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.

Comparison of oxidative stress induced by ciprofloxacin and pyoverdin in bacteria and in leukocytes to evaluate toxicity.

Oxidative stress induced by ciprofloxacin and pyoverdin, a leukotoxic pigment, was studied by comparing their effect in bacteria and leukocytes. Chemiluminescence (CL) assays with lucigenin or luminol were adapted to measure the stimuli of superoxide anion (O2 -) and other reactive species of oxygen (ROS) in bacteria. Ciprofloxacin principally induced the production of O2 - in the three species studied: Staphylococcus aureus, Enterococcus faecalis and Escherichia coli. Lucigenin CL assay showed high oxidative stress in S. aureus due to its low superoxide dismutase (SOD) activity, whereas E. coli exhibited important SOD activity, responsible for little production of O2 - in absence or presence of ciprofloxacin. Reduction of nitroblue of tetrazolium (NBT) was applied. This assay indicated that there was higher oxidative stress in S. aureus and E. faecalis than in E. coli. The comparison of oxidative stress generated in bacteria and leukocytes was used to check the selective toxicity of ciprofloxacin in comparison with pyoverdin. Ciprofloxacin did not generate significant stimuli of O2 - in neutrophils, while pyoverdin duplicated the production of O2 -. CL and NBT were useful to study the leukotoxicity of ciprofloxacin. Oxidative stress caused by the antibiotic and the leukotoxic pigment was similar in bacteria.

Selection of bacteriocin producer strains of lactic acid bacteria from a dairy environment.

Two strains showing bacteriocin production were selected from a total of 206 lactic acid bacteria isolated from samples of milk, milk serum, whey and homemade cheeses in Southern Cordoba, Argentina. This property was detected by means of well diffusion assays. The strains were identified as Enterococcus hirae and Enterococcus durans. The protein nature of those substances was proved by showing their sensitivity to type IV and XXV proteases, papaine, trypsin, pepsin and K proteinase. The bacteriocins inhibited the growth of Listeria monocytogenes, Bacillus cereus, Clostridium perfringes and two strains of Staphylococcus aureus, an A-enterotoxin and a B-enterotoxin producers. All of these bacteria are common pathogens usually associated with food borne diseases (ETA). These lactic acid bacteria or their bacteriocins could be suitable candidates for food preservation and specially useful in the our regional dairy industry.

Leukotoxicity of pyoverdin, production of reactive oxygen species, and effect of UV radiation.

Pyoverdin was purified by solvent extraction, gel filtration, and ionic exchange chromatography. Assays of cytotoxic of pyoverdin were done with human leukocytes and macrophages from the peritoneum of mice. Both cell quantities showed a significant reduction. Death was followed by lysis in a dose-dependent form. The mechanism of action of pyoverdin involved the stimulation of reactive oxygen species (ROS) measured by Nitroblue Tetrazolium (NBT) reaction and chemiluminescence (CL). UV radiation at 368 nm increased the leukotoxicity; expositions of 5 min were enough to photostimulate the effect of pyoverdin on cellular oxydative metabolism, which increased between 35.4 and 53.2%. Genestein, an inhibitor of tyrosine kinases, counteracted the ROS stimuli of pyoverdin, suggesting endocytic mechanism of action for this pigment. The little chloroquine interference on oxydative stress indicated that intraphagosomal pH and the stimuli of reactive nitrogen intermediaries (RNI) seem to be of less importance than ROS in pyoverdin action on leukocytes.

Elevation of alanine amino transferase and aspartate amino transferase produced by pyoverdin, a photolabile pigment of Pseudomonas fluorescens.

The effect of three forms pyoverdin on mouse liver was studied. Significant increases of alanine amino transferase (ALT) and aspartate amino transferase (AST) were obtained in mice after ingestion of water with forms A and C. The effect on liver was more evident with A than with C. Pyoverdin was purified by means of salt saturation, solvent extractions and ion-exchange chromatography. Fluorescent peaks obtained in the presence of light were different from those eluted under dark conditions. The relative amounts of pyoverdin A, B and C varied when dark purification procedure was employed. Form A decreased while C increased in the absence of light. Optimum conditions for C were in the dark without iron. When C was exposed to light, it changed to form A. Fast Atom Bombardment (FAB) mass spectrometry of pyoverdin form C gave a form at M+ = 1324 m.u., which is 9 m.u. less than pyoverdin purified in the presence of light. The results suggest that light can influence pyoverdin stability and toxicity.

Antibiotic activity of isoxazolylnaphthoquinone imines on mice infected with Staphylococcus aureus.

The antibiotic activity of new synthetic isoxazolylnaphthoquinone imines was studied. Pseudomonas aeruginosa ATCC 27853 and Escherichia coli ATCC 25922 were resistant to the four compounds studied (MIC > 128 micrograms ml-1), but Staphylococcus aureus ATCC 25923, ATCC 29213 and 30 clinical isolates of Staph. aureus were inhibited by 2-hydroxy-N-(3,4-dimethyl-5-isoxazolyl)-1,4-naphthoquinone-4-imine (I). This compound diminished bloodstream infection of mice injected i.m. with Staph. aureus; septicaemia decayed significantly when I was applied at the beginning of the infection while when I was given 3 d after bacterial challenge, a significant protection was afforded. Bactericidal activity in serum increased during the 5 h after I was administered i.p. The acetyl derivative of I had a high MIC but when inoculated orally in mice decreased the Staph. aureus counts in circulation. This protection occurred only when the schedule of administration started close to the bacterial challenge. Antibiotic activity in vivo may be associated with in vitro effects.

Staphylococcal protease assay with dye hide powder, elastin, collagen and udder extract.

Colorimetric determinations of proteolytic activity were performed to measure the effects on dye protean substrates including tissue powders. The substrates were assayed with 98 strains obtained from the milk of cows with mastitis. Trypsin was employed as positive control and it verified the susceptibility of the method. Enzymatic activity was estimated in trypsin units per milliliter of incubation mixture. The percentages of strains active on specific proteins were 47.8% for elastin, 61.6% for collagen and when hide powder and udder extract were used as dye substrates, the proteolytic staphylococci were 76.5 and 92.4% respectively. There was no significant difference in hydrolytic activity on proteins between coagulase positive and coagulase negative cocci.

Klebocin activity of Klebsiella pneumoniae strains and its heterologous effect on Staphylococcus sp.

Thirty six Klebsiella pneumoniae strains were assayed in a test to determine bacterial interference; two of them had bacteriocinic effects on homologous species, strains 6 and 150 inhibited sensitive K. pneumoniae and they also acted on positive and negative Staphylococci coagulase. All cocci were sensitive to the bacteriocinogenic K. pneumoniae and none of the non-bacteriocinogenic strains inhibited them. Klebocin with homologous activity on K. pneumoniae seemed to be undistinguishable from the compound with heterologous action on Staphylococci in the aspects that were characterized in this work; both were heat labile to the same degree; optimum pH was 7, acidity decreased klebocin activity more intensely than alkaline pH. The antagonistic substance was not produced in the synthetic medium employed and was developed in tryptic soy, nutrient agar, brain heart agar and blood agar; tryptone-beef extract agar complex medium neither permitted the homologous activity nor allowed the interference on Staphylococci. The compound (or compounds) responsible for homologous antimicrobial effect had a low molecular weight as demonstrated by the fact that it pierces a dialysis membrane with molecular weight of 10,000 D cut-offs. Ethidium bromide treatment of strains 6 and 150 produced five strains without bacteriocinic activity which simultaneously lost their homologous and heterologous inhibitory capacity.

Infeccion natural de Akodon dolores,Thomas, 1916 (Rodentia, Cricetidae) por el Trypanosoma cruzi. / Natural infection of Akodon dolores,Thomas,1916 (Rodentia, Cricetidae) by Trypanosoma cruzi

El estudio morfobiometrico y biologico de tres cepas de tripanosomas de una poblacion de Akodon dolores de la zona periurbana del municipio de Las Higueras, provincia de Cordoba, Argentina, demostro que se trataba de T. cruzi. Como el 15% de 134 A. dolores examinados en la zona habia demostrado tripanosomas cruziformes, se concluye que este roedor e sun reservorio frecuente del parasito.El A. dolores es la especie numericamente dominante en la comunidad de roedores que vive en los habitats de mayor estabilidad en la region. Debe investigarse el ciclo de transmision silvestre asi como su relacion con el domiciliario en esta zona de baja endemicidad de enfermedad de Chagas en el hombre

[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)

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

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 [quot ]in vitro[quot ] 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)

[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.

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

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.