Este trabajo fue apoyado por los EE.UU. Institutos Nacionales de Salud [de subvención R01AI-39108 de ASB] y la American Heart Association [SDG subvenciones 0630219N y la AID 09GRNT2180065 de YQX].

1. Preparación de las cepas de SARM para la infección <ol><li> Inocular un asa de la cultura MRSA de un tubo de valores a -80 ° C a una sangre de oveja agar tripticasa soya (TSA) placa (ver Tabla de reactivos y equipos específicos), y se incuba a 37 ° C durante la noche. </li><li> Comprobar la pureza de la cultura en la placa de agar sangre (fenotipos similares colonia) para asegurarse de que no hay contaminación. </li><li> Elige una colonia de la placa de la sangre de oveja TSA e inocular la colonia en 5 ml de caldo de soja tripticasa (TSB) en un 15 ml con tapa complemento tubo. </li><li> Incubar a 37 ° C durante la noche con agitación a 200 rpm. </li></ol> 2. Preparación de catéteres Cirugía Corte el tubo de polietileno (PE10;. Becton Dickinson, n º de pedido 427 401) a 10 cm de longitud, y se funden uno de los extremos al presionar la punta con unas pinzas esterilizadas. El propósito de sellar un extremo del catéter es para evitar el sangrado durante la cateterización. 3. Pre-sPreparación ratamientos quirúrgicos y anestesia <ol><li> Lugar rata Sprague-Dawley (Harlan, Indianapolis, IN femenino, 250-300 gramos) en una cámara que contiene oxígeno isofluorano-mezcla de gas (50%: 50%), hasta que la anestesia haga efecto (por ejemplo, los músculos se relajan y los reflejos están ausentes del pedal ), y mantener los animales en un estado de anestesia durante la cirugía con la mezcla de gases. </li><li> Limpie la zona del cuello desde la barbilla hasta justo debajo del esternón con Betadine y el 70% de etanol. </li></ol> 4. Procedimiento Quirúrgico <ol><li> Utilice una técnica estéril durante toda la cirugía. Hacer una incisión (1-1.5 cm) verticalmente a través de sólo la capa de piel del cuello por encima del esternón. </li><li> Con disección roma, separar la fascia para exponer la arteria carótida derecha con 2 pares de pinzas de dientes curvos. </li><li> Tire suavemente de la arteria hasta fuera de la cavidad del cuello, y coloque dos de 10 cm de longitud de la sutura de seda debajo de la arteria y atar la arteria en el extremo expuesto cefálica, colocar un clip en el arteria para prevenir el sangrado. </li><li> Hacer un pequeño agujero en la parte superior de la arteria mediante un catéter introductor (Becton Dickinson, no. 406.999), insertar un catéter con una pinza a través del agujero en la arteria, retire el clip y empuje hacia abajo el catéter hacia el corazón hasta que la resistencia se cumple. </li><li> Ate la sutura suelta alrededor del extremo caudal de la arteria y asegurar el catéter en su lugar con la sutura de seda cuando el catéter está en su lugar según lo determinado por: i) la longitud de catéter (4-5 cm), ii) la resistencia a un mayor avance , y iii) la pulsación de la sonda con el latido del corazón. Dejar el catéter en su lugar durante el resto del experimento. </li><li> Corte el exceso de sutura y termina con catéter, y asegurarse de que no hay sangrado por el catéter. Meta los extremos sueltos bajo la piel del cuello y cerrar la piel por medio de grapas. </li><li> Coloque la rata en la jaula en un lugar cálido hasta que se recupere de la anestesia, y proporcionar alimentos y agua. Ver la rata y con frecuencia durantedespués de la recuperación de la anestesia. </li><li> Esta cirugía es una &quot;Categoría E&quot; procedimiento. Los procedimientos producen el dolor no se alivia con analgésicos ya que las infecciones por SARM que acompañan a la angustia de los animales. </li></ol> 5. La infección por SARM <ol><li> La infección se puede realizar entre 1 y 7 días después de la cirugía, pero mantenga coherente dentro de los experimentos. </li><li> Limpieza cola de rata con 70% de etanol, e inyectar 0,5 ml de MRSA en un recuento de células deseada (abril 10-06 10 ufc / animal para la mayoría de las cepas de S. aureus). Con 27 aguja T 1/2 pulgada por vía intravenosa de la vena de la cola Precaución : la cultura MRSA pertenece al nivel de bioseguridad 2 (BSL2) y tiene peligro potencial moderado para el personal y el medio ambiente. </li><li> Mantenga la presión en el sitio hasta la hemostasia se produce antes de regresar a la rata de la jaula. </li></ol> 6. Las ratas de sacrificio y de tejidos cultura de destino <ol><li> Sacrificar las ratas por inyección ip de pentobarbital sódico (200 mg/ Kg) después de 1 a 6 días después de la infección. </li><li> Coloque el ratón sobre la espalda y limpie el pecho con el 70% de etanol. </li><li> Hacer una incisión en forma de V en el pecho debajo del esternón, y cortar el cartílago de las costillas a cada lado del esternón para exponer el corazón. </li><li> Tire el corazón con suavidad, y el clip a través del tejido del corazón cerca de la aorta y la disección hasta liberar el corazón. </li><li> Ponga el corazón en una placa de Petri estéril con el interior de gasa de 4x4 pulgadas, hacer un corte a través de la pared del ventrículo izquierdo interno y abrir la cámara del lado izquierdo. </li><li> Compruebe visualmente para la colocación del catéter. Examinar y eliminar las vegetaciones de la válvula con las tijeras y pinzas. </li><li> Pesar y homogeneizar las vegetaciones, y hacer diluciones seriadas con PBS para el cultivo cuantitativo. </li></ol> 7. Los resultados representativos Inmediatamente después de insertar el catéter en la arteria y empujando hacia abajo sobre el catéter 4-5 cm hacia el corazón, una resistencia será expeced. Si no se detecta la resistencia, el catéter no puede haber sido insertada con éxito en el lado izquierdo de la cámara del corazón, lo que puede afectar a la colocación del catéter. Una correcta colocación del catéter publicado previamente 9 se muestra en la Figura 1. Parte de las muestras de la infección por SARM tienen que ser cultivadas cuantitativa del asegúrese de que el exacto número de viables para la infección bacteriana y la pureza de la muestra de la infección. Además, los organismos recuperados de vegetaciones deben ser los mismos que los utilizados en el inóculo. La Tabla 1 presenta un ejemplo de la virulencia de una S. aureus cepa en el modelo de endocarditis rata que fue publicado previamente 9. Todos los animales desafiados con inóculos de 10 5 y 10 6 ufc y se sacrificaron entre 3 a 6 días después de las infecciones por endocarditis desarrollados con alta S. densidades de S. aureus en las vegetaciones cardíacas, así como los riñones y el bazo ( <strong&gt; Tabla 1). Las ratas con las culturas de las válvulas estériles se consideran no infectados. <img alt="Figura 1" src="/files/ftp_upload/3863/3863fig1.jpg" /> Figura 1. El catéter está en un lugar correcto (del lado izquierdo de la cámara del corazón), y numerosas vegetaciones son visibles alrededor de la válvula aórtica 9. <table border="1"><tbody><tr><td> Inóculo (número de ánimas) </td><td colspan="3"> El promedio diario de 10 UFC / g de tejido ± desviación estándar de una: </td></tr><tr><td></td><td> Vegetación </td><td> Riñón </td><td> Bazo </td></tr><tr><td> 10 6 UFC / animal (9) </td><td> 10,36 ± 0,85 </td><td> 7,30 ± 0,64 </td><td> 6,70 ± 0,57 </td></tr><tr><td> 10 5 UFC / animal (8) </td><td> 9,93 ± 0,53 </td><td> 7,14 ± 0,53 </td><td> 6,44 ± 0,63</td></tr><tr><td> 10 4 UFC / animal (7) </td><td> 3,46 ± 0,50 * </td><td> 1,81 ± 0,74 * </td><td> 1,58 ± 0,59 * </td></tr></tbody></table> una desviación SD estándar. P &lt;0,001 con respecto a 5 o 10 10 6 UFC-desafió animales. Tabla 1. S. Staphylococcus densidad en vegetaciones cardíacas con inóculos diferente en el modelo de rata endocarditis 9.

<ol>
	<li>Petti, C. A., Fowler, V. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Staphylococcus+aureus+bacteremia+and+endocarditis.">Staphylococcus aureus bacteremia and endocarditis.</a> Cardiol. Clin. 21, 219-233 (2003).</li><li>Bashore, T. M., Cabell, C., Fowler, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Update+on+infective+endocarditis.">Update on infective endocarditis.</a> Curr. Probl. Cardiol. 31, 274-352 (2006).</li><li>Fowler, V. G., Justice, A., Moore, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Risk+factors+for+hematogenous+complications+of+intravascular+catheter-associated+Staphylococcus+aureus+bacteremia.">Risk factors for hematogenous complications of intravascular catheter-associated Staphylococcus aureus bacteremia.</a> Clin. Infect. Dis. 17, 313-320 (1993).</li><li>Wisplinghoff, H., Bischoff, T., Tallent, S. M., Seifert, H., Wenzel, R. P., Edmond, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nosocomial+bloodstream+infections+in+US+hospitals:+analysis+of+24,179+cases+from+a+prospective+nationwide+surveillance+study.">Nosocomial bloodstream infections in US hospitals: analysis of 24,179 cases from a prospective nationwide surveillance study.</a> Clin. Infect. Dis. 39, 309-317 (2004).</li><li>Hoen, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Special+issues+in+the+management+of+infective+endocarditis+caused+by+gram-positive+cocci.">Special issues in the management of infective endocarditis caused by gram-positive cocci.</a> Infect Dis. Clin. North Am. 16, 437-452 (2002).</li><li>Moise, P. A., Hershberger, E., Amodio-Groton, M. I., Lamp, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Safety+and+clinical+outcomes+when+utilizing+high-dose+(&gt;+or+=8+mg/kg)+daptomycin+therapy.">Safety and clinical outcomes when utilizing high-dose (&gt; or =8 mg/kg) daptomycin therapy.</a> Ann. Pharmacother. 43, 1211-1219 (2009).</li><li>Sakoulas, G., Brown, J., Lamp, K. C., Friedrich, L. V., Lindfield, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+outcomes+of+patients+receiving+daptomycin+for+the+treatment+of+Staphylococcus+aureus+infections+and+assessment+of+clinical+factors+for+daptomycin+failure:+a+retrospective+cohort+study+utilizing+the+Cubicin+Outcomes+Registry+and+Experience.">Clinical outcomes of patients receiving daptomycin for the treatment of Staphylococcus aureus infections and assessment of clinical factors for daptomycin failure: a retrospective cohort study utilizing the Cubicin Outcomes Registry and Experience.</a> Clin. Ther. 31, 1936-1945 (2009).</li><li>Xiong, Y. Q., Willard, J., Kadurugamuwa, J. L., Yu, J., Francis, K. P., Bayer, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+in+vivo+bioluminescent+imaging+for+evaluating+the+efficacy+of+antibiotics+in+a+rat+Staphylococcus+aureus+endocarditis+model.">Real-time in vivo bioluminescent imaging for evaluating the efficacy of antibiotics in a rat Staphylococcus aureus endocarditis model.</a> Antimicrob. Agents Chemother. 49, 380-387 (2005).</li><li>Peerschke, E. I., Bayer, A. S., Ghebrehiwet, B., Xiong, Y. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=gC1qR/p33+blockade+reduces+Staphylococcus+aureus+colonization+of+target+tissues+in+an+animal+model+of+infective+endocarditis.">gC1qR/p33 blockade reduces Staphylococcus aureus colonization of target tissues in an animal model of infective endocarditis.</a> Infect. Immun. 74, 4418-4423 (2006).</li><li>Ganesh, V. K., Rivera, J. J., Smeds, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+structural+model+of+the+Staphylococcus+aureus+ClfA-fibrinogen+interaction+opens+new+avenues+for+the+design+of+anti-staphylococcal+therapeutics.">A structural model of the Staphylococcus aureus ClfA-fibrinogen interaction opens new avenues for the design of anti-staphylococcal therapeutics.</a> PLoS Pathog. 4, e1000226 (2008).</li><li>Boles, B. R., Horswill, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Staphylococcal+biofilm+disassembly.">Staphylococcal biofilm disassembly.</a> Trends Microbiol. , (2011).</li><li>Tseng, C. W., Sanchez-Martinez, M., Arruda, A., Liu, G. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subcutaneous+Infection+of+Methicillin+Resistant+Staphylococcus+Aureus+(MRSA).">Subcutaneous Infection of Methicillin Resistant Staphylococcus Aureus (MRSA).</a> J. Vis. Exp. (48), e2528 (2011).</li></ol>

No hay conflictos de interés declarado.

La endocarditis rata es un modelo animal importante y bien caracterizado-para estudios in vivo en la patogénesis y los agentes antimicrobianos en el tratamiento de infecciones bacterianas 9-11. Además, el modelo de rata endocarditis representa una combinación de las infecciones agudas y sub-aguda, y se asemeja mucho a la humana y la endocarditis de válvula nativa humana. Por otra parte, acompañado de un catéter en la vivienda, que representa una infección clásica asociada a la biopelícula, u...

<table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Número de catálogo </td><td> Comentarios </td></tr><tr><td> Placa de agar sangre, de carnero al 5% de sangre en agar tripticasa soya (TSA) </td><td> Diagnóstico Hardy </td><td> A10BX </td><td></td></tr><tr><td> Caldo de soja tripticasa (TSB) </td><td> Becton Dickinson </td><td> 211825 </td><td></td></tr><tr><td> Incubadora de agitación </td><td> Labnet </td><td> I5311-DS </td><td></td></tr><tr><td> Polietileno tubo </td><td> Becton Dickinson </td><td> 427401 </td><td></td></tr><tr><td> Catéter introductor </td><td> Becton Dickinson </td><td> 6999 </td><td></td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td> Isofluorance </td><td> Médico Nacional de Occidente SuppY, Inc </td><td> 2147 </td><td> Siga las instrucciones de seguridad y manejo </td></tr><tr><td> Los instrumentos quirúrgicos </td><td> Herramientas de Bellas Ciencia e Instrumentos de Investigación Biomédica, Inc. </td><td></td><td> Encuentra los instrumentos de las dos empresas para sus necesidades </td></tr></tbody></table>

experimental endocarditis model of methicillin resistant staphylococcus aureus (mrsa) in rat

Infecciones endovasculares, incluyendo la endocarditis, son peligrosas para la vida síndromes infecciosos 1-3. Staphylococcus aureus es el más común en todo el mundo la causa de los síndromes de ese tipo con la morbilidad y la mortalidad inaceptablemente alta, incluso con los tratamientos adecuados de agentes antimicrobianos 4-6. El aumento de las infecciones por S. aureus resistente a meticilina Staphylococcus aureus (MRSA), las altas tasas de fracasos de tratamiento clínico a la vancomicina y crecientes problemas de linezolid y la resistencia a la daptomicina tiene todo se complica aún más el manejo de pacientes con este tipo de infecciones, y dio lugar a altos costos de atención médica 7, 8. Además, cabe destacar que los estudios más recientes con los resultados del tratamiento con antibióticos se han basado en la práctica clínica, y por lo tanto podría estar influido por factores del huésped que varían de paciente a paciente. Por lo tanto, un modelo animal adecuado de la infección endovascular en la que los factores del huésped son similares a partir de animal a animal es máscrucial para investigar patogénesis microbiana, así como la eficacia de nuevos agentes antimicrobianos. La endocarditis en la rata es un modelo bien establecido animal de experimentación que se aproxima la endocarditis de válvula nativa humana. Este modelo ha sido utilizado para examinar el papel de determinados factores de virulencia estafilococos y la eficacia de los regímenes de tratamiento antibiótico para la endocarditis por estafilococos. En este reporte se describe el modelo de endocarditis experimental por MRSA que podrían ser utilizados para investigar la patogénesis bacteriana y la respuesta al tratamiento con antibióticos.

Watch this Scientific Journal Video about Experimental Endocarditis Model of Methicillin Resistant Staphylococcus aureus MRSA in Rat at JoVE.com

Experimental Endocarditis Model of Methicillin Resistant Staphylococcus aureus MRSA in Rat

Modelo experimental de rata endocarditis por estafilococos resistentes a<em&gt; S. Staphylococcus</em&gt;.

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 ...

experimental-endocarditis-model-methicillin-resistant-staphylococcus

Research

JoVE Journal

Immunology and Infection

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

15.0K vistas.  Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center. Modelo experimental de rata endocarditis por estafilococos resistentes a S. Staphylococcus.

Video: Experimental Endocarditis Model of Methicillin Resistant Staphylococcus aureus MRSA in Rat

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 ...

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 ...

Overcoming Unresponsiveness in Experimental Autoimmune Encephalomyelitis (EAE) Resistant Mouse Strains by Adoptive Transfer and Antigenic Challenge

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory disease of the central nervous system (CNS) and has been used as an animal model for study of the human demyelinating disease, multiple sclerosis (MS). EAE is characterized by pathologic infiltration of mononuclear cells into the CNS and by clinical manifestation of paralytic disease. Similar to MS, EAE is also under genetic control in that certain mouse strains are susceptible to disease induction while others are resistant. Typically, C57BL/6 (H-2b) mice immunized with myelin basic protein (MBP) fail to develop paralytic signs. This unresponsiveness is certainly not due to defects in antigen processing or antigen presentation of MBP, as an experimental protocol described here had been used to induce severe EAE in C57BL/6 mice as well as other reputed resistant mouse strains. In addition, encephalitogenic T cell clones from C57BL/6 and Balb/c mice reactive to MBP had been successfully isolated and propagated.
The experimental protocol involves using a cellular adoptive transfer system in which MBP-primed (200 &mu;g/mouse) C57BL/6 donor lymph node cells are isolated and cultured for five days with the antigen to expand the pool of MBP-specific T cells. At the end of the culture period, 50 million viable cells are transferred into naive syngeneic recipients through the tail vein. Recipient mice so treated normally do not develop EAE, thus reaffirming their resistant status, and they can remain normal indefinitely. Ten days post cell transfer, recipient mice are challenged with complete Freund adjuvant (CFA)-emulsified MBP in four sites in the flanks. Severe EAE starts to develop in these mice ten to fourteen days after challenge. Results showed that the induction of disease was antigenic specific as challenge with irrelevant antigens did not induce clinical signs of disease. Significantly, a titration of the antigen dose used to challenge the recipient mice showed that it could be as low as 5 &mu;g/mouse. In addition, a kinetic study of the timing of antigenic challenge showed that challenge to induce disease was effective as early as 5 days post antigenic challenge and as long as over 445 days post antigenic challenge. These data strongly point toward the involvement of a "long-lived" T cell population in maintaining unresponsiveness. The involvement of regulatory T cells (Tregs) in this system is not defined.

Overcoming Unresponsiveness in Experimental Autoimmune Encephalomyelitis EAE Resistant Mouse Strains by Adoptive Transfer and Antigenic Challenge

Certain mouse strains are able to resist induction of experimental autoimmune encephalomyelitis (EAE) with myelin basic protein. Described here is a simple immunization protocol that reverses the unresponsiveness and induces paralytic disease in several typical EAE resistant mouse stains.

Superar la falta de respuesta en encefalomielitis autoinmune experimental (EAE) cepas de ratones resistentes a través de transferencia adoptiva y la estimulación antigénica

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory disease of the central nervous system (CNS) and has been used as an animal model for ...

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 ...

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 ...