El factor de transcripción bZIP Afap1 afecta al estrés oxidativo y la biosíntesis de aflatoxinas en Aspergillus flavus / The bZIP transcription factor Afap1 mediates the oxidative stress response and aflatoxin biosynthesis in Aspergillus flavus

Abstract Aflatoxin is a carcinogenic secondary metabolite produced mainly by Aspergillus flavus and Aspergillus parasiticus, which can seriously endanger the health of humans and animals. Oxidative stress is a common defense response, and it is known that reactive oxygen species (ROS) can induce the synthesis of a series of secondary metabolites, including aflatoxin. By using mutants lacking the afap 1 gene, the role of afap 1 gene in oxidative stress and aflatoxin synthesis was assessed. The growth of the mutant strains was significantly inhibited by the increase in the concentration of H2O2, inhibition was complete at 40mmol/l. However, in the quantitative analysis by HPLC, the concentration of AFB1 increased with the increased H 2O 2 until 10mmol/l. Following an analysis based on the information provided by the NCBI BLAST analysis, it was assumed that Afap1, a basic leucine zipper (bZIP) transcription factor, was associated with the oxidative stress in this fungus. Treatment with 5mmol/l H 2O 2 completely inhibited the growth of the mutant strains in afap 1 but did not affect the growth of the CA14PTs strain (non-mutant strain). In addition, the concentration of AFB 1 in the mutant strains was approximately V of that observed in the CA14PTs strain. These results suggested that Afap1 plays a key role in the regulation of oxidative stress and aflatoxin production in A. flavus. ©2018 Published by Elsevier España, S.L.U. on behalf of Asociación Argentina de Microbiología. This is an open access article under the CC BY-NC-ND license (https://creativecommons.org/ licenses/by-nc-nd/4.0/).

Resumen La aflatoxina es un metabolito secundario cancerígeno producido principalmente por Aspergillus flavus y Aspergillus parasiticus, que pone en riesgo grave a la salud de los humanos y los animales. El estrés oxidativo es una respuesta de defensa común, y es sabido que las especies reactivas de oxígeno (ROS) pueden inducir la síntesis de una serie de metabolitos secundarios, incluida la aflatoxina. Empleando mutantes carentes del gen afap1 se evaluó el papel de Afap1 en el estrés oxidativo y la síntesis de aflatoxinas. El crecimiento de las cepas mutadas se vio significativamente inhibido con el aumento de la concentración de H 2O 2, la inhibición fue completa a 40mmol/l. Sin embargo, en el análisis cuantitativo por HPLC, la concentración de la aflatoxina AFBi aumentó con el aumento de la concentración de H 2O 2 hasta 10mmol/l. Tras un análisis apoyado en la información provista por la herramienta NCBI BLAST, se supuso que Afap1, un factor de transcripción de la cremallera de leucina básica (bZIP), estaba asociado con el estrés oxidativo en este hongo. El tratamiento con 5mmol/l de H 2O 2 inhibió completamente el crecimiento de las cepas mutantes en afap1, pero no afectó el crecimiento de la cepa CA14PTs (cepa no mutada). Además, la concentración de AFB 1 en las cepas mutadas fue de aproximadamente 1/4 de la observada en CA14PTs. Estos resultados sugieren que Afap1 juega un papel clave en la regulación del estrés oxidativo y la producción de aflatoxinas en A. flavus.

Desarrollo y capacidad de remoción de los pesticidas atrazina, clorpirifós y endosulfán por parte de cepas de Aspergillus sección Flavi no toxigénicas / Development and removal ability of non-toxigenic Aspergillus section Flavi in presence of atrazine, chlorpyrifos and endosulfan

This study evaluated the in vitro effect of three concentrations of atrazine, chlorpyrifos and endosulfan on the growth parameters of four non-toxigenic Aspergillus section Flavi strains. The ability of the strains to remove these pesticides in a synthetic medium was also determined. Growth parameters were measured on soil extract solid medium supplied with 5,10 and 20mg/l of each pesticide, and conditioned to -0.70, -2.78, -7.06 and -10.0 water potential (MPa). Removal assays were performed in Czapek Doc medium (CZD) supplied with 20mg/l of each pesticide under optimal environmental conditions (-2.78 of MPa and 25 °C). The residual levels of each pesticide were detected by the reversed-phase HPLC/fluorescence detection system. The lag phases of the strains significantly decreased in the presence of the pesticides with respect to the control media. This result indicates a fast adaptation to the conditions assayed. Similarly, the mycelial growth rates in the different treatments increased depending on pesticide concentrations. Aspergillus oryzae AM 1 and AM 2 strains showed high percentages of atrazine degradation (above 90%), followed by endosulfan (56 and 76%) and chlorpyrifos (50 and 73%) after 30 days of incubation. A significant (p <0.001) correlation (r = 0.974) between removal percentages and growth rate was found. This study shows that non-toxigenic Aspergillus section Flavi strains from agricultural soils are able to effectively grow in the presence of high concentrations of atrazine, chlorpyrifos and endosulfan under a wide range of MPa conditions. Moreover, these strains have the ability to remove high levels of these pesticides in vitro in a short time.

En este estudio se evaluó los efectos in vitro de 3 concentraciones de atrazina, clorpirifós y endosulfán sobre los parámetros de crecimiento de 4 cepas no toxigénicas de Aspergillus sección Flavi. También se evaluó la capacidad de las cepas de remover los pesticidas. Los parámetros de crecimiento se ensayaron en medio agar extracto de suelo suplementado con 5, 10 y 20mg/l de cada pesticida y acondicionado a -0.70, -2.78, -7.06 y -10.0 de potencial de agua (MPa). Los ensayos de remoción se realizaron en medio Czapek Dox con 20mg/l de cada pesticida bajo condiciones óptimas de crecimiento (-2.78 de MPa y 25 °C). Los niveles residuales de atrazina, clorpirifós y endosulfán se detectaron en un sistema HPLC con detección por fluorescencia. La fase de latencia de las cepas disminuyó significantemente en presencia de los pesticidas, indicando una rápida adaptación a dichas condiciones. La velocidad de crecimiento se incrementó considerablemente dependiendo de la concentración de pesticida. Las cepas Aspergillus oryzae AM1 y AM2 mostraron porcentajes elevados de degradación de atrazina (aproximadamente el 90%), seguidos por endosulfán (56 y 76%) y clorpirifós (50 y 73%). Se observó una correlación (r = 0.974) significante (p <0.001) entre el porcentaje de pesticida removido y la velocidad de crecimiento. Este estudio muestra que cepas no-toxigénicas de Aspergillus sección Flavi aisladas de suelos agrícolas desarrollan eficientemente en presencia de altas concentraciones de atrazina, clorpirifós y endosulfán en un amplio rango de MPa. Además, presentan capacidad de remover in vitro altos niveles de pesticidas en corto tiempo.

Differential role of gpaB and sidA gene expressions in relation to virulence in Aspergillus species from patients with invasive aspergillosis

Abstract The virulence genes in invasive aspergillosis (IA) have not been analyzed adequately. The present study was designed to evaluate the expression of gpaB and sidA genes, which are important virulence genes in Aspergillus spp. from bronchoalveolar lavage (BAL) samples. Direct examination and culture on Czapek Agar and Sabouraud Dextrose Agar media were performed for 600 BAL specimens isolated from patients with possible aspergillosis. A Galactomannan ELISA assay was also carried out. The expression levels of the gpaB and sidA genes in isolates were analyzed using quantitative real-time PCR (qRT-PCR). We identified 2 species, including Aspergillus flavus (A. flavus) and Aspergillus fumigatus (A. fumigatus) in 25 positive samples for invasive aspergillosis as validated using GM-ELISA. A. flavus is the main pathogen threatening transplant recipients and cancer patients worldwide. In this study, A. flavus had low levels of the gpaB gene expression compared to A. fumigatus (p = 0.006). The highest sidA expression was detected in transplant recipients (p = 0.05). There was no significant correlation between sidA expression and underlying disease (p = 0.15). The sidA and gpaB gene expression patterns may provide evidence that these virulence genes play important roles in the pathogenicity of Aspergillus isolates; however, there are several regulatory genes responsible for the unexpressed sidA and gpaB genes in the isolates.

Hongos productores de micotoxinas asociados a hierbas medicinales en la Ciudad de São Paulo, Brasil / Mould and mycotoxins associated in medicinal plants in São Paulo, Brazil

Antecedentes: la fitoterapia es una de las más antiguas prácticas utilizadas por la humanidad. Hasta mediados del siglo XIX, cuando se introdujeron los medicamentos, la formulación de estos generalmente era basada en plantas medicinales. Objetivos: Determinar la micobiota y los niveles de aflatoxinas originadas de Aspergillus sección Flavi aislados de las 50 muestras de medicamentos fitoterápicos comercializados actualmente en la ciudad de São Paulo, Brasil. Métodos: Cincuenta (50) muestras de medicamentos fitoterápicos en la forma de hojas (té-25) y cápsulas (25) fueron colectadas de agosto de 2000 a julio de 2001. Los hongos filamentosos aislados fueron identificados al nivel de género de acuerdo con las características morfológicas y criterios taxonómicos. El análisis de aflatoxinas fue realizada por cromatografía de capa fina (TLC). Resultados: El análisis microbiológico mostró que 41 (82 por ciento) de los medicamentos fitoterápicos presentaron un crecimiento fúngico sobre las 100 UFC/g. Un total de 106 especies de seis diferentes géneros fueron aislados (Aspergillus, Penicillium, Mucor, Rhizopus y Alternaria). El género Aspergillus fue el predominante (60.5 por ciento) seguido por Penicillium (20,0 por ciento). Aspergillus niger (30 por ciento) A. flavus (22 por ciento), A. fumigatus (6,5 por ciento) y A. parasiticus fueron las especies de Aspergillus identificadas. Se observó que 13 (56,5 por ciento), de los 23 A. flavus aislados y dos aislados de A. parasiticus produjeron aflatoxinas. Conclusiones: La contaminación observada en la mayoría de los productos y el alto nivel de cepas productoras de aflatoxinas justifica un análisis más cuidadoso de los medicamentos fitoterápicos comercializados y la aplicación de leyes más rigurosas son necesarias para garantizar la calidad de los productos.

Background: phytotherapy is one of the most ancient practices used by humanity. In Antiquity until the middle of the XIX century, when chemotherapeutic drugs were introduced, formulation of medicines was usually based on medicinal plants. Objective: To determine mycobiota and levels of Aspergillus section Flavi aflatoxins isolated from 50 samples of phytotherapeutic remedies currently commercialized in São Paulo, Brazil. Methods: Fifty (50) samples of phytotherapeutic remedies in the form of leaves (teas-25) and powders (capsules-25) were collected from August 2000 to July 2001. Filamentous fungi isolates were identified at the genera level in accordance with morphological characteristics and taxonomic criteria. Aflatoxins were performed by Thin-layer chromatography (TLC). Results: The microbiological analysis showed that 41 (82 percent) of phytotherapeutic remedies presented a fungal growth over 100 CFU/g. A total of 106 species of six different genera were isolated (Aspergillus, Penicillium, Mucor, Rhizopus and Alternaria). The genus Aspergillus was the predominant (60.5 percent) followed by Penicillium genus (20.0 percent). Aspergillus niger (30 percent) A. flavus (22 percent), A. fumigatus (6.5 percent) and A. parasiticus were the species of Aspergillus identified. It was observed that 13 (56.5 percent) of 23 A. flavus isolates and two A. parasiticus isolates produced aflatoxins. Conclusions: The contamination observed in most products and the high level of aflatoxigenic strains justify the concern regarding the execution of more careful analyzes of the commercialized phytotherapeutic remedies and the application of more rigorous laws that may warrant the quality of these products.

Intoxicação experimental por aflatoxina em bezerros / Experimental aflatoxin poisoning in calves

Foram realizados dois experimentos para determinar os efeitos tóxicos de diferentes doses de aflatoxinas em bezerros, considerando-se aspectos clínicos, produtivos e patológicos. ... No primeiro experimento, o ganho de peso dos bezerros recebendo AFB1 foi equivalente ao do grupo controle durante todo período experimental. ...Durante o período experimental, e três semanas após o término desse período, não foram observados sinais clínicos e alterações histopatológicas associadas ao consumo de aflatoxinas, em qualquer dos bezerros do grupo tratamento do primeiro experimento.... Níveis alterados da atividade sérica de FA e GGT foram observados em todos os bezerros do grupo tratamento durante grande parte do período experimental. Queda acentuada do nível da AS sérica foi observada na coleta do 49º dia do experimento no bezerro que recebia a maior dose de aflatoxina. Não foram observadas variações no hematócrito e na atividade sérica da AST, nem nos níveis séricos de proteína total, bilirrubina total e bilirrubina direta em qualquer dos bezerros desse experimento. Alterações histopatológicas nos bezerros intoxicados incluíram proliferação de ductos biliares, degeneração citoplasmática vacuolar consistente com acumulação hepatocelular de lipídios, fibrose periportal, ou em ponte, megalocitose, fibrose subendotelial das veias hepáticas terminais e edema. Achados de necropsia do bezerro recebendo a maior dose de AFB1 incluíram fígado levemente aumentado de tamanho, difusamente amarelo-claro e firme, discreta ascite, edema de mesentério e submucosa do abomaso. Os dados obtidos nesses experimentos permitem afirmar que doses de 500±100 ppb de AFB1 não causam alterações patológicas e produtivas em bezerros em condições experimentais, mas podem estar associadas à mínimas alterações bioquímicas, enquanto doses de 1.250, 2.500 e 5.000 ppb de aflatoxina B1 causam doença hepática crônica em bezerros em condições experimentais.

Two experiments were performed in order to determine the toxic effects of varying doses of aflatoxins in calves. Clinical, productive and pathologic aspects of affected calves were considered….During all the experimental period of the first experiment, the weight gain of the calves receiving AFB1 was equivalent to that of the control group.…During the whole experimental period and up to three weeks after the final of the experiment, no clinical signs or histopathological changes associated with the consumption of aflatoxins were observed in any of the calves of the first experiment….Increased activity of AF and GGT were observed in all the calves of the treated group during most part of the experimental period. A marked drop in the serum levels of SA was observed in the serum sampled on the 49º day of the experiment in the calf receiving. the largest dose of aflatoxin. No changes were observed regarding PCV, TP, total bilirubin, direct bilirubin and in the serum activity of AST in any of the calves of the second experiment. Histopathological changes in intoxicated calves included bile duct proliferation, cytoplasmic vacuolar hepatocelular degeneration consistent with hepatocelular deposit of lipids, periportal to bridging fibrosis, megalocytosis, subendothelial edema and fibrosis in terminal hepatic veins. Necropsy findings in the euthanatized calf which receive de largest doses of AFB1 included slight enlargement of the liver which was firm and diffusely light-yellow, mild ascites, and edema of the mesentery and of abomasal folds. Data stemmed from these two experiments allow to conclude that AFB1 doses of 500±100 in the ration do not cause pathologic changes or decrease in productivity in calves kept in experimental conditions, but can be associated to minimal serum biochemistry; while AFB1 doses of 1.250, 2.500 e 5.000 ppb in the ration cause chronic hepatic disease in calves in kept in experimental conditions.

Reportes clínicos: infección probada por Aspergillus flavus y probable infección pulmonar por Emericella nidulans en pacientes inmunodeprimidos / Clinical reports: tested rhinosinusal infection by Aspergillus flavus and probable pulmonary infection by Emericella nidulans in immunodepressed patients

En los últimos 20 años la incidencia de aspergilosis invasiva ha aumentado entre 5 y 10 veces, presentando una mortalidad que varía desde un 60 a 98 por ciento dependiendo de su localización, enfermedad de base (inmunodepresión), diagnóstico y tratamiento precoz. A pesar que se ha logrado un importante avance en estos dos últimos decenios, aproximadamente un 50 por ciento de los casos se diagnostican post mortem. Se reportan dos casos de aspergilosis (probada y probable) en pacientes inmunodeprimidos de 21 y 44 años respectivamente. El primer caso corresponde a una infección rinosinusal en una paciente con leucemia linfoblástica, con un cuadro clínico caracterizado por aumento de volumen facial izquierdo, compromiso progresivo del estado general y equimosis en región periorbitaria. La endoscopia nasal detectó abundante secreción y tejido friable amarillento sugerente de infección micótica. Las muestras de tejidos enviadas al laboratorio para examen directo y cultivos arrojaron presencia de hifas en la mucosa y crecimiento abundante de Aspergillus flavus. Se inició terapia con itraconazol, cambiándose posteriormente a voriconazol, con respuesta favorable y disminución del dolor y volumen facial. Sin embargo, la paciente fallece al 10º día del ingreso debido a su mal estado general, avance de la falla medular y posterior insuficiencia respiratoria. El segundo caso corresponde a una probable infección pulmonar en una paciente con cáncer de mama en tratamiento quimioterápico. La paciente ingresó con el diagnóstico de neutropenia febril y probable neumonía de etiología bacteriana, fue tratada con antimicrobianos tanto en el hospital comunal como en el de base. Sin embargo, presentó una evolución tórpida, cursando posteriormente con hemoptisis, insuficiencia respiratoria y compromiso de conciencia. Se tomaron muestras de secreción endotraqueal las que fueron enviadas para estudio bacteriológico(TBC) y micológico, además de hemo y urocultivos. Los resultados de los...

In the lately twenty years the incidence of invasive spergillosis has increased five and ten times, causing a mortality rate ranging from 60 to 98 percent, depending on its location, nature of disease (immunodepression), diagnosis and forward treatment. In spite of the fact that there has been a significant progress in this lately period of twenty years, about 50 percent of cases were detected post mortem. Two cases of spergillosis (tested and probable) in 21 and 44 aged immunodepressed patients respectively are herein reported. The first case involves a rhinosinusal infection in a female patient diagnosed with lymphoblastic leukemia, whose clinical symptoms were an increase in the left volume of her face, a progressivecompromise in her overall state of health and ecchymosis in her periorbitary region. Nasal endoscopy revealed abundant secretion and yellowish friable tissues which suggested a mycotic infection. Samples of tissues sent to the lab for direct exam and cultures revealed the presenceof hypha in the mucus and an abundant growth of Aspergillus flavus. The first therapy was with itraconazol, changing later on to voriconazol what resulted in adecrease of pain and face volume. However, the patient dies on the tenth day of her ingress due to her bad state ofhealth, progress in glandular failure and ultimate respiratory insufficiency. The second case involves a probable pulmonary infection in a patient diagnosed with breast cancer and receiving chemotherapy treatment. She was ingressed with a febrile neutropenia and a probable bacterial etiology pneumonia and she was treated with antimicrobial medication both in the community hospital and in the base building. However, she showed a torpid evolution followed furthermore by hemoptisis, respiratory insufficiencyand consciousness compromise. Samples of endotraqueal secretion were taken in order to submit them to bacteriological and mycological (TBC) studies, together with hemo...

Toxicidad de cepas de aspergillus flavus aisladas del maíz / Toxicity of aspergillus flavus strains isolated from corn

En muestras de granos de maíz de la zona centro norte de la Provincia de Santa Fe, Argentina, se seleccionaron 10 cepas de Aspergillus flavus, para estudiar sus características morfotaxonómicas y toxicogénicas. Para los ensayos taxonómicos se efectuaron observaciones macro y microscópicas de los cultivos puros en CYA y MEA, mientras los ensayos de toxicidad en el medio YES, registrandose la producción de aflatoxina en algunas cepas a los 7, 14 y 21 días de incubación. Mediante el empleo de cromatografía en capa fina, se determinó la presencia de aflatoxinas con los estandares correspondientes (B1, B2, G1, G2). Los resultados obtenidos en base a la morfología y a la producción de aflatoxinas, permitió afirmar que la totalidad de las cepas ensayadas, pertenecían a las especie A.flavus Link:Fr. Dos cepas produjeron aflatoxinas B1 y B2, a los 14 y 21 días de incubación. Solo 1 de ellas produjo cantidades significativas de ambas toxinas, pero ninguna produjo aflatoxinas G. Se destaca el valor quimiotaxonómico de los estudios de toxigenicidad, para la identificación a nivel de especie dentro de la sección Flavi

Aislamiento de diversas especies del género aspergillus de la tierra de plantas ornamentales intrahospitalarias / Isolation of different aspergillus strains fron the soil of ornamental plants located at hospital

The earth of ornamental plants is one of the main reservoirs of Aspergillus type of fungi in hospital areas. We studied 174 ornamental interior plants from a hospital at Santiago. Samples were obtained from the soil surface and sowed in Sabouraud-glucose agar, adding streptomycin and G-penicillin. After 72 h of culture, at least one strain of Aspergillus was isolated from 140 samples (80.5 percent). The most frequently isolated strain was A fumigatus (129 samples), followed by A miger (75 samples). A fumigatus and A niger were the only isolated strains in 65 and 11 samples respectively. These findings confirm that ornamental plants can be important reservoirs of Aspergillus strains, a potential infectious agent for immunocompromised patients in hospital areas

Histological study of the effect of contaminated food by mycotoxin AFBI and CPA in albino rats

Fungal flora of mixed food belonging to Aspergillus, Mucor, Penicillium and Rbizopus species were the most frequently isolated, from mixed poultry feed, especially A. flvus This study was conducted to determine whether exposure to cyclopiazonic acid [CPA] and Aflatoxin[B1] [AFB1] wouId alter the toxicity with exposure to either toxin individually. Seventy-two adult rats of both sexes were used, classified into 6 groups, 12 for each. The groups were dosed 0, 0.1 and 4 mg/kg B. W. [CPA: cyclopiazonic acid] and 0, 0.1 and 0.1 mg/kg B. W. [AFB1: Aflatoxin [B1]] daily for 3 successive doses for each group. 6 rats of each group were sacrificed alter 4 days from the initial dose, the remainder were left for a recovery period of 4 days prior to being killed. Small pieces of liver, kidney and spleen were processed histologically and paraffin sections were stained, then examined microscopically. Grossly, loss of weight of the rats receiving 0.2 mg/kg B. W. aflatoxin within 24 hours from the first dose and food consumption was 60% of controls. Rats of recovery lost 31-38 gm of their weights, the food consumption was 50%. In groups receiving CPA, food consumption was 75 of controls. Microscopically, the liver showed severe interstitial haemorrhages and lymphocytosis in the portal tracts in [lethal doses]. In animals receiving low doses, focal areas of haemorrhages were discerned with some lymphocytic infiltration, while in animals of [one dose], there were hypertrophied liver cells with many pyknotic nuclei of many cells. The kidneys of one dose had congested cortical blood vessels with pyknotic nuclei in the epithelial cell of the renal tubules. In [lethal doses], the congestion of renal tissue blood vessels was not so extensive as in the liver which was the most organ to be affected. The spleen, revealed delayed red pulp area on the expense of the white pulp in the lethal dose. In other doses, only vacuolation of many large Iympbocytes were seen in the splenic follicles