Protocolo para la identificación rápida y sensible de ricina en muestras ambientales ante una alerta biológica / Protocol for rapid and sensitive identification of ricin in environmental samples during a biological alert

Introducción: La ricina es una toxina muy potente que ha adquirido importancia por su potencial uso como arma biológica, fundamentalmente en forma pulverulenta. El desarrollo de métodos que permitan una detección rápida y temprana tras la exposición a la toxina permitiría reducir las tasas de morbilidad y mortalidad asociadas. Ante una alerta/amenaza biológica con una muestra sospechosa de contener ricina, la Red de Laboratorios de Alerta Biológica (RELAB), infraestructura de naturaleza científico-técnica creada mediante la Orden PRE/305/2009, autoriza el envío de la muestra al Instituto Nacional de Técnica Aerospacial (INTA), uno de los laboratorios de referencia que integran esta Red. Objetivos: Desarrollo de un protocolo para la detección de ricina basado en una doble detección, un ensayo inmunológico y un análisis proteico, para aumentar la fiabilidad del diagnóstico además de acortar sensiblemente el tiempo de respuesta del laboratorio. Material y Métodos: El diagnóstico inmunológico con anticuerpos in house combinados en un ELISA tipo Sandwich. El diagnóstico proteico mediante la técnica SDS-PAGE (electroforesis en gel de poliacrilamida con dodecilsulfato sódico) para determinar el tamaño y la estructura de las distintas proteínas de la muestra. Resultados: La optimización del marcado de los anticuerpos de detección, así como la preparación y almacenamiento de placas previamente tapizadas/bloqueadas permite conseguir un ensayo muy sensible (<1 ng/mL) en un tiempo inferior a cuatro horas. Conclusión: La realización de ambos ensayos en paralelo permite disponer de un método diagnóstico robusto, sensible y rápido (AU)

Introduction: Ricin is a powerful toxin wich has gained importance due to its potential use as biological weapon, essentially in powder form. The development of methods that promote a rapid detection after the toxin exposure will allow us to reduce its associated rates of both mobility and mortality. In the face of biological threat with samples suspected of containing ricin, the Biological-Alert-Laboratory-Network (RELAB), which is a scientific-technical infrastructure created by the Order of the Ministry of the Presidency PRE/305/2009, authorizes the shipment of this type of samples to the National Institute of Aerospace Technique (INTA), one of the Reference Laboratory belonging to this Network. Aim: Development of one protocol to detect ricin based on both an immunological assay and a protein analysis focused to reduce noticeably the response time of the laboratory. Material and Methods: The Immunological diagnosis of ricin uses in house antibodies combined in an ELISA sandwich. Protein diagnosis through the SDS-PAGE technique defines the size and structure of different proteins contained in the sample. Results: The optimization of detection antibody labelling and the development of precoated-and-preblocked stored plates has allowed us to achieve a very sensitive assay (<1 ng/mL) less than four hours. Conclusion: Carrying out both assays in paralell enables us to have a hardy, sensitive and rapid method to identify ricin (AU)

Microbiología del agua mineromedicinal del Balneario de San Nicolás / Microbiology of the natural mineral water of San Nicolás Spa

Se han estudiado los microorganismos autóctonos y alóctonos de las aguas mineromedicinales del Balneario San Nicolás (Alhama de Almería). No se han encontrado bacterias patógenas ni indicadores fecales en 250 mL de agua. La microbiota autóctona está constituida, principalmente, por bacterias oligotrofas Gram positivas de la clase Firmicutes. El sondeo Sillero, utilizado en los tratamientos, presenta un número muy bajo de bacterias viables (<10 ufc/mL) que corresponden en su mayoría a cocos Gram positivos (93,3%) y a la especie Staphylococcus lugdunensis (40%). El sondeo San Marcos tiene una mayor diversidad microbiana, predominando los bacilos Gram positivos de la especie Bacillus licheniformis (32,4%) y los bacilos Gram negativos de la especie Cupriavidus pauculus (16,2%). En todas las muestras se han detectado microorganismos proteolíticos, amilolíticos, nitrificantes y amonificantes, así como bacterias halófilas y del hierro. También se han estudiado los biotapetes formados en las fuentes utilizadas para bebida, constituidos por una asociación de cianobacterias filamentosas y esféricas, así como del alga Cosmarium (AU)

Both the autochthonous and the allochthonous microorganisms of minero‐medicinal water from the San Nicolas spa (Alhama of Almería, Spain) have been analyzed. No pathogen bacteria or faecal indicators have been found in 250 mL of water. The autochthonous microbiota is mainly composed of oligotrophic Grampositive bacteria of the Firmicutes phylum. The Sillero sampling, used in the spa treatments, presents a very low count of viable bacteria (< 10 cfu / mL), being the most part of them Grampositive cocci (93.3%) of the Staphylococcus lugdunensis (40%) species. The San Marcos sampling presents a higher microbial diversity, predominantly Gram‐positive bacilli of the Bacillus licheniformis (32.4%) species and Gram‐negative bacilli of the Cupriavidus pauculus (16.2%) species. Proteolytic, amylolitic, nitrifying and ammonifying microorganisms have been detected in all samples, altogether with halophilic and iron bacteria. An analysis of the microbial mats growing in the spa drinking water sources shows that these consist of an association of spherical and filamentous cyanobacteria and the Cosmarium algae (AU)

Enriquecimiento-mejora en la eficacia de la realización de la reacción en cadena de la polimerasa de patógenos en alimentos / Enrichment-improvement in the effectiveness of the implementation of the polymerase chain reaction of pathogens in food

Objetivo. Mejorar la eficacia de los ensayos cualitativos por reacción en cadena de la polimerasa (PCR) de patógenos en alimentos. Al realizar un enriquecimiento consistente en hacer una dilución inferior a la 1/10 se pretende adelantar en tiempo, reducir el gasto de medios, se procura posibilitar el completar hasta el 100% las muestras a realizar en una PCR y no disminuir la carga celular diana del alimento. Métodos. Investigación de la presencia o ausencia de una bacteria patógena en un alimento y su concentración por mililitro de disolución, siguiendo el método tradicional a distintas diluciones, en dos matrices. Resultados. Los resultados obtenidos en el experimento con un patógeno en alimentos, evidencia que con diluciones inferiores a 1/10, la concentración bacteriana para un mínimo de horas de incubación adelanta la detección por PCR del ácido desoxirribonucleico (ADN) diana, con valores de ufc/ml por encima de 1x103 . Por tanto el tiempo de incubación en un caldo de enriquecimiento se acorta, así como los volúmenes de las disoluciones a incubar. Y de este modo solo es necesario un operario y una estufa de incubación para realizar todas las tareas. Conclusiones. Mayor eficacia al necesitarse menos medios materiales y horas de trabajo. Realización de mayor número de muestras en una misma jornada de trabajo (se posibilita realizar el total de muestras que admite el termociclador por cada puesta en funcionamiento), se acorta el tiempo de análisis en siete horas como mínimo

Objective. Improve the effectiveness of the tests by qualitative polymerase chain reaction (PCR) of pathogens in food. When performing a enrichment consists in making a lower dilution to the 1/10 is intends to bring forward in time, reduce the cost of media, it seeks to enable the complete up to 100% of the samples to perform in a PCR and does not decrease the load cell Diana of the food. Methods. Investigation of the presence or absence of a pathogenic bacterium in food and its concentration per millilitre of dissolution, following the traditional method in different dilutions, in two different types of food. Results. The results obtained in the experiment with a pathogen in food, evidence that using dilutions lower than 1/10, the bacterial concentration for a minimum number of hours of incubation anticipates the detection by PCR of deoxyribonucleic acid (DNA) target, with values of cfu/ml above 1x103 . Therefore the incubation time in an enrichment broth and the volumes of the solutions are reduced. And it is only needed one operator and one incubator for all tasks. Conclusions. The efficacy increases because are needed less material and working hours. Testing more samples in the same working day (makes possible to perform all samples that supports the thermal cycler for each start-up) the analysis time decreases in seven hours at least

Removal of soluble microbial products as the precursors of disinfection by-products in drinking water supplies.

Water pollution worsens the problem of disinfection by-products (DBPs) in drinking water supply. Biodegradation of wastewater organics produces soluble microbial products (SMPs), which can be important DBP precursors. In this laboratory study, a number of enhanced water treatment methods for DBP control, including enhanced coagulation, ozonation, and activated carbon adsorption, were evaluated for their effectiveness in treating SMP-containing water for the DBP reduction purpose. The results show that enhanced coagulation with alum could remove SMPs only marginally and decrease the DBP formation potential (DBPFP) of the water by less than 20%. Although ozone could cause destruction of SMPs in water, the overall DBPFP of the water did not decrease but increased after ozonation. In contrast, adsorption by granular activated carbon could remove the SMP organics from water by more than 60% and reduce the DBPFP by more than 70%. It is apparent that enhanced coagulation and ozonation are not suitable for the removal of SMPs as DBP precursors from polluted water, although enhanced coagulation has been commonly used to reduce the DBP formation caused by natural organic matter. In comparison, activated carbon adsorption is shown as a more effective means to remove the SMP content from water and hence to control the wastewater-derived DBP problem in water supply.

Estudo longitudinal da infecção por enteropatógenos em bezerros neonatos, com diarreia, sob diferentes estratégias de aleitamento / Longitudinal study of infection by enteropathogens in newborn calves with diarrhea under different feeding strategies

Foram utilizados 17 bezerros, recém nascidos, da raça Holandesa, com o objetivo de avaliar a influência do volume de sucedâneo nos principais patógenos causadores de diarreia neonatal. [...] Foram coletadas amostras de sangue dos bezerros com cinco dias de idade para dosagem da proteína total. A média da proteína total foi 6,33 e 6,21g/dL nos grupos 1 e 2 respectivamente. O grupo 2 apresentou tendência (p<0,1) de maior consumo de sucedâneo no período avaliado. A quantidade de sucedâneo oferecida aos animais não influenciou a incidência de diarreia e sua etiologia, ou seja, não foi observada diferença (p>0,05) na frequência das amostras positivas para cada agente entre os grupos. A frequência dos enteropatógenos nas amostras foi de 100 e 75 por cento para Cryptosporidium spp.; 28,5 e 43,7 por cento para Salmonella spp.; 28,5 e 15,6 por cento para patotipos de E. coli; 3,5 e 6,2 por cento para Rotavírus e 10,7 e 9,4 por cento para Giardia sp. nos grupos 1 e 2 respectivamente. Foram encontrados os sorotipos de Salmonella infantis e muenster. Os patotipos de E. coli isolados foram classificados como E. coli enterohemorrágica, enteropatogênica, enterotoxigênica e produtoras de toxinas Shiga 1 e 2. Foi observada associação entre o Cryptosporidium spp. e os patotipos de E. coli em 30 por cento das amostras do grupo 1 e Cryptosporidium spp. e Salmonella spp. em 45,5 por cento no grupo 2. Os resultados do presente trabalho demonstraram que o fornecimento de diferentes volumes de sucedâneo não apresentou influência sobre a incidência e etiologia da diarreia neonatal. A avaliação longitudinal dos enteropatógenos durante o período de patência da diarreia demonstrou que a associação entre eles ocorre a partir do primeiro dia da doença e destacou a importância da infecção pelo Cryptosporidium spp. agente encontrado em todos os momentos e animais.

Seventeen Holstein newborn calves were used with the objective of evaluating the influence of milk replacer volume in the pattern of pathogens causing neonatal diarrhea. […] Were collected blood samples from calves with five days of age for determination of total protein. The average total protein was 6.33 and 6.21g/dL in groups 1 and 2, respectively. The group 2 tended (p<0.1) higher consumption of milk replacer during the study period. The volume of milk replacer did not influenced the incidence of diarrhea and the frequency of positive samples for each etiologic agent between the two groups (p>0.05). Also, there was no difference (p>0.05) on the pattern of the frequency of positive samples for evaluated pathogens. The frequency of pathogens in the samples was 100 and 75 percent for Cryptosporidium, 28.5 and 43.7 percent for Salmonella spp., 28.5 and 15.6 percent for E. coli pathotypes, 3.5 and 6.2 percent for Rotavirus and 10.7 and 9.4 percent for Giardia in groups 1 and 2, respectively. Serotypes of Salmonella infantis and muenster were found. The isolated pathotypes of E. coli isolates were classified as Escherichia coli enteropathogenic, enterotoxigenic and Shiga-toxin-producing 1 and 2. Associations between Cryptosporidium spp. and E. coli pathotypes, and between Cryptosporidium spp. and Salmonella spp. were found in 30 percent of the samples in group 1 and in 45.5 percent in group 2, respectively. Our results showed that the different volumes of milk replacer did not influence the incidence and etiology of neonatal diahrrea. Longitudinal evaluation of enteropathogens during patency demonstrated that the association between the etiologic agents starts from the first day of disease. This result highlighted the great importance of the infection by Cryptosporidium spp. which was present in every moments and animals evaluated.

Bacterial microbiota present in the gallbladder of cattle and antimicrobial resistance of Staphylococcus isolates / Microbiota bacteriana presente na vesícula biliar de bovinos e resistência antimicrobiana de isolados de Staphyloccocus

Pathogenic microorganisms can reside transiently or permanently in the gallbladder of cattle. Thus, during slaughter, more attention should be given to the gastrointestinal tract, especially to the accessory organ, the gallbladder. The main aim of this study was to characterize the bacterial microbiota present in bile and gallbladder epithelium of cattle slaughtered in a slaughtering plant under sanitary conditions and to evaluate the antimicrobial resistance in strains of the genus Staphylococcus. Thirty intact gallbladders were collected and the in bile and epithelium were researched for the presence of Aerobic Mesophilic Heterotrophic Bacteria (AMHB), Staphylococcus spp., total Enterobacteriaceae, Enterococcus spp. and Salmonella spp.The frequency of isolation of the microorganism mentioned above were, respectively: 23.02 percent, 14.39 percent, 13.67 percent, 24.46 percent, 0 percent and 24.46 percent. Concerning both gallbladder environments, the frequency of isolation of the microorganisms in the epithelium was 64.03 percent, and in the bile 35.97 percent, with no statistical difference, but with significant difference between the population averages. In antimicrobial susceptibility testing, strains of Staphylococcus from both bile and gallbladder epithelium showed sensitivity to the antimicrobials: penicillin G, ceftriaxone, chloramphenicol and gentamicin. The observation that the gallbladder supports a high frequency of microorganisms brings us to the possible fact that cattle might be a persistent carrier of pathogens of great importance to public health...

Microrganismos patogênicos podem residir temporariamente ou permanentemente na vesícula biliar de bovinos. Assim, durante o abate, maior atenção deve ser dada ao trato gastrointestinal, especialmente para o órgão acessório, a vesícula biliar. O principal objetivo deste estudo foi caracterizar a microbiota bacteriana presente na bile e no epitélio de vesículas biliares de bovinos abatidos em matadouro frigorífico sob inspeção sanitária e avaliar a resistência antimicrobiana de estirpes do gênero Staphyloccocus. Foram coletadas 30 vesículas biliares íntegras e foi pesquisada na bile e no epitélio do órgão a presença de bactérias heterotróficas aeróbias mesófilas (BHAM), Staphylococcus spp. e Enterobacteriaceae totais, Escherichia coli, Enterococcus spp. e Salmonella spp. A frequência de isolamento dos microrganismos citados acima foi, respectivamente: 23,02 por cento, 14,39 por cento, 13,67 por cento, 24,46 por cento, 0por cento e 24,46 por cento. Em relação aos dois ambientes da vesícula, a frequência de isolamento dos microrganismos no epitélio foi de 64,03 por cento, e na bile 35,97 por cento, não sendo diferente estatisticamente, mas com diferença significativa entre as médias populacionais.No teste de susceptibilidade aos antimicrobianos, as estirpes de Staphylococcus isoladas a partir da bile e do epitélio da vesícula biliar apresentaram sensibilidade a: penicilina G, ceftriaxona, cloranfenicol e gentamicina. A observação de que a vesícula biliar comporta microrganismos em elevadas frequências atenta para o fato de que o bovino possa ser um portador persistente de patógenos de grande importância em saúde pública...

Aerially transmitted human fungal pathogens: what can we learn from metagenomics and comparative genomics? / Hongos de transmisión aérea patógenos para el ser humano: conocimientos adquiridos a partir de su metagenómica y genómica comparativa

In the last few decades, aerially transmitted human fungal pathogens have been increasingly recognized to impact the clinical course of chronic pulmonary diseases, such as asthma, cystic fibrosis or chronic obstructive pulmonary disease. Thanks to recent development of culture-free high-throughput sequencing methods, the metagenomic approaches are now appropriate to detect, identify and even quantify prokaryotic or eukaryotic microorganism communities inhabiting human respiratory tract and to access the complexity of even low-burden microbe communities that are likely to play a role in chronic pulmonary diseases. In this review, we explore how metagenomics and comparative genomics studies can alleviate fungal culture bottlenecks, improve our knowledge about fungal biology, lift the veil on cross-talks between host lung and fungal microbiota, and gain insights into the pathogenic impact of these aerially transmitted fungi that affect human beings. We reviewed metagenomic studies and comparative genomic analyses of carefully chosen microorganisms, and confirmed the usefulness of such approaches to better delineate biology and pathogenesis of aerially transmitted human fungal pathogens. Efforts to generate and efficiently analyze the enormous amount of data produced by such novel approaches have to be pursued, and will potentially provide the patients suffering from chronic pulmonary diseases with a better management. This manuscript is part of the series of works presented at the "V International Workshop: Molecular genetic approaches to the study of human pathogenic fungi" (Oaxaca, Mexico, 2012) (AU)

En las últimas décadas se ha reconocido cada vez más la influencia de los hongos patógenos para el ser humano, y cuya transmisión es aérea, en el curso clínico de afecciones pulmonares crónicas, como el asma, la fibrosis quística o la enfermedad pulmonar obstructiva crónica. Gracias al desarrollo reciente de métodos de secuenciación de alto rendimiento, que no requieren cultivo, en la actualidad los análisis metagenómicos permiten detectar, identificar e incluso cuantificar comunidades de microorganismos procariotas o eucariotas que habitan en las vías respiratorias del ser humano, y acceder a la complejidad de las comunidades microbianas cuya población es de baja densidad, que posiblemente desempeñan un papel en las enfermedades pulmonares crónicas. En la presente revisión examinamos cómo los estudios metagenómicos y genómicos comparativos pueden ayudar a superar los obstáculos de los cultivos de hongos, mejorar nuestros conocimientos sobre la biología fúngica, desvelar el diálogo cruzado (crosstalk) entre el pulmón del huésped y la microbiota fúngica asociada, y adquirir información sobre la influencia patogénica de estos hongos transmitidos por el aire que afectan al ser humano. Revisamos los estudios metagenómicos y los análisis genómicos comparativos de microorganismos cuidadosamente seleccionados, y confirmamos la utilidad de estas estrategias para definir mejor la biología y la patogenia de hongos de transmisión aérea que son patógenos para el ser humano. Los esfuerzos por generar y analizar eficientemente la ingente cantidad de datos obtenidos con estos nuevos métodos deberán continuar, y es posible que ofrezcan un mejor tratamiento de los pacientes portadores de enfermedades pulmonares crónicas.Este manuscrito forma parte de la serie de artículos presentados en el «V International Workshop: Molecular genetic approaches to the study of human pathogenic fungi» (Oaxaca, México, 2012) (AU)