We thank Saran Kupul for technical assistance. This work was supported in part by NIH grant R01-AI104952 to FW. Further support was provided by the University of Alabama at Birmingham (UAB) Center for AIDS Research (CFAR), an NIH funded program (P30 AI027767) that was made possible by the following NIH Institutes: NIAID, NIMH, NIDA, NICHD, NHLDI, NIA.

1. Iniciación de biopelículas <ol><li> Hacer crecer una cultura de producción de biofilm organismo en un medio rico en nutrientes. Inocular Staphylococcus aureus cepa Newman en 10 ml de medio Mueller-Hinton de un stock de glicerol. Realizar todos los trabajos que impliquen la manipulación de S. aureus con guantes y dentro de una cabina de bioseguridad. </li><li> Incubar durante 16 horas a 37 ° C en un agitador rotatorio (100 a 200 rpm). </li><li> Determinar DO600 del cultivo usando un espectrofotómetro y una cubeta estándar (longitud de recorrido: 1 cm). </li><li> Calcular el volumen (V) de la cultura necesaria para preparar 20 ml de inóculo con OD 600 (inóculo) de 0,1 en chelexed medios Roswell Park Memorial Institute (cRPMI) 9 usando la fórmula: [V = 20 ml * 0,1 / OD 600 (cultivo )] Nota: Referencia 9 proporciona detalles sobre la preparación cRPMI. <ol><li> Añadir el volumen de cultivo calculado (desde arriba) a un tubo de centrífuga y las células de pellets por centrifugación durante 1 min a 10.000 x g. para prepare inóculo biofilm, aspirar el pellet de células medio de cultivo y se resuspenden en 20 ml pasado cRPMI. </li></ol></li><li> Verter inóculo en un depósito de 25 ml. </li><li> Retirar con cuidado la tapa de una placa de PEG de 96 pocillos estériles tirando de él hacia arriba. Nota: Se debe tener cuidado de no contaminar las clavijas que cuelgan por debajo de la tapa. La tapa se puede colocar boca abajo sobre una superficie limpia dentro de una cabina de bioseguridad. </li><li> Usando una pipeta multicanal l 200, añadir 125 l de inóculo a cada pocillo de las filas A a G de la placa inferior. </li><li> Llenar medio cRPMI 125 l estéril en cada pocillo de la fila H como un control de esterilidad. </li><li> Tome la clavija-tapa y mantenerla por encima del fondo del plato con las clavijas hacia abajo. alinee con cuidado las clavijas individuales con sus respectivos pozos. Evitar el contacto físico entre la placa y las clavijas. Una vez alineado, baje la tapa con cuidado hacia abajo. Evitar desajustes como reajustes realizados después de las clavijas han tocado la placa inferior puede contcontroles de esterilidad aminar en la fila H. </li><li> Sellar la tapa a la placa con película de parafina para evitar la evaporación y colocar en una bolsa de plástico con cierre, sellado herméticamente. Mantenga el volumen de aire en la bolsa lo más bajo posible para minimizar la evaporación. </li><li> Se incuba sin agitación a 37 ° C durante 48 horas en un incubador de CO2 al 5%. Nota: 48 h de crecimiento ha sido óptima para S. aureus, pero variará basado en el organismo utilizado. Nota: Aunque la incubación estática es un buen punto de partida para optimizar el ensayo, agitando suavemente a 150 rpm en un agitador de microplacas es una posible variación que pueden mejorar la formación de biopelículas de algunos S. aureus aislados. </li></ol> 2. Preparación de la placa de biofilm Challenge <ol><li> En el día del desafío biopelícula tomar una placa de 96 pocillos fresca y añadir 100 l de cRPMI, se equilibraron a temperatura ambiente, a cada pocillo de las filas B a H. Para evitar inconsistencias en los resultados, utilizar una placa de 96 pocillos que tiene capacidad para 200 lde medios de comunicación cuando las clavijas están en el interior, y cuenta con pozos con dimensiones idénticas a las de la placa de iniciación biofilm. </li><li> Diluir hasta 4 compuestos de ensayo por separado en 1.0 ml de cRPMI a 2x la concentración final deseada, con el fin de dar cuenta de una dilución final en el paso 2.7. Nota: Un buen punto de partida es 8 veces por encima de la concentración inhibitoria de las células planctónicas. </li><li> Añadir 200 l de compuesto 1 en los pocillos A1 a A3, compuesto 2: pocillos A4 a A6, el compuesto 3 en pozos A7 a A9 y el compuesto 4 en pocillos A10 a A12. </li><li> Diluciones seriadas de los compuestos de ensayo utilizando una pipeta multicanal y la transferencia de 100 l de la fila A a la fila B y mezclar. </li><li> De esa manera, continúe el traspaso de 100 l pozo en pozo. Mezclar después de cada traslado y la parada en la fila F. </li><li> Después de mezclar, expulsar a 100 l de la fila F en un contenedor de residuos. No transfiera cualquier líquido a las filas G y H. </li><li> Añadir 100 l de cRPMI a cada pocillo de la placa usando una pipeta multicanal allevar el volumen a 200 l. Ver Figura 1 para la disposición final de la placa. </li></ol> 3. El biofilm Challenge <ol><li> Añadir 200 l de solución salina tamponada con fosfato (PBS) a una placa de 96 pocillos estéril fresco que cuenta con pozos con dimensiones idénticas a las de la placa de iniciación biofilm. </li><li> Retire la bolsa de plástico y lámina de parafina de la placa se incubó la iniciación de biopelículas. </li><li> Levante la tapa hacia arriba. Evitar el contacto entre las clavijas y el fondo del plato, ya que podría dañar la biopelícula. Mantenga la parte inferior para su posterior análisis OD 600 (véase la sección 3.8). </li><li> Enjuague las células planctónicas mediante la colocación de la clavija-tapa sobre la placa que contenía PBS (del paso 3.1). Sumergir las clavijas durante 5 segundos, sacar de la PBS, y se sumerja de nuevo, teniendo cuidado de no golpear las clavijas contra los lados así. </li><li> Coloque la clavija-tapa se enjuaga en la placa de desafío. Evitar el contacto innecesario entre las clavijas y la placa inferior, ya que dañará la biopelícula que lleva aresultados inconsistentes. </li><li> La placa del sello con la película de parafina y colocar en una bolsa de plástico con cierre, como se describe en el paso 1.10. </li><li> Se incuba la placa sin agitación durante 24 horas a 37 ° C en una incubadora de CO2 al 5%. </li><li> Tomar parte inferior de la placa de biofilm iniciación de 3,3. Volver a suspender las bacterias en cada pocillo usando una pipeta multicanal y medir el diámetro exterior 600 con un lector de microplacas adecuado para confirmar el crecimiento se produce en todos los pozos, pero los controles de esterilidad en la fila H. </li></ol> 4. Determinación de biopelículas <ol><li> Use un lector de placas equipado con una cámara de incubación y capaz de tomar lecturas de fluorescencia cinéticos para la detección de resazurina conversión. Configurar una medición de fluorescencia cinética de 24 horas usando una excitación de 530 nm y 590 nm de emisión. </li><li> Ajuste la temperatura de la cámara a 37 ° C y tiene la placa de leer cada 20 minutos. Ajuste el lector de placas para medir desde la parte inferior de la placa de acuerdo con instrucciones del fabricanteciones. </li><li> Preparar la placa de lavado mediante la adición de 200 l de PBS con una pipeta multicanal a una placa de 96 pocillo estéril. </li><li> Preparar medio de recuperación de biopelícula mediante la adición de 400 l de 0,8 mg / ml de solución madre de resazurina a 20 ml de medio cRPMI a una concentración final de 16 mg de resazurina / ml. Nota: La resazurina es un irritante conocido piel. Use una bata de laboratorio, gafas protectoras contra salpicaduras y guantes durante la manipulación. CRPMI como el medio de recuperación de biopelícula se utilizó ya que proporciona la señal de fondo más baja, pero puede ser sustituido por medios de Mueller Hinton si es apropiado. </li><li> Añadir 150 l de medio de recuperación de la biopelícula con una pipeta multicanal a cada pocillo de una placa de 96 pocillos fresca. </li><li> Transferencia placa desafío de la incubadora en una cabina de bioseguridad y retirar con cuidado bolsa de plástico y lámina de cierre. </li><li> Retire la clavija-tapa de la placa de desafío y enjuagar las células planctónicas en PBS como se describe en 3.4. Mantenga la parte inferior de la placa de desafío para la posterior OD 600 analisis (véase 4.10). </li><li> Después de aclarar, coloque la clavija-tapa en la parte inferior placa que contiene el medio de recuperación de biopelícula. </li><li> Envolver el lado de la placa con película de parafina, teniendo cuidado de no obstruir el fondo de los pocillos de perímetro. Inmediatamente después de concluir la placa, colocarla en el lector de placas y empezar una lectura cinética durante un período de 24 horas, iniciando el hecho previamente resazurina protocolo cinética desde el paso 4.1. </li><li> Para cuantificar el crecimiento de las células planctónicas que pueden o no pueden ocurrir durante desafío, leer la OD 600 de toda la placa inferior desafío usando un lector de micro placa. Siga las instrucciones dadas en el paso 3.8. Nota: resuspender cuidadosamente el contenido de cada pocillo como células vertimiento de la biopelícula tienden a crecer en grupos en la parte inferior del pozo. Cualquier burbuja dentro del pozo serán fuertemente interferir con las lecturas de DO a 600 y deben ser evitadas o eliminadas antes de la lectura. </li></ol>

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The authors have nothing to disclose.

Aquí, hemos descrito un ensayo de biopelícula modificado para determinar la actividad de los inhibidores de la prueba sobre S. biopelículas aureus que se centran en el estado metabólico de las células del biofilm asociado. Mientras que la iniciación de la biopelícula se describe y procedimientos reto recomendaciones del fabricante sobre todo imitaba, el uso de resazurina tinte para detectar y cuantificar las células del biofilm-asociado que sobreviven a un desafío inhibidor de 24 hr simplifica...

Los microbios patógenos pueden formar biopelículas in vivo que conducen a infecciones crónicas no agudos 1. Infección por biofilm asociado es un factor de riesgo grave de procedimientos médicos que implican la implantación de cuerpos extraños (por ejemplo, substituciones de huesos artificiales, implantes de pecho) o la instalación de los tubos traqueales o catéteres urinarios 2. En estos contextos, la terapia anti-infecciosa es casi siempre necesario como infecciones basado en biopelícula rara vez se borran por su cuenta, incluso en individuos inmunocompetentes. Staphylococcus aureus es uno de los patógenos más frecuentemente observados implicados en las complicaciones relacionadas con la biopelícula que se producen durante el uso de dispositivos médicos invasivos 3. Desafortunadamente, la naturaleza misma de biofilms como una barrera protectora hace más resistentes al tratamiento que las células planctónicas 1,4, y la evaluación de la eficacia clínica predicho es una parte crítica de tanto inicialel desarrollo de fármacos, así como vigilar la resistencia. Existe una creciente reconocimiento de que las condiciones de laboratorio, se centró en las culturas planctónicos, pueden no representar fielmente la enfermedad en el mundo real 5. Agravando aún más el problema, la replicación de los fenotipos de biopelícula es difícil, y los modelos de biopelículas existentes son tediosos y sufren de alta variabilidad inter e intra-ensayo 6. Por lo tanto, muchos investigadores, por necesidad, por defecto a ensayos de células planctónicas de susceptibilidad a los fármacos, lo que potencialmente descuidar un aspecto importante de la virulencia bacteriana y la enfermedad. Aquí se describe el protocolo para el ensayo de bacterias, específicamente S. aureus, en los biofilms pre-crecido que utilizan un sistema de biopelícula 7,8 basadas en 96 pocillos. Mientras que el protocolo para la formación de biopelículas y el desafío sigue esencialmente la recomendación del fabricante, se presenta una metodología rica en información alternativa para cuantificar la viabilidad de la bioFILM después de la exposición. Brevemente, las bacterias se cultivan en la placa de clavija, donde las biopelículas se forman en las clavijas sobresalientes unidos a la tapa de la placa. Después de la formación de biopelículas, las clavijas se sumergen suavemente en pocillos de una placa fresca llena con PBS para eliminar las células planctónicas. El PEG-tapa, con biofilms adjuntos, se transfiere a una nueva placa de desafío, que contiene varias concentraciones de antibióticos a ensayar. Después de una segunda incubación, las tapas se retiran de nuevo, se lavaron y se transfirieron a una placa de recuperación que contiene resazurina tinte, donde se someten a una incubación final. conversión resazurina se puede grabar cinéticamente o toma como una lectura de punto final después de un período de recuperación definido. Este método basado en el colorante de la cuantificación de la viabilidad de biofilms difiere considerablemente de las CFU (unidades formadoras de colonias) tediosas metodología basada en contar se describe en el protocolo original 7. OD 600 mediciones de la placa desafío de las drogas y resazurina cinética de conversión sirven como lectura viabilidadouts de células planctónicas y biofilm, respectivamente, que ofrecen un ensayo rápido, fiable, rico en información y técnicamente simple para la supervivencia de biopelículas.

<table border="0" cellpadding="0" cellspacing="0" width="612">
	<tbody>
		<tr height="61">
			<td height="61" style="height:61px;width:227px;">&#160;Mueller-Hinton Medium</td>
			<td style="width:89px;">Oxoid</td>
			<td style="width:103px;">CM0405</td>
			<td style="width:195px;">Follow recommendations of manufacturer</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:227px;">RPMI-medium</td>
			<td style="width:89px;">Corning&#160;</td>
			<td style="width:103px;">17-105-CV</td>
			<td style="width:195px;"></td>
		</tr>
		<tr height="101">
			<td height="101" style="height:101px;width:227px;">CRPMI</td>
			<td style="width:89px;">Ref 9</td>
			<td style="width:103px;"></td>
			<td style="width:195px;">RPMI-1640 medium chelexed for 1h with Chelex 100 resin and then supplemented with 10% unchelexed RPMI-1640</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:227px;">Chelex 100 Resin</td>
			<td style="width:89px;">Bio Rad</td>
			<td style="width:103px;">142-2822</td>
			<td style="width:195px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:227px;">MBEC-plates</td>
			<td style="width:89px;">Innovotech</td>
			<td style="width:103px;">19111</td>
			<td style="width:195px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:227px;">Resazurin Sodium Salt</td>
			<td style="width:89px;">Sigma</td>
			<td style="width:103px;">R7017</td>
			<td style="width:195px;">800&#181;g/ml in DI water&#160;&#160;&#160;&#160; Filter sterile</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:227px;">Micro Plate Shaking Platform&#160;</td>
			<td style="width:89px;">Heidolph Titramax 1000</td>
			<td style="width:103px;"></td>
			<td style="width:195px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:227px;">Cytation 3 Plate Reader</td>
			<td style="width:89px;">Biotek</td>
			<td style="width:103px;"></td>
			<td style="width:195px;"></td>
		</tr>
		<tr height="57">
			<td height="57" style="height:58px;width:227px;">Gen5 software</td>
			<td style="width:89px;">Biotek</td>
			<td style="width:103px;"></td>
			<td style="width:195px;">Recording and analysis of resazurin conversion</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:227px;">Neocuproine</td>
			<td style="width:89px;">Sigma</td>
			<td style="width:103px;">N1501&#160;</td>
			<td style="width:195px;">prepare 10 mM stock in 100% Ethanol, store at -80&#186;C</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:227px;">Copper sulfate</td>
			<td style="width:89px;">Acros Organics</td>
			<td style="width:103px;">7758-99-8</td>
			<td style="width:195px;">prepare a 100 mM stock solution in water, store at 4&#186;C</td>
		</tr>
		<tr height="121">
			<td height="121" style="height:121px;width:227px;">Cu-Neocuproine</td>
			<td style="width:89px;">Self-Made</td>
			<td style="width:103px;"></td>
			<td style="width:195px;">Generated by mixing equal molarities of neocuproine and copper sulfate. Mix was diluted in CRPMI medium to desired concentration.</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:227px;">Gentamicin</td>
			<td style="width:89px;">Sigma</td>
			<td style="width:103px;">G3632-1G</td>
			<td style="width:195px;"></td>
		</tr>
	</tbody>
</table>

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.

El ensayo de la resazurina es suficientemente sensible para detectar con fiabilidad muy pocas células viables capaces de replicarse en medio libre de fármaco después de la exposición. En esta metodología, se define el biofilm erradicar la concentración mínima como la concentración más baja a la que no se observa una conversión de resazurina dentro de las 24 horas. El ensayo se basa en la resazurina moléculas oxidativas que se encuentran en las células metabólicamente activas...

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

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

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.

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

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Research

JoVE Journal

Immunology and Infection

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

13.0K vistas.  University of Alabama at Birmingham. 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. 

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

Microtiter Dish Biofilm Formation Assay

Biofilms are communities of microbes attached to surfaces, which can be found in medical, industrial and natural settings. In fact, life in a biofilm probably represents the predominate mode of growth for microbes in most environments. Mature biofilms have a few distinct characteristics. Biofilm microbes are typically surrounded by an extracellular matrix that provides structure and protection to the community. Microbes growing in a biofilm also have a characteristic architecture generally comprised of macrocolonies (containing thousands of cells) surrounded by fluid-filled channels. Biofilm-grown microbes are also notorious for their resistance to a range of antimicrobial agents including clinically relevant antibiotics.
The microtiter dish assay is an important tool for the study of the early stages in biofilm formation, and has been applied primarily for the study of bacterial biofilms, although this assay has also been used to study fungal biofilm formation. Because this assay uses static, batch-growth conditions, it does not allow for the formation of the mature biofilms typically associated with flow cell systems. However, the assay has been effective at identifying many factors required for initiation of biofilm formation (i.e, flagella, pili, adhesins, enzymes involved in cyclic-di-GMP binding and metabolism) and well as genes involved in extracellular polysaccharide production. Furthermore, published work indicates that biofilms grown in microtiter dishes do develop some properties of mature biofilms, such a antibiotic tolerance and resistance to immune system effectors.
This simple microtiter dish assay allows for the formation of a biofilm on the wall and/or bottom of a microtiter dish. The high throughput nature of the assay makes it useful for genetic screens, as well as testing biofilm formation by multiple strains under various growth conditions. Variants of this assay have been used to assess early biofilm formation for a wide variety of microbes, including but not limited to, pseudomonads, Vibrio cholerae, Escherichia coli, staphylocci, enterococci, mycobacteria and fungi.
In the protocol described here, we will focus on the use of this assay to study biofilm formation by the model organism Pseudomonas aeruginosa. In this assay, the extent of biofilm formation is measured using the dye crystal violet (CV). However, a number of other colorimetric and metabolic stains have been reported for the quantification of biofilm formation using the microtiter plate assay. The ease, low cost and flexibility of the microtiter plate assay has made it a critical tool for the study of biofilms.

The assay describes a rapid means to measure early biofilm formation in bacteria and fungi. This method uses a microtiter plate as the substratum for microbial biofilm formation, and the biofilm is visualized using crystal violet strain. The assay provides either a qualitative or quantitative assay for early biofilm formation.

Plato de microtitulación de formación de biopelículas de ensayo

Biofilms are communities of microbes attached to surfaces, which can be found in medical, industrial and natural settings.  In fact, life in a biofilm ...

Investigación

Inmunología e Infección

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

Most microbes in nature are thought to exist as surface-associated communities in biofilms.1 Bacterial biofilms are encased within a matrix and attached to a surface.2 Biofilm formation and development are commonly studied in the laboratory using batch systems such as microtiter plates or flow systems, such as flow-cells. These methodologies are useful for screening mutant and chemical libraries (microtiter plates)3 or growing biofilms for visualization (flow cells)4. Here we present detailed protocols for growing Staphylococcus aureus in two additional types of flow system biofilms: the drip flow biofilm reactor and the rotating disk biofilm reactor.
Drip flow biofilm reactors are designed for the study of biofilms grown under low shear conditions.5 The drip flow reactor consists of four parallel test channels, each capable of holding one standard glass microscope slide sized coupon, or a length of catheter or stint. The drip flow reactor is ideal for microsensor monitoring, general biofilm studies, biofilm cryosectioning samples, high biomass production, medical material evaluations, and indwelling medical device testing.6,7,8,9
The rotating disk reactor consists of a teflon disk containing recesses for removable coupons.10 The removable coupons can by made from any machinable material. The bottom of the rotating disk contains a bar magnet to allow disk rotation to create liquid surface shear across surface-flush coupons. The entire disk containing 18 coupons is placed in a 1000 mL glass side-arm reactor vessel. A liquid growth media is circulated through the vessel while the disk is rotated by a magnetic stirrer. The coupons are removed from the reactor vessel and then scraped to collect the biofilm sample for further study or microscopy imaging. Rotating disc reactors are designed for laboratory evaluations of biocide efficacy, biofilm removal, and performance of anti-fouling materials.9,11,12,13

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

Protocols for utilizing open system flow biofilms with drip flow reactors and rotating disk reactors are presented in detail.

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 ...

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. ...

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 ...

A Mouse Model to Assess Innate Immune Response to Staphylococcus aureus Infection

Staphylococcus aureus (S. aureus)&#160;infections, including methicillin resistant stains, are an enormous burden on the healthcare system. With incidence rates of S. aureus infection climbing annually, there is a demand for additional research in its&#160;pathogenicity. Animal models of infectious disease advance our understanding of the host-pathogen response and lead to the development of effective therapeutics. Neutrophils play a primary role in the innate immune response that controls S. aureus infections by forming an abscess to wall off the infection and facilitate bacterial clearance; the number of neutrophils that infiltrate an S. aureus skin infection often correlates with disease outcome. LysM-EGFP mice, which possess the enhanced green fluorescent protein (EGFP) inserted in the Lysozyme M (LysM) promoter region (expressed primarily by neutrophils), when used in conjunction with in vivo whole animal fluorescence imaging (FLI) provide&#160;a means of quantifying neutrophil emigration noninvasively and longitudinally into wounded skin. When combined with a bioluminescent S. aureus strain and sequential in vivo whole animal bioluminescent imaging (BLI), it is possible to longitudinally monitor both the neutrophil recruitment dynamics and in vivo bacterial burden at the site of infection in anesthetized mice from onset of infection to resolution or death. Mice are more resistant to a number of virulence factors produced by S. aureus that facilitate effective colonization and infection in humans. Immunodeficient mice provide a more sensitive animal model to examine persistent S. aureus infections and the ability of therapeutics to boost innate immune responses. Herein, we characterize responses in LysM-EGFP mice that have been bred to MyD88-deficient mice (LysM-EGFP&#215;MyD88-/- mice) along with wild-type LysM-EGFP mice to investigate S. aureus skin wound infection. Multispectral simultaneous detection enabled study of neutrophil recruitment dynamics by using in vivo FLI, bacterial burden by using in vivo BLI, and wound healing longitudinally and noninvasively over time.

A Mouse Model to Assess Innate Immune Response to Staphylococcus aureus Infection

An approach is described for real-time detection of the innate immune response to cutaneous wounding and Staphylococcus aureus infection of mice. By comparing LysM-EGFP mice (which possess fluorescent neutrophils) with a LysM-EGFP crossbred immunodeficient mouse strain, we advance our understanding of infection and the development of approaches to combat infection.

Un modelo de ratón para evaluar respuesta inmune innata a la infección por Staphylococcus aureus

Staphylococcus aureus (S. aureus)&#160;infections, including methicillin resistant stains, are an enormous burden on the healthcare system. With incidence ...

Orientación de Biofilm Asociadas

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Plato de microtitulación de formación de biopelículas de ensayo

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

Subcutánea infección de Staphylococcus aureus resistentes a meticilina (MRSA)

Modelo experimental de endocarditis Resistente a la Meticilina

Staphylococcus aureus Crecimiento con hemoglobina humana como una fuente de hierro

Multiplex PCR Ensayo para la tipificaci

El estudio de las interacciones de

genotipado de

Metodología para el Estudio de la transferencia horizontal de genes en

Un modelo de ratón para evaluar respuesta inmune innata a la infección por Staphylococcus aureus