Esta investigación fue apoyada por los EE.UU. Servicio de Salud Pública de subvenciones AI69233 y AI073843 del Instituto Nacional de Alergias y Enfermedades Infecciosas. EPS es un compañero Burroughs Wellcome en la patogénesis de las enfermedades infecciosas. KPH fue financiado por el Celular y Molecular Microbiología Programa de Entrenamiento de subvención T32 5 A107611-10.

1. La purificación de la hemoglobina de la sangre fresca <ol><li> Adquirir sangre humana fresca suplementada con un anticoagulante. Mantener la sangre en hielo oa 4 ° C durante toda la purificación. </li><li> Centrifugar la sangre durante 20 min a 1.500 x g. Los glóbulos rojos (GR) será en la parte inferior del tubo. Aspirar con cuidado el sobrenadante y resuspender el sedimento suavemente en helado 0,9% (w / v) de solución de NaCl. Repetir la centrifugación y lavar 3 veces. </li><li> Resuspender el precipitado en 1 volumen de helado de 10 mM Tris-HCl (pH 8,0). Esto induce la lisis de los glóbulos rojos debido a la presión osmótica. Añadir tolueno a ~ 20% del volumen final. </li><li> Incubar a 4 ° C durante la noche en un asador. </li><li> Se centrifuga el lisado a 20.000 xg durante 1 hr. Recoger la fracción hemolizado medio dejando intacto el tolueno (flotante en la parte superior) y pellet. Utilizar una pipeta de cuello largo para recoger la fracción intermedia. </li><li> Pasar a través de filtro de jeringa de 0,44 m. Si la solución contiene partículasla materia y no se puede pasar a través del filtro, repita el paso 1,5. </li><li> Purificar la hemoglobina (Hb) con una cromatografía líquida de alto rendimiento (HPLC) en columna de intercambio aniónico (Varian, PL-SAX 1.000 Å 8 micras, 150 mm x 4,6 mm). La fase móvil A es 10 mM Tris-HCl (pH 8,0) y la fase móvil B es 10 mM Tris-HCl (pH 8,0) + 0,5 M NaCl. Un 0% -100% de gradiente de disolvente B se ejecuta más de 2 min a una velocidad de flujo de 2,0 ml / min. La elución se controla sobre la base de la absorción (λ: 410 nm y 280). Recoger sólo la fracción caracteriza por un color rojo brillante y un pico de absorción prominente (Figura 1). </li><li> Dializar la elución contra salina tamponada con fosfato (PBS) durante la noche y luego durante unas pocas horas más. Esterilizar mediante el paso a través de un filtro de 0,22 micras jeringa. </li><li> Para medir la concentración de Hb, preparar las soluciones estándar de concentraciones conocidas de Hb en PBS. Determinar la concentración de Hb en la muestra mediante la mezcla de las soluciones patrón (véase la tabla con reactivos nosotrosed) o la solución de la muestra con el reactivo de Drabkin 2x (preparado a partir de polvo) en una relación 1:1. Por ejemplo, mezclar 100 l solución de Hb con 100 l de reactivo de Drabkin 2x en placas de 96 pocillos. Incubar durante 15 min y la absorbancia medida a 540 nm. Trazar una curva estándar y determinar la concentración de Hb en la muestra. Cinco a quince mg / ml rendimientos son típicos. </li><li> Ejecutar 15-20 g hemoglobina purificada en un 15% SDS-PAGE por duplicado. Mancha de uno de los geles; transferir las proteínas a partir de otro gel a una membrana de nitrocelulosa y de inmunotransferencia para la hemoglobina (Figura 2). </li><li> Congelar y almacenar alícuotas de 1 ml de hemoglobina en nitrógeno líquido. </li></ol> 2. Preparación de hierro agotan Medios de Crecimiento <ol><li> Preparar Roswell Park Memorial Institute (RPMI) caldo disolviendo el polvo RPMI en agua, añadir bicarbonato de sodio según lo recomendado por el fabricante y el 1% de ácidos cassamino (CA) (w / v). Esterilizar mediante el paso a través de un filtro de 0,2 micras y refrigerador tiendaerated. </li><li> Preparar metálica empobrecida en RPMI (NRPMI) mediante la adición de 7% (w / v) Chelex 100 y mezcla durante la noche en una placa de agitación. Eliminar Chelex 100 pasando a través de un filtro de 0,2 micras y almacén refrigerado. Suplemento medios imprescindibles de metales no ferrosos: 25 mM ZnCl 2, 25 mM MnCl 2, 100 mM CaCl 2 y 1 mM MgCl 2, preparado de antemano como una solución estéril x 1.000. Use recipientes desechables de plástico para este paso para evitar la contaminación de hierro de re-utilizables suministros. </li></ol> 3. Staphylococcus aureus Crecimiento utilizando hemoglobina como una única fuente de hierro <ol><li> Streak S. para el aislamiento de S. aureus en agar tripticasa soja (TSA) de un stock congelado. Incubar a 37 ° C durante 20-24 h. </li><li> Resuspender etilendiamina-N, N&#39;-bis (2-hidroxifenilacético) (EDDHA) en etanol anhidro a 100 mM. EDDHA no entra en solución, pero se esteriliza por etanol. </li><li> Añadir EDDHA de RPMI a una concentración final de 0,5 mM. PermitirEDDHA para disolver durante al menos 30 min antes de proceder al siguiente paso. Debido a la variación de lote a lote, la concentración final EDDHA puede ser necesario disminuir a 0,25 mm para permitir el crecimiento bacteriano. </li><li> Inocular colonias individuales de S. aureus en 5 ml de RPMI que contiene EDDHA en 15 ml con tapón de rosca tubos cónicos. Incubar a 37 ° C con agitación a 180 revoluciones por minuto (rpm) durante 16-20 h. </li><li> Centrifugar los cultivos de una noche durante 5 min a 7.500 xg y resuspender el precipitado en NRPMI que contiene 0,5 mM EDDHA. Normalizar OD 600 a ~ 3. </li><li> Preparar NRPMI que contiene 2,5 g por ml Hb y EDDHA 0.1-1.0 mM. Debido a la variación entre los lotes, la concentración de EDDHA requerido para quelar el hierro libre en NRPMI puede variar. </li><li> Subculture 10 l de suspensión bacteriana de la etapa 1 en 3,5 ml NRPMI + EDDHA + Hb en un 15 ml con tapón de rosca tubo cónico. </li><li> Incubar los cultivos a 37 ° C durante un máximo de 48 horas con agitación a 180 rpm o en un tambor de enrollamiento. </li><li&gt; Cada hora 12.06 tomar lecturas de OD 600 mediante la eliminación de 50 l de la cultura y la mezcla con 150 ml de PBS en placas de 96 pocillos. </li></ol>

<ol>
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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+battle+for+iron:+host+sequestration+and+Staphylococcus+aureus+acquisition.">A battle for iron: host sequestration and Staphylococcus aureus acquisition.</a> Microbes and infection. Institut Pasteur. 14, 217-227 (2012).</li><li>Krishna Kumar, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+basis+for+hemoglobin+capture+by+Staphylococcus+aureus+cell-surface+protein.">Structural basis for hemoglobin capture by Staphylococcus aureus cell-surface protein.</a> IsdH. The Journal of biological chemistry. 286, 38439-38447 (2011).</li><li>Grigg, J. C., Mao, C. X., Murphy, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Iron-coordinating+tyrosine+is+a+key+determinant+of+NEAT+domain+heme+transfer.">Iron-coordinating tyrosine is a key determinant of NEAT domain heme transfer.</a> Journal of Molecular Biology. 413, 684-698 (2011).</li><li>Villareal, V. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transient+weak+protein-protein+complexes+transfer+heme+across+the+cell+wall+of+Staphylococcus+aureus.">Transient weak protein-protein complexes transfer heme across the cell wall of Staphylococcus aureus.</a> Journal of the American Chemical Society. 133, 14176-14179 (2011).</li><li>Muryoi, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Demonstration+of+the+iron-regulated+surface+determinant+(Isd)+heme+transfer+pathway+in+Staphylococcus+aureus.">Demonstration of the iron-regulated surface determinant (Isd) heme transfer pathway in Staphylococcus aureus.</a> J. Biol. Chem. 283, 28125-28136 (2008).</li><li>Reniere, M. L., Skaar, E. P. <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+haem+oxygenases+are+differentially+regulated+by+iron+and+haem.">Staphylococcus aureus haem oxygenases are differentially regulated by iron and haem.</a> Mol. Microbiol. 69, 1304-1315 (2008).</li><li>Liu, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+hemin+transfer+from+IsdA+to+IsdC+in+the+iron-regulated+surface+determinant+(Isd)+heme+acquisition+system+of+Staphylococcus+aureus.">Direct hemin transfer from IsdA to IsdC in the iron-regulated surface determinant (Isd) heme acquisition system of Staphylococcus aureus.</a> J. Biol. Chem. 283, 6668-6676 (2008).</li><li>Pishchany, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specificity+for+human+hemoglobin+enhances+Staphylococcus+aureus+infection.">Specificity for human hemoglobin enhances Staphylococcus aureus infection.</a> Cell Host Microbe. 8, 544-550 (2010).</li><li>Pishchany, G., Dickey, S. E., Skaar, E. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subcellular+localization+of+the+Staphylococcus+aureus+heme+iron+transport+components+IsdA+and+IsdB.">Subcellular localization of the Staphylococcus aureus heme iron transport components IsdA and IsdB.</a> Infect. Immun. 77, 2624-2634 (2009).</li><li>Torres, V. J., Pishchany, G., Humayun, M., Schneewind, O., Skaar, E. P. <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+IsdB+is+a+hemoglobin+receptor+required+for+heme+iron+utilization.">Staphylococcus aureus IsdB is a hemoglobin receptor required for heme iron utilization.</a> J. Bacteriol. 188, 8421-8429 (2006).</li><li>Kim, H. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IsdA+and+IsdB+antibodies+protect+mice+against+Staphylococcus+aureus+abscess+formation+and+lethal+challenge.">IsdA and IsdB antibodies protect mice against Staphylococcus aureus abscess formation and lethal challenge.</a> Vaccine. 28, 6382-6392 (2010).</li><li>Cheng, A. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+requirements+for+Staphylococcus+aureus+abscess+formation+and+persistence+in+host+tissues.">Genetic requirements for Staphylococcus aureus abscess formation and persistence in host tissues.</a> Faseb J. 23, 3393-3404 (2009).</li><li>Andreini, C., Bertini, I., Cavallaro, G., Holliday, G. L., Thornton, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metal+ions+in+biological+catalysis:+from+enzyme+databases+to+general+principles.">Metal ions in biological catalysis: from enzyme databases to general principles.</a> J. Biol. Inorg. Chem. 13, 1205-1218 (2008).</li><li>Weinberg, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Iron+availability+and+infection.">Iron availability and infection.</a> Biochimica et Biophysica Acta (BBA) - General Subjects. 1790, 600-605 (2009).</li><li>Drabkin, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolism+of+the+Hemin+Chromoproteins.">Metabolism of the Hemin Chromoproteins.</a> Physiological Reviews. 31, 345-431 (1951).</li><li>Graversen, J. H., Madsen, M., Moestrup, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD163:+a+signal+receptor+scavenging+haptoglobin-hemoglobin+complexes+from+plasma.">CD163: a signal receptor scavenging haptoglobin-hemoglobin complexes from plasma.</a> The international journal of biochemistry &amp; cell biology. 34, 309-314 (2002).</li><li>Torres, V. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Staphylococcus+aureus+Fur+regulates+the+expression+of+virulence+factors+that+contribute+to+the+pathogenesis+of+pneumonia.">Staphylococcus aureus Fur regulates the expression of virulence factors that contribute to the pathogenesis of pneumonia.</a> Infect. Immun. 78, 1618-1628 (2010).</li><li>Hammer, N. D., Skaar, E. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+Mechanisms+of+Staphylococcus+aureus+Iron+Acquisition.">Molecular Mechanisms of Staphylococcus aureus Iron Acquisition.</a> Annu. Rev. Microbiol. , (2011).</li><li>Hurd, A. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+iron-regulated+surface+proteins+IsdA,+IsdB,+and+IsdH+are+not+required+for+heme+iron+utilization+in+Staphylococcus+aureus.">The iron-regulated surface proteins IsdA, IsdB, and IsdH are not required for heme iron utilization in Staphylococcus aureus.</a> Fems. Microbiology Letters. 329, 93-100 (2012).</li><li>Boys, B. L., Kuprowski, M. C., Konermann, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Symmetric+behavior+of+hemoglobin+alpha-+and+beta-+subunits+during+acid-induced+denaturation+observed+by+electrospray+mass+spectrometry.">Symmetric behavior of hemoglobin alpha- and beta- subunits during acid-induced denaturation observed by electrospray mass spectrometry.</a> Biochemistry. 46, 10675-10684 (2007).</li><li>Williams, R. C., Tsay, K. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+convenient+chromatographic+method+for+the+preparation+of+human+hemoglobin.">A convenient chromatographic method for the preparation of human hemoglobin.</a> Analytical Biochemistry. 54, 137-145 (1973).</li><li>Shen, T. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+unmodified+human+adult+hemoglobin+in+Escherichia+coli.">Production of unmodified human adult hemoglobin in Escherichia coli.</a> Proc. Natl. Acad. Sci. U.S.A. 90, 8108-8112 (1993).</li><li>Manjula, B. N., Acharya, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+and+molecular+analysis+of+hemoglobin+by+high-performance+liquid+chromatography.">Purification and molecular analysis of hemoglobin by high-performance liquid chromatography.</a> Methods Mol. Med. 82, 31-47 (2003).</li><li>Neilands, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbial+envelope+proteins+related+to+iron.">Microbial envelope proteins related to iron.</a> Annual review of microbiology. 36, 285-309 (1982).</li><li>Chart, H., Buck, M., Stevenson, P., Griffiths, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Iron+regulated+outer+membrane+proteins+of+Escherichia+coli:+variations+in+expression+due+to+the+chelator+used+to+restrict+the+availability+of+iron.">Iron regulated outer membrane proteins of Escherichia coli: variations in expression due to the chelator used to restrict the availability of iron.</a> Journal of General Microbiology. 132, 1373-1378 (1986).</li><li>Rogers, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Iron-Binding+Catechols+and+Virulence+in+Escherichia+coli.">Iron-Binding Catechols and Virulence in Escherichia coli.</a> Infection and Immunity. 7, 445-456 (1973).</li></ol>

No tenemos nada que revelar.

El hierro es un nutriente esencial requerido por los organismos de todos los reinos de la vida 15. En los vertebrados, el hierro es retenido para evitar la toxicidad causada por este elemento. Este secuestro también oculta de hierro de los microbios invasores en un proceso conocido como inmunidad nutricional 16. En respuesta, los patógenos han desarrollado estrategias que evitan la inmunidad nutricional. Uno de estos mecanismos depende de la hemoglobina, que es la fuente más abundante de hierro ...

<table border="1"><tbody><tr><td> Nombre del reactivo / material </td><td> Empresa </td><td> Número de catálogo </td><td> Comentarios </td></tr><tr><td> HPLC columna de intercambio de aniones </td><td> Varian </td><td> PL1551-3802 </td><td></td></tr><tr><td> Reactivo de Drabkin </td><td> Sigma </td><td> D5941-6VL </td><td></td></tr><tr><td> La hemoglobina normal </td><td> Pointe Scientific </td><td> H7506-STD </td><td></td></tr><tr><td> RPMI </td><td> HyClone </td><td> SH30011.02 </td><td></td></tr><tr><td> Chelex 100 sodio forma </td><td> Sigma </td><td> C7901 </td><td></td></tr><tr><td> EDDHA </td><td> LGC Standards GmbH </td><td> ANC 001 </td><td></td></tr><tr><td> Hemoglobina un anticuerpo </td><td> Santa Cruz Biotechnology, Inc </td><td> SC-21005 </td><td> &lt;/ Td&gt; </tr><tr><td> Agar de soja tríptico </td><td> BD </td><td> 236920 </td><td></td></tr></tbody></table>

staphylococcus aureus growth using human hemoglobin as an iron source

S. aureus es una bacteria patógena que requiere de hierro para llevar a cabo las funciones metabólicas vitales y causan enfermedades. El depósito más abundante de hierro en el interior del huésped humano es heme, que es el cofactor de la hemoglobina. Para adquirir el hierro de la hemoglobina, S. aureus utiliza un complejo sistema conocido como el determinante superficie regulada por hierro (Isd) del sistema 1. Los componentes del sistema Isd primero hemoglobina anfitrión bind, a continuación, extraer e importar hemo, y, finalmente, liberar el hierro del grupo hemo en el citoplasma bacteriano 2,3. Esta vía ha sido diseccionado a través de numerosos estudios in vitro 4-9. Además, la contribución del sistema de Isd a la infección se ha demostrado repetidamente en modelos de ratón 8,10-14. El establecimiento de la contribución del sistema de Isd a la hemoglobina derivada de la adquisición de hierro y crecimiento ha demostrado ser más difícil. Ensayos de crecimiento usando hemoglobina como única fuente de hierro son complicadas by la inestabilidad de la hemoglobina disponible comercialmente, contaminando hierro libre en el medio de crecimiento, y la toxicidad asociada con quelantes de hierro. Aquí presentamos un método que supera estas limitaciones. Hemoglobina de alta calidad se preparó a partir de sangre fresca y se almacena en nitrógeno líquido. Hemoglobina purificada se completa en hierro-agotan medio que imitan el entorno pobre en hierro encontrada por patógenos dentro del huésped vertebrado. Al privar S. aureus de hierro libre y se completa con una forma mínimamente manipulados de hemoglobina que inducir el crecimiento de una manera que es totalmente dependiente de la capacidad de unirse a la hemoglobina, extraer hemo, heme pasar a través de la envoltura celular bacteriana y degradar hemo en el citoplasma. Este ensayo será útil para los investigadores que buscan para dilucidar los mecanismos de adquisición de hierro hemoglobin-/heme-derived en S. aureus y posiblemente otros patógenos bacterianos.

Hemos purificado hemoglobina humana de hemolizado con HPLC (Protocolo de paso 1,7). Figura 1 muestra registró la absorbancia del eluato a 280 y 410 nm longitud de onda. La fracción 5 se recogió y otras fracciones fueron descartados. Los rendimientos de cinco a quince miligramos de hemoglobina por mililitro de eluato se adquieren. Hemoglobina purificada se analizó por SDS-PAGE por duplicado y los geles se tiñeron para las proteínas o se trasladaron a nitrocelulosa y se inmunotransfirieron (Protocol...

Watch this Scientific Journal Video about Staphylococcus aureus Growth using Human Hemoglobin as an Iron Source at JoVE.com

Staphylococcus aureus Growth using Human Hemoglobin as an Iron Source

Aquí se describe un ensayo de crecimiento para<em&gt; Staphylococcus aureus</em&gt; Usando hemoglobina como la única fuente de hierro nutrientes disponibles. Este ensayo establece el papel de los factores bacterianos implicados en la hemoglobina derivada de la adquisición de hierro.

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 ...

staphylococcus-aureus-growth-using-human-hemoglobin-as-an-iron-source

Research

JoVE Journal

Immunology and Infection

Staphylococcus aureus Growth using Human Hemoglobin as an Iron Source

16.4K vistas.  Vanderbilt University Medical School. Aquí se describe un ensayo de crecimiento para Staphylococcus aureus Usando hemoglobina como la única fuente de hierro nutrientes disponibles. Este ensayo establece el papel de los factores bacterianos implicados en la hemoglobina derivada de la adquisición de hierro.

Video: Staphylococcus aureus Growth using Human Hemoglobin as an Iron Source

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 ...

Cost-effective Method for Microbial Source Tracking Using Specific Human and Animal Viruses

Microbial contamination of the environment represents a significant health risk. Classical bacterial fecal indicators have shown to have significant limitations, viruses are more resistant to many inactivation processes and standard fecal indicators do not inform on the source of contamination. The development of cost-effective methods for the concentration of viruses from water and molecular assays facilitates the applicability of viruses as indicators of fecal contamination and as microbial source tracking (MST) tools. Adenoviruses and polyomaviruses are DNA viruses infecting specific vertebrate species including humans and are persistently excreted in feces and/or urine in all geographical areas studied. In previous studies, we suggested the quantification of human adenoviruses (HAdV) and JC polyomaviruses (JCPyV) by quantitative PCR (qPCR) as an index of human fecal contamination. Recently, we have developed qPCR assays for the specific quantification of porcine adenoviruses (PAdV) and bovine polyomaviruses (BPyV) as animal fecal markers of contamination with sensitivities of 1-10 genome copies per test tube. In this study, we present the procedure to be followed to identify the source of contamination in water samples using these tools. As example of representative results, analysis of viruses in ground water presenting high levels of nitrates is shown.
Detection of viruses in low or moderately polluted waters requires the concentration of the viruses from at least several liters of water into a much smaller volume, a procedure that usually includes two concentration steps in series. This somewhat cumbersome procedure and the variability observed in viral recoveries significantly hamper the simultaneous processing of a large number of water samples.
In order to eliminate the bottleneck caused by the two-step procedures we have applied a one-step protocol developed in previous studies and applicable to a diversity of water matrices. The procedure includes: acidification of ten-liter water samples, flocculation by skimmed milk, gravity sedimentation of the flocculated materials, collection of the precipitate and centrifugation, resuspension of the precipitate in 10 ml phosphate buffer. The viral concentrate is used for the extraction of viral nucleic acids and the specific adenoviruses and polyomaviruses of interest are quantified by qPCR. High number of samples may be simultaneously analyzed using this low-cost concentration method.
The procedure has been applied to the analysis of bathing waters, seawater and river water and in this study, we present results analyzing groundwater samples. This high-throughput quantitative method is reliable, straightforward, and cost-effective.

The study describes a cost-effective method for the identification of the source of fecal/urine contamination or contamination by nitrates in water using qPCR for the specific quantification of human/porcine/bovine DNA viruses, adenoviruses and polyomaviruses, proposed as MST tools.

Método rentable para el Rastreo de uso microbiana específica virus humanos y animales

Microbial contamination of the environment represents a significant health risk. Classical bacterial fecal indicators have shown to have significant ...

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 ...

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 ...

Quantifying the Cytotoxicity of Staphylococcus aureus Against Human Polymorphonuclear Leukocytes

Staphylococcus aureus is capable of secreting a wide range of leukocidins that target and disrupt the membrane integrity of polymorphonuclear leukocytes (PMNs or neutrophils). This protocol describes both the purification of human PMNs and the quantification of S. aureus cytotoxicity against PMNs in three different sections. Section 1 details the isolation of PMNs and serum from human blood using density centrifugation. Section 2 tests the cytotoxicity of extracellular proteins produced by S. aureus against these purified human PMNs. Section 3 measures the cytotoxicity against human PMNs following the phagocytosis of live S. aureus. These procedures measure disruption of PMN plasma membrane integrity by S. aureus leukocidins using flow cytometry analysis of PMNs treated with propidium iodide, a DNA binding fluorophore that is cell membrane impermeable. Collectively, these methods have the advantage of rapidly testing S. aureus cytotoxicity against primary human PMNs and can be easily adapted to study other aspects of host-pathogen interactions.

Quantifying the Cytotoxicity of Staphylococcus aureus Against Human Polymorphonuclear Leukocytes

This protocol describes a method for the purification of polymorphonuclear leukocytes from whole human blood and two distinct assays that quantify the cytotoxicity of Staphylococcus aureus against these important innate immune cells.

Cuantificación de la citotoxicidad de Staphyloccus aureus contra leucocitos polimorfonucleares humanos

Staphylococcus aureus is capable of secreting a wide range of leukocidins that target and disrupt the membrane integrity of polymorphonuclear leukocytes ...