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.

[Frequency of pulmonary and extrapulmonary tuberculosis in a reference hospital in Córdoba province, 1991-2003]. / Frecuencia de tuberculosis pulmonar y extrapulmonar en un hospital de referencia de la provincia de Córdoba, 1991-2003.

Tuberculosis continues to be a serious problem of public health causing nearly three million deaths per year all over the world. Despite technologic improvements in the diagnostic methods, it is not possible to control the disease in the absence of surveillance and treatment follow-up programs supervising the ending of treatments, and definitive cure of patients. The frequency of pulmonary and extrapulmonary tuberculosis, and simultaneous pulmonary and extrapulmonary tuberculosis localization among patients assisted at Tránsito Cáceres de Allende Hospital during thirteen years (1991-2003), was determined. The benefit of inoculating the specimens on Stonebrink medium for the best recuperation of Mycobacterium bovis was herein observed, and the contribution of Ziehl Neelsen staining in extrapulmonary materials was tested as well. Out of 790 cases of tuberculosis diagnosed, 723 were pulmonary, and 48 were extrapulmonary localization (pleural 31, renal 7, ganglionar 5, meningeal 2, genital 1, pericardial 1 and digestive 1), and 19 patients presented both, pulmonary and extrapulmonary tuberculosis. Out of the 723 pulmonary cases, 9 were caused by M. bovis. All M. bovis isolates grew on Stonebrink medium, and only one grew also on Lowenstein Jensen. Smear microscopy using Ziehl Neelsen staining resulted positive in 4 extrapulmonary specimens.

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.

Frecuencia de tuberculosis pulmonar y extrapulmonar en un hospital de referencia de la provincia de Córdoba, 1991-2003 / [Frequency of pulmonary and extrapulmonary tuberculosis in a reference hospital in Córdoba province, 1991-2003]

Tuberculosis continues to be a serious problem of public health causing nearly three million deaths per year all over the world. Despite technologic improvements in the diagnostic methods, it is not possible to control the disease in the absence of surveillance and treatment follow-up programs supervising the ending of treatments, and definitive cure of patients. The frequency of pulmonary and extrapulmonary tuberculosis, and simultaneous pulmonary and extrapulmonary tuberculosis localization among patients assisted at Tránsito Cáceres de Allende Hospital during thirteen years (1991-2003), was determined. The benefit of inoculating the specimens on Stonebrink medium for the best recuperation of Mycobacterium bovis was herein observed, and the contribution of Ziehl Neelsen staining in extrapulmonary materials was tested as well. Out of 790 cases of tuberculosis diagnosed, 723 were pulmonary, and 48 were extrapulmonary localization (pleural 31, renal 7, ganglionar 5, meningeal 2, genital 1, pericardial 1 and digestive 1), and 19 patients presented both, pulmonary and extrapulmonary tuberculosis. Out of the 723 pulmonary cases, 9 were caused by M. bovis. All M. bovis isolates grew on Stonebrink medium, and only one grew also on Lowenstein Jensen. Smear microscopy using Ziehl Neelsen staining resulted positive in 4 extrapulmonary specimens.

Frecuencia de tuberculosis pulmonar y extrapulmonar en un hospital de referencia de la provincia de Córdoba, 1991-2003. / [Frequency of pulmonary and extrapulmonary tuberculosis in a reference hospital in Córdoba province, 1991-2003]

Tuberculosis continues to be a serious problem of public health causing nearly three million deaths per year all over the world. Despite technologic improvements in the diagnostic methods, it is not possible to control the disease in the absence of surveillance and treatment follow-up programs supervising the ending of treatments, and definitive cure of patients. The frequency of pulmonary and extrapulmonary tuberculosis, and simultaneous pulmonary and extrapulmonary tuberculosis localization among patients assisted at Tránsito Cáceres de Allende Hospital during thirteen years (1991-2003), was determined. The benefit of inoculating the specimens on Stonebrink medium for the best recuperation of Mycobacterium bovis was herein observed, and the contribution of Ziehl Neelsen staining in extrapulmonary materials was tested as well. Out of 790 cases of tuberculosis diagnosed, 723 were pulmonary, and 48 were extrapulmonary localization (pleural 31, renal 7, ganglionar 5, meningeal 2, genital 1, pericardial 1 and digestive 1), and 19 patients presented both, pulmonary and extrapulmonary tuberculosis. Out of the 723 pulmonary cases, 9 were caused by M. bovis. All M. bovis isolates grew on Stonebrink medium, and only one grew also on Lowenstein Jensen. Smear microscopy using Ziehl Neelsen staining resulted positive in 4 extrapulmonary specimens.

Prevalencia de micobacteriosis y de tuberculosis en pacientes de un hospital de referencia de la provincia de Córdoba / Prevalence of mycobacteriosis and tuberculosis in a reference hospital, Cordoba province

Las micobacterias ambientales (MA) constituyen un importante grupo de especies bacterianas que se encuentran en el medio ambiente, pueden colonizar y ocasionalmente producir enfermedad enel hombre. En este trabajo se investigó la frecuencia de casos de micobacteriosis en relación con los de tuberculosis durante un período de diez años (1.991-2.000). Se estudiaron 16.700 muestras de 9.300 pacientes adultos de ambos sexos asistidos en el Hospital Regional de Tuberculosis de la Provincia de Córdoba, por consulta espontánea. Los aislamientos se realizaron por cultivo en los medios de Lowenstein Jensen y Stonebrink. Las colonias de bacilos ácidoalcohol resistentes (BAAR) se identificaron por pruebas bioquímicas y moleculares. El total de casos diagnosticados fue de 716, de los cuales 684 (95,5%) correspondieron a al complejo Mycobacterium tuberculosis y a micobacterias ambientales 32 (4,5%). Los casos de micobacteriosis se definieron por reiterados aislamientos con desarrollo representativo de una micobacteria ambiental, sospecha clínica y radiológica. De los 32 casos de micobacteriosis, el 75% del total correspondió aMycobacterium avium-intracellulare,15,6% a Mycobacterium fortuitum, 3,1% a Mycobacterium kansasii y 6,3% a Mycobacterium chelonae.Los casos de tuberculosis fueron 94,5% de localización pulmonar y 5,5% extrapulmonar.

Environmental mycobacteria (EM) constitute an important group of bacteria species found in the environment. They can colonize and occasionally produce disease in man. Sixteen thousand three hundred samples from 9300 adult symptomatic patients from the Hospital Regional of Tuberculosis in Cordoba were bacteriolocally investigated. The isolations were performed by culture on Lowenstein Jensen and Stonebrink culture media. The colonies of acid fast bacilli (AFB) were identified by biochemical and molecular tests. Among 716 culture positive cases, 684 (95.5%) were due to Mycobacterium tuberculosis complex and 32 to environmental mycobacteria.Serial samples allowed the confirmation of the etiologicalagent in culture and correlated with consistent clinical and radiological abnormalities. Seventy-five percente of these patients were affected by M. avium complex, 15.6% by M. fortuitum, 3.1% Mycobacterium kansasii and 6.3% Mycobacterium chelonae. Among tuberculosis cases, 94.5% and 5.5% had pulmonary and extrapulmonary disease respectively.

[Prevalence of mycobacteriosis and tuberculosis in a reference hospital, Cordoba province]. / Prevalencia de micobacteriosis y de tuberculosis en pacientes de un hospital de referencia de la provincia de Córdoba.

Environmental mycobacteria (EM) constitute an important group of bacteria species found in the environment. They can colonize and occasionally produce disease in man. Sixteen thousand three hundred samples from 9300 adult symptomatic patients from the Hospital Regional of Tuberculosis in Cordoba were bacteriolocally investigated. The isolations were performed by culture on Lowenstein Jensen and Stonebrink culture media. The colonies of acid fast bacilli (AFB) were identified by biochemical and molecular tests. Among 716 culture positive cases, 684 (95.5%) were due to Mycobacterium tuberculosis complex and 32 to environmental mycobacteria. Serial samples allowed the confirmation of the etiological agent in culture and correlated with consistent clinical and radiological abnormalities. Seventy-five percent of these patients were affected by M. avium complex, 15.6% by M. fortuitum, 3.1% Mycobacterium kansasii and 6.3% Mycobacterium chelonae. Among tuberculosis cases, 94.5% and 5.5% had pulmonary and extrapulmonary disease respectively.

Prevalencia de micobacteriosis y de tuberculosis en pacientes de un hospital de referencia de la provincia de Córdoba. / [Prevalence of mycobacteriosis and tuberculosis in a reference hospital, Cordoba province]

Environmental mycobacteria (EM) constitute an important group of bacteria species found in the environment. They can colonize and occasionally produce disease in man. Sixteen thousand three hundred samples from 9300 adult symptomatic patients from the Hospital Regional of Tuberculosis in Cordoba were bacteriolocally investigated. The isolations were performed by culture on Lowenstein Jensen and Stonebrink culture media. The colonies of acid fast bacilli (AFB) were identified by biochemical and molecular tests. Among 716 culture positive cases, 684 (95.5

) were due to Mycobacterium tuberculosis complex and 32 to environmental mycobacteria. Serial samples allowed the confirmation of the etiological agent in culture and correlated with consistent clinical and radiological abnormalities. Seventy-five percent of these patients were affected by M. avium complex, 15.6

by M. fortuitum, 3.1

Mycobacterium kansasii and 6.3

Mycobacterium chelonae. Among tuberculosis cases, 94.5

and 5.5

had pulmonary and extrapulmonary disease respectively.

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.

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.