Damos las gracias a los técnicos del laboratorio de Bacteriología del Centro Hospitalario Universitario de Lausana por su ayuda para poner en práctica las técnicas en el laboratorio.

Este protocolo ha sido desarrollado y validado después de los procesos de investigación y desarrollo y las normas éticas de nuestra institución antes de ser implementado como una herramienta rutinaria. 1 Preparación de un cultivo de sangre bacteriana Pellet por Amonio Procedimiento Cloruro de eritrocitos-lisis <ol><li> Preparación de una cultura positiva de sangre para su posterior centrifugación. <ol><li> Esterilizar la tapa hemocultivo. Añadir 70% de etanol en la tapa de la botella y quemarlo. NOTA: No realice este paso en una campana de flujo laminar. </li><li> Mover la botella a una campana de flujo laminar. Recoger 5 ml del cultivo de la sangre con una jeringa 20 G. </li><li> Añadir los 5 ml de cultivo de sangre en un tubo de centrífuga de 50 ml que contiene 45 ml de agua estéril (H 2 O) y mezclar la muestra. </li></ol></li><li> Preparación de un cultivo de sangre sedimento bacteriano por lisis centrifugación. <ol><li> Centrifugar la muestra a 1000 xg durante 10 min a TA. </li><li> Remove el sobrenadante (que contiene principalmente H 2 O y células de la sangre) con una bomba de vacío de laboratorio. </li><li> Resuspender el precipitado en 1 ml de cloruro de amonio casero solución de lisis (0,15 M NH 4 Cl, 1 mM KHCO3). Divida el sedimento por raspado y agitando con vórtex el tubo y centrifugar la muestra a 140 xg durante 10 min a TA. </li><li> Descartar el sobrenadante que contiene principalmente los escombros células rojas de la sangre. <ol><li> Si el sedimento se mantuvo hemorrágico, resuspender el precipitado en 2 ml de H destilada estéril y 2 0 para lisar los glóbulos rojos más residuales y para lavar el sedimento. Centrifugar la muestra a 140 xg durante 10 min a TA y desechar el sobrenadante. </li></ol></li><li> Resuspender el sedimento en 200 l de H 2 O </li></ol></li><li> Subcultura de cultivo de sangre en diversos medios de agar selectivo y no selectivo. <ol><li> Recoge 1 ml del cultivo de sangre con una jeringa de 20 G. </li><li> Inocular 1 gota de hemocultivo into cuatro cuadrantes en agar sangre, agar chocolate, agar McConkey y agar Schaedler. </li><li> Incubar a 37 ° C el agar sangre y placas de agar de chocolate en una atmósfera de 5% de CO 2, el agar McConkey en un ambiente normal y la placa de agar Schaedler en condiciones anaeróbicas. </li><li> Siga el crecimiento bacteriano en los distintos medios. </li><li> Compruebe la pureza bacteriana. </li></ol></li></ol> 2. identificación por MALDI-TOF MS <ol><li> La identificación directa del sedimento de sangre. <ol><li> Transferencia de 1 l de la pastilla de sangre en una placa de MALDI objetivo y dejar secar en una plataforma de calentamiento 42 ° C. </li><li> Superposición de la muestra con ácido fórmico 1 l 70% y dejar secar en una plataforma de calentamiento 42 ° C. </li><li> Superposición de la muestra con 1 l de matriz MALDI y dejar secar en una plataforma de calentamiento 42 ° C (solución de α-ciano-4-hidroxicinámico en acetonitrilo al 50%-un 2,5% de ácido trifluoroacético saturada). </li><li> Proceda a MALDI-TOF MS análisis con un sistema MALDI-TOF MS como se describe por los fabricantes. </li><li> Si la puntuación de identificación no es válido a nivel de especie (por ejemplo, con una puntuación &lt;2), lleve a cabo una extracción de proteínas de la muestra de sedimento de sangre. </li></ol></li><li> Identificación de la proteína después de la extracción del sedimento cultivo de sangre. <ol><li> Mezclar 20 l de la pastilla con 1 ml de etanol al 70% y se centrifuga a 13.000 xg durante 2 min a TA. </li><li> Descartar el sobrenadante y añadir 25 l de ácido fórmico al 70% y 25 l de acetonitrilo al 100% a la pastilla. Vortex vigorosamente para volver a suspender los pellets. Resuspender bolitas duras por romper hacia abajo con puntas de pipeta antes de vórtice. </li><li> Se centrifuga a 13.000 xg durante 2 minutos a temperatura ambiente. Transferencia de 1 l del sobrenadante (que contiene proteínas extraídas) en una placa de MALDI objetivo y dejar secar en una plataforma de calentamiento 42 ° C. Proceda como se describe en 2.1.2. </li></ol></li></ol> 3. bacteriana Identificación y Antibiótico Pruebas de Susceptibilidad con un Sistema Automatizado Microbiana <ol><li> Preparación de una suspensión bacteriana de identificación bacteriana y AST con un sistema automatizado microbiana. <ol><li> Preparar un tubo de plástico que contiene 3 ml de solución estéril de NaCl al 0,45% compatible con el sistema automatizado microbiana. </li><li> Añadir un volumen suficiente de la pastilla de cultivo de sangre en la solución de NaCl 0,45% para obtener una suspensión bacteriana correspondiente a una turbidez McFarland de 0,6 a 0,8. Medir la turbidez usando una máquina de densitómetro. NOTA: El volumen del sedimento bacteriano cultivo de sangre varía entre 50 y 200 l para lograr una turbidez McFarland de 0,6 a 0,8. </li></ol></li><li> Inocular las automatizados Gram-positivo (GP) o Gram-negativo (GN) tarjetas de identificación y las tarjetas AST automatizados para la prueba de susceptibilidad antimicrobiana de bacterias cocos Gram-positivos y Gram-negativos como se describe por el fabricante. NOTA: Identificación con GP o tarjetas GN no se aplica si MALDI-TOF MS valor de la puntuación de identificación es&gt; 2. Comprobar la pureza de la suspensión bacteriana mediante subcultivo la suspensión bacteriana en agar sangre (GP y GN) y agar MacConkey (GN) placas. </li></ol> 4. antibióticos Pruebas de Susceptibilidad por difusión con discos de ensayo El ensayo de difusión en disco es descrito por el Comité Europeo de las pruebas de sensibilidad a los antimicrobianos (EUCAST, Versión 3.0, abril de 2013, www.eucast.org). <ol><li> Preparación de una suspensión bacteriana para realizar pruebas de susceptibilidad antimicrobiana mediante el ensayo de difusión en disco. <ol><li> Preparar un tubo de vidrio que contiene 3 ml de solución estéril de NaCl al 0,9%. </li><li> Añadir un volumen suficiente de la pastilla de cultivo de sangre en la solución de NaCl al 0,9% para obtener una suspensión bacteriana correspondiente a una turbidez McFarland de 0,5. Medir la turbidez usando una máquina de densitómetro. NOTA: El volumen del cultivo de sangresedimento bacteriano varía entre 50 y 200 l para lograr una turbidez McFarland de 0,6 a 0,8. </li><li> Vórtex con la suspensión bacteriana. </li></ol></li><li> La inoculación de suspensión bacteriana sobre agar Mueller-Hinton (MH) o agar Mueller-Hinton agar-organismos fastidiosos (MHF) (MH suplementado con 5% de sangre desfibrinada de caballo y 20 mg / l de adenina dinucleótido nicotinamida-β). <ol><li> Sumergir un hisopo de algodón estéril en la suspensión bacteriana y retirar suavemente el exceso de fluido girando el hisopo en la pared interior del tubo de vidrio. </li><li> Corre la suspensión bacteriana de manera uniforme sobre toda la superficie de la placa de agar MH / MHF en muestras de secreciones en tres direcciones o mediante el uso de una placa de rotador automático. </li></ol></li><li> Aplicación de los discos antimicrobianos en placas de agar inoculadas. <ol><li> Aplicar de cerca los discos en la superficie de las placas de agar inoculadas secas manualmente o mediante un dispensador de disco de susceptibilidad. </li><li> Incubar la placa a la tempe apropiadaratura ambiente y como se describe en el método de difusión en disco EUCAST las pruebas de sensibilidad a los antimicrobianos (versión 3.0, abril de 2013, www.eucast.org). </li></ol></li></ol> 5. automatizado PCR-basado de prueba de diagnóstico para la detección de MRSA <ol><li> Usando una micropipeta, transferir 50 l de la pastilla de cultivo de sangre en el frasco de reactivo de ejemplo proporcionado con el kit de prueba de MRSA automatizado de diagnóstico basado en la PCR y agitar durante 20 seg. </li><li> Usando una micropipeta de 1 ml, dispensar la muestra en el puerto de muestra del cartucho. Insertar el cartucho en el sistema de PCR automatizado e iniciar el ensayo como se describe por el fabricante. </li></ol>

<ol>
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Los autores declaran que no tienen intereses financieros en competencia.

En comparación con los métodos de diagnóstico de hemocultivos positivos convencionales, la rápida adquisición de un sedimento bacteriano utilizando el cloruro de enfoque centrifugación lisis de amonio a reducir el tiempo para informar de identificación en un 16 a 24 horas y el tiempo de reportar AST por 24 a 48 horas (Figuras 1 y 3). Rápida introducción de la terapia antibiótica adecuada es fundamental para mejorar el pronóstico de los pacientes que sufren de infe...

Infecciones del torrente sanguíneo y la sepsis en pacientes hospitalizados son una causa importante de morbilidad y mortalidad. Por lo tanto, se observa la mortalidad relacionada con infecciones del torrente sanguíneo en aproximadamente 14% a 37% de los pacientes hospitalizados y puede aumentar hasta el 35% en las unidades de cuidados intensivos de los pacientes 1-3. La identificación rápida del agente infeccioso es fundamental para orientar el tratamiento antimicrobiano óptimo y para aumentar el éxito de la terapia antimicrobiana 4,5. El rápido análisis de la tinción de Gram de hemocultivo positivo ya tiene un impacto significativo en la adaptación de la terapia antimicrobiana 6,7 pero la identificación precisa del agente infeccioso que se requiere para proporcionar el tratamiento antibiótico más adecuado a las pacientes. Por ejemplo, diferentes regímenes de tratamiento con antibióticos tienen que ser aplicadas tras bacteriemia con enterococos y estreptococos que son difíciles de distinguir por la tinción de Gram. Del mismo modo, la identificación de las especies LevEL es necesario para detectar enterobacterias Gram negativo que codifica un gen cromosómico ampC que confieren una mayor resistencia a β-lactámicos 8. Con un hemocultivo positivo, el enfoque de diagnóstico convencional es subcultura del agente infeccioso en diferentes placas de agar, que requiere varias horas de incubación adicional previa identificación con diversos enfoques, incluyendo pruebas bioquímicas, el crecimiento en diferentes medios selectivos y sistemas de identificación microbiana automatizados. El tiempo de los resultados de un enfoque de diagnóstico convencional es de aproximadamente 1 a 3 días. La aparición de la tecnología de desorción láser asistida por matriz / espectrometría de masas de tiempo de vuelo-ionización (MALDI-TOF) para la identificación rápida de microorganismos ha proporcionado una nueva herramienta para identificar rápidamente los microorganismos de colonias crecidas en placas de agar sino también directamente a partir de sangre positivo culturas (Figura 1) 9-12. The uso de MALDI-TOF para identificar un agente infeccioso a partir de cultivos de sangre se ha reducido significativamente el tiempo a los resultados a unos pocos minutos en lugar de las horas y los días requeridos por los métodos tradicionales. Como se discutió por Croxatto et al. 13, la eficiencia de identificación MALDI-TOF se basa en diferentes parámetros, incluyendo la pureza y la cantidad de microorganismo. Estos dos criterios se obtienen fácilmente a partir de colonias discretas cultivadas en placas de agar, pero requiere un tratamiento de pre-analítica para el enriquecimiento bacteriana y purificación a partir de muestras complejas, tales como cultivo de sangre, que contienen múltiples componentes celulares y proteínas que pueden interferir con la identificación MALDI-TOF. Varios métodos de aislamiento de microorganismos a partir de cultivo de sangre se han utilizado en una serie de estudios que incluyen saponina u otro método detergentes suaves para la extracción bacteriana 9,14, el método de separador de suero 10, la lisis métodos de centrifugación 12 </sarriba&gt; y soluciones comercialmente, tales como el kit de sepsityper. Nuestro laboratorio de diagnóstico bacteriológico ha desarrollado una preparación sedimento bacteriano sencilla hemocultivo a base de cloruro de amonio-lisis de los eritrocitos, que permiten la identificación rápida de bacterias y levaduras por MALDI-TOF y los sistemas de identificación automáticos (Figura 2) 15. Esta preparación de pellets de hemocultivo también proporcionar una muestra para otras aplicaciones posteriores directos tales como la tinción de Gram, pruebas de diagnóstico basados ​​en PCR automatizados, como el POCT-PCR para la detección rápida de la meticilina-resistente Staphylococcus aureus (MRSA), y las pruebas de sensibilidad a los antibióticos con sistemas automatizados de AST y / o por ensayos de difusión de disco en placas de agar (Figura 3). En este trabajo se describen los diferentes pasos para la preparación del sedimento bacteriano hemocultivos según ha explicado Prod&#39;hom et al. 15 (Figura 4). También vamos a desescriba los protocolos para tres de las principales aplicaciones que se pueden realizar en el sedimento de la cultura de la sangre: La identificación por MALDI-TOF 15, la identificación (ID) y las pruebas de sensibilidad a los antibióticos (AST) con los sistemas automatizados 16 para enterobacterias y estafilococos y PCR automatizada prueba de diagnóstico basado en la detección de MRSA 17.

<table border="0" cellpadding="0" cellspacing="0" width="1130">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:268px;">20 needle gauge&#160;</td>
			<td style="width:268px;">Terumo, Leuven, Belgium</td>
			<td style="width:239px;">NN-2038R</td>
			<td style="width:355px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">50 ml Falcon tube</td>
			<td>BD, Franklin Lakes, NJ, USA</td>
			<td>352070</td>
			<td>50 ml centrifuge tubes</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Ammonium chlorure</td>
			<td>Merck, Darmstadt, Germany</td>
			<td>101145</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Potassium hydrogen carbonate</td>
			<td>Fluka, St. Louis, MO, USA&#160;</td>
			<td>60340</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Formic acid</td>
			<td>Sigma-Aldrich, St. Louis, MO, USA&#160;</td>
			<td>F0507</td>
			<td>Flammable, corrosive</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">a-Cyano-4-hydroxycinnamic acid</td>
			<td>Fluka, St. Louis, MO, USA&#160;</td>
			<td>70990</td>
			<td>Acute toxicity</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Acetonitrile</td>
			<td>Sigma-Aldrich, St. Louis, MO, USA&#160;</td>
			<td>271004</td>
			<td>Flammable, acute toxicity</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Trifluoroacetic acid</td>
			<td>Sigma-Aldrich, St. Louis, MO, USA&#160;</td>
			<td>T6508</td>
			<td>Corrosive, acute toxicity</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Vitek 2 60 instrument</td>
			<td>Biom&#233;rieux, Marcy-l&#39;Etoile, France</td>
			<td>27202</td>
			<td>automated microbial system instrument</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Vitek 2 Gram-positive (GP) card</td>
			<td>Biom&#233;rieux, Marcy-l&#39;Etoile, France</td>
			<td>21342</td>
			<td>automated GP&#160; identification card</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Vitek 2 AST-P580 card</td>
			<td>Biom&#233;rieux, Marcy-l&#39;Etoile, France</td>
			<td>22233</td>
			<td>automated microbial AST system</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Vitek 2 Gram-negative (GN) card</td>
			<td>Biom&#233;rieux, Marcy-l&#39;Etoile, France</td>
			<td>21341</td>
			<td>automated GN&#160; identification card</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Vitek 2 AST-N242 card</td>
			<td>Biom&#233;rieux, Marcy-l&#39;Etoile, France</td>
			<td>413391</td>
			<td>automated microbial AST system</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Xpert MRSA</td>
			<td>Cepheid, Sunnyvale, Ca, USA</td>
			<td>&#160;GXMRSA-100N-10</td>
			<td>nucleic acid amplification technology MRSA</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">GeneXpert IV instrument</td>
			<td>Cepheid, Sunnyvale, Ca, USA</td>
			<td>GXIV-4-D</td>
			<td>nucleic acid amplification technology instrument</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Microflex LT MALDI-TOF MS instrument</td>
			<td>Bruker Daltonics, Bremen, Germany</td>
			<td>BDAL microflex LT/SH</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">MSP 96 target&#160; steel BC</td>
			<td>Bruker Daltonics, Bremen, Germany</td>
			<td>280799</td>
			<td>MALDI target plate</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Densitometer Densicheck instrument</td>
			<td>Biom&#233;rieux, Marcy-l&#39;Etoile, France</td>
			<td>27208</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">MALDI Sepsityper kit 50</td>
			<td>Bruker Daltonics, Bremen, Germany</td>
			<td>8270170</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Mac Conkey agar</td>
			<td>Biolife, Milano, Italy</td>
			<td>4016702</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Mueller-Hinton agar</td>
			<td>Oxoid, Hampshire, England</td>
			<td>CM0337</td>
			<td>Mueller-Hinton agar (MH)&#160;</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">MHF agar</td>
			<td>Biom&#233;rieux, Marcy-l&#39;Etoile, France</td>
			<td>43901</td>
			<td>Mueller-Hinton agar-fastidious organisms agar (MHF)</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">BD columbia III agar</td>
			<td>BD, Franklin Lakes, NJ, USA</td>
			<td>254071</td>
			<td>blood agar</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">BD chocolate agar</td>
			<td>BD, Franklin Lakes, NJ, USA</td>
			<td>254089</td>
			<td>chocolate agar</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">BD schaedler agar</td>
			<td>BD, Franklin Lakes, NJ, USA</td>
			<td>254084</td>
			<td>Schaedler agar</td>
		</tr>
	</tbody>
</table>

preparation of a blood culture pellet for rapid bacterial identification and antibiotic susceptibility testing

Infecciones del torrente sanguíneo y sepsis son una causa importante de morbilidad y mortalidad. El resultado exitoso de los pacientes que sufren de bacteremia depende de una rápida identificación del agente infeccioso para guiar el tratamiento antibiótico óptimo. El análisis de la tinción de Gram de hemocultivo positivo puede llevar a cabo con rapidez y ya afecta significativamente el régimen de antibióticos. Sin embargo, todavía se requiere la identificación precisa del agente infeccioso para establecer el tratamiento óptimo de destino. Se presenta aquí una preparación sedimento bacteriano simple y rápido de un cultivo de sangre positivo que puede ser utilizado como una muestra para varias aplicaciones posteriores esenciales tales como la identificación por MALDI-TOF MS, las pruebas de sensibilidad a los antibióticos (AST) por ensayo de difusión en disco o sistemas automatizados de AST y por PCR automatizado basado en pruebas de diagnóstico. El rendimiento de estos diferentes sistemas de identificación y AST aplicados directamente sobre los sedimentos bacterianos de cultivo de sangre es muy SIMilar con el rendimiento que se obtiene normalmente a partir de colonias aisladas cultivadas en placas de agar. En comparación con los métodos convencionales, la rápida adquisición de un sedimento bacteriano se reduce significativamente el tiempo para reportar tanto la identificación y AST. Así, siguiendo la positividad del cultivo de sangre, identificación por MALDI-TOF puede ser reportado en menos de 1 hora mientras que los resultados de AST por sistemas automatizados AST o ensayos de difusión en disco dentro de 8 a 18 horas, respectivamente. Del mismo modo, los resultados de un ensayo rápido basado en la PCR se pueden comunicar a los médicos a menos de 2 horas tras el informe de una bacteriemia. Juntos, estos resultados demuestran que la rápida preparación de un sedimento bacteriano cultivo de sangre tiene un impacto significativo en el tiempo de identificación y de respuesta AST y por tanto en el resultado exitoso de pacientes que sufren de infecciones del torrente sanguíneo.

En el estudio realizado por Prod&#39;hom et al. 15, sedimentos bacterianos obtenidos por cloruro de amonio lisis centrifugación de 122 hemocultivo positivo de 78 pacientes se analizaron por MALDI-TOF MS. Fuera de 122 hemocultivos positivos, 95 (77,9%) fueron correctamente identificados a nivel de especie y uno (0,8%) a nivel de género. Los 26 (21,3%) Bolitas de hemocultivo restantes no dieron ninguna identificación fiable por MALDI-TOF. Entre ellos, 21 eran bacterias gram positivas incluyendo 13 e...

Watch this Scientific Journal Video about Preparation of a Blood Culture Pellet for Rapid Bacterial Identification and Antibiotic Susceptibility Testing at JoVE.com

Preparation of a Blood Culture Pellet for Rapid Bacterial Identification and Antibiotic Susceptibility Testing

Una preparación rápida sedimento bacteriano de un cultivo de sangre positivo puede ser utilizado como una muestra para aplicaciones tales como la identificación por MALDI-TOF, la tinción de Gram, la prueba de susceptibilidad a los antibióticos y prueba basada en PCR. Los resultados pueden ser comunicados rápidamente a los médicos a mejorar el resultado de pacientes que sufren de infecciones del torrente sanguíneo.

Preparación de una Cultura de pellets de sangre para Rapid bacteriana Identificación y Prueba de Susceptibilidad a los antibióticos

Bloodstream infections and sepsis are a major cause of morbidity and mortality. The successful outcome of patients suffering from bacteremia depends on a ...

preparation-blood-culture-pellet-for-rapid-bacterial-identification

Research

JoVE Journal

Immunology and Infection

35.4K vistas.  University Hospital Center and University of Lausanne.  Una preparación rápida sedimento bacteriano de un cultivo de sangre positivo puede ser utilizado como una muestra para aplicaciones tales como la identificación por MALDI-TOF, la tinción de Gram, la prueba de susceptibilidad a los antibióticos y prueba basada en PCR. Los resultados pueden ser comunicados rápidamente a los médicos a mejorar el resultado de pacientes que sufren de infecciones del torrente sanguíneo. 

Video: Preparation of a Blood Culture Pellet for Rapid Bacterial Identification and Antibiotic Susceptibility Testing

A 96 Well Microtiter Plate-based Method for Monitoring Formation and Antifungal Susceptibility Testing of Candida albicans Biofilms

Candida albicans remains the most frequent cause of fungal infections in an expanding population of compromised patients and candidiasis is now the third most common infection in US hospitals. Different manifestations of candidiasis are associated with biofilm formation, both on host tissues and/or medical devices (i.e. catheters). Biofilm formation carries negative clinical implications, as cells within the biofilms are protected from host immune responses and from the action of antifungals. We have developed a simple, fast and robust in vitro model for the formation of C. albicans biofilms using 96 well microtiter-plates, which can also be used for biofilm antifungal susceptibility testing. The readout of this assay is colorimetric, based on the reduction of XTT (a tetrazolium salt) by metabolically active fungal biofilm cells. A typical experiment takes approximately 24 h for biofilm formation, with an additional 24 h for antifungal susceptibility testing. Because of its simplicity and the use of commonly available laboratory materials and equipment, this technique democratizes biofilm research and represents an important step towards the standardization of antifungal susceptibility testing of fungal biofilms.

A 96 Well Microtiter Plate-based Method for Monitoring Formation and Antifungal Susceptibility Testing of Candida albicans Biofilms

We describe a simple, rapid and robust method for the formation of Candida albicans biofilms using 96 well microtiter plates and its utility in antifungal susceptibility testing of cells within biofilms.

A 96 de microtitulación y placa basada en el método para la formación de Control y pruebas de sensibilidad antifúngica Candida albicans Las biopelículas

Candida albicans remains the most frequent cause of fungal infections in an expanding population of compromised patients and candidiasis is now the third ...

Investigación

Inmunología e Infección

Protocol for Recombinant RBD-based SARS Vaccines: Protein Preparation, Animal Vaccination and Neutralization Detection

Based on their safety profile and ability to induce potent immune responses against infections, subunit vaccines have been used as candidates for a wide variety of pathogens 1-3. Since the mammalian cell system is capable of post-translational modification, thus forming properly folded and glycosylated proteins, recombinant proteins expressed in mammalian cells have shown the greatest potential to maintain high antigenicity and immunogenicity 4-6.
Although no new cases of SARS have been reported since 2004, future outbreaks are a constant threat; therefore, the development of vaccines against SARS-CoV is a prudent preventive step and should be carried out. The RBD of SARS-CoV S protein plays important roles in receptor binding and induction of specific neutralizing antibodies against virus infection 7-9. Therefore, in this protocol, we describe novel methods for developing a RBD-based subunit vaccine against SARS. Briefly, the recombinant RBD protein (rRBD) was expressed in culture supernatant of mammalian 293T cells to obtain a correctly folded protein with proper conformation and high immunogenicity 6. The transfection of the recombinant plasmid encoding RBD to the cells was then performed using a calcium phosphate transfection method 6,10 with some modifications. Compared with the lipid transfection method 11,12, this modified calcium phosphate transfection method is cheaper, easier to handle, and has the potential to reach high efficacy once a transfection complex with suitable size and shape is formed 13,14. Finally, a SARS pseudovirus neutralization assay was introduced in the protocol and used to detect the neutralizing activity of sera of mice vaccinated with rRBD protein. This assay is relatively safe, does not involve an infectious SARS-CoV, and can be performed without the requirement of a biosafety-3 laboratory 15.
The protocol described here can also be used to design and study recombinant subunit vaccines against other viruses with class I fusion proteins, for example, HIV, respiratory syncytial virus (RSV), Ebola virus, influenza virus, as well as Nipah and Handra viruses. In addition, the methods for generating a pseudovirus and subsequently establishing a pseudovirus neutralization assay can be applied to all these viruses.

This protocol describes a general procedure for studying recombinant receptor-binding domain (RBD)-based subunit vaccines against SARS. It includes methods for transfection and expression of RBD protein in 293T cells, immunization of mice with RBD and detection of neutralization activity of mouse sera using an established SARS pseudovirus neutralization assay.

Protocolo para las vacunas recombinantes para RBD basada en el SRAS: Preparación de la proteína, la vacunación de animales y la detección de neutralización

Based on their safety profile and ability to induce potent immune responses against infections, subunit vaccines have been used as candidates for a wide ...

A 1.5 Hour Procedure for Identification of Enterococcus Species Directly from Blood Cultures

Enterococci are a common cause of bacteremia with E. faecalis being the predominant species followed by E. faecium. Because resistance to ampicillin and vancomycin in E. faecalis is still uncommon compared to resistance in E. faecium, the development of rapid tests allowing differentiation between enterococcal species is important for appropriate therapy and resistance surveillance. The E. faecalis OE PNA FISH assay (AdvanDx, Woburn, MA) uses species-specific peptide nucleic acid (PNA) probes in a fluorescence in situ hybridization format and offers a time to results of 1.5 hours and the potential of providing important information for species-specific treatment. Multicenter studies were performed to assess the performance of the 1.5 hour E. faecalis/OE PNA FISH procedure compared to the original 2.5 hour assay procedure and to standard bacteriology methods for the identification of enterococci directly from a positive blood culture bottle.

A 1.5 Hour Procedure for Identification of Enterococcus Species Directly from Blood Cultures

A rapid protocol for the direct identification of Enterococcus faecalis and other Enterococcus species from a positive blood culture using a Peptide Nucleic Acid fluorescent in situ hybridization assay (PNA FISH).

Un procedimiento de 1,5 horas para la identificación de Enterococcus Especies directamente los cultivos de sangre

Enterococci are a common cause of bacteremia with E. faecalis being the predominant species followed by E. faecium.  Because resistance to ampicillin and ...

Antimicrobial Susceptibility Testing of Mycobacterium Tuberculosis Complex for First and Second Line Drugs by Broth Dilution in a Microtiter Plate Format

The rapid detection of antimicrobial resistance is important in the effort to control the increase in resistant Mycobacterium 
tuberculosis (Mtb). Antimicrobial susceptibility testing (AST) of Mtb has traditionally been performed by the agar method of proportion or by 
macrobroth testing on an instrument such as the BACTEC (Becton Dickinson, Sparks, MD), VersaTREK (TREK Diagnostics, Cleveland, OH) or BacT/ALERT (bioM&eacute;rieux, Hazelwood, MO). The agar proportion method, while considered the &#x201C;gold&#x201D; standard of AST, is labor intensive and requires calculation of resistance by performing colony counts on drug-containing agar as compared to drug-free agar. If there is &ge;1% growth on the drug-containing medium as compared to drug-free medium, the organism is considered resistant to that drug. The macrobroth methods require instrumentation and test break point ("critical") drug concentrations for the first line drugs (isoniazid, ethambutol, rifampin, and pyrazinamide). The method described here is commercially available in a 96 well microtiter plate format [MYCOTB (TREK Diagnostics)] and contains increasing concentrations of 12 antimicrobials used for treatment of tuberculosis including both first (isoniazid, rifampin, ethambutol) and second line drugs (amikacin, cycloserine, ethionamide, kanamycin, moxifloxacin, ofloxacin, para-aminosalicylic acid, rifabutin, and streptomycin). Pyrazinamide, a first line drug, is not included in the microtiter plate due to its need for acidic test conditions. Advantages of the microtiter system include both ease of set up and faster turn around time (14 days) compared with traditional agar proportion (21 days). In addition, the plate can be set up from inoculum prepared using either broth or solid medium. Since the microtiter plate format is new and since Mtb presents unique safety challenges in the laboratory, this protocol will describe how to safely setup, incubate and read the microtiter plate.

Antimicrobial susceptibility testing of Mycobacterium tuberculosis is challenging but critical for patient care. This microtiter plate format offers testing of 12 antimycobacterial drugs and provides minimum inhibitory concentration (MIC) values, which will aid in appropriate treatment.

Pruebas de susceptibilidad antimicrobiana de Mycobacterium tuberculosis complex para drogas de primera línea y la segunda por dilución en caldo en un formato de placa de microtitulación

The rapid detection of antimicrobial resistance is important in the effort to control the increase in resistant Mycobacterium 
tuberculosis (Mtb). ...

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

La introducción de la tensión de cizallamiento en el Estudio de la adhesión bacteriana

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

One-day Workflow Scheme for Bacterial Pathogen Detection and Antimicrobial Resistance Testing from Blood Cultures

Bloodstream infections are associated with high mortality rates because of the probable manifestation of sepsis, severe sepsis and septic shock1. Therefore, rapid administration of adequate antibiotic therapy is of foremost importance in the treatment of bloodstream infections. The critical element in this process is timing, heavily dependent on the results of bacterial identification and antibiotic susceptibility testing. Both of these parameters are routinely obtained by culture-based testing, which is time-consuming and takes on average 24-48 hours2, 4. The aim of the study was to develop DNA-based assays for rapid identification of bloodstream infections, as well as rapid antimicrobial susceptibility testing. The first assay is a eubacterial 16S rDNA-based real-time PCR assay complemented with species- or genus-specific probes5. Using these probes, Gram-negative bacteria including Pseudomonas spp., Pseudomonas aeruginosa and Escherichia coli as well as Gram-positive bacteria including Staphylococcus spp., Staphylococcus aureus, Enterococcus spp., Streptococcus spp., and Streptococcus pneumoniae could be distinguished. Using this multiprobe assay, a first identification of the causative micro-organism was given after 2 h.
Secondly, we developed a semi-molecular assay for antibiotic susceptibility testing of S. aureus, Enterococcus spp. and (facultative) aerobe Gram-negative rods6. This assay was based on a study in which PCR was used to measure the growth of bacteria7. Bacteria harvested directly from blood cultures are incubated for 6 h with a selection of antibiotics, and following a Sybr Green-based real-time PCR assay determines inhibition of growth. The combination of these two methods could direct the choice of a suitable antibiotic therapy on the same day (Figure 1). In conclusion, molecular analysis of both identification and antibiotic susceptibility offers a faster alternative for pathogen detection and could improve the diagnosis of bloodstream infections.

The design of a straightforward one-day workflow scheme for bacterial pathogen diagnostics enables the rapid recognition of bloodstream infections. The inclusion of eight clinically relevant bacterial targets and their antibiotic resistance profiles offers the clinician an initial insight on the same day, which can lead to more adequate therapy.

Un día de flujo de trabajo Método de detección de patógenos bacterianos y pruebas de resistencia a los antimicrobianos de cultivos de sangre

Bloodstream infections are associated with high mortality rates because of the probable manifestation of sepsis, severe sepsis and septic shock1. ...

Rapid Identification of Gram Negative Bacteria from Blood Culture Broth Using MALDI-TOF Mass Spectrometry

An important role of the clinical microbiology laboratory is to provide rapid identification of bacteria causing bloodstream infection. Traditional identification requires the sub-culture of signaled blood culture broth with identification available only after colonies on solid agar have matured. MALDI-TOF MS is a reliable, rapid method for identification of the majority of clinically relevant bacteria when applied to colonies on solid media. The application of MALDI-TOF MS directly to blood culture broth is an attractive approach as it has potential to accelerate species identification of bacteria and improve clinical management. However, an important problem to overcome is the pre-analysis removal of interfering resins, proteins and hemoglobin&#160;contained in blood culture specimens which, if not removed, interfere with the MS spectra and can result in insufficient or low discrimination identification scores. In addition it is necessary to concentrate bacteria to develop spectra of sufficient quality. The presented method describes the concentration, purification, and extraction of Gram negative bacteria allowing for the early identification of bacteria from a signaled blood culture broth.

The application of matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry (MS) directly to blood culture broth expedites the identification of bacteria. The presented method is a rapid and reliable method for identification of Gram negative bacteria directly from blood culture broth.

La identificación rápida de bacterias gram negativas de Cultura sangre caldo de cultivo utilizando MALDI-TOF espectrometría de masas

An important role of the clinical microbiology laboratory is to provide rapid identification of bacteria causing bloodstream infection. Traditional ...

A Reference Broth Microdilution Method for Dalbavancin In Vitro Susceptibility Testing of Bacteria that Grow Aerobically

Antimicrobial susceptibility testing (AST) is performed to assess the in vitro activity of antimicrobial agents against various bacteria. The AST results, which are expressed as minimum inhibitory concentrations (MICs) are used in research for antimicrobial development and monitoring of resistance development and in the clinical setting for antimicrobial therapy guidance. Dalbavancin is a semi-synthetic lipoglycopeptide antimicrobial agent that was approved in May 2014 by the Food and Drug Administration (FDA) for the treatment of acute bacterial skin and skin structure infections caused by Gram-positive organisms. The advantage of dalbavancin over current anti-staphylococcal therapies is its long half-life, which allows for once-weekly dosing. Dalbavancin has activity against Staphylococcus aureus (including both methicillin-susceptible S. aureus [MSSA] and methicillin-resistant S. aureus [MRSA]), coagulase-negative staphylococci, Streptococcus pneumoniae, Streptococcus anginosus group, &#946;-hemolytic streptococci and vancomycin susceptible enterococci. Similar to other recent lipoglycopeptide agents, optimization of CLSI and ISO broth susceptibility test methods includes the use of dimethyl sulfoxide (DMSO) as a solvent when preparing stock solutions and polysorbate 80 (P80) to alleviate adherence of the agent to plastic. Prior to the clinical studies and during the initial development of dalbavancin, susceptibility studies were not performed with the use of P-80 and MIC results tended to be 2-4 fold higher and similarly higher MIC results were obtained with the agar dilution susceptibility method. Dalbavancin was first included in CLSI broth microdilution methodology tables in 2005 and amended in 2006 to clarify use of DMSO and P-80. The broth microdilution (BMD) procedure shown here is specific to dalbavancin and is in accordance with the CLSI and ISO methods, with step-by-step detail and focus on the critical steps added for clarity.

A Reference Broth Microdilution Method for Dalbavancin In Vitro Susceptibility Testing of Bacteria that Grow Aerobically

Here, we present a protocol for determination of dalbavancin susceptibility of clinically relevant Gram-positive bacteria using a broth microdilution reference methodology.

Una referencia Caldo método de microdilución para Dalbavancina<em&gt; In Vitro</em&gt; Pruebas de Susceptibilidad de bacterias que crecen en aerobiosis

Antimicrobial susceptibility testing (AST) is performed to assess the in vitro activity of antimicrobial agents against various bacteria. The AST results, ...

A RAPID Method for Blood Processing to Increase the Yield of Plasma Peptide Levels in Human Blood

Research in the field of food intake regulation is gaining importance. This often includes the measurement of peptides regulating food intake. For the correct determination of a peptide&#39;s concentration, it should be stable during blood processing. However, this is not the case for several peptides which are quickly degraded by endogenous peptidases. Recently, we developed a blood processing method employing Reduced temperatures, Acidification, Protease inhibition, Isotopic exogenous controls and Dilution (RAPID) for the use in rats. Here, we have established this technique for the use in humans and investigated recovery, molecular form and circulating concentration of food intake regulatory hormones. The RAPID method significantly improved the recovery for 125I-labeled somatostatin-28 (+39%), glucagon-like peptide-1 (+35%), acyl ghrelin and glucagon (+32%), insulin and kisspeptin (+29%), nesfatin-1 (+28%), leptin (+21%) and peptide YY3-36 (+19%) compared to standard processing (EDTA blood on ice, p&#160;&#60;0.001). High performance liquid chromatography showed the elution of endogenous acyl ghrelin at the expected position after RAPID processing, while after standard processing 62% of acyl ghrelin were degraded resulting in an earlier peak likely representing desacyl ghrelin. After RAPID processing the acyl/desacyl ghrelin ratio in blood of normal weight subjects was 1:3 compared to 1:23 following standard processing (p&#160;= 0.03). Also endogenous kisspeptin levels were higher after RAPID compared to standard processing (+99%, p&#160;= 0.02). The RAPID blood processing method can be used in humans, yields higher peptide levels and allows for assessment of the correct molecular form.

The RAPID blood processing method can be used in humans and yields higher peptide levels as well as allows for assessment of the correct molecular form. Therefore, this method will be a valuable tool in peptide research.

Un método rápido para el procesamiento de sangre para aumentar el rendimiento de los niveles plasmáticos de péptido en la sangre humana

Research in the field of food intake regulation is gaining importance. This often includes the measurement of peptides regulating food intake. For the ...

Culture of Macrophage Colony-stimulating Factor Differentiated Human Monocyte-derived Macrophages

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. For initiation of experiments, fresh or frozen monocytes are cultured in flasks for 1 week with M-CSF to induce their differentiation into macrophages. Then, the macrophages can be harvested and seeded into culture wells at required cell densities for carrying out experiments. The use of defined numbers of macrophages rather than defined numbers of monocytes to initiate macrophage cultures for experiments yields macrophage cultures in which the desired cell density can be more consistently attained. Use of cryopreserved monocytes reduces dependency on donor availability and produces more homogeneous macrophage cultures.

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. The protocol utilizes cryopreservation of monocytes coupled with their bulk differentiation into macrophages. Then harvested macrophages can then be seeded into culture wells at required cell densities for carrying out experiments.

La cultura de los macrófagos derivados de monocitos-macrófagos factor estimulante de colonia humana diferenciada

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. For initiation ...