In vitro activity of N-benzenesulfonylbenzotriazole on Trypanosoma cruzi epimastigote and trypomastigote forms.

Chagas disease is still an important health problem in Central and South America. However, the only drugs currently available for specific treatment of this disease may induce toxic side effects in the host. The aim of this work was to determine the activity of N-benzenesulfonylbenzotriazole (BSBZT) against the protozoan parasite Trypanosoma cruzi. The effects of BSBZT and benzotriazole (BZT) were compared to those of benznidazole (BZL) on epimastigote and trypomastigote forms. BSBZT was found to have an in vitro growth inhibitory dose-dependent activity against epimastigotes, with flow cytometry analysis confirming that the treated parasites presented size reduction. BSBZT showed an IC(50) of 21.56 µg/mL (81.07 µM) against epimastigotes at 72 h of incubation, whereas BZT did not affect the growth of this parasite form. Furthermore, the toxic effect of BSBZT, was stronger and appeared earlier (at 24h) in trypomastigotes than in epimastigotes, with the LC(50) of this compound being 28.40 µg/mL (106.79 µM) against trypomastigotes. The concentrations of BSBZT used in this study presented low hemolytic activity and cytotoxicity. Consequently, at concentrations near IC(50) and LC(50) (25µg/mL), BSBZT caused only 2.4% hemolysis and 15% of RAW 264.7 cell cytotoxicity. These results reveal the potential of BSBZT as a prototype in drug design for developing new anti-T. cruzi compounds.

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.

New aspect of the synergistic antibacterial action of ampicillin and gentamicin.

A new aspect in the action of ampicillin and gentamicin was detected in Enterococcus faecalis. Reactive oxygen species (ROS) increased in sensitive strains during treatment with each antibiotic up to a certain concentration of antibiotic, above which ROS diminished as a consequence of oxidative stress. Tiron, a scavenger of the superoxide anion O(2)(-), counteracted the effect of the generated ROS. The oxidative stress was a consequence of an increase in ROS in the cytoplasm of bacteria, as observed by the nitroblue tetrazolium reaction. The viability of sensitive strains was significantly reduced at concentrations of antibiotics that increased the ROS, and this increment was parallel to the bactericidal effect. Sensitive E. faecalis strains showed an immediate increase of ATP in the presence of both antibiotics, thus an energy-dependent process had been triggered, indicating a bacterial reaction against the stress. The combination of both antibiotics augmented the effect of ROS, which helps to explain the synergism between ampicillin and gentamicin.

Primary isolation of Trypanosoma cruzi by hemoculture: influence of glucose concentration.

Isolation of Trypanosoma cruzi in hemoculture was done in media with different glucose concentrations. The three strains studied developed into epimastigotes 2 weeks after seeding in media with less than 4.5 mM hexose. Inhibition of growth of trypanosomes in media containing a greater amount of glucose was not caused by the higher osmotic pressures. Cultures containing more than 4.5 mM glucose showed a greater depletion of pH and higher lactic acid production than did those with lower concentrations, resulting in damage to amastigote clusters or to initial epimastigote development. The maximum recommended level of glucose in the culture medium is determined by the detrimental acidification resulting from excessive quantities.

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.

[Klebocins K6 and K150: production and activity in various experimental conditions]. / Klebocinas K6 y K150: producción y actividad en diferentes condiciones experimentales.

Klebsiella is a common agent in hospital-acquired infections and its importance in disease transmission was evident in the isolation obtained in a health center in the city of Rio Cuarto. Bacteriocinogenic strains by the cross-streak method in tryptic-soy agar were investigated. Two Klebsiella produced bacteriocins with broad patterns of sensitivity among the tested strains. The K150 klebocin was more active than the K6 klebocin, but this one was more heat-stable than K150. The klebocins were not detected in the synthetic media employed and they caused inhibitory areas in complex media, except in triptose-beef extract. K150 was not active in eosine-methylene blue and nutrient agar. The bacteriocins were associated to lipase activity and hemolytic effect on chicken erythrocytes. The strains 6 and 150 were multiresistant to antimicrobial agents, with a pattern of sensitivity different from that of other multiresistant strains.

[Production of pyoverdin by Pseudomonas fluorescens in human blood at 4 degrees C: association with proteins and the cytotoxic effect of the pigment]. / Producción de pioverdina por Pseudomonas fluorescens en sangre humana a 4 grados C: asociación a proteínas y efecto citotóxico del pigmento.

Pyoverdin was purified from Pseudomonas fluorescens cultured in a synthetic medium. Cytotoxic effect on human leukocytes was assayed. Death and lysis was observed depending on concentration and time. Sub-lytic dose decreased leukocyte phagocytosis. The pigment was produced by P. fluorescens in blood stored at 4 degrees C. Saline precipitation of plasma showed that globulin and albumin fraction retained 46% and 37% of pyoverdin, respectively. By means of dialysis it was possible to determine that albumin retained more pigment than the other fractions either bound to the protein or aggregated.

[Presence of extracellular proteases in Pseudomonas aeruginosa]. / Presencia de proteasas extracelulares en Pseudomonas aeruginosa.

Proteolytic activity on hide power azure (HPA) and elastin was assayed in 32 Pseudomonas strains. Dye substrates were incubated with culture supernatants for 30 min at 37 degrees C and then released dye was measured photocolometrically. 100% of strains showed proteolytic activity on HPA and 56.25% were elastase positive, the values obtained with the first substrate were the highest. No relation with strain origin was established.

[Utilization by Escherichia coli and Pseudomonas fluorescens of a siderophore from Pseudomonas fluorescens strain PAB]. / Utilización por Escherichia coli y Pseudomonas fluorescens de un sideróforo de Pseudomonas fluorescens cepa PAB.

Pseudomonas fluorescens PAB strain produced pyoverdine in a synthetic medium. Pigment was purified by solvent extraction and ion exchange, and sterilized and used as siderophore for E. coli, P. fluorescens, ATCC 13.525, ATCC 17.400, W and PAB strains. Bacteria were grown in iron-free medium and medium with iron. Free--pyoverdine and pyoverdine bound to Fe+3 were added in different concentrations. P. fluorescens PAB siderophore did not stimulate E. coli growth while it was selective for other P. fluorescens strains. Paper disks impregnated with pyoverdine did not inhibit E. coli growth.

[Primary isolation of Trypanosoma cruzi using hemoculture: effect of media composition on epimastigote differentiation]. / Aislamiento primario de Trypanosoma cruzi por hemocultivo: efecto de la composición del medio en la diferenciación a epimastigote.

It was investigated the modifications of culture medium which facilitated the differentiation of blood tripomastigotes of Trypanosoma cruzi to epimastigotes and its further reproduction. Trypanosomas were obtained "in vitro" from country rodents with parasites, caught in Las Higueras Municipality, Río Cuarto Department. They were differentiated and developed faster in mediums poorer in nutrients (N.N.N. and Tobie) than in enriched ones, generally used to mantain epimastigotes in culture (LIT and Medium Base). Different mediums were tested: a) Novy and Mc Neal medium modified by Nicole (N.N.N.); b) N.N.N. medium modified by the addition of glucose (10 g/l); c) Tobie medium with different mediums as liquid phase; d) Tobie medium modified by the addition of glucose (10 g/l); e) Warren medium; f) Warren medium modified by the addition of glucose (10 g/l); g) LIT medium; h) Medium Base (M.B.); i) 16 mediums obtained from M.B. modified by changing only one of its components, either quantitative or quantitatively, so that the difference with the original one was in a sole component. All mediums were assayed with blood of albino BALB/c mice infected with T. cruzi: Tulahuén strain and two wild strains isolated from country mice. To observe the effect on results, certain working conditions were changed: a) cultivated tripomastigotes density; b) blood from different rabbits, to enrich the cultures; c) trademark of each component used in medium preparations; d) bleeding of infected mice in different days post injection; e) mediums in liquid state or diphasic. These technical modifications did not alter the results. Only glucose proved to influence the differentiation to epimastigote.(ABSTRACT TRUNCATED AT 250 WORDS)

[A new oxygen-labile hemolysin in Klebsiella pneumoniae]. / Nueva hemolisina oxígeno-lábil en Klebsiella pneumoniae.

The hemolytic activity of sixty K. pneumoniae strains was investigated in tryptic soy agar with rabbit, dog, sheep, human, chicken and mouse blood. All of them were lytic only for rabbit red cells. In liquid medium it was necessary a 2 mercaptoethanol treatment to detect a good degree of hemolysis. Cultures in tryptic soy broth gave 100% hemolysis in assays with rabbit erythrocytes and only when hemolysin was concentrated by purification was it active on dog and sheep but in a lesser degree (7.5% hemolysis). Supernatants of cultures were precipitated at different saline concentrations; the fraction obtained with 30-50% (NH4)2SO4 had hemolytic activity after dialysis and 2 mercaptoethanol treatment. Then this fraction was eluted in a Sephadex G-100 column, but electrophoresis in polyacrylamide gel showed that the hemolytic molecules obtained by gel filtration were contaminated with protein structures which had different electrophoretic migration. Ion-exchange chromatography showed best purification index and the recovery of activity was over 100%, it was possible to explain this good recovery once an inhibitor was detected. Two rabbit red cell lysins were purified, both shared several properties: SH-activation, pH optimum, thermolability, selectivity for rabbit red cells, mechanism of action, inhibition by cholesterol and divalent cations.

[Outbreak of hospital infection, due to members of the Klebsielleae tribe, in an intensive care unit for infants]. / Brote de infección hospitalaria, producido por miembros de la tribu Klebsielleae, en una unidad de cuidados intensivos para lactantes.

At the Pediatric Intensive Care Unit of the Provincial Regional Hospital, in Río Cuarto, Argentina, nearly all hospitalized infants showed clinical symptoms of septicaemia and gastroenteritis. Neither Salmonella nor Shigella were found in the stool cultures, but Klebsiella pneumoniae was isolated as predominant flora. Three haemocultures displayed K. pneumoniae and Enterobacter cloacae; the other three developed only E. cloacae. Since the infants came from different places and it was possible to isolate members of the Klebsielleae tribe from all of them, a hospital infection was suspected. Searching for the infectious source, K. pneumoniae was detected in the water bath used to keep the feeding-bottles at 37 degrees C. To clarify the existence of any relationship between the strains isolated from patients and from the water bath, several characteristics were compared: biotypes, haemolityc activity, antibiotic sensibility patterns, and pathogenicity, assessed as lethal dose 50%. Identical results were found for the biochemical tests of all the strains belonging to the same species. The antibiotic sensibility patterns and LD 50% showed quite similar values. All bacteria displayed haemolityc activity for rabbit and lamb erythrocytes. It could be considered that the septicaemia had an intestinal origin, and that the infection spread was due to the contamination of the water bath where the feeding bottles were kept.

[In vitro antibacterial activity of isoxazolyl++ naphthoquinones. I]. / Actividad antibacteriana in vitro de isoxazolilnaftoquinonas. I.

The in vitro antibacterial activity of 2-hydroxy-N-(3,4-dimethyl-5-isoxazolyl) 1,4 naphthoquinone-4-imine (I) and three of this derivatives against Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Proteus mirabilis y Morganella morganii was investigated. From the four naphthoquinone-imine studied, compound I exhibited activity against S. aureus. This effect was observed even in oxygen atmosphere with 5% CO2 and in the presence of human albumin. The development of drug-resistant strains by serial passage in media with concentrations lower than the C.I.M. was determined in 31 strains of S. aureus from clinical material and one from collection. No significant differences in C.I.M. were observed.

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.

Increased advanced oxidation of protein products and enhanced total antioxidant capacity in plasma by action of toxins of Escherichia coli STEC.

Shiga toxin (Stx) and hemolysin (Hly) of Escherichia coli O157:H7 produced an increase of reactive oxygen species (ROS) in normal human blood. In vitro assays showed that stimuli of ROS with these toxins oxidized proteins to carbonyls in plasma and raised the degradation of oxidized macromolecules, with the AOPP/carbonyl relationship also increasing. The oxidative stress generated by toxins during the Hemolytic Uremic Syndrome (HUS) produced oxidation of blood proteins with a rise in advanced oxidation protein products (AOPP) in children with HUS. There was a response from the antioxidant system in these patients, evaluated through the determination of the total antioxidant capacity of plasma by the Ferric Reducing Antioxidant Power (FRAP), which reduced the stimuli of ROS during in vitro incubation with Stx or Hly. The application of natural antioxidants was sufficient to reduce in vitro the oxidative stress provoked by both toxins in blood.

Antibacterial activity of anthraquinone derivatives from Heterophyllaea pustulata (Rubiaceae).

Photosensitizing anthraquinones isolated from Heterophyllaea pustulata Hook f. (Rubiaceae), namely soranjidiol, rubiadin, damnacanthal and 5,5'-bisoranjidiol, showed antibacterial activity (bacteriostatic/bactericide) on Staphylococcus aureus. The mechanism of action seems to involve an increase in the levels of superoxide anion (O(2)(·-)) and/or singlet molecular oxygen ((1)O(2)). Moreover, the effect of actinic irradiation as a boosting agent for the production of both reactive species of oxygen as well as its influence on antibacterial activity was assessed. The routine susceptibility assay (minimum inhibitory concentration determination) was carried out by means of the broth macrodilution method. Bactericide activity was determined counting the colony-forming units per milliliter (cfu/mL) in plate. The O(2)(·-) production was determined by means of an indirect photobiological assay (Nitroblue Tetrazolium test), and the production of (1)O(2) was followed using an indirect steady-state method, with methionine as the (1)O(2) chemical quencher.