<html> 1. Hemocultivos Caldos Marcar como &quot;positiva&quot; <ol><li> Retire el frasco de hemocultivo señalado desde el gabinete de incubación monitoreo continuo y colóquelo en una cabina de seguridad biológica. Nota: Las botellas pueden contener microorganismos peligrosos y precauciones universales deben ser seguidas. Debido al riesgo de aerosoles infecciosos en el muestreo, los procedimientos de muestreo deben realizarse en una cabina de flujo laminar de bioseguridad de Clase II. </li></ol> 2. Tinción de Gram se prepara <ol><li> Preparar una tinción de Gram del caldo de cultivo de sangre señalizado según los protocolos institucionales locales. Nota: Cuando los organismos Gram negativos se identifican en la microscopía del caldo de cultivo de sangre se procesa de acuerdo con el siguiente método. Cuando se identifican los organismos Gram positivos, un método molecular alternativa focalización identificación genética y los marcadores de resistencia se aplica al caldo (no se aborda en este artículo) 13. </li></ol>e &quot;&gt; 3. Transferencia de Marcado Blood caldo de cultivo a un tubo de separación de suero <ol><li> Mezcle suavemente el frasco de hemocultivo invirtiendo 2-3x. </li><li> Dentro de la cabina de bioseguridad adjuntar una jeringa de 10 ml a un dispositivo de transferencia de sangre seguridad. </li><li> Una el dispositivo de transferencia de sangre a la botella de cultivo de sangre y retirar 5 ml de caldo en la jeringa. Transferir el caldo BC aspirado en un tubo de separar el suero. </li></ol> 4. Concentración de Cultura sangre Caldo <ol><li> Centrifugar el tubo de separación de suero a 1250 xg durante 15 min que elimina un gran volumen de células rojas de la sangre. </li><li> Aspirar y descartar el sobrenadante usando una pipeta de transferencia estéril teniendo cuidado de dejar aproximadamente 1 ml de la capa leucocitaria inmediatamente por encima de la interfaz de gel / líquido. Nota: Las botellas aeróbicas muestran una clara separación de las células rojas de la sangre a la parte inferior del tubo con la interfaz de gel / líquido que aparece de un color blanco opaco. En contraste, cuandoaplicado al caldo de cultivo de sangre anaeróbica lítico la interfaz de gel / líquido aparece como un color rojo intenso debido a los componentes de las células rojas de la sangre lisadas permaneciendo en suspensión en el sobrenadante. </li></ol> 5. Repita los pasos de centrifugación Wash <ol><li> Mezcle suavemente los últimos 1 ml de líquido de la capa leucocitaria por encima de la interfaz de gel con una pipeta estéril y luego transferir todo el volumen en un tubo de microcentrífuga de 1,5 ml. Centrifugar el nuevo tubo a 288 g durante 30 seg. </li><li> Transferir el sobrenadante usando una pipeta de transferencia desechable de 1 ml de plástico en un nuevo tubo de microcentrífuga de 1,5 ml y desechar el tubo con el sedimento. Nota: Es muy importante en este paso que se tiene cuidado de evitar la transferencia de la pastilla. Esto es particularmente importante para un espécimen que se procesa de la botella antes de Cristo anaeróbica como el sobrenadante permanece pigmentada y el sedimento no se ve siempre claramente. </li></ol> 6. La lisis de las células residuales </p&gt; <ol><li> Centrifugar la muestra a 18.407 xg durante 1 min y luego aspirado (y de descarte) el sobrenadante usando una pipeta de transferencia de punta fina para dejar tan poco líquido residual como sea posible sin perturbar el sedimento. </li><li> Resuspender el pellet residual en 1 ml de DNAsa RNAsa estéril y libre de agua con la pipeta hacia arriba y abajo. Nota: Algunos autores han descrito el uso de soluciones de alcohol en lugar de agua estéril para volver a suspender el sedimento que hace que las bacterias no viables. </li><li> Centrifugar la solución se resuspendieron a 18.407 xg durante 1 min. </li></ol> 7. Extracción de Proteínas Bacterianas <ol><li> Aspirar y desechar el sobrenadante, utilizando de nuevo la aspiración con una pipeta de punta fina asegurar que se elimina la mayor cantidad de líquido posible. </li><li> Resuspender el precipitado en 10 l de ácido fórmico (70% v / v). Nota: El ácido fórmico puede afectar al organismo si es inhalado o si entra en contacto con la piel. Al preparar o manipular soluciones es recommended que el personal utilizan un gabinete de humos, además de los equipos de protección personal. </li><li> Mezclar bien usando la pipeta para asegurar una solución homogénea resuspendido. Uso de la punta de la pipeta para romper físicamente la pastilla puede ser necesario para asegurar una solución más homogénea. </li></ol> 8. Preparación de MALDI-TOF Target Plate <ol><li> Inocular una placa objetivo clean MALDI-TOF con 2 l de la suspensión dispuesta por lugar de destino. Preparar 3 sitios diana para cada muestra para contrarrestar el problema ocasional de lecturas fallado. </li><li> Deje que la placa del objetivo MALDI-TOF se seque. La velocidad de secado se puede aumentar mediante la colocación de la placa en el borde de la campana de humos para maximizar el flujo de aire a través de la placa. </li><li> Superposición de cada punto se secó con 1 l de la solución de matriz (10 mg / ml de ácido α-ciano-4-hidroxicinámico (HCCA), 50% de acetonitrilo, ácido trifluoroacético 2,5%). Nota: La solución de la matriz puede afectar al organismo si es inhalado o si entra en contact con la piel. Al preparar o manipular soluciones, se recomienda que el personal utiliza un armario de humos, además de los equipos de protección personal. </li><li> Deje que la placa del objetivo MALDI-TOF se seque sobre el borde de la campana extractora de humos como se describió anteriormente. Asegurar una preparación homogénea llena cada uno de los lugares de destino en el plato. </li></ol> 9. Place MALDI-TOF Target Plate en el espectrómetro de masas <ol><li> Inserte la placa del objetivo MALDI-TOF en el espectrómetro de masas MALDI-TOF. Asegúrese de que la placa está sentado a ras con las placas de resorte. Nota: No inserte la placa del objetivo hasta que todos los puntos de destino están secos. </li><li> Asegúrese de que la junta de goma esté libre de pelusa, polvo y el pelo para que las condiciones de vacío requeridas pueden ser creados. </li><li> Cierre la tapa etapa MALDI-TOF y luego presione el botón &quot;in / out&quot; en la parte frontal del instrumento. La tapa ahora bloquear y permitir que el vacío necesario que se creará. </li></ol> 10. MALDI-TOF MS SPECTRA Adquisición <ol><li> Abra el MALDI biotipificación y software FlexControl. </li><li> Dentro del software Typing seleccionar &quot;nueva clasificación&quot; en el menú desplegable titulado &quot;archivo&quot;. Asigne un nombre al proyecto y, a continuación, seleccione &quot;nuevo&quot;. Seleccione &quot;Siguiente&quot; para completar la configuración en el software de mecanografía. </li><li> Dentro de la ventana del software de control de identificar el gráfico en pantalla de la tablilla de puntería. Resalte los puntos objetivo MALDI-TOF que están en uso, moviendo el ratón sobre los puntos inoculados mientras mantiene pulsado el botón izquierdo del ratón. </li><li> Utilice el botón derecho del ratón para hacer clic en cualquier lugar de las posiciones de destino seleccionados y luego seleccione &quot;agregar analitos&quot; en el menú desplegable. </li><li> Añadir sanguíneos cultura datos identificativos de la columna &quot;ID&quot; para cada uno de los lugares de destino y haga clic en &quot;Siguiente&quot; cuando haya terminado. </li><li> Seleccione la &quot;taxonomía&quot; base de datos de espectros comerciales y seleccione &quot;siguiente&quot;. Nota: Laborios pueden utilizar adicional en-casa o bases de datos comerciales para aumentar el número de espectros de referencia. </li><li> Haga clic en &quot;Finalizar&quot; y el MALDI-TOF MS se iniciará la generación de espectros. Nota: Los ajustes de MALDI-TOF fueron según la configuración de la producción (modo lineal positiva, 60 Hz de frecuencia láser, 20 kV de tensión de aceleración, 16,7 kV de tensión IS2, 170 de retardo de extracción de nanosegundos, y 2,000-20,137 rango m / z). </li><li> Si el software de control de MALDI no genera picos, se puede realizar la adquisición manual de los espectros (método no se describe). </li></ol> 11. Interpretación Análisis del anuncio de MALDI-TOF MS Partituras <ol><li> Una vez que los espectros son adquiridos y el análisis esté completo, seleccione el botón &quot;+&quot; al lado de la identificación de especies en el software de mecanografía para expandir la lista de identificación para cada destino. </li><li> Estudie los 5 mejores identificaciones, observando si el top 5 son similares. Top discordantes 5 géneros con puntuaciones ≥ 1.7 del mismo objetivopunto planteó la posibilidad de caldos mixtos. Nota: la tecnología MALDI-TOF tiene poca sensibilidad para la detección de caldos mixtos. Por ejemplo, cuando un caldo contiene especies mixtas una puntuación alta confianza puede ser identificado por sólo una de las especies mixtas. </li><li> Cuando una puntuación ≥ 2,0 se caracteriza por el mayor partido en el lugar de destino, el informe de la especie. </li><li> Cuando la puntuación más alta en un punto de destino es ≥ 1,7, pero &lt;2,0, sólo informamos de los géneros. Si los criterios adicionales de los 5 mejores identificaciones son concordantes, a continuación, informe a nivel de especie. </li></ol> 12. La eliminación de la placa de MALDI-TOF Target <ol><li> Cuando todos los espectros son el botón de &quot;in / out&quot; en la máquina MALDI-TOF de prensa. </li><li> Abra el MALDI-TOF receptáculo tablilla de puntería y retire la placa del objetivo MALDI-TOF. </li><li> Si se utiliza una placa reutilizable diana MALDI-TOF, limpie todos los puntos de destino mediante el alcohol y almacenar la placa de MALDI-TOF a TA una vez que esté limpio y seco. </li></ol>

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Los autores declaran que no tienen intereses financieros en competencia.

Es importante aplicar el MALDI-TOF MS de caldo de cultivo de sangre que los pasos posteriores a la centrifugación se llevan a cabo con el cuidado suficiente para no volver a mezclar los componentes separados. Es particularmente importante para eliminar los constituyentes de cultivos de sangre y proteínas celulares humanos, incluyendo la hemoglobina, que pueden producir picos de interferencia con los espectros de MALDI-TOF. Aunque MS fabrica recomendar cortar de puntuación de ≥ 2,0 para ...

Los pacientes con infección del torrente sanguíneo (BSI), debido a las bacterias continúan teniendo una alta mortalidad en el hospital, que van 6-48% 1. La entrega de antibiótico empírico adecuado promueve la supervivencia y en el subconjunto de pacientes con sepsis grave, cada retardo horas a la terapia apropiada se correlaciona con una menor supervivencia 2,3. En consecuencia, un objetivo clave del laboratorio clínico es detectar rápidamente, identificar y comunicar la presencia de bacterias en cultivos de sangre para informar las decisiones clínicas. Se ha demostrado que el laboratorio de microbiología tiene la mayor influencia en la terapia antimicrobiana en el momento de la presentación de informes de la tinción de Gram 4 y, recientemente, un estudio observacional demostró que el tiempo de desorción por láser asistida por matriz / ionización de vuelo espectrometría de masas (MALDI-TOF MS) realizado directamente en caldos de cultivo de sangre influir en la prescripción en más de una tercera parte de BSI causadas por bacterias Gram negativas 5. 6,7. La tecnología está ahora bien establecida y se ha integrado en muchos laboratorios para la identificación rápida y precisa de los microorganismos aislados en el 6,8 medios sólidos. La aplicación directa de MALDI-TOF MS para hemocultivo (BC) de caldo que se han señalado &quot;positivo&quot; para los microorganismos apela a los médicos y directores de laboratorio debido a la posibilidad de obtener una identificación más temprana de los microorganismos a bajo costo. La utilidad clínica de la aplicación directa de MALDI-TOF MS de caldo de cultivo de sangre ha sido limitado por la amplia gama de sensibilidades observados cuando se compara con los métodos basados ​​en la cultura fenotípica estándar de identificación, con los informes de la identificación con éxito de las bacterias Gram negativas que van desde 47 hasta 98,9 9-11%. La variación en la sensibilidad probablese refiere a la composición antes de Cristo caldo, la concentración bacteriana inicial, la variación en los métodos de preparación de muestras, así como la gama de organismos Gram negativos encontrados en las poblaciones de estudio 9. En comparación con estos otros protocolos publicados el método presentado aquí evita el uso de etanol, cloruro de amonio o (no-matriz) acetonitrilo adicional. Como resultado, el sedimento bacteriano se permanecer viable (hasta el punto de extracción de la proteína) que permite métodos potenciales de prueba de susceptibilidad fenotípica a ser aplicados directamente a estos organismos en el caldo. Además, el método que se presenta se ha demostrado para ser de bajo costo, confiable y rápida con la identificación de bacterias disponibles dentro de 25 min de los hemocultivos Gram resultados de la tinción, con un mínimo de &quot;manos en&quot; el tiempo 12. Este método es un simple protocolo de spin-lisis de la casa utilizando la extracción con ácido fórmico aplicado directamente a los caldos de cultivo de sangre positivo para identificar Gram negativo bacteria con la tecnología MALDI-TOF MS.

<table border="0" cellpadding="0" cellspacing="0" width="1319">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:275px;">BACTEC Plus Aerobic/F Medium</td>
			<td style="width:307px;">Becton Dickinson; BD, Franklin Lakes, NJ, USA</td>
			<td align="right" style="width:188px;">442192</td>
			<td style="width:551px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">BACTEC Lytic/10 Anaerobic /F Medium</td>
			<td>Becton Dickinson; BD, Franklin Lakes, NJ, USA</td>
			<td align="right">442265</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">BACTEC Peds Plus Medium</td>
			<td>Becton Dickinson; BD, Franklin Lakes, NJ, USA</td>
			<td align="right">442194</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Vacutainer - Blood transfer device</td>
			<td>Becton Dickinson; BD, Franklin Lakes, NJ, USA</td>
			<td align="right">364880</td>
			<td>Single use sampling device reducing the risk of needlestick injury</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Vacutainer SST 5.0mL, Advance plus</td>
			<td>Becton Dickinson; BD, Franklin Lakes, NJ, USA</td>
			<td align="right">367954</td>
			<td>Serum separating tube</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Syringe 10 mL</td>
			<td>Becton Dickinson; BD, Franklin Lakes, NJ, USA</td>
			<td align="right">302143</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Transfer pipette</td>
			<td>Samco, USA</td>
			<td>222-20S</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Transfer pipette (fine tipped)</td>
			<td>Samco, USA</td>
			<td>232-20S</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microcentrifuge tube (2.0 mL)</td>
			<td>Eppendorf, Hamburg, Germany</td>
			<td>0030.120.094</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sterile water (DNAse and RNAse free)</td>
			<td>Life Technologies, Carlsbad, California, USA</td>
			<td>10977-015</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Formic acid</td>
			<td>Sigma-Aldrich, St. Louis, Missouri, USA</td>
			<td>F0507</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Matrix solution&#160;</td>
			<td>Bruker Daltonics, Bremen Germany</td>
			<td align="right">285074</td>
			<td>10 mg/ml &#945;-cyano-4-hydroxycinnamic acid, 50% acetonitrile, 2.5% trifluoroacetic acid</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Benchtop microflex LT MALDI-TOF MS</td>
			<td>Bruker Daltonics, Bremen Germany</td>
			<td></td>
			<td>Utilizing BioTyper 3.1 (Build 65) and FlexControl 3.3 (Build 99) software</td>
		</tr>
	</tbody>
</table>

rapid identification of gram negative bacteria from blood culture broth using maldi-tof mass spectrometry

Una función importante de los laboratorios de microbiología clínica es proporcionar una rápida identificación de bacterias causantes de infección del torrente sanguíneo. Identificación tradicional requiere la sub-cultura de la señalada caldo de cultivo de sangre con identificación disponible sólo después de colonias en agar sólido han madurado. MALDI-TOF MS es un método fiable, rápido para la identificación de la mayoría de bacterias clínicamente relevantes cuando se aplica a las colonias en medios sólidos. La aplicación de MALDI-TOF MS directamente al caldo de cultivo de sangre es un enfoque atractivo, ya que tiene el potencial de acelerar la identificación de especies de bacterias y mejorar la gestión clínica. Sin embargo, un problema importante a superar es la eliminación de pre-análisis de interferencia resinas, proteínas y hemoglobina contenidas en las muestras para cultivo de sangre que, si no se elimina, interfieren con los espectros de MS y puede resultar en las puntuaciones de identificación de discriminación insuficientes o bajos. Además, es necesario concentrar bacteria para desarrollar espectros de calidad suficiente. El método que se presenta describe la concentración, purificación y extracción de las bacterias Gram negativas que permitan la identificación temprana de las bacterias de un caldo de cultivo de sangre señalado.

La generada MALDI-TOF MS espectros se comparan con la base de datos de referencia integrado de los espectros. Una puntuación logarítmica es asignado por la confianza del partido entre el aislado de prueba y la base de datos de referencia de los aislamientos, con la recomendación de un puntaje ≥ 1,7 requerida para su identificación a nivel de género probable (Figura 1) y ≥ 2,0 para la identificación probable de especies (Figura 2). Informe a nivel de especie cuando la puntuaci?...

Watch this Scientific Journal Video about Rapid Identification of Gram Negative Bacteria from Blood Culture Broth Using MALDI-TOF Mass Spectrometry at JoVE.com

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

La aplicación de láser asistida por matriz de desorción / ionización tiempo de vuelo (MALDI-TOF) espectrometría de masas (MS) directamente a caldo de cultivo de sangre acelera la identificación de bacterias. El método presentado es un método rápido y fiable para la identificación de bacterias Gram negativas directamente desde el caldo de cultivo de sangre.

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 ...

rapid-identification-gram-negative-bacteria-from-blood-culture-broth

Research

JoVE Journal

Immunology and Infection

23.8K vistas.  Westmead Hospital.  La aplicación de láser asistida por matriz de desorción / ionización tiempo de vuelo (MALDI-TOF) espectrometría de masas (MS) directamente a caldo de cultivo de sangre acelera la identificación de bacterias. El método presentado es un método rápido y fiable para la identificación de bacterias Gram negativas directamente desde el caldo de cultivo de sangre. 

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

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 ...

Investigación

Inmunología e Infección

Purification and Visualization of Lipopolysaccharide from Gram-negative Bacteria by Hot Aqueous-phenol Extraction

Lipopolysaccharide (LPS) is a major component of Gram-negative bacterial outer membranes. It is a tripartite molecule consisting of lipid A, which is embedded in the outer membrane, a core oligosaccharide and repeating O-antigen units that extend outward from the surface of the cell1, 2. LPS is an immunodominant molecule that is important for the virulence and pathogenesis of many bacterial species, including Pseudomonas aeruginosa, Salmonella species, and Escherichia coli3-5, and differences in LPS O-antigen composition form the basis for serotyping of strains. LPS is involved in attachment to host cells at the initiation of infection and provides protection from complement-mediated killing; strains that lack LPS can be attenuated for virulence6-8. For these reasons, it is important to visualize LPS, particularly from clinical isolates. Visualizing LPS banding patterns and recognition by specific antibodies can be useful tools to identify strain lineages and to characterize various mutants. 
In this report, we describe a hot aqueous-phenol method for the isolation and purification of LPS from Gram-negative bacterial cells. This protocol allows for the extraction of LPS away from nucleic acids and proteins that can interfere with visualization of LPS that occurs with shorter, less intensive extraction methods9. LPS prepared this way can be separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and directly stained using carbohydrate/glycoprotein stains or standard silver staining methods. Many anti-sera to LPS contain antibodies that cross-react with outer membrane proteins or other antigenic targets that can hinder reactivity observed following Western immunoblot of SDS-PAGE-separated crude cell lysates. Protease treatment of crude cell lysates alone is not always an effective way of removing this background using this or other visualization methods. Further, extensive protease treatment in an attempt to remove this background can lead to poor quality LPS that is not well resolved by any of the aforementioned methods. For these reasons, we believe that the following protocol, adapted from Westpahl and Jann10, is ideal for LPS extraction.

We describe a modified hot aqueous-phenol extraction method for purifying lipopolysaccharide (LPS) from Gram-negative bacteria. Once extracted, the LPS can be subsequently analyzed by SDS-PAGE and visualized by direct staining or Western immunoblot.

La purificación y la visualización de lipopolisacárido de bacterias Gram-negativas por Hot acuosa de fenol-Extracción

Lipopolysaccharide (LPS)  is a major component of Gram-negative bacterial outer membranes. It is a tripartite molecule consisting of  lipid A, which is ...

Detection of Bacteria Using Fluorogenic DNAzymes

Outbreaks linked to food-borne and hospital-acquired pathogens account for millions of deaths and hospitalizations as well as colossal economic losses each and every year. Prevention of such outbreaks and minimization of the impact of an ongoing epidemic place an ever-increasing demand for analytical methods that can accurately identify culprit pathogens at the earliest stage. Although there is a large array of effective methods for pathogen detection, none of them can satisfy all the following five premier requirements embodied for an ideal detection method: high specificity (detecting only the bacterium of interest), high sensitivity (capable of detecting as low as a single live bacterial cell), short time-to-results (minutes to hours), great operational simplicity (no need for lengthy sampling procedures and the use of specialized equipment), and cost effectiveness. For example, classical microbiological methods are highly specific but require a long time (days to weeks) to acquire a definitive result.1 PCR- and antibody-based techniques offer shorter waiting times (hours to days), but they require the use of expensive reagents and/or sophisticated equipment.2-4 Consequently, there is still a great demand for scientific research towards developing innovative bacterial detection methods that offer improved characteristics in one or more of the aforementioned requirements. Our laboratory is interested in examining the potential of DNAzymes as a novel class of molecular probes for biosensing applications including bacterial detection.5
DNAzymes (also known as deoxyribozymes or DNA enzymes) are man-made single-stranded DNA molecules with the capability of catalyzing chemical reactions.6-8 These molecules can be isolated from a vast random-sequence DNA pool (which contains as many as 1016 individual sequences) by a process known as "in vitro selection" or "SELEX" (systematic evolution of ligands by exponential enrichment).9-16 These special DNA molecules have been widely examined in recent years as molecular tools for biosensing applications.6-8
Our laboratory has established in vitro selection procedures for isolating RNA-cleaving fluorescent DNAzymes (RFDs; Fig. 1) and investigated the use of RFDs as analytical tools.17-29 RFDs catalyze the cleavage of a DNA-RNA chimeric substrate at a single ribonucleotide junction (R) that is flanked by a fluorophore (F) and a quencher (Q). The close proximity of F and Q renders the uncleaved substrate minimal fluorescence. However, the cleavage event leads to the separation of F and Q, which is accompanied by significant increase of fluorescence intensity.
More recently, we developed a method of isolating RFDs for bacterial detection.5 These special RFDs were isolated to "light up" in the presence of the crude extracellular mixture (CEM) left behind by a specific type of bacteria in their environment or in the media they are cultured (Fig. 1). The use of crude mixture circumvents the tedious process of purifying and identifying a suitable target from the microbe of interest for biosensor development (which could take months or years to complete). The use of extracellular targets means the assaying procedure is simple because there is no need for steps to obtain intracellular targets.
Using the above approach, we derived an RFD that cleaves its substrate (FS1; Fig. 2A) only in the presence of the CEM produced by E. coli (CEM-EC).5 This E. coli-sensing RFD, named RFD-EC1 (Fig. 2A), was found to be strictly responsive to CEM-EC but nonresponsive to CEMs from a host of other bacteria (Fig. 3).
Here we present the key experimental procedures for setting up E. coli detection assays using RFD-EC1 and representative results.

We have recently reported a novel approach for generating fluorogenic DNAzyme probes that can be applied to set up a simple, "mix-and-read" fluorescent assay for bacterial detection. These special DNA probes catalyze the cleavage of a chromophore-modified DNA-RNA chimeric substrate in the presence of crude extracellular mixture (CEM) produced by a specific bacterium, thereby translating bacterial detection into fluorescence signal generation. In this report we will describe key experimental procedures where a specific DNAzyme probe denoted "RFD-EC1" is employed for the detection of the model bacterium, Escherichia coli (E. coli).

La detección de bacterias mediante ADNzimas fluorogénicos

Outbreaks linked to food-borne and hospital-acquired pathogens account for millions of deaths and hospitalizations as well as colossal economic losses ...

Whole-cell MALDI-TOF Mass Spectrometry is an Accurate and Rapid Method to Analyze Different Modes of Macrophage Activation

MALDI-TOF is an extensively used mass spectrometry technique in chemistry and biochemistry. It has been also applied in medicine to identify molecules and biomarkers. Recently, it has been used in microbiology for the routine identification of bacteria grown from clinical samples, without preparation or fractionation steps. We and others have applied this whole-cell MALDI-TOF mass spectrometry technique successfully to eukaryotic cells. Current applications range from cell type identification to quality control assessment of cell culture and diagnostic applications. Here, we describe its use to explore the various polarization phenotypes of macrophages in response to cytokines or heat-killed bacteria. It allowed the identification of macrophage-specific fingerprints that are representative of the diversity of proteomic responses of macrophages. This application illustrates the accuracy and simplicity of the method. The protocol we described here may be useful for studying the immune host response in pathological conditions or may be extended to wider diagnostic applications.

This protocol describes the use of whole-cell MALDI-TOF mass spectrometry on eukaryotic cells. Here, we illustrate the accuracy of this technique by analyzing the multiple activation states of macrophages in response to their microenvironment.

MALDI-TOF is an extensively used mass spectrometry technique in chemistry and biochemistry. It has been also applied in medicine to identify molecules and ...

Isolation of Double Negative &#945;&#946; T Cells from the Kidney

There is currently no standard protocol for the isolation of DN T cells from the non-lymphoid tissues despite their increasingly reported involvement in various immune responses. DN T cells are a unique immune cell type that has been implicated in regulating immune and autoimmune responses and tolerance to allotransplants1-6. DN T cells are, however, rare in peripheral blood and secondary lymphoid organs (spleen and lymph nodes), but are major residents of the normal kidney. Very little is known about their pathophysiologic function7 due to their paucity in the periphery. We recently described a comprehensive phenotypic and functional analysis of this population in the kidney8 in steady state and during ischemia reperfusion injury. Analysis of DN T cell function will be greatly enhanced by developing a protocol for their isolation from the kidney.
Here, we describe a novel protocol that allows isolation of highly pure ab CD4+ CD8+ T cells and DN T cells from the murine kidney. Briefly, we digest kidney tissue using collagenase and isolate kidney mononuclear cells (KMNC) by density gradient. This is followed by two steps to enrich hematopoietic T cells from 3% to 70% from KMNC. The first step consists of a positive selection of hematopoietic cells using a CD45+ isolation kit. In the second step, DN T cells are negatively isolated by removal of non-desired cells using CD4, CD8, and MHC class II monoclonal antibodies and CD1d &#945;-galcer tetramer. This strategy leads to a population of more than 90% pure DN T cells. Surface staining with the above mentioned antibodies followed by FACs analysis is used to confirm purity.

Isolation of Double Negative &amp;#945;&amp;#946; T Cells from the Kidney

DNabT cells are rare among peripheral T cells; however, they are abundant in certain non-lymphoid tissues. Difficulty of isolating DN T cells from non-lymphoid tissue hinders their functional analysis despite increasing recognized pathophysiologic significance. We describe a novel protocol for isolation of highly purified DN T cells from murine kidney.

Aislamiento de doble negación células T αβ del Riñón

There is currently no standard protocol for the isolation of DN T cells from the non-lymphoid tissues despite their increasingly reported involvement in ...

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

Bloodstream infections and sepsis are a major cause of morbidity and mortality. The successful outcome of patients suffering from bacteremia depends on a rapid identification of the infectious agent to guide optimal antibiotic treatment. The analysis of Gram stains from positive blood culture can be rapidly conducted and already significantly impact the antibiotic regimen. However, the accurate identification of the infectious agent is still required to establish the optimal targeted treatment. We present here a simple and fast bacterial pellet preparation from a positive blood culture that can be used as a sample for several essential downstream applications such as identification by MALDI-TOF MS, antibiotic susceptibility testing (AST) by disc diffusion assay or automated AST systems and by automated PCR-based diagnostic testing. The performance of these different identification and AST systems applied directly on the blood culture bacterial pellets is very similar to the performance normally obtained from isolated colonies grown on agar plates. Compared to conventional approaches, the rapid acquisition of a bacterial pellet significantly reduces the time to report both identification and AST. Thus, following blood culture positivity, identification by MALDI-TOF can be reported within less than 1 hr whereas results of AST by automated AST systems or disc diffusion assays within 8 to 18 hr, respectively. Similarly, the results of a rapid PCR-based assay can be communicated to the clinicians less than 2 hr following the report of a bacteremia. Together, these results demonstrate that the rapid preparation of a blood culture bacterial pellet has a significant impact on the identification and AST turnaround time and thus on the successful outcome of patients suffering from bloodstream infections.

A rapid bacterial pellet preparation from a positive blood culture can be used as a sample for applications such as identification by MALDI-TOF, Gram staining, antibiotic susceptibility testing and PCR-based test. The results can be rapidly communicated to clinicians to improve the outcome of patients suffering from bloodstream infections.

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 ...

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, ...

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 ...

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 ...

Dextran Enhances the Lentiviral Transduction Efficiency of Murine and Human Primary NK Cells

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining the role of the gene of interest in the development, differentiation, and function of NK cells. Recent advances related to chimeric antigen receptors (CARs) in cancer immunotherapy accentuate the need for an efficient method to deliver exogenous genes to effector lymphocytes. The efficiencies of lentiviral-mediated gene transductions into primary human or mouse NK cells remain significantly low, which is a major limiting factor. Recent advances using cationic polymers, such as polybrene, show an improved gene transduction efficiency in T cells. However, these products failed to improve the transduction efficiencies of NK cells. This work shows that dextran, a branched glucan polysaccharide, significantly improves the transduction efficiency of human and mouse primary NK cells. This highly reproducible transduction methodology provides a competent tool for transducing human primary NK cells, which can vastly improve clinical gene delivery applications and thus NK cell-based cancer immunotherapy.

The goal of this study was to formulate technologies that allow for successful gene transduction in primary natural killer (NK) cells. The dextran-mediated lentiviral transduction of human or mouse primary NK cells results in higher gene expression efficiencies. This method of gene transduction will vastly improve NK cell genetic manipulation.

Dextrano mejora la eficiencia de transducción lentivirales de las células NK primaria murinas y humanas

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining ...