Comparison of Microcystis aeruginosa (PCC7820 and PCC7806) growth and intracellular microcystins content determined by liquid chromatography-mass spectrometry, enzyme-linked immunosorbent assay anti-Adda and phosphatase bioassay.

Cyanobacteria are able to produce several metabolites that have toxic effects on humans and animals. Among these cyanotoxins, the hepatotoxic microcystins (MC) occur frequently. The intracellular MC content produced by two strains of Microcystis aeruginosa, PCC7806 and PCC7820, and its production kinetics during the culture time were studied in order to elucidate the conditions that favour the growth and proliferation of these toxic strains. Intracellular MC concentrations measured by liquid chromatography (LC) coupled to electrospray ionization mass spectrometer (MS) were compared with those obtained by enzyme-linked immunosorbent assay (ELISA) anti-Adda and protein phosphatase 2A (PP2A) inhibition assays. It has been demonstrated there are discrepancies in the quantification of MC content when comparing ELISA and LC-MS results. However, a good correlation has been obtained between PP2A inhibition assay and LC-MS. Three MC were identified using LC-MS in the PCC7806 strain: MC-LR, demethylated MC-LR and a new variant detected for the first time in this strain, [L-MeSer(7)] MC-LR. In PCC7820, MC-LR, D-Asp(3)-MCLR, Dglu(OCH3)-MCLR, MC-LY, MC-LW and MC-LF were identificated. The major one was MC-LR in both strains, representing 81 and 79% of total MC, respectively. The total MC content in M. aeruginosa PCC7820 was almost three-fold higher than in PCC7806 extracts.

Escarlatina: una enfermedad actual (revisión de 165 casos) / Scarlet fever: a present-day disease. Review of 165 cases

Objetivos: Evaluar las características epidemiológicas y clínicas de los niños diagnosticados de escarlatina en nuestro medio. Metodología: Estudio retrospectivo de niños menores de 15 años diagnosticados de escarlatina en el Servicio de Pediatría del Hospital Universitario Príncipe de Asturias entre julio de 1997 y junio de 2000. Los criterios diagnósticos fueron: a) clínicos (exantema típico, faringoamigdalitis y fiebre), y b) microbiológicos (cultivo faríngeo o enzimoinmunoanálisis para detección rápida del estreptococo del grupo A positivo). Resultados: Se revisaron 239 informes. De ellos, 165 cumplían los criterios diagnósticos. La incidencia fue de 19,5 casos por 100.000 habitantes. El rango de edad estaba entre 8 meses y 14 años. El 83 por ciento de los casos eran menores de cinco años; el 23 por ciento menores de tres. El 46 por ciento de los casos sucedieron en primavera. Dos pacientes fueron diagnosticados de escarlatina recurrente. Cuatro casos precisaron ingreso, uno de ellos al cumplir, además, criterios clínicos de enfermedad de Kawasald. No se halló ningún caso de fiebre reumática ni glomerulonefritís postestreptocócica. Conclusiones: La escarlatina es una enfermedad que aparece en niños en edad preescolar. La incidencia actual en nuestra área es superior a lo referido en la bibliografia. Los métodos rápidos de enzimoinmunoanálisis son útiles para establecer el diagnóstico. El hallazgo de estreptococo del grupo A en frotis faríngeo no debería ser criterio de exclusión para el diagnóstico de enfermedad de Kawasald. Las enfermedades lesivas por estreptococo pueden aparecer en la evolución de una escarlatina (AU)

Hospitalización por infecciones bacterianas cutáneas / Bacterial skin infections requiring hospitalization

Objetivo: Análisis epidemiológico de las infecciones bacterianas cutáneas que requirieron hospitalización en nuestra área; se valoraron: diagnósticos, etiología, asociación con otras enfermedades, tasa de hospitalización, y parámetros clínicos y analíticos. Métodos: Análisis retrospectivo de los casos hospitalizados en el servicio de Pediatría del Hospital Universitario “Príncipe de Asturias” desde enero de 1993 a diciembre de 2000. Diagnósticos revisados: celulitis, impétigo, erisipela y fascitis necrotizante. Resultados: Se hallaron 128 casos; 81% celulitis, 17% impétigo y 2% erisipela. El 59,5% de los niños eran menores de 5 años. Ningún hemocultivo fue positivo y en el 45,5% de los casos en que se cultivaron exudados se aisló Staphylococcus aureus. La asociación más frecuente fue con celulitis y varicela. Dos casos evolucionaron a fascitis necrotizante, ambos asociados a varicela. El 50% presentó fiebre al ingreso. La principal localización fue en extremidades inferiores. En el 29,9% de los casos se observó leucocitosis (leucocitos >15 x 103/µL); el valor medio de la proteína C reactiva (PCR) fue 108 mg/dL (normal <5). Se evidenció una tendencia creciente en la hospitalización por infecciones cutáneas bacterianas. Conclusiones: Las infecciones cutáneas bacterianas son más frecuentes en niños del sexo masculino, menores de 5 años y en extremidades inferiores. El hemocultivo no ayudó a esclarecer el diagnóstico en ningún caso. La leucocitosis y la PCR pueden ayudar al diagnóstico y seguimiento. La varicela es la enfermedad más frecuentemente asociada. Es recomendable la antibioticoterapia parenteral y la hospitalización en infecciones cutáneas bacterianas asociadas con varicela, ante la posibilidad de complicaciones (AU)

Objective. Epidemiological analysis of bacterial skin infections requiring hospitalization in our area. The factors studied were diagnoses, etiology, association with other diseases, rate of hospitalization and clinical and analytical parameters. Methods. Retrospective analysis of patients admitted to the Department of Pediatrics of “Principe de Asturias” University Hospital from January 1993 to December 2000. Diagnoses reviewed included cellulitis, impetigo, erysipleas and necrotizing fasciitis. Results. Eighty-one percent of the 128 patients reviewed had cellulitis, 17% impetigo and 2% erysipelas. Fifty-nine percent of the patients were under five years age. None of the blood cultures were positive, and Staphylococcus aureus was isolated from the skin lesion in 45.5% of cases. Cellulitis and varicella constituted the most frequent association. Two patients developed necrotizing fasciitis, associated with varicella in both cases. Fifty percent of the patients presented fever at the time of admission. The main site of the infection was the lower extremities. Leukocytosis (WBC count >15 x 103/µL) was found in 29,5% of the patients. The mean value for C-reactive protein was 108 mg/dl (normal value <5). We observed an increasing trend in hospitalization due to bacterial skin infections. Conclusions. Bacterial skin infections occur primarily in boys under five years of age and in the lower extremities. Blood culture was not useful for diagnosis in any patient. The WBC count and the C-reactive protein level can be of help in the diagnosis and follow-up. Varicella is the most frequently associated disease. To prevent complications, parenteral antibiotic therapy and hospitalization are recommended in those cases associated with varicella (AU)

Activation of the Anabaena nir operon promoter requires both NtcA (CAP family) and NtcB (LysR family) transcription factors.

A region of the genome of the heterocyst-forming cyanobacterium Anabaena sp. PCC 7120 containing the ntcB gene was identified. This region is located upstream from the nir operon involved in nitrate assimilation in this cyanobacterium. An Anabaena ntcB mutant was able to use ammonium and dinitrogen as sources of nitrogen for growth but was unable to assimilate nitrate. Enzymes of the nitrate reduction system were not synthesized in the ntcB mutant under derepression conditions. The transcription start-point of the Anabaena nir operon, which has been shown to be subjected to ammonium-stimulated repression and whose expression requires the global nitrogen regulator NtcA, was only weakly used in the ntcB mutant. The expression of the ntcB gene in strain PCC 7120 was also subjected to repression by ammonium and was found to take place from an NtcA-activated promoter located 31 bp upstream from the start of the ntcB gene. NtcB binds to the nir promoter region in vitro and protects a region localized just upstream from the NtcA-binding site in footprinting assays. These results showed that NtcB, a LysR-family protein, is required in addition to NtcA, a CAP-family protein, for the expression of genes encoding proteins specifically involved in nitrate assimilation in Anabaena sp. PCC 7120.

Nitrate assimilation gene cluster from the heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120.

A region of the genome of the filamentous, nitrogen-fixing, heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120 that contains a cluster of genes involved in nitrate assimilation has been identified. The genes nir, encoding nitrite reductase, and nrtABC, encoding elements of a nitrate permease, have been cloned. Insertion of a gene cassette into the nir-nrtA region impaired expression of narB, the nitrate reductase structural gene which together with nrtD is found downstream from nrtC in the gene cluster. This indicates that the nir-nrtABCD-narB genes are cotranscribed, thus constituting an operon. Expression of the nir operon in strain PCC 7120 is subjected to ammonium-promoted repression and takes place from an NtcA-activated promoter located 460 bp upstream from the start of the nir gene. In the absence of ammonium, cellular levels of the products of the nir operon are higher in the presence of nitrate than in the absence of combined nitrogen.

Requirement of the regulatory protein NtcA for the expression of nitrogen assimilation and heterocyst development genes in the cyanobacterium Anabaena sp. PCC 7120.

The cyanobacterial ntcA gene encodes a DNA-binding protein that belongs to the Crp family of bacterial transcriptional regulators. In this work, we describe the isolation of an ntcA insertional mutant of the dinitrogen-fixing, heterocyst-forming cyanobacterium Anabaena sp. PCC 7120. The Anabaena ntcA mutant was able to use ammonium as a source of nitrogen for growth, but was unable to assimilate atmospheric nitrogen (dinitrogen) or nitrate. Nitrogenase and enzymes of the nitrate reduction system were not synthesized in the ntcA mutant under derepressing conditions, and glutamine synthetase levels were lower in the mutant than in the wild-type strain. In the ntcA mutant, in response to removal of ammonium, accumulation of mRNA of the genes encoding nitrogenase (nifHDK), nitrite reductase (nir, the first gene of the nitrate assimilation operon), and glutamine synthetase (glnA) was not observed. A transcription start point of the Anabaena glnA gene (corresponding to RNAl), that has been shown to be used preferentially after nitrogen step-down, was not used in the ntcA insertional mutant. Heterocyst development (which is necessary for the aerobic fixation of dinitrogen) and induction of hetR (a regulatory gene that is required for heterocyst development) were also impaired in the ntcA mutant. These results showed that the ntcA gene product, NtcA, is required in Anabaena sp. PCC 7120 for the expression of genes encoding proteins involved in the assimilation of nitrogen sources alternative to ammonium including dinitrogen and nitrate, and that the process of heterocyst development is also controlled by NtcA.

General distribution of the nitrogen control gene ntcA in cyanobacteria.

The ntcA gene from Synechococcus sp. strain PCC 7942 encodes a regulatory protein which is required for the expression of all of the genes known to be subject to repression by ammonium in that cyanobacterium. Homologs to ntcA have now been cloned by hybridization from the cyanobacteria Synechocystis sp. strain PCC 6803 and Anabaena sp. strain PCC 7120. Sequence analysis has shown that these ntcA genes would encode polypeptides strongly similar (77 to 79% identity) to the Synechococcus NtcA protein. Sequences hybridizing to ntcA have been detected in the genomes of nine other cyanobacteria that were tested, including strains of the genera Anabaena, Calothrix, Fischerella, Nostoc, Pseudoanabaena, Synechococcus, and Synechocystis.