oxidative metabolism of neutrophils, but over 50 microg toxin/ml the stimulus ceased as it was shown by NBT assay due to cell death. Chemiluminescence evidenced an increase in superoxide anion generation, but an excess of toxin interfered with this stimulus, as was previously observed in HlyA Escherichia coli toxin. Cross-reaction was found by immunoblotting with this HlyA. E. cloacae toxin presented higher amounts of proline, valine, aspartic and glutamic acids than HlyA. E. cloacae toxin was similar to HlyA in the prescence of a glycine-rich DNA sequence and in the observed effect of calcium on toxin activity. E. cloacae toxin did not cross-react by immunoblotting with hemolysin HmpA of Proteus.

Conservation in probiotic preparations of Lactobacillus with inhibitory capacity on other species.

Strains of Lactobacillus isolated from dairy products and genital tract competed with Candida albicans through a membrane of 12000 dalton cut-off. This inhibition was due to hydrogen peroxide and was trypsin-stable, heat-sensitive and antagonized by catalase. Lactobacillus coming from "starters" showed antimicrobial activity against fungus isolated in a yogurt factory. Penicillium, Alternaria, Phialophora, Microsporum and Candida spp. were inhibited when 10(2) spores were inoculated in the assay. No inhibition was observed with 10(5) spores. Besides, one of 21 Lactobacillus strains isolated from the vaginas of healthy women inhibited pathogenic bacteria by means a bacteriocin trypsin-sensitive, heat-stable and retained by dialysis membrane. Tablets for future probiotic use were prepared and the viability of bacteria was assayed using media with different compositions. Pharmaceutical preparations with polyethyleneglycol was the best formulation for the Lactobacillus viability, the counts remained between 10(7) and 10(6) cfu/tablet for up to 1 year.

Induction of oxidative stress in leukocytes by an Enterobacter cloacae toxin able to form oligomers and binding to proteins.

A leukotoxic and hemolytic toxin was purified from cultures of Enterobacter cloacae. Stimulation of oxidative stress was observed and the production of reactive oxidant species was measured in leukocytes treated with toxin by means of nitroblue tetrazolium and chemiluminescence assays. Molecular weight of toxin was estimated by chromatography and SDS-PAGE. Two protean peaks with toxic activity were found in Sephadex G-100 (P1, 42.0 kDa; and P2, 13.3 kDa). The relative amounts between the peaks (P1/P2 = 0.36) changed when 2-mercaptoethanol was employed (P1/P2 = 0.59). When Sephadex G-200 chromatography was performed, a protean peak of Ve = 113 mL (100 kDa) was found; its was dissociated with 3 M urea in toxic proteins of lower mass: 42, 27, and 13.3 kDa. SDS-PAGE (15%) showed a single toxin band of purified monomer (13.3 kDa), but electrophoresis of a 42-kDa toxin with urea presented three bands of trimer, dimer, and monomer. An increase of casein hydrolysate and albumin molecular weight was observed by chromatography after incubation with toxin due to the binding of both proteins with toxin.

Purification and characterization of a cytotoxin from Enterobacter cloacae.

Leukotoxic activity was assayed in clinical isolates of Enterobacter cloacae. Two strains were selected out of 38 by their greater hemolytic activity in blood agar plates. Leukotoxin was purified by salt precipitation, dialysis, chromatography by gel filtration, and high pressure liquid chromatography (HPLC). Human leukocytes, when incubated with purified E. cloacae toxin, showed high percentages of death and lysis, with time and dose dependence. The chromatographic profile of gel filtration presented three protein peaks and toxic activity was detected in the second peak. After HPLC, leukotoxin coeluted with the hemolytic activity and both activities were detected only after 2-mercaptoethanol treatment. Coomassie-stained sodium dodecyl sulfate--polyacrylamide gels showed a single band. This band was estimated to represent a protein of 13300 Da on the basis of both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration chromatography.