Agradecemos Pr. Bernard La Scola para consejos técnicos útiles y discusión interesante sobre el co-cultivo de amebas y enriquecimiento de amebas. También agradecemos a la Dra. Vincent Thomas por su ayuda en la implementación de la técnica en nuestro laboratorio.

1. Amebas Coculture 1.1 Preparación de la muestra <ol><li> Muestra Ambiental <ol><li> Las muestras de agua Se filtra la muestra de agua (500 ml a 1 L) a través de una membrana de tamaño de poro 0,22 micras. Luego, sacuda la membrana en solución salina ameba medianas PAS de Página (120 mg de NaCl, 4 mg de MgSO4 • 7H 2 O, 4 mg de CaCl2 • 2H 2 O, 142 mg de Na 2 HPO 4, y 136 mg de KH 2 PO 4 en 1 L de agua destilada). </li><li> Las muestras sólidas Resuspender muestras sólidas como muestras de suelo o de arena y las muestras semi-sólidas, tales como lodos activados en agua destilada o PBS y filtrar a través de una membrana de tamaño de poro 0,22 micras. Luego, sacuda la membrana en el PAS. </li><li> Las muestras altamente contaminadas con amebas endógeno y protozoos Primera sedimentar las muestras mediante centrifugación a baja velocidad (180 xg) foR 10 minutos o por filtración a través de una membrana de tamaño de poro 5 micras. Además procesar el sobrenadante (filtrado, respectivamente), como en 1.1.1. Nota: Otras técnicas de descontaminación podrían utilizarse para descontaminar más muestras ambientales: Se calienta la muestra a 50 ° C durante 30 minutos o tratar con soluciones ácidas o básicas 14. </li></ol></li><li> Muestra clínica Procesar las muestras clínicas en función de sus propiedades físico-químicas. Filtrar o centrifugar líquidos para eliminar las impurezas grandes. Resuspender muestras sólidas. Para utilizar los tejidos, se muelen, por ejemplo mediante el uso de un homogeneiser Dounce o perlas de vidrio, y lisar las células para liberar las bacterias intracelulares. </li></ol> 1.2 Preparación Amebas <ol><li> Preparación Caldo Prepare los siguientes medios: a ricos medios que contienen peptona, extracto de levadura y glucosa (PYG; 100 g de peptona proteasa, 10 g de extracto de levadura, 4,9 g de MgSO4 • 7H 2O, 5 g de citrato de sodio • 2H 2 O, 0,1 g de Fe (NH4) 2 (SO4) 2 • 6H 2 O, 1,7 g de KH 2 PO 4, 1,97 g de Na 2 HPO 4 • 7H 2 O , 45 g de glucosa, y 0,295 g de CaCl2 en 5 L de agua destilada) y un medio no nutritivo tales como el PAS para las especies de Acanthamoeba. </li><li> Cultura amebas <ol><li> Cultivar amebas (preferentemente Acanthamoeba castellanii ATCC 30010 o A. polyphaga Linc-AP1) a 25 ° C en frascos de cultivo de células que contienen 30 ml de medio PYG. </li><li> Se recoge el amebas por agitación vigorosa del matraz y se centrifuga la suspensión celular durante 10 min a 1500 x g. Lavar el pellet dos veces con medio PAS. Contar las células en un portaobjetos Kova y ajustar el volumen para obtener una suspensión de 5 x 10 5 células por ml. </li><li> Transferir la suspensión de amebas en microplacas. Utilice 1 ml por pocillo de 12 - y 24-wplacas de ELL, 500 l de placas de 48 pocillos y 300 l de placas de 96 pocillos. Incubar la microplaca durante al menos 2 horas a 25 ° C. Esto permite la sedimentación y la fijación de las amebas a la parte inferior de cada pocillo. </li></ol></li></ol> 1.3 Coculture <ol><li> La inoculación de la muestra <ol><li> Inocular la placa de 2.3.3 con diluciones seriadas de la muestra de 1,1 o 1,2, por lo general con la serie de dilución de 10 veces, comenzando con 100 l de muestra no diluida. </li><li> Centrifugar la microplaca a 1800 xg durante 10 min a sedimentos en el césped de amebas los microorganismos potencialmente presentes en la muestra. Esto aumenta el contacto y la fagocitosis de los microorganismos por amebas. </li><li> Las placas se incuban durante 45 minutos a 25 ° C y lavar tres veces con PAS mediante la sustitución de los medios de comunicación con el PAS fresco. Añadir 1 ml por pocillo de PAS con o sin adición de antibióticos (estreptomicina, penicilina, gentamicina y / o vancomicina), dependiendo de las bacteriuriacol especies que se buscan. </li><li> Incubar la microplaca a 32 ° C en una atmósfera humidificada para evitar el enquistamiento de las amebas. Observar cada pocillo a diario con un objetivo 20X para detectar la presencia de bacterias invasoras y de lisis amebas. </li><li> En el caso de la lisis, realizar un subcultivo en amebas fresco mediante la inoculación de 100 l de cocultivos a una monocapa de aproximadamente 10 5 amebas / cm 2. Para aislar específicamente una determinada especie bacteriana, inocular también medios de agar específico diseñado para estas bacterias (por ejemplo, agar BCYE por Legionella spp.). </li><li> En ausencia de lisis, tome 100 l de cocultivos cuatro a siete días después de la primera inoculación y inocular un cultivo fresco de amebas de 900 l en una placa de 24 pocillos. Si se observa la lisis rápida de las amebas sin proliferación de bacterias, un virus puede estar presente. En este caso, se filtra el sobrenadante a 0,22 micras y utilizar la suspensión se filtró para infectar amebas fresco. </li></ol&gt; </li></ol> 1.4 Aislamiento bacteriano y caracterización <ol><li> Tinción bacteriana Realice cocultivos directamente en cubreobjetos de vidrio en 24-así microplacas. Retire el medio y llevar a cabo la tinción o inmunofluorescencia. <ol><li> Tinción de Romanowsky Modificado <ol><li> Deje que el portaobjetos seco. Sumergir el cubreobjetos cinco veces en la solución de fijación (2 mg / L de Fast Green en metanol). </li><li> Sumergir el cubreobjetos cinco veces en la solución de tinción I (1,22 g / L de Eosina G en tampón fosfato pH 6.6) </li><li> Finalmente, sumerja el cubreobjetos 5 veces en la solución de tinción II (1,1 g / L de tiazina en tampón fosfato pH 6,6). </li><li> Enjuague la muestra con agua destilada. Dejar secar y observar por microscopía. </li></ol></li><li> La tinción de Ziehl-Neelsen <ol><li> Deje que el portaobjetos seco. Cubra la muestra con Ziehl fucsina. Calentar el tinte con una llama hasta que aparezcan vapores. </li><li> Enfriar a temperatura ambiente durante al menos 5 miny enjuague con agua destilada. Cubra la muestra con solución de ácido clorhídrico al 3% en isopropanol durante 2 minutos y enjuagar con agua destilada. </li><li> Cubra durante 30 segundos con azul de metileno y enjuague con agua destilada. Dejar secar y observar por microscopía 22. </li></ol></li><li> Tinción Gimenez <ol><li> Preparar una solución de fucsina básica de stock mediante la mezcla de 100 ml de 10% fucsina básica (10 g de fucsina básica en 100 ml de etanol al 95%), fenol acuosa 250 ml de 4% y 650 ml de agua destilada. Incubar a 37 ° C durante 48 horas antes de su uso. </li><li> Deje que el portaobjetos se seque y fije haciéndolo pasar a través de la llama. Cubra la muestra con fucsina básica recién filtrada (4 ml de solución de fucsina básica de stock en 10 ml de 0,1 M de tampón de fosfato de sodio, pH 7,45) durante 2 min. </li><li> Enjuague la muestra con agua y se incuban en verde de malaquita (0,8% en agua destilada) durante 10 seg. Enjuagar nuevamente con agua y repita la tinción de verde malaquita. Enjuagar nuevamente con agua. Deje que el cubreobjetos secos, mporte, y observar por microscopía 23. </li></ol></li><li> Inmunofluorescencia <ol><li> Fijar el cubreobjetos incubando en metanol durante 5 min o con paraformaldehído al 4% durante 10 min. </li><li> Lavar tres veces con PBS y se incuba durante 2 horas en solución de bloqueo (5% de BSA, 0,1% de saponina en PBS) a temperatura ambiente. </li><li> Incubar el portaobjetos durante 1 hora en solución de bloqueo que contiene anticuerpos producidos contra el microorganismo de interés. </li><li> Lavar de nuevo tres veces en PBS e incubar durante 1 hora con un anticuerpo secundario dirigido contra el anticuerpo primario y enlazada a un fluoróforo. Lavar tres veces con PBS, montar el cubreobjetos y observar por microscopía de fluorescencia. </li></ol></li></ol></li><li> La detección de ADN por PCR Extracto de ADN de 100 a 200 l de cocultivo de amebas. Detectar microorganismos con cebadores universales dirigidos al gen 16S rRNA o cebadores específicos para especies de interés tales como micobacterias 24, Legionella 25 o los miembros de la 26 Chlamydiales. </li></ol> 2. Enriquecimiento amebas 2.1. Preparación de la muestra Resuspender muestras sólidas y semi-sólidos en el PAS por agitación. Centrifugar la suspensión a baja velocidad (180 xg) durante 10 min. Esto permite el enriquecimiento de las amebas de vida libre en el sedimento. El sobrenadante puede ser utilizado para el cocultivo de amebas y el sedimento para el enriquecimiento de amebas 21. 2.2. Preparación Mediana <ol><li> Añadir 1,5 g de agar a 100 ml de PAS y autoclave el medio 15 minutos a 121 ° C. Verter el medio caliente en placas de Petri y dejar que se solidifique a temperatura ambiente. </li><li> Crecer Escherichia coli (ATCC 25922) en LB o caldo de tioglicolato la noche a 37 ° C. Lavar las bacterias dos veces con PBS y resuspender en medio de PAS. Diluir 10 veces en PAS y difundir 2-3 ml de esta dilución en una placa de agar PAS y dejar secar. </li></ol> Añadir una gota de muestra (o un trozo de filtro) en un lado de la placa y dejar que fluya sobre la placa de Petri para formar una línea en el centro del plato. 2.4. Crecimiento amebas amebas y la subcultura <ol><li> Observar la placa de Petri diaria. Si se detecta un frente de migración amebas, cortar un pequeño trozo de agar en el frente de migración e inocular un fresco plato de Petri NNA cubierto con un césped de E. coli. </li><li> Repita la reinoculación varias veces dependiendo de la pureza de la muestra, con el fin de tener un cultivo puro de una cepa de amebas dado. </li></ol> 2.5. Las amebas y bacterias caracterización <ol><li> Raspar las células y resuspender en PAS. </li><li> Extraer el ADN y determinar la identidad de las amebas y / o endosimbiontes bacterianos por PCR y secuenciación (amplificación de 16S rRNA para las bacterias, resp. 18S ARNr de las amebas y secuenciación, por ejemplo). </li> &lt;li&gt; Utilice estas células tomadas por raspado en PAS para inocular amebas nuevo y hacer una co-cultivo de amebas para detectar bacterias posiblemente presentes en la muestra. </li></ol>

<ol>
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Dis. 9, 815-821 (2003).</li><li>Collingro, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recovery+of+an+environmental+Chlamydia+strain+from+activated+sludge+by+co-cultivation+with+Acanthamoeba+sp.">Recovery of an environmental Chlamydia strain from activated sludge by co-cultivation with Acanthamoeba sp.</a> Microbiology. 151, 301-309 (2005).</li><li>Lienard, J., Croxatto, A., Prod'hom, G., Greub, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estrella+lausannensis,+a+new+star+in+the+Chlamydiales+order.">Estrella lausannensis, a new star in the Chlamydiales order.</a> Microbes Infect. 13, 1232-1241 (2011).</li><li>La Scola, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+giant+virus+in+amoebae.">A giant virus in amoebae.</a> Science. 299, 2033 (2003).</li><li>Boyer, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Giant+Marseillevirus+highlights+the+role+of+amoebae+as+a+melting+pot+in+emergence+of+chimeric+microorganisms.">Giant Marseillevirus highlights the role of amoebae as a melting pot in emergence of chimeric microorganisms.</a> Proc. Natl. Acad. Sci. U.S.A. 106, 21848-21853 (2009).</li><li>Thomas, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lausannevirus,+a+giant+amoebal+virus+encoding+histone+doublets.">Lausannevirus, a giant amoebal virus encoding histone doublets.</a> Environ. Microbiol. 13, 1454-1466 (2011).</li><li>Raoult, D., Renesto, P., Brouqui, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laboratory+infection+of+a+technician+by+mimivirus.">Laboratory infection of a technician by mimivirus.</a> Ann. Internal Med. 144, 702-703 (2006).</li><li>Greub, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parachlamydia+acanthamoebae,+an+emerging+agent+of+pneumonia.">Parachlamydia acanthamoebae, an emerging agent of pneumonia.</a> Clin. Microbiol. Infect. 15, 18-28 (2009).</li><li>Lamoth, F., Greub, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amoebal+pathogens+as+emerging+causal+agents+of+pneumonia.">Amoebal pathogens as emerging causal agents of pneumonia.</a> FEMS Microbiol. Rev. 34, 260-280 (2010).</li><li>Lienard, J. G., Ashbolt, K., Sen, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ch.+6.">Ch. 6.</a> Environmental microbiology, current technology and water applications. , 143-162 (2011).</li><li>Boughalmi, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+isolation+of+giant+viruses+of+the+Mimiviridae+and+Marseilleviridae+families+in+the+Tunisian+environment.">High-throughput isolation of giant viruses of the Mimiviridae and Marseilleviridae families in the Tunisian environment.</a> Environ. Microbiol. , (2012).</li><li>Ovrutsky, A. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cooccurrence+of+Free-Living+Amoebae+and+Nontuberculous+Mycobacteria+in+Hospital+Water+Networks,+and+Preferential+Growth+of+Mycobacterium+avium+in+Acanthamoeba+lenticulata.">Cooccurrence of Free-Living Amoebae and Nontuberculous Mycobacteria in Hospital Water Networks, and Preferential Growth of Mycobacterium avium in Acanthamoeba lenticulata.</a> Appl. Environ. Microbiol. 79, 3185-3192 (2013).</li></ol>

Los autores declaran que no tienen intereses financieros en competencia.

Cocultivo amebas y enriquecimiento amebas son métodos eficientes que permitieron el aislamiento de muchas nuevas especies de bacterias y amebas. Los resultados obtenidos con estos métodos confirman la presencia ubicua de los dos amebas y bacterias ameba-resistencia en el medio ambiente, y lo más interesante de las redes de agua artificiales que se consideran para ser controlada por tratamientos químicos tales como cloración y ozonización. Cocultivo amebas y enriquecimiento amebas son herramientas esenciales para a...

Antes de la llegada del diagnóstico molecular, los microorganismos presentes en los nichos ambientales o en las muestras clínicas se detectan generalmente mediante su cultivo en diferentes medios selectivos, principalmente en agar en placas de Petri. El fenotipo de las colonias bacterianas y su actividad metabólica después se dejó clasificación bacteriana a nivel de especies. Caldo también puede utilizarse para aumentar la sensibilidad de detección. Sin embargo, ambas técnicas no permiten la recuperación de bacterias que crecen lentamente o en absoluto en estos medios. Esta es la razón por qué los enfoques moleculares se usan tan ampliamente hoy en día. Sin embargo, la detección de ADN no proporciona ninguna pista sobre la viabilidad de las bacterias. Por otra parte, contrariamente a la cultura, enfoques moleculares no dan lugar a una cepa que puede caracterizarse adicionalmente. El estudio de los patógenos que crecen pobremente en medios sólidos o que las células necesitan para crecer es complicado. La mayoría de estos &quot;difíciles de cultivar&quot; las bacterias son intr fastidiosobacterias acelulares, a menudo descubiertas y caracterizadas siguiente brotes grandes como lo fue el caso de la Legionella pneumophila. Esta bacteria se caracterizó después de un brote que se produjo durante una convención de la Legión Americana. Nada menos que 182 personas fueron infectadas y 29 murieron a causa de una neumonía grave 1,2. Se demostró más tarde que las amebas son los huéspedes naturales de esta bacteria y que su presencia en las redes hotel de sistemas de aire acondicionado y el agua estaba en el origen del brote de la enfermedad de la llamada del legionario 3. Las amebas están presentes en todo el mundo y fueron aisladas de suelo, el aire, el agua y la mucosa nasal de voluntarios humanos (revisado en 4). Estas amebas &quot;vida libre&quot; generalmente se divide de forma autónoma en el medio ambiente, pero en ocasiones puede invadir anfitriones permisivas 5. Las amebas de alimentación en varios microorganismos a través de la fagocitosis y la posterior digestión lisosomal por Hydrolases 6. Muchas bacterias intracelulares facultativas o obligar son capaces de resistir la digestión y así infectar y dividir en amebas como por ejemplo bacterias o micobacterias relacionadas con clamidia Legionella, (revisado en 7 y 8). Amebas de vida libre, probablemente, representan un importante reservorio potencial de bacterias intracelulares que aún no han sido descubiertos. Esto llevó a nuestro grupo para implementar en Lausana dos técnicas principales, llamados co-cultivo de amebas amebas y enriquecimiento, lo que permitió los diferentes grupos para aislar varios nuevos microorganismos intracelulares obligados de diversas muestras ambientales 9-15. Desde amebas son fagocitos profesionales que pastan en las bacterias, una bacteria capaz de resistir la fagocitosis y de crecer dentro de estos protistas también puede colonizar los fagocitos humanos y ser patógeno para los seres humanos. Esto fue parcialmente demostrada por algunas bacterias relacionadas con la clamidia, como Waddlia chondrophila. W. chondrophila puede crecer no sólo en las amebas, sino también en varios tipos de células, tales como células de mamífero epiteliales, macrófagos, y las líneas celulares de peces 16-18. El cocultivo de amebas también aparece relevante para la detección de bacterias intracelulares en muestras clínicas 19,20, incluyendo las heces, que están fuertemente contaminados con diferentes especies bacterianas 21. Aquí se describen los pasos principales de cocultivo de amebas y el enriquecimiento de amebas, incluyendo (a) el tratamiento de muestras ambientales o clínicos; (b) el crecimiento de las amebas en medios axénicos y en un césped bacteriano de Escherichia coli y (c) la selección y caracterización de bacterias intracelulares.

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent/ Equipment</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr>
			<td>Glucose monohydrate</td>
			<td>Merck, Darmstadt, Germany</td>
			<td>108342</td>
			<td></td>
		</tr>
		<tr>
			<td>0.22 &#956;m pore size membrane</td>
			<td>Merck Millipore, Darmstadt, Germany</td>
			<td>SCVPU11RE</td>
			<td></td>
		</tr>
		<tr>
			<td>proteose peptone</td>
			<td>Becton-Dickinson, Franklin Lakes, NJ</td>
			<td>211693</td>
			<td></td>
		</tr>
		<tr>
			<td>yeast extract</td>
			<td>Becton-Dickinson, Franklin Lakes, NJ</td>
			<td>212750</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture flasks</td>
			<td>Becton-Dickinson, Franklin Lakes, NJ</td>
			<td>353135</td>
			<td></td>
		</tr>
		<tr>
			<td>Kova slide</td>
			<td>Hycor, Indianapolis, IN</td>
			<td>87144</td>
			<td></td>
		</tr>
		<tr>
			<td>cell culture microplates</td>
			<td>Corning Inc, Corning, NY</td>
			<td>3524</td>
			<td></td>
		</tr>
		<tr>
			<td>Diff-Quik staining kit</td>
			<td>Siemens Healthcare diagn., Munich, Germany</td>
			<td>130832</td>
			<td></td>
		</tr>
		<tr>
			<td>Ziehl fuchsin</td>
			<td>Fluka, St-Louis, MI</td>
			<td>21820</td>
			<td></td>
		</tr>
		<tr>
			<td>basic fuchsin</td>
			<td>Sigma, St-Louis, MI</td>
			<td>857843</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenol</td>
			<td>Sigma, St-Louis, MI</td>
			<td>P1037</td>
			<td>Corrosive and mutagenic</td>
		</tr>
		<tr>
			<td>malachite green oxalate</td>
			<td>Fluka, St-Louis, MI</td>
			<td>63160</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde 16% solution</td>
			<td>Electron Microscopy Sciences, Hatfield, PA</td>
			<td>15710</td>
			<td></td>
		</tr>
		<tr>
			<td>Saponin</td>
			<td>Sigma, St-Louis, MI</td>
			<td>84510</td>
			<td></td>
		</tr>
	</tbody>
</table>

discovery of new intracellular pathogens by amoebal coculture and amoebal enrichment approaches

Los patógenos intracelulares tales como legionella, micobacterias y organismos Chlamydia como son difíciles de aislar porque a menudo crecen poco o nada en absoluto en un medio selectivo que por lo general se utilizan para cultivar bacterias. Por esta razón, muchos de estos patógenos fueron descubiertas sólo recientemente o después de los brotes importantes. Estos patógenos se asocian a menudo con las amebas, que sirven como la célula huésped y permitir la supervivencia y el crecimiento de las bacterias. Pretendemos aquí para proporcionar una demostración de dos técnicas que permiten el aislamiento y caracterización de patógenos intracelulares presentes en muestras clínicas o ambientales: el co-cultivo de amebas y el enriquecimiento de amebas. Cocultivo de amebas permite la recuperación de bacterias intracelulares mediante la inoculación de la muestra investigada en un césped de amebas que puede ser infectado y se lisaron por las bacterias intracelulares presentes en la muestra. Enriquecimiento de amebas permite la recuperación de las amebas presente en una muestra clínica o ambiental. Thse puede conducir al descubrimiento de nuevas especies de amebas, pero también de nuevas bacterias intracelulares que crecen específicamente en estas amebas. Juntas, estas dos técnicas ayudan a descubrir nuevas bacterias intracelulares capaces de crecer en las amebas. Debido a su capacidad para infectar las amebas y resistir la fagocitosis, estas bacterias intracelulares también pueden escapar de la fagocitosis por los macrófagos y, por tanto, ser patógenos para los eucariotas superiores.

Uso de cocultivo de amebas y el enriquecimiento de amebas, toda una gama de bacterias del medio ambiente y / o patógenos se descubrió (Tabla 1). Cocultivo amebas fue utilizado por nuestro grupo y otros para analizar las muestras ambientales, plantas de tratamiento de agua y sistemas de distribución de agua. Una amplia gama de microorganismos puede ser aislado con esta técnica. Las bacterias más comunes aisladas por cocultivo amebas son miembros del género Mycobacte...

Watch this Scientific Journal Video about Discovery of New Intracellular Pathogens by Amoebal Coculture and Amoebal Enrichment Approaches at JoVE.com

Discovery of New Intracellular Pathogens by Amoebal Coculture and Amoebal Enrichment Approaches

Cocultivo de amebas es un sistema de cultivo celular usando amebas adherente a crecer selectivamente a los patógenos intracelulares capaces de resistir las células fagocíticas tales como amebas y macrófagos. Por lo tanto, representa una herramienta clave para descubrir nuevos agentes infecciosos. Enriquecimiento amebas permite el descubrimiento de nuevas especies de amebas y de sus bacterias intracelulares específicos.

Descubrimiento de nuevos patógenos intracelulares por amebas Coculture y Enfoques amebas Enriquecimiento

Intracellular pathogens such as legionella, mycobacteria and Chlamydia-like organisms are difficult to isolate because they often grow poorly or not at ...

discovery-new-intracellular-pathogens-amoebal-coculture-amoebal

Research

JoVE Journal

Immunology and Infection

14.4K vistas.  University Hospital Center and University of Lausanne.  Cocultivo de amebas es un sistema de cultivo celular usando amebas adherente a crecer selectivamente a los patógenos intracelulares capaces de resistir las células fagocíticas tales como amebas y macrófagos. Por lo tanto, representa una herramienta clave para descubrir nuevos agentes infecciosos. Enriquecimiento amebas permite el descubrimiento de nuevas especies de amebas y de sus bacterias intracelulares específicos. 

Video: Discovery of New Intracellular Pathogens by Amoebal Coculture and Amoebal Enrichment Approaches

Infection of Zebrafish Embryos with Intracellular Bacterial Pathogens

Zebrafish (Danio rerio) embryos are increasingly used as a model for studying the function of the vertebrate innate immune system in host-pathogen interactions 1. The major cell types of the innate immune system, macrophages and neutrophils, develop during the first days of embryogenesis prior to the maturation of lymphocytes that are required for adaptive immune responses. The ease of obtaining large numbers of embryos, their accessibility due to external development, the optical transparency of embryonic and larval stages, a wide range of genetic tools, extensive mutant resources and collections of transgenic reporter lines, all add to the versatility of the zebrafish model. Salmonella enterica serovar Typhimurium (S. typhimurium) and Mycobacterium marinum can reside intracellularly in macrophages and are frequently used to study host-pathogen interactions in zebrafish embryos. The infection processes of these two bacterial pathogens are interesting to compare because S. typhimurium infection is acute and lethal within one day, whereas M. marinum infection is chronic and can be imaged up to the larval stage 2, 3. The site of micro-injection of bacteria into the embryo (Figure 1) determines whether the infection will rapidly become systemic or will initially remain localized. A rapid systemic infection can be established by micro-injecting bacteria directly into the blood circulation via the caudal vein at the posterior blood island or via the Duct of Cuvier, a wide circulation channel on the yolk sac connecting the heart to the trunk vasculature. At 1 dpf, when embryos at this stage have phagocytically active macrophages but neutrophils have not yet matured, injecting into the blood island is preferred. For injections at 2-3 dpf, when embryos also have developed functional (myeloperoxidase-producing) neutrophils, the Duct of Cuvier is preferred as the injection site. To study directed migration of myeloid cells towards local infections, bacteria can be injected into the tail muscle, otic vesicle, or hindbrain ventricle 4-6. In addition, the notochord, a structure that appears to be normally inaccessible to myeloid cells, is highly susceptible to local infection 7. A useful alternative for high-throughput applications is the injection of bacteria into the yolk of embryos within the first hours after fertilization 8. Combining fluorescent bacteria and transgenic zebrafish lines with fluorescent macrophages or neutrophils creates ideal circumstances for multi-color imaging of host-pathogen interactions. This video article will describe detailed protocols for intravenous and local infection of zebrafish embryos with S. typhimurium or M. marinum bacteria and for subsequent fluorescence imaging of the interaction with cells of the innate immune system.

Transparent zebrafish embryos have proved useful model hosts to visualize and functionally study interactions between innate immune cells and intracellular bacterial pathogens, such as Salmonella typhimurium and Mycobacterium marinum. Micro-injection of bacteria and multi-color fluorescence imaging are essential techniques involved in the application of zebrafish embryo infection models.

La infección de embriones de pez cebra con patógenos bacterianos intracelulares

Zebrafish (Danio rerio) embryos are increasingly used as a model for studying the function of the vertebrate innate immune system in host-pathogen ...

Investigación

Inmunología e Infección

Single Cell Measurements of Vacuolar Rupture Caused by Intracellular Pathogens

Shigella flexneri are pathogenic bacteria that invade host cells entering into an endocytic vacuole. Subsequently, the rupture of this membrane-enclosed compartment allows bacteria to move within the cytosol, proliferate and further invade neighboring cells. Mycobacterium tuberculosis is phagocytosed by immune cells, and has recently been shown to rupture phagosomal membrane in macrophages. We developed a robust assay for tracking phagosomal membrane disruption after host cell entry of Shigella flexneri or Mycobacterium tuberculosis. The approach makes use of CCF4, a FRET reporter sensitive to &beta;-lactamase that equilibrates in the cytosol of host cells. Upon invasion of host cells by bacterial pathogens, the probe remains intact as long as the bacteria reside in membrane-enclosed compartments. After disruption of the vacuole, &beta;-lactamase activity on the surface of the intracellular pathogen cleaves CCF4 instantly leading to a loss of FRET signal and switching its emission spectrum. This robust ratiometric assay yields accurate information about the timing of vacuolar rupture induced by the invading bacteria, and it can be coupled to automated microscopy and image processing by specialized algorithms for the detection of the emission signals of the FRET donor and acceptor. Further, it allows investigating the dynamics of vacuolar disruption elicited by intracellular bacteria in real time in single cells. Finally, it is perfectly suited for high-throughput analysis with a spatio-temporal resolution exceeding previous methods. Here, we provide the experimental details of exemplary protocols for the CCF4 vacuolar rupture assay on HeLa cells and THP-1 macrophages for time-lapse experiments or end points experiments using Shigella flexneri as well as multiple mycobacterial strains such as Mycobacterium marinum, Mycobacterium bovis, and Mycobacterium tuberculosis.

We describe a method for tracking the endomembrane rupture elicited by the intracellular bacteria Shigella flexneri and Mycobacterium tuberculosis upon host cell invasion. Our assay makes use of CCF4, a host cytoplasmic FRET probe in live or fixed cells. This reporter is degraded by an enzyme activity present on the bacterial surface.

Sola célula Medidas de Ruptura Vacuolar causadas por patógenos intracelulares

Shigella flexneri are pathogenic bacteria that invade host cells entering into an endocytic vacuole. Subsequently, the rupture of this membrane-enclosed ...

Monitoring Changes in Membrane Polarity, Membrane Integrity, and Intracellular Ion Concentrations in Streptococcus pneumoniae Using Fluorescent Dyes

Membrane depolarization and ion fluxes are events that have been studied extensively in biological systems due to their ability to profoundly impact cellular functions, including energetics and signal transductions. While both fluorescent and electrophysiological methods, including electrode usage and patch-clamping, have been well developed for measuring these events in eukaryotic cells, methodology for measuring similar events in microorganisms have proven more challenging to develop given their small size in combination with the more complex outer surface of bacteria shielding the membrane. During our studies of death-initiation in Streptococcus pneumoniae (pneumococcus), we wanted to elucidate the role of membrane events, including changes in polarity, integrity, and intracellular ion concentrations. Searching the literature, we found that very few studies exist. Other investigators had monitored radioisotope uptake or equilibrium to measure ion fluxes and membrane potential and a limited number of studies, mostly in Gram-negative organisms, had seen some success using carbocyanine or oxonol fluorescent dyes to measure membrane potential, or loading bacteria with cell-permeant acetoxymethyl (AM) ester versions of ion-sensitive fluorescent indicator dyes. We therefore established and optimized protocols for measuring membrane potential, rupture, and ion-transport in the Gram-positive organism S. pneumoniae. We developed protocols using the bis-oxonol dye DiBAC4(3) and the cell-impermeant dye propidium iodide to measure membrane depolarization and rupture, respectively, as well as methods to optimally load the pneumococci with the AM esters of the ratiometric dyes Fura-2, PBFI, and BCECF to detect changes in intracellular concentrations of Ca2+, K+, and H+, respectively, using a fluorescence-detection plate reader. These protocols are the first of their kind for the pneumococcus and the majority of these dyes have not been used in any other bacterial species. Though our protocols have been optimized for S. pneumoniae, we believe these approaches should form an excellent starting-point for similar studies in other bacterial species.

Monitoring Changes in Membrane Polarity, Membrane Integrity, and Intracellular Ion Concentrations in Streptococcus pneumoniae Using Fluorescent Dyes

Unlike that seen for eukaryotes, there is a paucity of studies that detail membrane depolarization and ion concentration changes in bacteria, primarily as their small size makes conventional methods of measurement difficult. Here, we detail protocols for monitoring such events in the significant Gram-positive pathogen Streptococcus pneumoniae utilizing fluorescence techniques.

Vigilar los cambios en la polaridad de la membrana, membrana Integridad, e intracelular de iones concentraciones en<em&gt; Streptococcus pneumoniae</em&gt; Uso de tintes fluorescentes

Membrane depolarization and ion fluxes are events that have been studied extensively in biological systems due to their ability to profoundly impact ...

Application of Fluorescent Nanoparticles to Study Remodeling of the Endo-lysosomal System by Intracellular Bacteria

Fluorescent nanoparticles (NPs) with desirable chemical, optical and mechanical properties are promising tools to label intracellular organelles. Here, we introduce a method using gold-BSA-rhodamine NPs to label the endo-lysosomal system of eukaryotic cells and monitor manipulations of host cellular pathways by the intracellular pathogen Salmonella enterica. The NPs were readily internalized by HeLa cells and localized in late endosomes/lysosomes. Salmonella infection induced rearrangement of the vesicles and accumulation of NPs in Salmonella-induced membrane structures. We deployed the Imaris software package for quantitative analyses of confocal microscopy images. The number of objects and their size distribution in non-infected cells were distinct from the ones in Salmonella-infected cells, indicating extremely remodeling of the endo-lysosomal system by WT Salmonella.

This article describes methods for the synthesis and fluorescent labeling of nanoparticles (NPs). The NPs were applied in pulse-chase experiments to label the endo-lysosomal system of eukaryotic cells. Manipulation of the endo-lysosomal system by activities of the intracellular pathogen Salmonella enterica were followed by live cell imaging and quantified.

Aplicación de nanopartículas fluorescentes para estudiar Remodelación del Sistema Endo-lisosomal por bacterias intracelulares

Fluorescent nanoparticles (NPs) with desirable chemical, optical and mechanical properties are promising tools to label intracellular organelles. Here, we ...

Biolistic Transformation of a Fluorescent Tagged Gene into the Opportunistic Fungal Pathogen Cryptococcus neoformans

The basidiomycete Cryptococcus neoformans, an invasive opportunistic pathogen of the central nervous system, is the most frequent cause of fungal meningitis worldwide resulting in more than 625,000 deaths per year worldwide.&#160;Although electroporation has been developed for the transformation of plasmids in Cryptococcus, only&#160;biolistic&#160;delivery provides&#160;an effective means to transform&#160;linear&#160;DNA&#160;that can be integrated into the genome by homologous recombination.&#160;
Acetate has been shown to be a major fermentation product during cryptococcal infection, but the significance of this is not yet known.&#160;A bacterial pathway composed of the enzymes xylulose-5-phosphate/fructose-6-phosphate phosphoketolase (Xfp) and acetate kinase (Ack) is one of three potential pathways for acetate production in C. neoformans. Here, we demonstrate the biolistic transformation of a construct, which has the gene encoding Ack fused to the fluorescent tag mCherry, into&#160;C. neoformans. We then confirm integration of the ACK-mCherry fusion into the&#160;ACK&#160;locus.

Biolistic Transformation of a Fluorescent Tagged Gene into the Opportunistic Fungal Pathogen Cryptococcus neoformans

Biolistic transformation is a method used to generate stable integration of DNA into the genome of the opportunistic pathogen Cryptococcus neoformans through homologous recombination. We will demonstrate biolistic transformation of a construct, which has the gene encoding acetate kinase fused to the fluorescent tag mCherry into&#160;C. neoformans.

Transformación biolística de un fluorescente Tagged gen en el oportunista por hongos patógenos<em&gt; Cryptococcus neoformans</em

The basidiomycete Cryptococcus neoformans, an invasive opportunistic pathogen of the central nervous system, is the most frequent cause of fungal ...

Identification of Rare Bacterial Pathogens by 16S rRNA Gene Sequencing and MALDI-TOF MS

There are a number of rare and, therefore, insufficiently described bacterial pathogens which are reported to cause severe infections especially in immunocompromised patients. In most cases only few data, mostly published as case reports, are available which investigate the role of such pathogens as an infectious agent. Therefore, in order to clarify the pathogenic character of such microorganisms, it is necessary to conduct epidemiologic studies which include large numbers of these bacteria. The methods used in such a surveillance study have to meet the following criteria: the identification of the strains has to be accurate according to the valid nomenclature, they should be easy to handle (robustness), economical in routine diagnostics and they have to generate comparable results among different laboratories. Generally, there are three strategies for identifying bacterial strains in a routine setting: 1) phenotypic identification characterizing the biochemical and metabolic properties of the bacteria, 2) molecular techniques such as 16S rRNA gene sequencing and 3) mass spectrometry as a novel proteome based approach. Since mass spectrometry and molecular approaches are the most promising tools for identifying a large variety of bacterial species, these two methods are described. Advances, limitations and potential problems when using these techniques are discussed.

Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) and molecular techniques (16S rRNA gene sequencing) permit the identification of rare bacterial pathogens in routine diagnostics. The goal of this protocol lies in the combination of both techniques which leads to more accurate and reliable data.

La identificación de patógenos bacterianos raras por 16S rRNA gen secuenciación y MALDI-TOF MS

There are a number of rare and, therefore, insufficiently described bacterial pathogens which are reported to cause severe infections especially in ...

High-throughput Detection of Respiratory Pathogens in Animal Specimens by Nanoscale PCR

Nanoliter scale real-time PCR uses spatial multiplexing to allow multiple assays to be run in parallel on a single plate without the typical drawbacks of combining reactions together. We designed and evaluated a panel based on this principle to rapidly identify the presence of common disease agents in dogs and horses with acute respiratory illness. This manuscript describes a nanoscale diagnostic PCR workflow for sample preparation, amplification, and analysis of target pathogen sequences, focusing on procedures that are different from microliter scale reactions. In the respiratory panel presented, 18 assays were each set up in triplicate, accommodating up to 48 samples per plate. A universal extraction and pre-amplification workflow was optimized for high-throughput sample preparation to accommodate multiple matrices and DNA and RNA based pathogens. Representative data are presented for one RNA target (influenza A matrix) and one DNA target (equine herpesvirus 1). The ability to quickly and accurately test for a comprehensive, syndrome-based group of pathogens is a valuable tool for improving efficiency and ergonomics of diagnostic testing and for acute respiratory disease diagnosis and management.

High-throughput testing of DNA and RNA based pathogens by nanoscale PCR is described using a syndromic canine and equine respiratory PCR panel.

La detección de alto rendimiento de los patógenos respiratorios de especímenes animales por PCR nanoescala

Nanoliter scale real-time PCR uses spatial multiplexing to allow multiple assays to be run in parallel on a single plate without the typical drawbacks of ...

Visualizing Intracellular SNARE Trafficking by Fluorescence Lifetime Imaging Microscopy

Soluble N-ethylmaleimide sensitive fusion protein (NSF) attachment protein receptor (SNARE) proteins are key for membrane trafficking, as they catalyze membrane fusion within eukaryotic cells. The SNARE protein family consists of about 36 different members. Specific intracellular transport routes are catalyzed by specific sets of 3 or 4 SNARE proteins that thereby contribute to the specificity and fidelity of membrane trafficking. However, studying the precise function of SNARE proteins is technically challenging, because SNAREs are highly abundant and functionally redundant, with most SNAREs having multiple and overlapping functions. In this protocol, a new method for the visualization of SNARE complex formation in live cells is described. This method is based on expressing SNARE proteins C-terminally fused to fluorescent proteins and measuring their interaction by F&#246;rster resonance energy transfer (FRET) employing fluorescence lifetime imaging microscopy (FLIM). By fitting the fluorescence lifetime histograms with a multicomponent decay model, FRET-FLIM allows (semi-)quantitative estimation of the fraction of the SNARE complex formation at different vesicles. This protocol has been successfully applied to visualize SNARE complex formation at the plasma membrane and at endosomal compartments in mammalian cell lines and primary immune cells, and can be readily extended to study SNARE functions at other organelles in animal, plant, and fungal cells.

This protocol describes a new method allowing for the quantitative visualization of complex formation of SNARE proteins, based on F&#246;rster resonance energy transfer, and fluorescence lifetime imaging microscopy.

Visualizar el lazo intracelular del tráfico de por vida de fluorescencia microscopía de imágenes

Soluble N-ethylmaleimide sensitive fusion protein (NSF) attachment protein receptor (SNARE) proteins are key for membrane trafficking, as they catalyze ...

Zika Virus Specific Diagnostic Epitope Discovery

High-density peptide microarrays allow screening of more than six thousand peptides on a single standard microscopy slide. This method can be applied for drug discovery, therapeutic target identification, and developing of diagnostics. Here, we present a protocol to discover specific Zika virus (ZIKV) diagnostic peptides using a high-density peptide microarray. A human serum sample validated for ZIKV infection was incubated with a high-density peptide microarray containing the entire ZIKV protein translated into 3,423 unique 15 linear amino acid (aa) residues with a 14-aa residue overlap printed in duplicate. Staining with different secondary antibodies within the same array, we detected peptides that bind to Immunoglobulin M (IgM) and Immunoglobulin G (IgG) antibodies present in serum. These peptides were selected for further validation experiments. In this protocol, we describe the strategy followed to design, process, and analyze a high-density peptide microarray.

In this protocol, we describe a technique to discover Zika virus specific diagnostic peptides using a high-density peptide microarray. This protocol can readly be adapted for other emerging infectious diseases.

Zika Virus específico diagnóstico del epítopo descubrimiento

High-density peptide microarrays allow screening of more than six thousand peptides on a single standard microscopy slide. This method can be applied for ...

Human Colonoid Monolayers to Study Interactions Between Pathogens, Commensals, and Host Intestinal Epithelium

Human 3-dimensional (3D) enteroid or colonoid cultures derived from crypt base stem cells are currently the most advanced ex vivo model of the intestinal epithelium. Due to their closed structures and significant supporting extracellular matrix, 3D cultures are not ideal for host-pathogen studies. Enteroids or colonoids can be grown as epithelial monolayers on permeable tissue culture membranes to allow manipulation of both luminal and basolateral cell surfaces and accompanying fluids. This enhanced luminal surface accessibility facilitates modeling bacterial-host epithelial interactions such as the mucus-degrading ability of enterohemorrhagic E. coli (EHEC) on colonic epithelium. A method for 3D culture fragmentation, monolayer seeding, and transepithelial electrical resistance (TER) measurements to monitor the progress towards confluency and differentiation are described. Colonoid monolayer differentiation yields secreted mucus that can be studied by the immunofluorescence or immunoblotting techniques. More generally, enteroid or colonoid monolayers enable a physiologically-relevant platform to evaluate specific cell populations that may be targeted by pathogenic or commensal microbiota.

Here, we present a protocol to culture human enteroid or colonoid monolayers that have intact barrier function to study host epithelial-microbiota interactions at the cellular and biochemical level.

Monocapas de Colonoid humano para estudiar las interacciones entre patógenos, comensales y epitelio Intestinal Host

Human 3-dimensional (3D) enteroid or colonoid cultures derived from crypt base stem cells are currently the most advanced ex vivo model of the intestinal ...