NIAID subvención K22AI081748.

1. El flujo de goteo Biofilm Reactor <ol><li> El flujo de goteo reactor biopelícula (disponible en las tecnologías de Biosurface o versiones personalizadas diseñadas por lo general se puede hacer por talleres mecánicos universidad, ver Figura 1) se monta en autoclave. Asamblea consiste en la colocación de bonos en las cámaras y asegurar las tapas de cámara. La cámara, junto con el medio de biofilm (caldo de soja tríptico 2 g / L de glucosa y 2 g / L), y el tubo de nutrientes del afluente son esterilizados en autoclave. </li><li> La inoculación del reactor de flujo de goteo se realiza por colocar el reactor en una superficie plana, las líneas de tubería de sujeción de efluentes, llenando cada cámara con 10 ml de caldo de soja tríptico y la adición de 10 l de una S. cultura aureus crecido durante la noche en caldo de soja tríptico. El reactor se inoculó a continuación, coloque en una incubadora a 37 ° C durante 18 horas. </li><li> Después de 18 horas de incubación, los tubos de aguas residuales se suelta y el reactor se coloca en un bloque de madera cortada en un ángulo de 10 °. </li><li> Asépticamente conectar la tubería de nutrientes del afluente a la botella con el caldo de flujo de nutrientes continuo. Alimentar la línea de tubería a través de la bomba y los tubos de la primera mediante la ejecución de la bomba a una velocidad máxima (varía según el modelo de la bomba). </li><li> Una vez que la tubería del afluente se prepara detener la bomba y colocar las agujas se conectan (calibre 22, 1 pulgada) del extremo de cada tubo. Limpie el tapón de entrada de la cámara con una mezcla de etanol y limpiar asépticamente insertar las agujas a través del tapón de entrada. </li><li> Encienda la bomba y permita que los medios de comunicación a gotear lentamente (caudal ~ 125 l / min) en los cupones. Los medios de comunicación debe fluir hacia abajo a lo largo del cupón desde el puerto de entrada de tapón en el puerto de efluentes. Operar el reactor en flujo continuo durante 2-5 días (dependiendo de la aplicación), de vez en cuando comprobar el reactor de un drenaje adecuado. </li><li> Para cosechar las biopelículas de flujo de goteo reactor, detenga la bomba y retire con cuidado las agujas del reactor. El reactor puede ser colocado sobre una superficie plana y los cupones pueden ser asépticamente usando una pinza estéril. Si la microscopía se desea, los cupones ya se pueden procesar en consecuencia (Figura 2B es una micrografía electrónica de barrido de una biopelícula S. aureus crecido en un reactor de biofilm por goteo). Si la cuantificación de la biomasa de biofilm o estudios de fisiología son el objetivo del estudio, el biofilm puede ser removido de el cupón con un rascador de células. Mientras mantiene los cupones con las pinzas, raspar suavemente el biofilm el cupón en un tubo cónico que contiene tampón fosfato salino usando un raspador de células. Nota: para cuantificar las unidades formadoras de colonias en las biopelículas, es necesario homogeneizar los biofilms cosechadas con un pañuelo de papel homogeneizador de desagregar grupos y formar una suspensión homogénea. Varios modelos de homogeneizadores de tejidos son adecuados para esta aplicación. Utilizamos un Fisher Scientific Tissuemiser homogeneizador (producto # 15-338-420) a toda velocidad por 1 minuto para homogeneizar las muestras de biofilm. La falta de homogeneización de la biopelícula se traducirá en una subestimación de las unidades formadoras de colonias presentes en la muestra. </li></ol> 2. El disco giratorio del reactor biopelícula <ol><li> El reactor de disco giratorio biofilm (disponible a partir de las tecnologías disponibles Biosurface o puede ser por encargo, ver Figura 3) está montado y autoclave. Montar el reactor por el primer lugar de la gira del disco cupones en las ranuras del disco giratorio y colocarlo en un vaso de vidrio de 1 litro con un puerto de desbordamiento. Un número de 15 tapón de goma con agujeros en ella para permitir el flujo de los medios de comunicación y la aireación se utiliza como la tapa del reactor. El reactor, biofilm medios (caldo de soja tríptico 2 g / L de glucosa y 2 g / L), y el tubo de entrada se esteriliza en autoclave. </li><li> El reactor de disco giratorio biofilm se inocula mediante la colocación de 250 ml de medio estéril en el reactor, y la adición de 0,5 ml de un día para otro S. cultura aureus crecido en caldo de soja tríptico. El reactor se coloca sobre una placa de agitación conjunto a 250 rpm y se incubaron durante la noche a la temperatura deseada. </li><li> Después de 16 horas de incubación conectar la tubería de entrada al puerto en medio de depósito y conectarlo a la bomba peristáltica. La bomba se encienda y el flujo ajustado a ~ 0.25 mL / min (flujo puede ser alterado en función de la tasa de crecimiento deseado). </li><li> Después de 24 horas parada del reactor y asépticamente retire el disco sin tocar los cupones de biofilm. Eliminar los cupones con pinzas estériles y baño cada una en tampón fosfato salino para eliminar las bacterias unen libremente. </li><li> Para las pruebas de compuestos antimicrobianos, los chips pueden ser colocados en los distintos pozos de una placa de 96 pocillos que contienen compuestos de interés. Después de la incubación, los chips se transfieren a tubos de 1,5 mL microcentrífuga que contiene 1 ml de solución salina tampón fosfato y se homogeneiza con un homogeneizador de tejidos para dispersar a la biopelícula intacto. </li><li> Las células se diluyen en serie y se colocaron en un medio de agar nutriente para determinar viable colonia Formiunidades ng. </li><li> Nota: muchas variaciones se pueden hacer en el protocolo anterior. Por ejemplo, haciendo girar el disco cupones pueden ser recubiertos con o hechos de potencial anti-biopelículas compuestos para probar su eficacia. Dispersantes biofilm también se puede evaluar que la incubación de los cupones de compuestos dispersantes y cuantificar las bacterias frente unido separado. </li></ol> 3. Resultados representante Un ejemplo de un montaje del reactor de goteo de flujo se muestra si la figura 1. Después de tres días de flujo de grandes cantidades de biofilm se acumulan en la superficie del cupón, la figura 2A. La biomasa total variará dependiendo de las cepas bacterianas y las condiciones precisas de crecimiento. A Micrografía electrónica de barrido de una S. biofilm aureus cultivados en el reactor de flujo de goteo se muestra en la Figura 2B. Un reactor de disco giratorio se muestra en la figura 3A. El protocolo descrito se puede adaptar a las necesidades específicas de general, cualquier microorganismo capaz de formar una biopelícula. Figura 3B muestra el disco giratorio con 18 discos de plástico colocada. Estas biopelículas crecido en disco son especialmente adecuados para las pruebas antimicrobianas y con unos resultados altamente reproducibles 11. <img src="/files/ftp_upload/2470/2470fig1.jpg" alt="Figura 1" /> Figura 1. El flujo de goteo de configuración del reactor. Componentes importantes son etiquetados. <img src="/files/ftp_upload/2470/2470fig2.jpg" alt="Figura 2" /> Figura 2. Ejemplo de un flujo de goteo S. aureus biofilm. A) El biofilm creció durante tres días siguiendo el protocolo descrito. Las tapas de los primeros dos cámaras del reactor se retiran para mostrar la amarilla S. aureus biofilm de la biomasa. B) Sacnning micrografía electrónica de una S. biofilm aureus cultivados en el reactor de flujo por goteo. <img src="/files/ftp_upload/2470/2470fig3.jpg" alt="Figura 3" /> Figura 3. El reactor de disco giratorio. A) Ejemplo de un reactor de disco giratorio corriendo. Componente clave están etiquetados. B) Cierre de vista de un disco giratorio.

<ol>
	<li>Costerton, J. W., Lewandowski, Z., Caldwell, D. E., Korber, D. R., Lappin-Scott, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbial+Biofilms.">Microbial Biofilms.</a> Annu. Rev. Microbiol. 49, 711-745 (1995).</li><li>Costerton, J. W., Cheng, K. J., Gessey, G. G., Ladd, T. I., Nickel, J. C., Dasgupta, M., Marrie, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+biofilms+in+nature+and+disease.">Bacterial biofilms in nature and disease.</a> Ann. Rev. Microbiol. 41, 435-464 (1987).</li><li>O'Toole, G. A., Kolter, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Initiation+of+biofilm+formation+in+Pseudomonas+fluorescens+WCS365+proceeds+via+multiple,+convergent+signaling+pathways:+a+genetic+analysis.">Initiation of biofilm formation in Pseudomonas fluorescens WCS365 proceeds via multiple, convergent signaling pathways: a genetic analysis.</a> Mol. Micro. 28, 449-461 (2002).</li><li>Boles, B. R., Horswill, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Agr-mediated+dispersal+of+Staphylococcus+aureus+biofilms.">Agr-mediated dispersal of Staphylococcus aureus biofilms.</a> PLoS Pathog. 4, e1000052-e1000052 (2008).</li><li>Goeres, D. M., Haamilton, M. A., Beck, N. A., Buckingham-Meyer, K., Hilyard, J., Loetterle, L. A., Walker, D. K., Stewart, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+growing+a+biofilm+under+low+shear+at+the+air-liquid+interface+using+the+drip+flow+biofilm+reactor.">A method for growing a biofilm under low shear at the air-liquid interface using the drip flow biofilm reactor.</a> Nature Protocols. 4, 783-788 (2009).</li><li>Fu, W., Forster, T., Mayer, O., Curtin, J. J., Lehman, S. M., Donlan, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacteriophage+cocktail+for+the+prevention+of+biofilm+formation+by+Pseudomonas+aeruginosa+on+catheters+in+an+in+vitro+model+system.">Bacteriophage cocktail for the prevention of biofilm formation by Pseudomonas aeruginosa on catheters in an in vitro model system.</a> Antimicrob Agents Chemother. 54, 397-404 (2010).</li><li>Xu, K. D., McFeters, G. A., Stewart, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biofilm+resistance+to+antimicrobial+agents.">Biofilm resistance to antimicrobial agents.</a> Microbiology. 146, 547-549 (2000).</li><li>Xu, K. D., Stewart, P. S., Xia, F., Huang, C. T., McFeters, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+physiological+heterogeneity+in+Pseudomonas+aeruginosa+biofilm+is+determined+by+oxygen+availability.">Spatial physiological heterogeneity in Pseudomonas aeruginosa biofilm is determined by oxygen availability.</a> Appl. Environ. Microbiol. 64, 4035-4039 (1998).</li><li>Boles, B. R., Thoendel, M., Singh, P. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-generated+diversity+produces+"insurance+effects"+in+biofilm+communities.">Self-generated diversity produces "insurance effects" in biofilm communities.</a> Proc. Natl. Acad. Sci. USA. 101, 16630-16635 (2004).</li><li>Pitts, B., Willse, A., McFeters, G. A., Hamilton, M. A., Zelver, N., Stewart, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+repeatable+laboratory+method+for+testing+the+efficacy+of+biocides+against+toilet+bowl+biofilms.">A repeatable laboratory method for testing the efficacy of biocides against toilet bowl biofilms.</a> J. Appl. Microbiol. 91, 117-11 (2001).</li><li>Boles, B. R., Thoendel, M., Singh, P. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rhamnolipids+mediate+detachment+of+Pseudomonas+aeruginosa+from+biofilms.">Rhamnolipids mediate detachment of Pseudomonas aeruginosa from biofilms.</a> Mol. Microbiol. 57, 1210-1223 (2005).</li><li>Hentzer, M., Teitzel, G. M., Balzer, G. J., Heydorn, A., Molin, S., Givskov, M., Parsek, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alginate+overproduction+affects+Pseudomonas+aeruginosa+biofilm+structure+and+function.">Alginate overproduction affects Pseudomonas aeruginosa biofilm structure and function.</a> J. Bacteriol. 183, 5395-5401 (2001).</li><li>Lin, H. Y., Chen, C. T., Huang, C. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+merocyanine+540+for+photodynamic+inactivation+of+Staphylococcus+aureus+planktonic+and+biofilm+cells.">Use of merocyanine 540 for photodynamic inactivation of Staphylococcus aureus planktonic and biofilm cells.</a> Appl. Environ. Microbiol. 70, 6453-6458 (2004).</li></ol>

Biofilms cultivados en reactores diferentes a menudo tienen características diferentes y cada uno tiene diferentes aplicaciones del reactor. En este trabajo se describe el uso de dos reactores de biopelícula: un flujo de goteo reactor biopelícula y un reactor de disco giratorio. Goteo de reactores de flujo son útiles para el cultivo bajo las biopelículas de corte en una interfase aire-líquido y son adaptables a una variedad de condiciones. Los encontramos muy conveniente para los estudios en los que una gran canti...

Erratum: The Use of Drip Flow and Rotating Disk Reactors for Staphylococcus aureus Biofilm Analysis

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Drip Flow Reactors</td>
<td>&nbsp;</td>
<td>BioSurface Technologies Corporation</td>
<td>DFR 110</td>
<td>&nbsp;</td>
<tr>
<td>Rotating Disk Reactors</td>
<td>&nbsp;</td>
<td>BioSurface Technologies Corporation</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

the use of drip flow and rotating disk reactors for staphylococcus aureus biofilm analysis

La mayoría de los microbios en la naturaleza se piensa que existen como asociados en la superficie las comunidades en las biopelículas. 1 biofilms bacterianos están encerrados dentro de una matriz y unido a una formación de biopelículas de la superficie. 2 y el desarrollo son comúnmente estudiados en el laboratorio con sistemas de lotes, tales como placas de microtitulación o el flujo de sistemas, como el flujo de las células. Estas metodologías son útiles para la selección de bibliotecas mutante y químicas (placas de microtitulación) 3 o en crecimiento para la visualización de biofilms (células de flujo) 4. Aquí se presentan los protocolos detallados para el Staphylococcus aureus que crece en otros dos tipos de biofilms sistema de flujo: el flujo de goteo reactor biopelícula y el reactor de biofilm disco giratorio. Goteo de reactores de biopelícula de flujo están diseñados para el estudio de las biopelículas se cultivan en condiciones de bajo cizallamiento. 5 El reactor de flujo de goteo consta de cuatro canales de prueba en paralelo, cada uno capaz de contener un microscopio estándar de vidrio de tamaño cupón de diapositivas, o un trozo de catéter o restricción. El reactor de flujo por goteo es ideal para el monitoreo de microsensores, los estudios generales de biofilm, las muestras de biofilm muestras criostáticas, alta producción de biomasa, material de las evaluaciones médicas y pruebas de mora en los dispositivos médicos. 6,7,8,9 El reactor de disco giratorio se compone de un disco de teflón que contienen huecos de cupones desprendibles. 10 Los cupones desprendibles de puede hacerse de cualquier material mecanizable. La parte inferior del disco giratorio contiene un imán de barra para permitir la rotación del disco de corte para crear la superficie del líquido a través de la superficie al ras-cupones. Todo el disco contiene 18 cupones se coloca en un recipiente de 1000 ml de vidrio del reactor brazo lateral. Un medio de cultivo líquido se distribuye a través del vaso, mientras que el disco es girado por un agitador magnético. Los cupones se retiran de la vasija del reactor y luego raspar para recoger la muestra de biofilm para un estudio más o imágenes de microscopía. Rotación de los reactores de disco están diseñados para las pruebas de laboratorio de la eficacia biocida, la eliminación de biopelículas, y el rendimiento de los materiales anti-incrustantes. 9,11,12,13

Watch this Scientific Journal Video about The Use of Drip Flow and Rotating Disk Reactors for Staphylococcus aureus Biofilm Analysis at JoVE.com

The Use of Drip Flow and Rotating Disk Reactors for Staphylococcus aureus Biofilm Analysis

Protocolos para la utilización de abrir las biopelículas de flujo del sistema de reactores de flujo por goteo y la rotación de los reactores de disco se presentan en detalle.

El uso de un flujo de goteo y la rotación del disco para reactores Staphylococcus aureus Análisis de Biofilm

Most microbes in nature are thought to exist as surface-associated communities in biofilms.1  Bacterial biofilms are encased within a matrix and attached ...

the-use-drip-flow-rotating-disk-reactors-for-staphylococcus-aureus

Research

JoVE Journal

Immunology and Infection

The Use of Drip Flow and Rotating Disk Reactors for Staphylococcus aureus Biofilm Analysis

17.0K vistas.  University of Michigan. Protocolos para la utilización de abrir las biopelículas de flujo del sistema de reactores de flujo por goteo y la rotación de los reactores de disco se presentan en detalle.

Video: The Use of Drip Flow and Rotating Disk Reactors for Staphylococcus aureus Biofilm Analysis

Pseudomonas aeruginosa and Saccharomyces cerevisiae Biofilm in Flow Cells

Many microbial cells have the ability to form sessile microbial communities defined as biofilms that have altered physiological and pathological properties compared to free living microorganisms. Biofilms in nature are often difficult to investigate and reside under poorly defined conditions1. Using a transparent substratum it is possible to device a system where simple biofilms can be examined in a non-destructive way in real-time: here we demonstrate the assembly and operation of a flow cell model system, for in vitro 3D studies of microbial biofilms generating high reproducibility under well-defined conditions2,3.
The system consists of a flow cell that serves as growth chamber for the biofilm. The flow cell is supplied with nutrients and oxygen from a medium flask via a peristaltic pump and spent medium is collected in a waste container. This construction of the flow system allows a continuous supply of nutrients and administration of e.g. antibiotics with minimal disturbance of the cells grown in the flow chamber. Moreover, the flow conditions within the flow cell allow studies of biofilm exposed to shear stress. A bubble trapping device confines air bubbles from the tubing which otherwise could disrupt the biofilm structure in the flow cell.
The flow cell system is compatible with Confocal Laser Scanning Microscopy (CLSM) and can thereby provide highly detailed 3D information about developing microbial biofilms. Cells in the biofilm can be labeled with fluorescent probes or proteins compatible with CLSM analysis. This enables online visualization and allows investigation of niches in the developing biofilm. Microbial interrelationship, investigation of antimicrobial agents or the expression of specific genes, are of the many experimental setups that can be investigated in the flow cell system.

Pseudomonas aeruginosa and Saccharomyces cerevisiae Biofilm in Flow Cells

Protocol describing the application of a flow cell system for growing and analyzing microbial biofilms for Confocal Laser Scanning Microscopy (CLSM).

Pseudomonas aeruginosa y Biofilm Saccharomyces cerevisiae en las celdas de flujo

Many microbial cells have the ability to form sessile microbial communities defined as biofilms that have altered physiological and pathological ...

Investigación

Inmunología e Infección

Subcutaneous Infection of Methicillin Resistant Staphylococcus Aureus (MRSA)

MRSA is a worldwide threat to public health, and MRSA skin and soft-tissue infections now account for more than half of all soft-tissue infections in the United States. Among soft-tissue infections, myositis, pyomyositis, and necrotizing fasciitis have been increasingly reported in association with MRSA arising from the community. To understand the interplay between MRSA and host immunity leading to more severe infection, the availability of animal models is critical, permitting the study of host and bacterial factors. Several infection models have been introduced to assess the pathogenesis of S. aureus during superficial skin infection. Here, we describe a subcutaneous infection model that examines the skin, subcutaneous, and muscle pathologies.

Subcutaneous Infection of Methicillin Resistant Staphylococcus Aureus MRSA

Murine skin and soft tissue infection model is utilized for assessing the virulence function of methicillin resistant Staphylococcus aureus (MRSA) and the host immunological responses. Here, we presented a subcutaneous infection model for skin and soft tissue infection.

Subcutánea infección de Staphylococcus aureus resistentes a meticilina (MRSA)

MRSA is a worldwide threat to public health, and MRSA skin and soft-tissue infections now account for more than half of all soft-tissue infections in the ...

Experimental Endocarditis Model of Methicillin Resistant Staphylococcus aureus (MRSA) in Rat

Endovascular infections, including endocarditis, are life-threatening infectious syndromes1-3. Staphylococcus aureus is the most common world-wide cause of such syndromes with unacceptably high morbidity and mortality even with appropriate antimicrobial agent treatments4-6. The increase in infections due to methicillin-resistant S. aureus (MRSA), the high rates of vancomycin clinical treatment failures and growing problems of linezolid and daptomycin resistance have all further complicated the management of patients with such infections, and led to high healthcare costs7, 8. In addition, it should be emphasized that most recent studies with antibiotic treatment outcomes have been based in clinical settings, and thus might well be influenced by host factors varying from patient-to-patient. Therefore, a relevant animal model of endovascular infection in which host factors are similar from animal-to-animal is more crucial to investigate microbial pathogenesis, as well as the efficacy of novel antimicrobial agents. Endocarditis in rat is a well-established experimental animal model that closely approximates human native valve endocarditis. This model has been used to examine the role of particular staphylococcal virulence factors and the efficacy of antibiotic treatment regimens for staphylococcal endocarditis. In this report, we describe the experimental endocarditis model due to MRSA that could be used to investigate bacterial pathogenesis and response to antibiotic treatment.

Experimental Endocarditis Model of Methicillin Resistant Staphylococcus aureus MRSA in Rat

Experimental rat endocarditis model due to methicillin-resistant S. aureus.

Modelo experimental de endocarditis Resistente a la Meticilina<em&gt; Staphylococcus aureus</em&gt; (SARM) en la rata

Endovascular infections, including endocarditis, are life-threatening infectious syndromes1-3. Staphylococcus aureus is the most common world-wide cause ...

Staphylococcus aureus Growth using Human Hemoglobin as an Iron Source

S. aureus is a pathogenic bacterium that requires iron to carry out vital metabolic functions and cause disease. The most abundant reservoir of iron inside the human host is heme, which is the cofactor of hemoglobin. To acquire iron from hemoglobin, S. aureus utilizes an elaborate system known as the iron-regulated surface determinant (Isd) system1. Components of the Isd system first bind host hemoglobin, then extract and import heme, and finally liberate iron from heme in the bacterial cytoplasm2,3. This pathway has been dissected through numerous in vitro studies4-9. Further, the contribution of the Isd system to infection has been repeatedly demonstrated in mouse models8,10-14. Establishing the contribution of the Isd system to hemoglobin-derived iron acquisition and growth has proven to be more challenging. Growth assays using hemoglobin as a sole iron source are complicated by the instability of commercially available hemoglobin, contaminating free iron in the growth medium, and toxicity associated with iron chelators. Here we present a method that overcomes these limitations. High quality hemoglobin is prepared from fresh blood and is stored in liquid nitrogen. Purified hemoglobin is supplemented into iron-deplete medium mimicking the iron-poor environment encountered by pathogens inside the vertebrate host. By starving S. aureus of free iron and supplementing with a minimally manipulated form of hemoglobin we induce growth in a manner that is entirely dependent on the ability to bind hemoglobin, extract heme, pass heme through the bacterial cell envelope and degrade heme in the cytoplasm. This assay will be useful for researchers seeking to elucidate the mechanisms of hemoglobin-/heme-derived iron acquisition in S. aureus and possibly other bacterial pathogens.

Staphylococcus aureus Growth using Human Hemoglobin as an Iron Source

Here we describe a growth assay for Staphylococcus aureus using hemoglobin as the sole source of available nutrient iron. This assay establishes the role of bacterial factors involved in hemoglobin-derived iron acquisition.

Staphylococcus aureus Crecimiento con hemoglobina humana como una fuente de hierro

S. aureus is a pathogenic bacterium that requires iron to  carry out vital metabolic functions and cause disease. The most abundant  reservoir of iron ...

Multiplex PCR Assay for Typing of Staphylococcal Cassette Chromosome Mec Types I to V in Methicillin-resistant Staphylococcus aureus

Staphylococcal Cassette Chromosome mec (SCCmec) typing is a very important molecular tool for understanding the epidemiology and clonal strain relatedness of methicillin-resistant Staphylococcus aureus (MRSA), particularly with the emerging outbreaks of community-associated MRSA (CA-MRSA) occurring on a worldwide basis. Traditional PCR typing schemes classify SCCmec by targeting and identifying the individual mec and ccr gene complex types, but require the use of many primer sets and multiple individual PCR experiments. We designed and published a simple multiplex PCR assay for quick-screening of major SCCmec types and subtypes I to V, and later updated it as new sequence information became available. This simple assay targets individual SCCmec types in a single reaction, is easy to interpret and has been extensively used worldwide. However, due to the sophisticated nature of the assay and the large number of primers present in the reaction, there is the potential for difficulties while adapting this assay to individual laboratories. To facilitate the process of establishing a MRSA SCCmec assay, here we demonstrate how to set up our multiplex PCR assay, and discuss some of the vital steps and procedural nuances that make it successful.

Multiplex PCR Assay for Typing of Staphylococcal Cassette Chromosome Mec Types I to V in Methicillin-resistant Staphylococcus aureus

We demonstrate a simple multiplex PCR assay for quick-screening and typing of Staphylococcal Cassette Chromosome mec (SCCmec) types I-V for methicillin-resistant Staphylococcus aureus, and provide some of the vital steps and procedural nuances that make it successful for adapting this assay to individual laboratories.

Multiplex PCR Ensayo para la tipificaci

Staphylococcal Cassette Chromosome mec (SCCmec) typing  is a very important molecular tool for understanding the epidemiology and  clonal strain ...

Studying Interactions of Staphylococcus aureus with Neutrophils by Flow Cytometry and Time Lapse Microscopy

We present methods to study the effect of phenol soluble modulins (PSMs) and other toxins produced and secreted by Staphylococcus aureus on neutrophils. To study the effects of the PSMs on neutrophils we isolate fresh neutrophils using density gradient centrifugation. These neutrophils are loaded with a dye that fluoresces upon calcium mobilization. The activation of neutrophils by PSMs initiates a rapid and transient increase in the free intracellular calcium concentration. In a flow cytometry experiment this rapid mobilization can be measured by monitoring the fluorescence of a pre-loaded dye that reacts to the increased concentration of free Ca2+. Using this method we can determine the PSM concentration necessary to activate the neutrophil, and measure the effects of specific and general inhibitors of the neutrophil activation.	
	To investigate the expression of the PSMs in the intracellular space, we have constructed reporter fusions of the promoter of the PSM&alpha; operon to GFP. When these reporter strains of S. aureus are phagocytosed by neutrophils, the induction of expression can be observed using fluorescence microscopy.

Studying Interactions of Staphylococcus aureus with Neutrophils by Flow Cytometry and Time Lapse Microscopy

We present methods to study the effect of PSMs and other toxins secreted by Staphylococcus aureus on neutrophils using flow cytometry and fluorescence microscopy.

El estudio de las interacciones de<em&gt; Staphylococcus aureus</em&gt; Con neutrófilos mediante citometría de flujo y microscopía Tiempo Lapso

We present methods to study the effect of phenol soluble modulins (PSMs)  and other toxins produced and secreted by Staphylococcus  aureus on neutrophils. ...

Targeting Biofilm Associated Staphylococcus aureus Using Resazurin Based Drug-susceptibility Assay

Most pathogenic bacteria are able to form biofilms during infection, but due to the difficulty of manipulating and assessing biofilms, the vast majority of laboratory work is conducted with planktonic cells. Here, we describe a peg plate biofilm assay as performed with Staphylococcus aureus. Bacterial biofilms are grown on pegs attached to a 96-well microtiter plate lid, washed through gentle submersion in buffer, and placed in a drug challenge plate. After subsequent incubation they are again washed and moved to a final recovery plate, in which the fluorescent dye resazurin serves as a viability indicator. This assay offers greatly increased ease-of-use, reliability, and reproducibility, as well as a wealth of data when conducted as a kinetic read. Moreover, this assay can be adapted to a medium-throughput drug screening approach by which an endpoint fluorescent readout is taken instead, offering a path for drug discovery efforts.

Targeting Biofilm Associated Staphylococcus aureus Using Resazurin Based Drug-susceptibility Assay

Most bacterial infections produce a biofilm. By virtue of their environment, biofilm associated bacteria are often phenotypically drug resistant. Novel antibacterial molecules that kill bacteria in biofilms are thus a high priority. We establish an assay to quickly screen for antimicrobial compounds that are effective at eradicating biofilms.

Orientación de Biofilm Asociadas<em&gt; Staphylococcus aureus</em&gt; Uso de Ensayo de susceptibilidad a fármacos Basado resazurina

Most pathogenic bacteria are able to form biofilms during infection, but due to the difficulty of manipulating and assessing biofilms, the vast majority ...

Genotyping of Staphylococcus aureus by Ribosomal Spacer PCR (RS-PCR)

The ribosomal spacer PCR (RS-PCR) is a highly resolving and robust genotyping method for S. aureus that allows a high throughput at moderate costs and is, therefore, suitable to be used for routine purposes. For best resolution, data evaluation and data management, a miniaturized electrophoresis system is required. Together with such an electrophoresis system and the in-house developed software (freely available here) assignment of the pattern of bands to a genotype is standardized and straight forward. DNA extraction is simple (boiling prep), setting-up of the reactions is easy and they can be run on any standard PCR machine. PCR cycling is common except prolonged ramping and elongation times. Compared to spa typing and Multi Locus Sequence Typing (MLST), RS-PCR does not require DNA sequencing what simplifies the analysis considerably and allows a high throughput. Furthermore, the resolution for bovine strains of S. aureus is at least as good as spa typing and better than MLST or pulsed-field gel electrophoresis (PFGE). The RS-PCR data base includes presently a total of 141 genotypes and variants. The method is highly associated with the virulence gene pattern, contagiosity and pathogenicity of S. aureus strains involved in bovine mastitis. S. aureus genotype B (GTB) is contagious and causes herds problems causing large costs in the Switzerland and other European countries. All the other genotypes observed in Switzerland infect individual cows and quarters. Genotyping by RS-PCR allows the reliable prediction of the epidemiological and the pathogenic potential of S. aureus involved in bovine intramammary infection (IMI), two key factors for clinical veterinary medicine. Because of these beneficial properties together with moderate costs and a high sample throughput the goal of this publication is to give a detailed, step-by-step protocol for easily establishing and running RS-PCR for genotyping S. aureus in other laboratories.

Genotyping of Staphylococcus aureus by Ribosomal Spacer PCR RS-PCR

Here, ribosomal spacer PCR (RS-PCR) is used together with a miniaturized electrophoresis system as a fast and high resolution method for genotyping S. aureus at moderate costs allowing a high throughput.

genotipado de<em&gt; Staphylococcus aureus</em&gt; por Ribosómica espaciador de PCR (RS-PCR)

The ribosomal spacer PCR (RS-PCR) is a highly resolving and robust genotyping method for S. aureus that allows a high throughput at moderate costs and is, ...

Methodology for the Study of Horizontal Gene Transfer in Staphylococcus aureus

One important feature of the major opportunistic human pathogen Staphylococcus aureus is its extraordinary ability to rapidly acquire resistance to antibiotics. Genomic studies reveal that S. aureus carries many virulence and resistance genes located in mobile genetic elements, suggesting that horizontal gene transfer (HGT) plays a critical role in S. aureus evolution. However, a full and detailed description of the methodology used to study HGT in S. aureus is still lacking, especially regarding natural transformation, which has been recently reported in this bacterium. This work describes three protocols that are useful for the in vitro investigation of HGT in S. aureus: conjugation, phage transduction, and natural transformation. To this aim, the cfr gene (chloramphenicol/florfenicol resistance), which confers the Phenicols, Lincosamides, Oxazolidinones, Pleuromutilins, and Streptogramin A (PhLOPSA)-resistance phenotype, was used. Understanding the mechanisms through which S. aureus transfers genetic materials to other strains is essential to comprehending the rapid acquisition of resistance and helps to clarify the modes of dissemination reported in surveillance programs or to further predict the spreading mode in the future.

Methodology for the Study of Horizontal Gene Transfer in Staphylococcus aureus

We describe here three different protocols for the in vitro investigation of conjugation, transduction, and natural transformation in Staphylococcus aureus.

Metodología para el Estudio de la transferencia horizontal de genes en<em&gt; Staphylococcus aureus</em

One important feature of the major opportunistic human pathogen Staphylococcus aureus is its extraordinary ability to rapidly acquire resistance to ...

Characterization of Human Monocyte Subsets by Whole Blood Flow Cytometry Analysis

Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can have pathological significance. An ideal method for examining alterations to monocytes is whole blood flow cytometry as the minimal handling of samples by this method limits artifactual cell activation. However, many different approaches are taken to gate the monocyte subsets leading to inconsistent identification of the subsets between studies. Here we demonstrate a method using whole blood flow cytometry to identify and characterize human monocyte subsets (classical, intermediate, and non-classical). We outline how to prepare the blood samples for flow cytometry, gate the subsets (ensure contaminating cells have been removed), and determine monocyte subset expression of surface markers &#8212; in this example M1 and M2 markers. This protocol can be extended to other studies that require a standard gating method for assessing monocyte subset proportions and monocyte subset expression of other functional markers.

Here we present a protocol for characterizing monocyte subsets by whole blood flow cytometry. This includes outlining how to gate the subsets and assess their expression of surface markers and giving an example of the assessment of the expression of M1 (inflammatory) and M2 markers (anti-inflammatory).

Caracterización de subconjuntos de monocitos humanos de sangre análisis de citometría de flujo

Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can ...