Damos las gracias a Michael y Jean Feiss Sippy para la orientación en la creación de fagos y purificación. Damos las gracias a Michael Elowitz para proporcionar el software de reconocimiento celular, Schnitzcell. El trabajo en el laboratorio de Golding es apoyado por becas de los Institutos Nacionales de Salud (R01GM082837), la National Science Foundation (082.265, PFC: Centro para la Física de las células vivas), la Fundación Welch (Grant Q-1759) y Ciencia Human Frontier Programa (PRG 70/2008).

<ol>
	<li>Oppenheim, A. B., Kobiler, O., Stavans, J., Court, D. L., Adhya, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Switches+in+bacteriophage+lambda+development.">Switches in bacteriophage lambda development.</a> Annu. Rev. Genet. 39, 409-429 (2005).</li><li>Ptashne, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> A genetic switch : phage lambda revisited. , (2004).</li><li>Hendrix, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Lambda II. , (1983).</li><li>Hershey, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Bacteriophage lambda. , (1971).</li><li>Zeng, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decision+making+at+a+subcellular+level+determines+the+outcome+of+bacteriophage+infection.">Decision making at a subcellular level determines the outcome of bacteriophage infection.</a> Cell. 141, 682-691 (2010).</li><li>Edgar, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacteriophage+infection+is+targeted+to+cellular+poles.">Bacteriophage infection is targeted to cellular poles.</a> Mol. Microbiol. , (2008).</li><li>Ausubel, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Current protocols in molecular biology. , (1994).</li><li>Sambrook, J., Russell, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular cloning : a laboratory manual. , (2001).</li><li>Fasman, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Practical handbook of biochemistry and molecular biology. , (1989).</li><li>Kaiser, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+internal+structure+of+bacteriophage+lambda.">On the internal structure of bacteriophage lambda.</a> J. Gen. Physiol. 49, 171-178 (1966).</li><li>Kobiler, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+kinetic+analysis+of+the+bacteriophage+lambda+genetic+network.">Quantitative kinetic analysis of the bacteriophage lambda genetic network.</a> Proc Natl Acad Sci. 102, 4470-4475 (2005).</li><li>Shaner, N. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+monomeric+red,+orange+and+yellow+fluorescent+proteins+derived+from+Discosoma+sp.+red+fluorescent+protein.">Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein.</a> Nat. Biotechnol. 22, 1567-1572 (2004).</li><li>Zeng, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Personal communication with M. Feiss. , .</li><li>Schwartz, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+adsorption+of+coliphage+lambda+to+its+host:+effect+of+variations+in+the+surface+density+of+receptor+and+in+phage-receptor+affinity.">The adsorption of coliphage lambda to its host: effect of variations in the surface density of receptor and in phage-receptor affinity.</a> J. Mol. Biol. 103, 521-536 (1976).</li></ol>

No hay conflictos de interés declarado.

Cepas bacterianas, fagos y plásmidos: La cepa LE392 es supF. Fue elegida para suprimir la mutación SAM7 en el genoma del fago (véase la Tabla 1 para detalles). Por lo tanto, lisógenos inducidos finalmente lisis y libera partículas de fagos, como células infectadas que han optado por la vía lítica. Células lisogénicas se cultivan a 30 ° C debido a la presencia de la temperatura cI sensible alelo 857 en el genoma del fago. Después de la inducción de calor, GPD-EYFP y de tipo salvaje grupo D se co-expresan a partir del genoma de λ LZ1 y la pPlate plásmido * D respectivamente. Como resultado, la cápside de la recién creada LZ2 fago λ contiene una mezcla de GPD-EYFP y proteínas GPD. Este fago mosaico es estructuralmente estable y fluorescente suficientemente para permitir la detección de fagos individuales 5. pP RE - mCherry es un plásmido utilizado para detectar elección de la pathwa lisogénicoy. El RE promotor P se activa por CII durante el establecimiento de la lisogenia 1,11. pP RE - mCherry 5 se deriva de pE-gfp 11 mediante la sustitución de las buenas prácticas agrarias con mCherry 12. Para más detalles vea nuestro trabajo anterior 5. Parámetros de Crecimiento Estado: Durante la inducción lisógeno (Sección 1), agitación suave a 180 rpm da un rendimiento bueno virus 13. El uso de la glucosa en el medio de crecimiento debe ser evitado como metabolismo de la glucosa genera productos metabólicos ácidos, y partículas maduras lambda son inestables a pH ácido 13. La adición de MgSO 4 está dirigido a la estabilización de la cápside del fago 3. Para los fagos portadores de tipo salvaje cI (en lugar de cI 857), el lisógeno puede ser inducida mediante el C daña el ADN Mitomicina agente 3. En el paso 1.3, la incubación a 37 ° C no debe ser superior a 90 minutos. Es USEF<html> ul para comprobar la densidad celular por OD 600 cada 30 min. Por un lisado buena, OD 600 cae a alrededor de 0,2 o menos, y el restante OD 600 es un resultado de los restos celulares. Incubando durante demasiado tiempo puede resultar en un rendimiento inferior fago desde el fago recién creada puede comenzar a inyectar su ADN en los restos celulares. Para obtener una banda de fago visible (por lo menos 1 x 10 11 partículas de fago) en los pasos 2,11 y 2,13, crecer al menos 500 ml de cultivo en el paso 1,2. La adición de 0,2% de maltosa en el medio de crecimiento en los Pasos 5,1 y 5,2 está dirigido a inducir la expresión de cordero, el receptor para el fago lambda adsorción 3,14. La dilución de 1000 veces en lugar de 100 veces en el paso 5.2 está dirigida a reducir el nivel de fondo de la mCherry RE reportero plásmido pP - mCherry. En el paso 5,5 para la inyección de ADN de fago de activación, 35 ° C se elige para evitar la inducción del alelo cI857 sensible a la temperatura. Fago Purificación: jove_content &quot;&gt; Las etapas de purificación de fagos (Pasos 2,1 a través de 2,11) se puede reemplazar con otros protocolos de purificación 5, pero el final a través de ultracentrifugación en gradiente de equilibrio de CsCl (Pasos 2,12 y 2,13) ​​es inevitable. Rotores basculantes son necesarios en los pasos 2,10 y 2,12 a asegurar afilados bandas de fagos visibles. Obtención de un stock de fago puro puede tardar hasta una semana, por lo que es necesario comprobar el título del fago en el camino para asegurarse de que nada va mal durante los pasos intermedios. Fago Manipulación: Durante todos los procedimientos de purificación en la Sección 2, que es crítico para manejar fago lisado suavemente para evitar la cizalladura colas de fago a partir de cabezas de fagos. Durante la infección de células en la Sección 5 (por ejemplo, a través de los pasos 5,5 5,7), también es crítica para evitar el cizallamiento de las partículas de fagos de la célula infectada. Tenga en cuenta que si el fago se cizalla de la célula infectada después de inyectar su ADN, el resultado es un &quot;oscuro&quot; infección, es decir, en lafection resultado se observó en el experimento pero el fago infecta no. Para minimizar tales problemas, se usa una punta de pipeta de ancho cuando el manejo de los fagos o de la mezcla fago / células. DAPI Pruebas: La tinción de los stock de fago con DAPI (Sección 4) es un método rápido y eficiente para comprobar la pureza de la población de fagos. También se puede utilizar para comprobar la posible degradación de un stock de fago existente en el tiempo. Para una acción pura, la co-localización de las señales de YFP y DAPI bajo el microscopio de fluorescencia debe ser cercana a 100%. Nos típicamente observar que menos del 1% de los puntos YFP no contienen DAPI (representando cápsidas sin el genoma viral), lo que indica que estas partículas no con éxito empaquetar el ADN viral o había inyectado ya su ADN en otro lugar. Menos del 1% de los puntos DAPI no contienen YFP (correspondiente a los fagos no fluorescentes). Si este no es el caso, a través de los pasos 2,12 2,14 necesidad a repetirse en OrDer para purificar de nuevo. Con respecto a los parámetros de imagen, la configuración de microscopio en el Paso 4,3 no es tan crítica como en la Sección 5 porque no a largo plazo de células vivas de imágenes se requiere aquí. Sin embargo, mantener la configuración de microscopía mismos como en la sección 5 es útil si se quiere calibrar la intensidad de fluorescencia de una partícula de fago único. Si la losa de PBS-agarosa no está muy limpio, o demasiado DAPI colorante se utiliza, algunos puntos correspondientes a DAPI ADN del fago puede estar rodeado de un &quot;halo&quot;. Si tinte DAPI se usa demasiado poco, la señal del canal de DAPI puede ser muy débil. Microscopio Sistema: Para la formación de imágenes en la sección 6, se utiliza un microscopio de epifluorescencia invertida comercial (Eclipse TE2000-E, Nikon) con un objetivo de 100x (Plan Fluo, apertura numérica 1,40, aceite de inmersión) y conjuntos de filtros estándar (Nikon). La fuente de luz fluorescente es una lámpara de arco con control de intensidad de luz. Las siguientes funciones están controladas por ordenador: x, y y z poción, campo claro y fluorescencia persianas y selección de filtro de fluorescencia. Una función de enfoque automático se requiere. De lo contrario, el enfoque puede fácilmente desviarse durante la película de lapso de tiempo (normalmente de 4 horas de duración). La capacidad de adquirir múltiples posiciones (x, y) en cada punto de tiempo es útil, ya que permite a los acontecimientos múltiples de infección en paralelo. Por lo general adquieren 8 posiciones de escena en cada película, después de un máximo de 100 eventos de infección. La cámara que utilizamos es una solución enfriada CCD con 512x512 píxeles de la cámara de 16x16 micras con un rango dinámico de 16 bits (Cascade512, Fotometría). La adquisición se realizó utilizando el software Metamorph (Molecular Devices). El microscopio debe ser colocado en una habitación con temperatura controlada, como alternativa, la platina del microscopio debe ser rodeado por una cámara de temperatura controlada. Adquisición de imágenes: En vivo imágenes de células, es fundamental para evitar la exposición innecesaria de la muestra, lo que podría conducir a la decoloración y phototoxicity. Por lo tanto, es mejor para caracterizar primero el sistema para encontrar una exposición óptima de la luz que permite la detección de fluorescencia, mientras que no conduce a un crecimiento celular excesivo blanqueamiento o inhibición. Para obtener una imagen de fluorescencia bueno, jugar con la intensidad de la luz excitante, tiempo de exposición y ganancia de la cámara. En los pasos 6.2-6.3, el intervalo de 10 min marco se elige con el fin de minimizar la exposición de la luz. En cada trama, sólo un único foco en-imagen que se necesita en contraste de fase (para el reconocimiento de células) y los canales fluorescentes (para determinar el destino de células). En el primer punto de tiempo, sin embargo, múltiples posición z imágenes a través del canal de YFP son necesarios para capturar todos los fagos que infectan en la superficie celular. El tiempo de exposición YFP en el marco inicial también puede necesitar ser mayor que el utilizado para la película de lapso de tiempo en los marcos de tiempo posteriores. Análisis de imagen: Muy cuidadosamente contar partículas de fago alrededor de la superficie de la célula en el paso 7,1. Comose señaló anteriormente, tomamos una serie de z-pilas a través del canal YFP en el Paso 6.2. Sin embargo, esto puede hacer que algunas partículas de fago fluorescentes fuera de foco, que pone a prueba el recuento. La longitud de la célula en el período de tiempo inicial se mide utilizando el software Metamorph. La longitud de la célula también se puede medir por ImageJ o otras herramientas de software. Adicionalmente, un hogar automatizado construido programa Matlab puede ser muy útil en la obtención de información tales como el cambio de fluorescencia con el tiempo a lo largo de los linajes celulares.

<table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Número de catálogo </td></tr><tr><td> Cloroformo </td><td> Fisher Scientific </td><td> C298-500 </td></tr><tr><td> NaCl </td><td> Fisher Scientific </td><td> S271-3 </td></tr><tr><td> DNasa I </td><td> Sigma </td><td> D4527-10KU </td></tr><tr><td> RNasa </td><td> Sigma </td><td> R4642-10MG </td></tr><tr><td> PEG8000 </td><td> Fisher Scientific </td><td> BP233-1 </td></tr><tr><td> Tampón SM </td><td> Teknova </td><td> S0249 </td></tr><tr><td> NZYM </td><td> Teknova </td><td> N2062 </td></tr><tr><td> CsCl </td><td> Sigma </td><td> C3011-250G </td></tr><tr><td> Jeringa </td><td> Becton Dickinson </td><td> 309585 </td></tr><tr><td> Aguja </td><td> Becton Dickinson </td><td> 305176 </td></tr><tr><td> Diálisis casete </td><td> Thermo Scientific </td><td> 66333 </td></tr><tr><td> Portaobjetos del microscopio </td><td> Corning </td><td> 2947-75x50 </td></tr><tr><td> Agarosa </td><td> Fisher Scientific </td><td> BP160-100 </td></tr><tr><td> SW40Ti ultra-transparente tubo </td><td> Beckman Coulter </td><td> 344060 </td></tr><tr><td> SW60Ti ultra-transparente tubo </td><td> Beckman Coulter </td><td> 344062 </td></tr><tr><td> SW40Ti rotor </td><td> Beckman Coulter </td><td> 331302 </td></tr><tr><td> SW60Ti rotor </td><td> Beckman Coulter </td><td> 335649 </td></tr><tr><td> Refractómetro </td><td> Fisher Scientific </td><td> 13-947 </td></tr><tr><td> Microscopio de epifluorescencia </td><td> Nikon </td><td> Eclipse TE2000-E </td></tr></tbody></table> Tabla 2. Reactivos y equipo.

following cell-fate in e. coli after infection by phage lambda

El sistema comprende bacteriófago (fago) lambda y la bacteria E. coli ha servido durante mucho tiempo como un paradigma para el 1,2 determinación de la celda de destino. Después de la infección simultánea de la célula por un número de fagos, una de las dos vías se elige: lítico (virulento) o lisogénico (inactivo) 3,4. Recientemente hemos desarrollado un método para fluorescentemente etiquetado fagos individuales, y fueron capaces de examinar la decisión después de la infección en tiempo real bajo el microscopio, en el nivel de fagos individuales y las células 5. A continuación, se describe el procedimiento completo para realizar los experimentos de infección descritos en nuestro anterior trabajo 5. Esto incluye la creación de fagos fluorescentes, infección de las células, formación de imágenes en el microscopio y el análisis de datos. El fago fluorescente es un &quot;híbrido&quot;, co-expresan versiones de tipo salvaje y YFP fusión de la proteína de la cápside GPD. Un lisado de fago en bruto se obtuvo por primera vez mediante la inducción de un lisógeno de la (GPD-EYFP Enhbrado Yellow Fluorescent Protein) de fagos, que alberga un plásmido que expresa salvaje grupo D tipo. Una serie de pasos de purificación se realiza a continuación, seguido por DAPI-etiquetado y la formación de imágenes en el microscopio. Esto se hace con el fin de verificar la uniformidad, la eficacia de empaquetamiento del ADN, la señal de fluorescencia y la estabilidad estructural del stock de fago. La adsorción inicial de fagos a las bacterias se realizaron en hielo, a continuación, seguido por una breve incubación a 35 ° C para provocar la inyección de ADN viral 6. El fago / mezcla de bacterias se mueve entonces a la superficie de una losa delgada de agar nutriente, cubierto con un cubreobjetos y fotografiado bajo un microscopio de epifluorescencia. El proceso de post-infección se siguió durante 4 horas, a intervalos de 10 min. Múltiples posiciones de fase se realiza un seguimiento de tal manera que ~ 100 infecciones de células se puede rastrear en un solo experimento. En cada punto de posición y el tiempo, las imágenes se adquieren en el contraste de fase y canales fluorescentes rojas y verdes. La imagen de contraste de fase se utiliza posteriormente para la automatización cell reconocimiento, mientras que los canales fluorescentes se utilizan para caracterizar la infección por resultado: la producción de nuevos fagos fluorescente (verde) seguido por la lisis celular, o la expresión de los factores de lisogenia (rojo) seguido por el crecimiento celular y la división se reanuda. Los adquiridos time-lapse películas se procesan utilizando una combinación de métodos manuales y automatizados. Resultados Los datos de análisis en la identificación de los parámetros de infección para cada evento infección (por ejemplo, número y posiciones de infectar a los fagos), así como resultado la infección (lisis / lisogenia). Los parámetros adicionales se puede extraer si se desea.

Watch this Scientific Journal Video about Following Cell-fate in E. coli After Infection by Phage Lambda at JoVE.com

Following Cell-fate in E. coli After Infection by Phage Lambda

Este artículo describe el procedimiento para la preparación de una versión marcado fluorescentemente de bacteriófago lambda, la infección de<em&gt; E. coli</em&gt; Bacterias, tras el resultado infección bajo el microscopio, y el análisis de los resultados de la infección.

A raíz de la celda de destino en<em&gt; E. coli</em&gt; Después de la infección por fagos Lambda

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the ...

following-cell-fate-in-e-coli-after-infection-by-phage-lambda

Research

JoVE Journal

Immunology and Infection

Following Cell-fate in E. coli After Infection by Phage Lambda

23.2K vistas.  University of Illinois at Urbana-Champaign. Este artículo describe el procedimiento para la preparación de una versión marcado fluorescentemente de bacteriófago lambda, la infección de E. coli Bacterias, tras el resultado infección bajo el microscopio, y el análisis de los resultados de la infección.

Video: Following Cell-fate in E. coli After Infection by Phage Lambda

Monitoring Immune Cells Trafficking Fluorescent Prion Rods Hours after Intraperitoneal Infection

Presence of an abnormal form a host-encoded prion protein (PrPC) that is protease resistant, pathologic and infectious characterizes prion diseases such as Chronic Wasting Disease (CWD) of cervids and scrapie in sheep. The Prion hypothesis asserts that this abnormal conformer constitutes most or all of the infectious prion. The role of the immune system in early events in peripheral prion pathogenesis has been convincingly demonstrated for CWD and scrapie 1-3. Transgenic and pharmacologic studies in mice revealed an important role of the Complement system in retaining and replicating prions early after infection 4-6. In vitro and in vivo studies have also observed prion retention by dendritic cells 7-10, although their role in trafficking remains unclear 11-16. Macrophages have similarly been implicated in early prion pathogenesis, but these studies have focused on events occurring weeks after infection 3,11,17. These prior studies also suffer from the problem of differentiating between endogenous PrPC and infectious prions. Here we describe a semiquantitative, unbiased approach for assessing prion uptake and trafficking from the inoculation site by immune cells recruited there. Aggregated prion rods were purified from infected brain homogenate by detergent solubilization of non-aggregated proteins and ultracentrifugation through a sucrose cushion. Polyacrylamide gel electrophoresis, coomassie blue staining and western blotting confirmed recovery of highly enriched prion rods in the pelleted fraction. Prion rods were fluorochrome-labeled then injected intraperitoneally into mice. Two hours later immune cells from peritoneal lavage fluid, spleen and mediastinal and mesenteric lymph nodes were assayed for prion rod retention and cell subsets identified by multicolor flow cytometry using markers for monocytes, neutrophils, dendritic cells, macrophages and B and T cells. This assay allows for the first time direct monitoring of immune cells acquiring and trafficking prions in vivo within hours after infection. This assay also clearly differentiates infectious, aggregated prions from PrPC normally expressed on host cells, which can be difficult and lead to data interpretation problems in other assay systems. This protocol can be adapted to other inoculation routes (oral, intravenous, intranervous and subcutaneous, e.g.) and antigens (conjugated beads, bacterial, viral and parasitic pathogens and proteins, egg) as well.

Here we describe a novel assay for monitoring prion uptake and trafficking by immune cells immediately following intraperitoneal inoculation by purifying and fluorescently labeling aggregated prion rods from infected brain material then monitoring their uptake and movement from the injection site and characterizing the cells mediating these events.

Monitoreo células inmunes Tráfico Varillas fluorescente priones horas de la infección intraperitoneal

Presence of an abnormal form a host-encoded prion protein (PrPC) that is protease resistant, pathologic and infectious characterizes prion diseases such ...

Investigación

Inmunología e Infección

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

There is growing concern about the relevance of in vitro antimicrobial susceptibility tests when applied to isolates of P. aeruginosa from cystic fibrosis (CF) patients. Existing methods rely on single or a few isolates grown aerobically and planktonically. Predetermined cut-offs are used to define whether the bacteria are sensitive or resistant to any given antibiotic1. However, during chronic lung infections in CF, P. aeruginosa populations exist in biofilms and there is evidence that the environment is largely microaerophilic2. The stark difference in conditions between bacteria in the lung and those during diagnostic testing has called into question the reliability and even relevance of these tests3.
 Artificial sputum medium (ASM) is a culture medium containing the components of CF patient sputum, including amino acids, mucin and free DNA. P. aeruginosa growth in ASM mimics growth during CF infections, with the formation of self-aggregating biofilm structures and population divergence4,5,6. The aim of this study was to develop a microtitre-plate assay to study antimicrobial susceptibility of P. aeruginosa based on growth in ASM, which is applicable to both microaerophilic and aerobic conditions.
 An ASM assay was developed in a microtitre plate format. P. aeruginosa biofilms were allowed to develop for 3 days prior to incubation with antimicrobial agents at different concentrations for 24 hours. After biofilm disruption, cell viability was measured by staining with resazurin. This assay was used to ascertain the sessile cell minimum inhibitory concentration (SMIC) of tobramycin for 15 different P. aeruginosa isolates under aerobic and microaerophilic conditions and SMIC values were compared to those obtained with standard broth growth. Whilst there was some evidence for increased MIC values for isolates grown in ASM when compared to their planktonic counterparts, the biggest differences were found with bacteria tested in microaerophilic conditions, which showed a much increased resistance up to a &gt;128 fold, towards tobramycin in the ASM system when compared to assays carried out in aerobic conditions.
 The lack of association between current susceptibility testing methods and clinical outcome has questioned the validity of current methods3. Several in vitro models have been used previously to study P. aeruginosa biofilms7, 8. However, these methods rely on surface attached biofilms, whereas the ASM biofilms resemble those observed in the CF lung9 . In addition, reduced oxygen concentration in the mucus has been shown to alter the behavior of P. aeruginosa2 and affect antibiotic susceptibility10. Therefore using ASM under microaerophilic conditions may provide a more realistic environment in which to study antimicrobial susceptibility.

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

Current diagnostic antimicrobial susceptibility testing relies on the planktonic growth of isolates in nutrient rich, aerobic conditions. Here, we employ an alternative artificial sputum medium to study antimicrobial susceptibility of Pseudomonas aeruginosa biofilms under both aerobic and microaerophilic conditions more representative of the cystic fibrosis lung.

Uso de un medio artificial de esputo para probar la eficacia de antibióticos contra la<em&gt; Pseudomonas aeruginosa</em&gt; En condiciones más pertinentes a la fibrosis quística pulmonar

There  is growing concern about the relevance of in vitro antimicrobial  susceptibility tests when applied to isolates of P. aeruginosa from  cystic ...

Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

The investigation of the intracellular protein levels of bacterial species is of importance to understanding the pathogenic mechanisms of diseases caused by these organisms. Here we describe a procedure for protein extraction from Burkholderia species based on mechanical lysis using glass beads in the presence of ethylenediamine tetraacetic acid and phenylmethylsulfonyl fluoride in phosphate buffered saline. This method can be used for different Burkholderia species, for different growth conditions, and it is likely suitable for the use in proteomic studies of other bacteria. Following protein extraction, a two-dimensional (2-D) gel electrophoresis proteomic technique is described to study global changes in the proteomes of these organisms. This method consists of the separation of proteins according to their isoelectric point by isoelectric focusing in the first dimension, followed by separation on the basis of molecular weight by acrylamide gel electrophoresis in the second dimension. Visualization of separated proteins is carried out by silver staining.

Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

Members of the Burkholderia genus are pathogens of clinical importance. We describe a method for total bacterial protein extraction, using mechanical disruption, and 2-D gel electrophoresis for subsequent proteomic analysis.

Extracción de proteínas totales y 2-D Métodos gel de electroforesis para<em&gt; Burkholderia</em&gt; Especies

The investigation of  the intracellular protein levels of bacterial species is of importance to  understanding the pathogenic mechanisms of diseases ...

Scalable High Throughput Selection From  Phage-displayed Synthetic Antibody Libraries

The demand for antibodies that fulfill the needs of both basic and clinical research applications is high and will dramatically increase in the future. However, it is apparent that traditional monoclonal technologies are not alone up to this task. This has led to the development of alternate methods to satisfy the demand for high quality and renewable affinity reagents to all accessible elements of the proteome. Toward this end, high throughput methods for conducting selections from phage-displayed synthetic antibody libraries have been devised for applications involving diverse antigens and optimized for rapid throughput and success. Herein, a protocol is described in detail that illustrates with video demonstration the parallel selection of Fab-phage clones from high diversity libraries against hundreds of targets using either a manual 96 channel liquid handler or automated robotics system. Using this protocol, a single user can generate hundreds of antigens, select antibodies to them in parallel and validate antibody binding within 6-8 weeks. Highlighted are: i) a viable antigen format, ii) pre-selection antigen characterization, iii) critical steps that influence the selection of specific and high affinity clones, and iv) ways of monitoring selection effectiveness and early stage antibody clone characterization. With this approach, we have obtained synthetic antibody fragments (Fabs) to many target classes including single-pass membrane receptors, secreted protein hormones, and multi-domain intracellular proteins. These fragments are readily converted to full-length antibodies and have been validated to exhibit high affinity and specificity. Further, they have been demonstrated to be functional in a variety of standard immunoassays including Western blotting, ELISA, cellular immunofluorescence, immunoprecipitation and related assays. This methodology will accelerate antibody discovery and ultimately bring us closer to realizing the goal of generating renewable, high quality antibodies to the proteome.

A method is described with visual accompaniment for conducting scalable, high throughput selections from phage-displayed combinatorial synthetic antibody libraries against hundreds of antigens simultaneously. Using this parallel approach, we have isolated antibody fragments that exhibit high affinity and specificity for diverse antigens that are functional in standard immunoassays.

Escalable de alto rendimiento seleccion de bibliotecas de anticuerpos sintéticos fagos

The demand for antibodies that fulfill the needs of both basic and clinical research applications is high and will dramatically increase in the future. ...

Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat

Investigation of the interactions between animal host and bacterial pathogen is only meaningful if the infection model employed replicates the principal features of the natural infection. This protocol describes procedures for the establishment and evaluation of systemic infection due to neuropathogenic Escherichia coli K1 in the neonatal rat. Colonization of the gastrointestinal tract leads to dissemination of the pathogen along the gut-lymph-blood-brain course of infection and the model displays strong age dependency. A strain of E. coli O18:K1 with enhanced virulence for the neonatal rat produces exceptionally high rates of colonization, translocation to the blood compartment and invasion of the meninges following transit through the choroid plexus. As in the human host, penetration of the central nervous system is accompanied by local inflammation and an invariably lethal outcome. The model is of proven utility for studies of the mechanism of pathogenesis, for evaluation of therapeutic interventions and for assessment of bacterial virulence.

Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat

Here, a procedure is described for the establishment of systemic infection in the neonatal rat with cultures of Escherichia coli K1. This non-invasive procedure permits colonization of the gastrointestinal tract, translocation of the pathogen to the systemic circulation, and invasion of the central nervous system at the choroid plexus.

Modelo no invasiva de neuropatogénico<em&gt; Escherichia coli</em&gt; La infección en Neonatología Rata

Investigation of the interactions between animal host and bacterial pathogen is only meaningful if the infection model employed replicates the principal ...

Phage Phenomics: Physiological Approaches to Characterize Novel Viral Proteins

Current investigations into phage-host interactions are dependent on extrapolating knowledge from (meta)genomes. Interestingly, 60 - 95% of all phage sequences share no homology to current annotated proteins. As a result, a large proportion of phage genes are annotated as hypothetical. This reality heavily affects the annotation of both structural and auxiliary metabolic genes. Here we present phenomic methods designed to capture the physiological response(s) of a selected host during expression of one of these unknown phage genes. Multi-phenotype Assay Plates (MAPs) are used to monitor the diversity of host substrate utilization and subsequent biomass formation, while metabolomics provides bi-product analysis by monitoring metabolite abundance and diversity. Both tools are used simultaneously to provide a phenotypic profile associated with expression of a single putative phage open reading frame (ORF). Representative results for both methods are compared, highlighting the phenotypic profile differences of a host carrying either putative structural or metabolic phage genes. In addition, the visualization techniques and high throughput computational pipelines that facilitated experimental analysis are presented.

Here, we present phenomic approaches for the functional characterization of putative phage genes. Techniques include a developed assay capable of monitoring host anabolic metabolism, the Multi-phenotype Assay Plates (MAPs), in addition to the established method of metabolomics, capable of measuring effects to catabolic metabolism.

Fenómica fagos: enfoques fisiológicos para caracterizar Novela Virales Proteínas

Current investigations into phage-host interactions are dependent on extrapolating knowledge from (meta)genomes. Interestingly, 60 - 95% of all phage ...

Transformation of Probiotic Yeast and Their Recovery from Gastrointestinal Immune Tissues Following Oral Gavage in Mice

Development of recombinant oral therapy would allow for more direct targeting of the mucosal immune system and improve the ability to combat gastrointestinal disorders. Adapting probiotic yeast in particular for this approach carries several advantages. These strains have not only the potential to synthesize a wide variety of complex heterologous proteins but are also capable of surviving and protecting those proteins during transit through the intestine. Critically, however, this approach requires expertise in many diverse laboratory techniques not typically used in tandem. Furthermore, although individual protocols for yeast transformation are well characterized for commonly used laboratory strains, emphasis is placed here on alternative approaches and the importance of optimizing transformation for less well characterized probiotic strains. Detailing these methods will help facilitate discussion as to the best approaches for testing probiotic yeast as oral drug delivery vehicles and indeed serve to advance the development of this novel strategy for gastrointestinal therapy.

Presented here is a unified description of techniques that can be used to develop, transform, administer, and test heterologous protein expression of the probiotic yeast Saccharomyces boulardii.

Transformación de la levadura probiótica y su recuperación de tejidos gastrointestinales inmune después de alimentación forzada oral en ratones

Development of recombinant oral therapy would allow for more direct targeting of the mucosal immune system and improve the ability to combat ...

Foodborne Pathogen Screening Using Magneto-fluorescent Nanosensor: Rapid Detection of E. Coli O157:H7

Enterohemorrhagic Escherichia coli O157:H7 has been linked to both waterborne and foodborne illnesses, and remains a threat despite the food- and water-screening methods used currently. While conventional bacterial detection methods, such as polymerase chain reaction (PCR) and enzyme-linked immunosorbent assays (ELISA) can specifically detect pathogenic contaminants, they require extensive sample preparation and lengthy waiting periods. In addition, these practices demand sophisticated laboratory instruments and settings, and must be executed by trained professionals. Herein, a protocol is proposed for a simpler diagnostic technique that features the unique combination of magnetic and fluorescent parameters in a nanoparticle-based platform. The proposed multiparametric magneto-fluorescent nanosensors (MFnS) can detect E. coli O157:H7 contamination with as little as 1 colony-forming unit present in solution within less than 1 h. Furthermore, the ability of MFnS to remain highly functional in complex media such as milk and lake water has been verified. Additional specificity assays were also used to demonstrate the ability of MFnS to only detect the specific target bacteria, even in the presence of similar bacterial species. The pairing of magnetic and fluorescent modalities allows for the detection and quantification of pathogen contamination in a wide range of concentrations, exhibiting its high performance in both early- and late-stage contamination detection. The effectiveness, affordability, and portability of the MFnS make them an ideal candidate for point-of-care screening for bacterial contaminants in a wide range of settings, from aquatic reservoirs to commercially packaged foods.

Foodborne Pathogen Screening Using Magneto-fluorescent Nanosensor: Rapid Detection of E. Coli O157:H7

The overall goal of this protocol is to synthesize functional nanosensors for the portable, cost-effective, and rapid detection of specifically targeted pathogenic bacteria through a combination of magnetic relaxation and fluorescence emission modalities.

Patógenos de transmisión alimentaria proyección utilizando Magneto-fluorescente Nanosensor: Detección rápida de e. Coli O157: H7

Enterohemorrhagic Escherichia coli O157:H7 has been linked to both waterborne and foodborne illnesses, and remains a threat despite the food- and ...

Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System

Enterohemorrhagic E. coli (EHEC) O157:H7, which is a foodborne pathogen that causesdiarrhea, hemorrhagic colitis (HS), and hemolytic uremic syndrome (HUS), colonize to the intestinal tract of humans. To study the detailed mechanism of EHEC colonization in vivo, it is essential to have animal models to monitor and quantify EHEC colonization. We demonstrate here a mouse-EHEC colonization model by transforming the bioluminescent expressing plasmid to EHEC to monitor and quantify EHEC colonization in living hosts. Animals inoculated with bioluminescence-labeled EHEC show intense bioluminescent signals in mice by detection with a non-invasive in vivo imaging system. After 1 and 2 days post infection, bioluminescent signals could still be detected in infected animals, which suggests that EHEC colonize in hosts for at least 2 days. We also demonstrate that these bioluminescent EHEC locate to mouse intestine, specifically in the cecum and colon, from ex vivo images. This mouse-EHEC colonization model may serve as a tool to advance the current knowledge of the EHEC colonization mechanism.

Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System

A detailed protocol of a mouse model for enterohemorrhagic E. coli (EHEC) colonization by using bioluminescence-labeled bacteria is presented. The detection of these bioluminescent bacteria by a non-invasive in vivo imaging system in live animals can advance our current understanding of EHEC colonization.

Detección de la colonización de Escherichia Coli enterohemorrágica en anfitrión murino no invasiva In Vivo el sistema de bioluminiscencia

Enterohemorrhagic E. coli (EHEC) O157:H7, which is a foodborne pathogen that causesdiarrhea, hemorrhagic colitis (HS), and hemolytic uremic syndrome ...

Creation of a Dense Transposon Insertion Library Using Bacterial Conjugation in Enterobacterial Strains Such As Escherichia Coli or Shigella flexneri

Transposon mutagenesis is a method that allows gene disruption via the random genomic insertion of a piece of DNA called a transposon. The protocol below outlines a method for high efficiency transfer between bacterial strains of a plasmid harboring a transposon containing a kanamycin resistance marker. The plasmid-borne transposase is encoded by a variant tnp gene that inserts the transposon into the genome of the recipient strain with very low insertional bias. This method thus allows the creation of large mutant libraries in which transposons have been inserted into unique genomic positions in a recipient strain of either Escherichia coli or Shigella flexneri bacteria. By using bacterial conjugation, as opposed to other methods such as electroporation or chemical transformation, large libraries with hundreds of thousands of unique clones can be created. This yields high-density insertion libraries, with insertions occurring as frequently as every 4-6 base pairs in non-essential genes. This method is superior to other methods as it allows for an inexpensive, easy to use, and high efficiency method for the creation of a dense transposon insertion library. The transposon library can be used in downstream applications such as transposon sequencing (Tn-Seq), to infer genetic interaction networks, or more simply, in mutational (forward genetic) screens.

Creation of a Dense Transposon Insertion Library Using Bacterial Conjugation in Enterobacterial Strains Such As Escherichia Coli or Shigella flexneri

Presented here is a simple method for creating a high-density transposon insertion library in Escherichia coli or Shigella flexneri using bacterial conjugation. This protocol allows the creation of a collection of hundreds of thousands of unique mutants in bacteria by the random genomic insertion of a transposon.

Creación de un denso Transposon inserción biblioteca usando Conjugación bacteriana en cepas enterobacterianas tales como Escherichia Coli o Shigella flexneri

Transposon mutagenesis is a method that allows gene disruption via the random genomic insertion of a piece of DNA called a transposon. The protocol below ...