Este trabajo fue apoyado por la Fundación Bill y Melinda Gates (proyecto PneuCarriage, Grant 52099) y el Programa de Apoyo a la Infraestructura Operativa del Gobierno de Victoria. Gracias a Eileen Dunne, Janet Strachan, y Kim Hare para la lectura crítica del manuscrito.

1. Preparación de la suspensión celular para la tipificación <ol><li> El uso de un bucle de 1 l desechable estéril, tomar un pequeño barrido de fresco durante la noche cultivo puro de neumococos crecido en agar sangre de caballo no selectivo sólido e inocular 100 l Heart Infusion (HI) de caldo. La suspensión debe aparecer ser apenas visiblemente turbia. Agitar suavemente el tubo para emulsionar. Nota: otros medios, como el caldo de infusión cerebro corazón también pueden ser adecuados. La adición de suero (por ejemplo, una concentración final de 10% (v / v) de suero de caballo) y una incubación a 37 ° C durante aproximadamente 1 hora antes de su uso se puede usar para mejorar la reacción. </li></ol> 2. Comprobación de la densidad de la suspensión celular La suspensión celular preparada en la etapa 1.1 también se utilizará como control negativo, y se debe comprobar para densidad celular antes de añadir los sueros a escribir. <ol><li> El uso de un bucle de 1 l, transferir una gota de inóculo preparado en la etapa 1.1 en un vasodiapositiva s microscopio. </li><li> Con un par de pinzas, coloque una pequeña cubreobjetos circular sobre la suspensión. </li><li> Usando ajuste de contraste de fase, observar la preparación al microscopio bajo magnificación 400X, para comprobar la densidad de la suspensión (Figura 1A). <ol><li> Si el inóculo es demasiado pesada (Figura 2A), añadir más caldo a la suspensión de células realizado en el paso 1.1 para diluirlo. Mezcle suavemente de nuevo. </li><li> Si el inóculo es demasiado clara, por ejemplo, hay &lt;50 células visibles en el campo microscópico (Figura 2B), añadir más bacterias a la preparación de la suspensión de células realizado en el paso 1.1 y mezclar suavemente. Nota: Con la experiencia, la densidad de la suspensión de células se puede juzgar por agitación y sosteniendo a la luz, en el que debe aparecer ser simplemente visiblemente turbia. </li></ol></li><li> Repetir los pasos 2.1 a 2.3 hasta que un número adecuado de células son visibles en el campo microscópico. Nota: diapositivas y coversl Vidrioips presentan un peligro de objetos punzantes. Después de su uso, diapositivas y cubreobjetos están contaminados con material infeccioso y deben desecharse en un recipiente previsto. </li></ol> 3. Mecanografía <ol><li> Transferencia ~ 1 l del inóculo preparado (por ejemplo, utilizando un bucle de 1 l) sobre el portaobjetos de microscopio de vidrio. Tire el asa en el recipiente previsto. </li><li> Añadir un volumen igual (1 l asa) del antisuero a temperatura ambiente en la diapositiva y mezclar dos gotas de bien con el bucle. </li><li> Con un par de pinzas, coloque una pequeña cubreobjetos circular sobre la suspensión, teniendo cuidado de no tocar la gota. Coloque el cubreobjetos sobre la suspensión inmediatamente después de la adición de antisueros para evitar que el fluido en la diapositiva de la desecación. </li><li> Usando un microscopio, observar la suspensión bajo contraste de fase con una ampliación de 400X. Se observa una reacción negativa Quellung cuando poco o nada de &#39;hinchazón&#39; de las células neumocócicas pueden verse en 40Magnificación 0X, similar a lo que se observa en la suspensión celular neumocócica sin antisueros añadió (control negativo). Se observa una reacción positiva de Quellung cuando las células aparecen ampliada o &quot;hinchado&quot; y más visible cuando se compara con las células en el control negativo. Nota: reacciones dudosos deberán investigarse más a fondo, por ejemplo mediante el examen de la reacción después de 5-10 minutos de incubación a temperatura ambiente, o mediante la repetición de la reacción con antisuero más. </li><li> Realizar la serotipificación neumocócica con pruebas sucesivas de piscinas antisueros individuales hasta que se observa una reacción positiva. Alternativamente, las piscinas se pueden aplicar en &#39;rondas&#39;, dependiendo de la incidencia local de la serotipos. Nota: Las pruebas en &quot;rounds&quot; asegura que las piscinas que contienen anticuerpos contra los serotipos más comunes se prueban primero, minimizando el esfuerzo y el costo. Ensayo simultáneo de varias piscinas también proporciona controles negativos adicionales, que puede ser útil en interpretation. Si todas las piscinas en un &#39;round&#39; particular, son negativos, probar el aislamiento con las siguientes rondas de piscinas. </li><li> Pruebe el aislado utilizando el tipo apropiado o antisuero grupo contenida dentro de la piscina reactiva. En su caso, utilizar factor de antisueros para diferenciar aún más el serotipo. Nota: Claves para antisueros neumocócica suministrados por el fabricante se utilizan en la selección de la piscina, el tipo y grupo o factor de antisueros y la interpretación de los resultados (incluidas posibles reacciones cruzadas). </li><li> Si se obtienen resultados negativos con todas las piscinas, probar el aislamiento con el reactivo Omniserum (contiene anticuerpos contra 91 serotipos 21). Si todavía no se observa una reacción positiva de Quellung cuando se prueba un neumocócica aislar con el reactivo Omniserum, esto puede ser debido a que el aislamiento está expresando poca o ninguna cápsula, o que los anticuerpos contra el serotipo no están representados en la Omniserum. Esta última categoría puede incluir nuevos serotipos. </li> &lt;/ Ol&gt;

<ol>
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Dis. 4, 144-154 (2004).</li><li>Henrichsen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Six+newly+recognized+types+of+Streptococcus+pneumoniae.">Six newly recognized types of Streptococcus pneumoniae.</a> J. Clin. Microbiol. 33, 2759-2762 (1995).</li><li>Calix, J. J., 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=Biochemical,+genetic,+and+serological+characterization+of+two+capsule+subtypes+among+Streptococcus+pneumoniae+Serotype+20+strains:+discovery+of+a+new+pneumococcal+serotype.">Biochemical, genetic, and serological characterization of two capsule subtypes among Streptococcus pneumoniae Serotype 20 strains: discovery of a new pneumococcal serotype.</a> J. Biol. Chem. 287, 27885-27894 (2012).</li><li>Hausdorff, W. P., Bryant, J., Paradiso, P. R., Siber, G. 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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Carriage+of+pneumococci+after+pneumococcal+vaccination.">Carriage of pneumococci after pneumococcal vaccination.</a> Lancet. 348, 271-272 (1996).</li><li>Mulholland, K., Satzke, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serotype+replacement+after+pneumococcal+vaccination.">Serotype replacement after pneumococcal vaccination.</a> Lancet. 379, 1388-1389 (2012).</li><li>Austrian, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+quellung+reaction,+a+neglected+microbiologic+technique.">The quellung reaction, a neglected microbiologic technique.</a> Mt. Sinai. J. Med. 43, 699-709 (1976).</li><li>Konradsen, H. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Validation+of+serotyping+of+Streptococcus+pneumoniae+in+Europe.">Validation of serotyping of Streptococcus pneumoniae in Europe.</a> Vaccine. 23, 1368-1373 (2005).</li><li>Lovgren, 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=Evolution+of+an+international+external+quality+assurance+model+to+support+laboratory+investigation+of+Streptococcus+pneumoniae,+developed+for+the+SIREVA+project+in+Latin+America,+from.">Evolution of an international external quality assurance model to support laboratory investigation of Streptococcus pneumoniae, developed for the SIREVA project in Latin America, from.</a> J. Clin. Microbiol. 45, 3184-3190 (2007).</li><li>Reasonover, 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=The+International+Circumpolar+Surveillance+interlaboratory+quality+control+program+for+Streptococcus+pneumoniae.">The International Circumpolar Surveillance interlaboratory quality control program for Streptococcus pneumoniae.</a> J. Clin. Microbiol. 49, 138-143 (2011).</li><li>Hare, K. 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=#34;Dodgy+6As&amp;#34;:+differentiating+pneumococcal+serotype+6C+from+6A+by+use+of+the+Quellung+reaction.">#34;Dodgy 6As&amp;#34;: differentiating pneumococcal serotype 6C from 6A by use of the Quellung reaction.</a> J. Clin. Microbiol. 47, 1981-1982 (2009).</li><li>Kong, F., Gilbert, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+cpsA-cpsB+sequence+polymorphisms+and+serotype-/group-specific+PCR+to+predict+51+Streptococcus+pneumoniae+capsular+serotypes.">Using cpsA-cpsB sequence polymorphisms and serotype-/group-specific PCR to predict 51 Streptococcus pneumoniae capsular serotypes.</a> J. Med. 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Los autores no tienen nada que revelar.

Un paso crítico en esta técnica es asegurar un cultivo puro se utiliza para preparar la suspensión junto con la adición de un número adecuado de células bacterianas al caldo. Cabe señalar que el crecimiento de colonias y la morfología en placas de agar varía entre diferentes aislamientos de neumococos. Algunas producen colonias seca y áspera, que pueden ser difíciles de recoger con un bucle y no emulsionar bien en el caldo. Por esta razón, un barrido más grande de las bacterias puede ser necesaria cuando la preparación de la suspensión de células. En cualquier caso, es imperativo que las células individuales, y no agregados, son evidentes en el control negativo de modo que su tamaño relativo se puede comparar con las células con antisueros añadido. Además, demasiadas células bacterianas pueden resultar en una reacción negativa falsa, donde un exceso de antígeno capsular puede inhibir la reacción de Quellung 10. Por esta razón, es importante para crear una suspensión bacteriana con una densidad celular apropiada (Figura 1). El SUSpensión se puede conservar durante varios meses mediante la adición de varias gotas de formalina concentrada. Esto permite pruebas de lotes más fácil y también esteriliza el caldo de reducir el riesgo de infecciones adquiridas en el laboratorio, aunque esto debe sopesarse frente a los riesgos de seguridad de la manipulación de formalina, que es tóxico, corrosivo, cancerígeno, sensibilizante, e inflamable. Además, hemos observado que algunos aislados muestran reacciones más débiles en la presencia de formalina (datos no mostrados). Reacciones de Quellung se examinaron usando un microscopio. El uso de contraste de fase mejora la visibilidad de la cápsula. En algunos laboratorios, la reacción se ve mediante inmersión en aceite a 1000 x Ampliación 14, 16. Además, algunos laboratorios utilizan una mancha contador (azul de metileno) para mejorar aún más la visibilidad de una reacción positiva 10, 16, 22. El método que aquí se presenta es visto sin aceite-immersion y bajo una ampliación de 400X 15, 17. Este método es rápido, sencillo y relativamente fácil de leer 23. Es importante mantener buenos procedimientos de control interno de la calidad para asegurar los procedimientos de antisueros y pruebas están en orden. La participación en un programa de garantía de calidad externa a menudo es beneficioso. Una limitación importante de la reacción de Quellung por el serotipado de neumococos es el alto costo de los antisueros requerido. Sin embargo, esto se reduce al mínimo en el protocolo anterior debido a la pequeña cantidad de antisuero usado para cada reacción. Varios otros métodos han sido descritos para la serotipificación neumococos 23, 24. Sin embargo, de Quellung sigue siendo el estándar de oro y el desarrollo de nuevos métodos requiere la normalización en contra de la reacción de Quellung 23. Utilizando antisueros adecuados, la reacción de Quellung también puede ser utilizado para identificar y escriba otras tapasUle la producción de bacterias 25. La reacción Quellung es fundamental en la caracterización de los serotipos de neumococo actuales y emergentes, así como proporcionar información sobre la eficacia de la vacuna y el impacto a largo plazo de la vacunación en el carro y la enfermedad.

Streptococcus pneumoniae (neumococo) es responsable de la muerte de unos 800.000 niños menores de cinco años anualmente, con la carga de morbilidad que pesa sobre todo en los países con pocos recursos 1. Las enfermedades neumocócicas varían en gravedad de las infecciones localizadas como la otitis media aguda a las infecciones que amenazan la vida graves como sepsis, meningitis y neumonía 2. ¡Más de 90 serotipos capsulares de neumococos se han descrito 3, 4. Vacunas neumocócicas actuales proporcionan protección contra los serotipos que son responsables de la mayoría de la enfermedad invasiva 5, 6. Sin embargo, el uso generalizado de las vacunas se ha asociado con el reemplazo de serotipos incluidos en la vacuna por los serotipos no vacunales, tanto en el transporte nasofaríngeo y enfermedad invasiva 7, 8. Esto pone de relieve la importancia de la serotipificación por epidemiológicala vigilancia y el impacto de la vacuna a largo plazo estudia 9. El método estándar de oro para la tipificación neumocócica es la prueba de reacción capsular, conocido como la reacción de Quellung, primero descrita por Neufeld en 1902 (ver austriaca 10). Se ha encontrado un alto grado de concordancia entre los laboratorios y entre la reacción Quellung y otros métodos de serotipificación 11-13. Existen diversas variaciones de la reacción de Quellung 10, 14-16. El método descrito aquí no requiere el uso de una mancha de venta libre o la lente de inmersión en aceite; más bien, una gota de muestra preparada es mezclada con el suero de escribir y examinó inmediatamente bajo un aumento de 400X. La reacción de Quellung se realiza generalmente utilizando antisueros disponibles comercialmente. El método descrito aquí usa menos antisueros en comparación con algunos otros métodos 16, 17, por lo que es más rentable. Una vez que un aisladorcomió se identifica como el neumococo, se secuencialmente probado con piscinas antisueros hasta que se observa una reacción positiva. Cada antisuero piscina contiene diferentes mezclas de antisueros producidos contra 91 serotipos de neumococo 18. Una vez establecida la piscina, el tipo individual y del grupo de antisueros que se incluyen en el grupo reactivo se prueban individualmente en secuencia. Tipo antisueros generalmente reaccionan con un solo serotipo (por ejemplo, tipo 1 antisuero reacciona con el serotipo 1), mientras que los antisueros de grupo reaccionan con todos los serotipos de un grupo en particular (por ejemplo, el Grupo 22 antisuero reacciona con los serotipos 22F y 22A). Algunos serotipos dentro de los grupos se diferencian adicionalmente usando antisueros de los factores (por ejemplo, el serotipo 22F reacciona con el factor 22b, pero no 22c). En este caso la prueba se continuó con todos los antisueros factor relevante hasta que se determina el serotipo 19, 20. La reacción de Quellung es simple y rápida 14 y se puede realizar en cualquier laboratory que está equipado con un microscopio de buena calidad y conjunto apropiado de antisueros.

<table border="0" cellpadding="0" cellspacing="0" width="1305">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;">Pneumococcus (Neufeld) antisera</td>
			<td>SSI Diagnostica</td>
			<td></td>
			<td><a href="http://www.ssi.dk/ssidiagnostica">http://www.ssi.dk/ssidiagnostica</a> 
			Bring all antisera to room temperature before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pneumococcus (Omni) antiserum</td>
			<td>SSI Diagnostica</td>
			<td>2438</td>
			<td>Bring all antisera to room temperature before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Optical Microscope</td>
			<td>Leica Microsystems</td>
			<td>Leica DML</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Calibrated Disposable Inoculating Loops 1 &#181;l</td>
			<td>Copan</td>
			<td>CD175S01</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Horse Blood Agar (HBA) plates</td>
			<td>Thermo Fisher Scientific</td>
			<td>PP2001</td>
			<td><a href="http://www.thermofisher.com.au/">http://www.thermofisher.com.au/</a> 
			Bring to room temperature before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Heart Infusion (HI) broth</td>
			<td>Media Preparation Unit (MPU), The University of Melbourne</td>
			<td>40HIN</td>
			<td>Bring to room temperature before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glass microscope slides 76 mm x 26 mm</td>
			<td>Lomb Scientific</td>
			<td>7101</td>
			<td>Sharps hazard: Dispose in a biohazard sharps container</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Circular glass coverslips</td>
			<td>NeuVitro&#160;</td>
			<td>GG-5</td>
			<td></td>
		</tr>
		<tr height="20">
			<td colspan="2" height="20" style="height:20px;">*Excluding the antisera, all other materials can be purchased from alternative suppliers</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;">*Plates prepared from defibranated sheep blood are suitable but blood plates prepared from citrated blood or human blood are not</td>
		</tr>
	</tbody>
</table>

capsular serotyping of streptococcus pneumoniae using the quellung reaction

Hay más de 90 diferentes serotipos capsulares de Streptococcus pneumoniae (neumococo). Además de ser una herramienta para la comprensión de la epidemiología de neumococo, serotipificación capsular puede proporcionar información útil para los estudios de eficacia y de impacto de la vacuna. La reacción de Quellung es el método estándar de oro para la serotipificación capsular de neumococo. El método implica la evaluación de una suspensión celular neumocócica con antisueros agrupada y específica dirigida contra el polisacárido capsular. Las reacciones antígeno-anticuerpo se observan microscópicamente. El protocolo tiene tres pasos principales: 1) Preparación de una suspensión de células bacterianas, 2) de mezcla de células y antisuero sobre un portaobjetos de vidrio, y 3) la lectura de la reacción de Quellung usando un microscopio. La reacción de Quellung es razonablemente fácil de realizar y se puede aplicar siempre que sea un microscopio y antisueros adecuados están disponibles.

<html> Una reacción positiva de Quellung se produce cuando anticuerpos de tipo específico se une a la cápsula del neumococo, que conduce a un cambio en su índice de refracción 10. Cuando se observa bajo un microscopio, las bacterias parecen &#39;hinchada&#39; y más visible 14. Figura 1A muestra células neumocócicas en caldo de HI. Cuando se añade antisuero de tipo específico a la suspensión bacteriana, neumococos aparece &#39;hinchada&#39;, de refracción y más redondeada (Figura 1B). Demasiados células bacterianas añadidas al caldo puede dar lugar a la agregación de las células o una reacción falsa negativa. Figura 2 muestra inóculos de células neumocócicas en caldo HI que son &quot;demasiado pesado&quot; (Figura 2) o &#39;demasiado ligero&#39; (Figura 2B). 08fig1highres.jpg &quot;src =&quot; / files/ftp_upload/51208/51208fig1.jpg &quot;/&gt; Figura 1. Reacción negativa y positiva de Quellung. Preparaciones de serotipo 9V neumocócica aislar sin antisuero (1A) y con el grupo 9 antisuero (1B), se observa bajo una ampliación de 400X. Este último muestra la &#39;inflamación&#39; y el redondeo de las células bacterianas típicamente visto en una reacción positiva de Quellung. <img alt="Figura 2" fo:content-width="5in" fo:src="/files/ftp_upload/51208/51208fig2highres.jpg" src="/files/ftp_upload/51208/51208fig2.jpg" /> Figura 2. Inóculos demasiado pesada y demasiado clara. Preparaciones de suspensiones celulares de neumococo que son demasiado pesados ​​(2A) o demasiado clara (2B), cuando se utiliza con una ampliación de 400X.

Watch this Scientific Journal Video about Capsular Serotyping of Streptococcus pneumoniae Using the Quellung Reaction at JoVE.com

Capsular Serotyping of Streptococcus pneumoniae Using the Quellung Reaction

La reacción de Quellung es la técnica estándar de oro para la serotipificación Streptococcus pneumoniae. Esta técnica utiliza un microscopio y antisueros neumocócica específica y se utiliza comúnmente en referencia y laboratorios de investigación en todo el mundo.

Capsular serotipificación de<em&gt; Streptococcus pneumoniae</em&gt; Uso de la Reacción Quellung

There are over 90 different capsular serotypes of Streptococcus pneumoniae (the pneumococcus). As well as being a tool for understanding pneumococcal ...

capsular-serotyping-streptococcus-pneumoniae-using-quellung

Research

JoVE Journal

Immunology and Infection

Capsular Serotyping of Streptococcus pneumoniae Using the Quellung Reaction

45.2K vistas.  Murdoch Childrens Research Institute.  La reacción de Quellung es la técnica estándar de oro para la serotipificación Streptococcus pneumoniae. Esta técnica utiliza un microscopio y antisueros neumocócica específica y se utiliza comúnmente en referencia y laboratorios de investigación en todo el mundo. 

Video: Capsular Serotyping of Streptococcus pneumoniae Using the Quellung Reaction

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of infection with influenza virus alone. Instead, most individuals are thought to have succumbed to a secondary bacterial infection, predominately caused by the bacterium Streptococcus pneumoniae (the pneumococcus). The synergistic relationship between infections caused by influenza virus and the pneumococcus has subsequently been observed during the 1957 Asian influenza virus pandemic, as well as during seasonal outbreaks of the virus (reviewed in 1, 2). Here, we describe a protocol used to investigate the mechanism(s) that may be involved in increased morbidity as a result of concurrent influenza A virus and S. pneumoniae infection. We have developed an infant murine model to reliably and reproducibly demonstrate the effects of influenza virus infection of mice colonised with S. pneumoniae. Using this protocol, we have provided the first insight into the kinetics of pneumococcal transmission between co-housed, neonatal mice using in vivo imaging 3.

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

A concurrent infection with influenza A virus is one of the factors implicated in the induction of invasive pneumococcal disease during asymptomatic Streptococcus pneumoniae carriage. Here we describe a mixed infection method using infant mice to investigate the synergism between these two respiratory pathogens.

Utilizando imágenes bioluminiscentes encargado de investigar la sinergia entre Streptococcus pneumoniae Y el Virus de la Influenza en ratones lactantes

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of ...

Investigación

Inmunología e Infección

Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy

During the last few years scientists became increasingly aware that average data obtained from microbial population based experiments are not representative of the behavior, status or phenotype of single cells. Due to this new insight the number of single cell studies rises continuously (for recent reviews see 1,2,3). However, many of the single cell techniques applied do not allow monitoring the development and behavior of one specific single cell in time (e.g. flow cytometry or standard microscopy).
Here, we provide a detailed description of a microscopy method used in several recent studies 4, 5, 6, 7, which allows following and recording (fluorescence of) individual bacterial cells of Bacillus subtilis and Streptococcus pneumoniae through growth and division for many generations. The resulting movies can be used to construct phylogenetic lineage trees by tracing back the history of a single cell within a population that originated from one common ancestor. This time-lapse fluorescence microscopy method cannot only be used to investigate growth, division and differentiation of individual cells, but also to analyze the effect of cell history and ancestry on specific cellular behavior. Furthermore, time-lapse microscopy is ideally suited to examine gene expression dynamics and protein localization during the bacterial cell cycle. The method explains how to prepare the bacterial cells and construct the microscope slide to enable the outgrowth of single cells into a microcolony. In short, single cells are spotted on a semi-solid surface consisting of growth medium supplemented with agarose on which they grow and divide under a fluorescence microscope within a temperature controlled environmental chamber. Images are captured at specific intervals and are later analyzed using the open source software ImageJ.

Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy

This protocol provides a step-by-step procedure to monitor single cell behavior of different bacteria in time using automated fluorescence time-lapse microscopy. Furthermore, we provide guidelines how to analyze the microscopy images.

Imágenes de células vivas de Bacillus subtilis Y Streptococcus pneumoniae Automatizada utilizando Time-lapse microscopía

During the last few years scientists became increasingly aware that average data obtained from microbial population based experiments are not ...

Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae

Nasopharyngeal colonization by Streptococcus pneumoniae is a prerequisite to invasion to the lungs or bloodstream1. This organism is capable of colonizing the mucosal surface of the nasopharynx, where it can reside, multiply and eventually overcome host defences to invade to other tissues of the host. Establishment of an infection in the normally lower respiratory tract results in pneumonia. Alternatively, the bacteria can disseminate into the bloodstream causing bacteraemia, which is associated with high mortality rates2, or else lead directly to the development of pneumococcal meningitis. Understanding the kinetics of, and immune responses to, nasopharyngeal colonization is an important aspect of S. pneumoniae infection models.
Our mouse model of intranasal colonization is adapted from human models3 and has been used by multiple research groups in the study of host-pathogen responses in the nasopharynx4-7. In the first part of the model, we use a clinical isolate of S. pneumoniae to establish a self-limiting bacterial colonization that is similar to carriage events in human adults. The procedure detailed herein involves preparation of a bacterial inoculum, followed by the establishment of a colonization event through delivery of the inoculum via an intranasal route of administration. Resident macrophages are the predominant cell type in the nasopharynx during the steady state. Typically, there are few lymphocytes present in uninfected mice8, however mucosal colonization will lead to low- to high-grade inflammation (depending on the virulence of the bacterial species and strain) that will result in an immune response and the subsequent recruitment of host immune cells. These cells can be isolated by a lavage of the tracheal contents through the nares, and correlated to the density of colonization bacteria to better understand the kinetics of the infection.

Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae

Colonization of the murine nasopharynx with Streptococcus pneumoniae and the subsequent extraction of adherent or recruited cells is described. This technique involves flushing the nasopharynx and collection of the fluid through the nares and is adaptable for various readouts, including differential cell quantification and analysis of mRNA expression in situ.

Caracterización De Las Respuestas Inflamatorias Durante La Colonización Intranasal Con Streptococcus pneumoniae

Nasopharyngeal colonization by Streptococcus pneumoniae is a prerequisite to invasion to the lungs or bloodstream1. This organism is capable of colonizing ...

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 ...

Capsular Serotyping of Streptococcus pneumoniae by Latex Agglutination

Latex agglutination reagents are widely used in microbial diagnosis, identification and serotyping. Streptococcus pneumoniae (the pneumococcus) is a major cause of morbidity and mortality world-wide. Current vaccines target the pneumococcal capsule, and there are over 90 capsular serotypes. Serotyping pneumococcal isolates is therefore important for assessing the impact of vaccination programs and for epidemiological purposes. The World Health Organization has recommended latex agglutination as an alternative method to the &#8216;gold standard&#8217; Quellung test for serotyping pneumococci. Latex agglutination is a relatively simple, quick and inexpensive method; and is therefore suitable for resource-poor settings as well as laboratories with high-volume workloads. Latex agglutination reagents can be prepared in-house utilizing commercially-sourced antibodies that are passively attached to latex particles. This manuscript describes a method of production and quality control of latex agglutination reagents, and details a sequential testing approach which is time- and cost-effective. 
This method of production and quality control may also be suitable for other testing purposes.

Capsular Serotyping of Streptococcus pneumoniae by Latex Agglutination

Latex agglutination testing is a simple, rapid and inexpensive method for serotyping Streptococcus pneumoniae, and has also been widely applied in diagnostic microbiology. This manuscript describes the in-house production of latex agglutination reagents, quality control procedures and the application of this technique to pneumococcal serotyping.

Capsular Serotipificación de<em&gt; Streptococcus pneumoniae</em&gt; Por partículas de látex

Latex agglutination reagents are widely used in microbial diagnosis, identification and serotyping. Streptococcus pneumoniae (the pneumococcus) is a major ...

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of causing systemic disease in both fish and humans. A better understanding of S. iniae disease pathogenesis requires an appropriate model system. The genetic tractability and the optical transparency of the early developmental stages of zebrafish allow for the generation and non-invasive imaging of transgenic lines with fluorescently tagged immune cells. The adaptive immune system is not fully functional until several weeks post fertilization, but zebrafish larvae have a conserved vertebrate innate immune system with both neutrophils and macrophages. Thus, the generation of a larval infection model allows the study of the specific contribution of innate immunity in controlling S. iniae infection.
The site of microinjection will determine whether an infection is systemic or initially localized. Here, we present our protocols for otic vesicle injection of zebrafish aged 2-3 days post fertilization as well as our techniques for fluorescent confocal imaging of infection. A localized infection site allows observation of initial microbe invasion, recruitment of host cells and dissemination of infection. Our findings using the zebrafish larval model of S. iniae infection indicate that zebrafish can be used to examine the differing contributions of host neutrophils and macrophages in localized bacterial infections. In addition, we describe how photolabeling of immune cells can be used to track individual host cell fate during the course of infection.

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

Here, we present a protocol for the generation and imaging of a localized bacterial infection in the zebrafish otic vesicle.

Imagen no invasiva de la respuesta inmune innata en un modelo de larvas de pez cebra de<em&gt; Streptococcus iniae</em&gt; Infección

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of ...

A Murine Model of Group B Streptococcus Vaginal Colonization

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of 10 - 30% of adults. In immune-compromised individuals, including neonates, pregnant women, and the elderly, GBS may switch to an invasive pathogen causing sepsis, arthritis, pneumonia, and meningitis. Because GBS is a leading bacterial pathogen of neonates, current prophylaxis is comprised of late gestation screening for GBS vaginal colonization and subsequent peripartum antibiotic treatment of GBS-positive mothers. Heavy GBS vaginal burden is a risk factor for both neonatal disease and colonization. Unfortunately, little is known about the host and bacterial factors that promote or permit GBS vaginal colonization. This protocol describes a technique for establishing persistent GBS vaginal colonization using a single &#946;-estradiol pre-treatment and daily sampling to determine bacterial load. It further details methods to administer additional therapies or reagents of interest and to collect vaginal lavage fluid and reproductive tract tissues. This mouse model will further the understanding of the GBS-host interaction within the vaginal environment, which will lead to potential therapeutic targets to control maternal vaginal colonization during pregnancy and to prevent transmission to the vulnerable newborn. It will also be of interest to increase our understanding of general bacterial-host interactions in the female vaginal tract.

A Murine Model of Group B Streptococcus Vaginal Colonization

The purpose of this protocol is to imitate human group B Streptococcus (GBS) vaginal colonization in a murine model. This method may be used to investigate host immune responses and bacterial factors contributing to GBS vaginal persistence, as well as to test therapeutic strategies.

Un modelo murino de Grupo B<em&gt; Streptococcus</em&gt; Colonización vaginal

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of ...

A Robust Pneumonia Model in Immunocompetent Rodents to Evaluate Antibacterial Efficacy against S. pneumoniae, H. influenzae, K. pneumoniae, P. aeruginosa or A. baumannii

Efficacy of candidate antibacterial treatments must be demonstrated in animal models of infection as part of the discovery and development process, preferably in models which mimic the intended clinical indication. A method for inducing robust lung infections in immunocompetent rats and mice is described which allows for the assessment of treatments in a model of serious pneumonia caused by S. pneumoniae, H. influenzae, P. aeruginosa, K. pneumoniae or A. baumannii. Animals are anesthetized, and an agar-based inoculum is deposited deep into the lung via nonsurgical intratracheal intubation. The resulting infection is consistent, reproducible, and stable for at least 48 h and up to 96 h for most isolates. Studies with marketed antibacterials have demonstrated good correlation between in vivo efficacy and in vitro susceptibility, and concordance between pharmacokinetic/pharmacodynamic targets determined in this model and clinically accepted targets has been observed. Although there is an initial time investment when learning the technique, it can be performed quickly and efficiently once proficiency is achieved. Benefits of the model include elimination of the neutropenic requirement, increased robustness and reproducibility, ability to study more pathogens and isolates, improved flexibility in study design and establishment of a challenging infection in an immunocompetent host.

A Robust Pneumonia Model in Immunocompetent Rodents to Evaluate Antibacterial Efficacy against S. pneumoniae, H. influenzae, K. pneumoniae, P. aeruginosa or  A. baumannii

A method for establishing lung infections in immunocompetent rodents is described. With proficiency, this method can be performed quickly and easily to induce stable infection with many isolates of S. pneumoniae, H. influenzae, P. aeruginosa, K. pneumoniae and A. baumannii.

Un modelo de neumonía en inmunocompetentes robusta roedores para evaluar Eficacia antibacteriana contra<i&gt; S. pneumoniae</i&gt;,<i&gt; H. influenzae</i&gt;,<i&gt; K. pneumoniae</i&gt;,<em&gt; P. aeruginosa</em&gt; o<em&gt; A. baumannii</em

Efficacy of candidate antibacterial treatments must be demonstrated in animal models of infection as part of the discovery and development process, ...

Intracranial Subarachnoidal Route of Infection for Investigating Roles of Streptococcus suis Biofilms in Meningitis in a Mouse Infection Model

Streptococcus suis is not only a major bacterial pathogen of pigs worldwide but also an emerging zoonotic agent. In humans and pigs, meningitis is a major manifestation of S. suis infections. A suitable infection model is an essential tool to understand the mechanisms of diseases caused by pathogens. Several routes of infection in mice have been developed to study the pathogenesis of S. suis infection. However, the intraperitoneal, intranasal, and intravenous routes of infection are not suitable for studying the roles of S. suis surface components in meningitis directly in the brain, such as the extracellular matrix from biofilms. Although intracisternal inoculation has been used for S. suis infection, the precise injection site has not been described. Here, the intracranial subarachnoidal route of infection was described in a mouse model to investigate the roles of biofilms in S. suis meningitis. S. suis planktonic cells or biofilm state cells were directly injected into the subarachnoid space of mice through the injection site located 3.5 mm rostral from the bregma. Histopathological analysis and increased mRNA expression of TLR2 and cytokines of the brain tissue from mice injected with biofilm state cells clearly indicated that S. suis biofilm plays definitive roles in S. suis meningitis. This route of infection has obvious advantages over other routes of infection, allowing the study of the host-bacterium interaction. Furthermore, it permits the effect of bacterial components on host immune responses directly in the brain to be assessed, and mimics bacterial entrance into the central nervous system. This route of infection can be extended for investigating the mechanisms of meningitis caused by other bacteria. In addition, it can also be used to test the efficacy of drugs against bacterial meningitis.

Intracranial Subarachnoidal Route of Infection for Investigating Roles of Streptococcus suis Biofilms in Meningitis in a Mouse Infection Model

Here, we describe the intracranial subarachnoidal route of infection in mice to study roles of biofilms in Streptococcus suis meningitis. This infection model is also suitable for studying the pathogenesis of other bacterial meningitis and the efficacy of new drugs against bacterial meningitis.

Ruta de subaracnoideo intracraneal de la infección para investigar funciones de biopelículas del Streptococcus suis en Meningitis en un modelo murino de infección

Streptococcus suis is not only a major bacterial pathogen of pigs worldwide but also an emerging zoonotic agent. In humans and pigs, meningitis is a major ...

Measuring Dengue Virus RNA in the Culture Supernatant of Infected Cells by Real-time Quantitative Polymerase Chain Reaction

At present, real-time polymerase chain reaction (PCR) technology is an indispensable tool for the detection and quantification of viral genomes in research laboratories, as well as for molecular diagnosis, because of its sensitivity, specificity, and convenience. However, in most cases, the quantitative PCR (qPCR) assay generally used to detect virus infection has relied on the purification of viral nucleic acid prior to the PCR step. In this study, the fluorescence-based reverse transcription qPCR (RT-qPCR) assay is developed through the combination of a processing buffer and a one-step RT-PCR reagent so that the whole process, from the harvest of the culture supernatant of virus-infected cells until real-time detection, can be performed without viral RNA purification. The established protocol enables the quantification of a wide range of RNA concentrations of dengue virus (DENV) within 90 min. In addition, the adaptability of the direct RT-qPCR assay to the evaluation of an antiviral agent is demonstrated by an in vitro experiment using a previously reported DENV inhibitor, mycophenolic acid (MPA). Moreover, other RNA viruses, including yellow fever virus (YFV), Chikungunya virus (CHIKV), and measles virus (MeV), can be quantified by direct RT-qPCR with the same protocol. Therefore, the direct RT-qPCR assay described in this report is useful for monitoring RNA virus replication in a simple and rapid manner, which will be further developed into a promising platform for a high-throughput screening study and clinical diagnosis.

Real-time quantitative polymerase chain reaction analysis combined with reverse transcription (RT-qPCR) has been widely used to measure the level of RNA virus infections. Here we present a direct RT-qPCR assay, which does not require an RNA purification step, developed for the quantification of several RNA viruses, including dengue virus.

Medición de ARN del Virus del Dengue en el sobrenadante de cultivo de las células infectadas por reacción en cadena de la polimerasa cuantitativo en tiempo real