These experiments have been supported by research grants from Merck and Co. and Summit, PLC.

1. Aislar C. difficile de diferentes fuentes ambientales o clínicas <ol><li> Aislados medioambientales: Utilizando un calibre de algodón preesterilizado (ligeramente humedecido con NaCl al 0,85%), limpie con un hisopo la superficie de cualquier área de interés (piso, puerta, manija, estante, etc. ). 8 Asegúrese de usar guantes estériles y coloque el hisopo en un tubo esterilizado una vez completado. </li><li> Aislamientos clínicos (heces): Placa de 10 a 100 mg de muestras clínicas de heces en agar de cefoxitina-cicloserina-fructosa (CCFA) utilizando un bucle de inoculación e incubar bajo condiciones anaeróbicas estrictas durante 48 - 72 h. Almacenar colonias aisladas de C. difficile stock en cryovials a -80 ° C para análisis adicionales. 7 , 9 , 10 </li><li> Enriquecer las muestras de hisopo ambiental en infusión de corazón cerebral (BHI) caldo con taurocolato de sodio al 0,05% y colocar en una cámara anaeróbica a 37 ° Cdurante 5 días. Centrifugar 1 ml del cultivo a 10.000 xg y resuspender el sedimento en 100 μl de etanol. </li><li> Se colocan las células resuspendidas (50 μl) en placas de agar de fructosa de cicloserinecefoxitina (CCFA) e incuban en una cámara anaeróbica a 37 ° C durante 40-48 h. Almacenar colonias aisladas de C. difficile stock en cryovials a -80 ° C para análisis adicionales. </li><li> Pruebe las colonias sospechosas de C. difficile utilizando el reactivo de aglutinación de látex o PCR. </li></ol> 2. Cultivo de C. difficile y Killing Cinética Procedimientos <ol><li> Cultivar cepas de C. difficile contaminadas con el medio ambiente o clínicas en placas de agar sangre en una cámara anaeróbica a 37 ° C durante 48 h. </li><li> Tomar una colonia aislada con un bucle de inoculación, transferirla a 5 mL de medio BHI en un tubo de 15 mL, y crecer durante 24 h en una cámara anaeróbica a 37 ° C. </li><li> Diluir el precultivo de 1: 100 a aproximadamente 10 6 unidades de formación de colonias(CFU) / ml en BHIS pre-reducido fresco (BHI más 5 g / l de extracto de levadura y 1% de L-cisteína) suplementado con taurocolato de sodio al 0,1% y la concentración apropiada de antibiótico (T0). </li><li> Recoger una muestra de 1 mL con una pipeta en cada punto de tiempo (T0, T6, T24, T48) y extender una pequeña alícuota (100 μl en diluciones en serie) sobre una placa de agar sangre. Deje que las células crezcan en la placa de agar sangre durante 48 h en una cámara anaeróbica a 37 ° C y cuente el número resultante de colonias para determinar CFU. </li></ol> 3. Preparación de muestras para la microscopía electrónica de barrido <ol><li> Recoger 1 mL de células de cada punto de tiempo en tubos de microcentrífuga y centrifugar a 10.000 xg durante 10 min. Desechar el sobrenadante y lavar las células en PBS. </li><li> Centrifugar las muestras a 10.000 xg durante 10 minutos y descartar el sobrenadante. Reducir las células en 1 mL de paraformaldehído al 4% e incubar durante 1 h a temperatura ambiente. </li><li> Centrifugar nuevamente las muestras durante 10 min a 10,000 xg y desechar el sobrenadante. Lavar las células dos veces con agua destilada y rediluir en 100 μl de agua destilada. Ajuste el volumen dependiendo de la turbidez de la solución. NOTA: Hacer múltiples diluciones en serie es una buena idea. </li><li> Marque los cubreobjetos y añada 40 μl de la muestra. Incubar durante 15 minutos en una campana de flujo para evaporar el líquido y permitir que las células se adhieran a la cubreobjetos. Si todavía hay líquido, use un soplador para quitar el líquido. </li><li> Coloque los cubreobjetos marcados con las células en una máquina de pulverización de escritorio y cinta hacia abajo. Asegure el oro puro en la máquina de pulverización. Encienda la máquina y comience a pulverizar a baja presión (50 mTorr). Recubrir las células durante 30 s a 80 mA, lo que se traduce en 20 nm de recubrimiento de oro. </li><li> Transferencia de células revestidas al microscopio electrónico de barrido. </li></ol> 4. Imagen de células C. difficile en un microscopio electrónico de barrido <ol><li> Ventilar el microscopio electrónico de barrido (SEM) correctamente por prEl botón de ventilación en el software de la computadora. </li><li> Usando cinta de carbón, asegure los cubreobjetos recubiertos sobre la platina metálica. Una vez que el SEM se ventila, la puerta debe abrirse fácilmente. Bloquee la etapa de metal en la cámara de SEM atornillándola. </li><li> Haga clic en el botón BOMBA en el software del ordenador. El SEM será utilizable cuando el sistema lea &quot;Vac OK&quot;. </li><li> Haga clic en el detector de SE bajo la pestaña de detectores. Encienda la viga haciendo clic en el botón que muestra el voltaje. Comience la imagen a una tensión inferior (5 kV) antes de aumentar la tensión (hasta 15 kV). Una imagen aparecerá después de encender el haz. </li><li> Usando la función de rastreo, encuentre un área en los cubreobjetos recubiertos a la imagen. Zoom en la región y encontrar en forma de barras estructuras; Estos son clostridium difficile . </li><li> Acercar y enfocar la imagen para calibrar el sistema. Esto debe hacerse a múltiples distancias de trabajo: 15, 9 y 5 mm. Imagen a una distancia de trabajo de 5 mm. </li><li> Encienda el uLtra modo de imagen de alta resolución y comenzar a enfocar y optimizar el astigmatismo. </li><li> Comience a enfocar con una ampliación alta. Utilice los botones de enfoque grueso y fino para esto. Ajuste el astigmatismo también para obtener una imagen más clara. <ol><li> Ajustar el astigmatismo con gran aumento. Para ello, ajuste las teclas de astigmatismo (parecen similares a las de enfoque fino / grueso) y compruebe la claridad de la imagen haciendo zoom digitalmente en el software del ordenador. </li></ol></li><li> La función de escaneado lento para recoger una imagen de alta calidad. Guarde la imagen recogida como un archivo .TIFF, que se utilizará para el análisis. Asegúrese de que la barra de datos esté seleccionada si se realizarán mediciones durante el análisis. </li><li> Recoja las imágenes en ángulos diferentes (hasta 52 ° girando manualmente el ángulo directamente sobre el SEM.) Las imágenes angulares tienden a revelar más información de profundidad. </li><li> Cambie el voltaje del haz dependiendo de las celdas que se están formando imágenes. Mantenga esto principalmente entre 5 - 15 kV para todos los experimentos. </li&gt; <li> Una vez completada la imagen, apague el haz y eleve la distancia de trabajo hasta 20 mm. La cámara puede ser ventilada y la etapa puede ser removida. Copie todas las imágenes en una unidad para un análisis posterior. </li></ol> 5. Procesamiento y análisis de imágenes <ol><li> Descargue e instale el software libremente disponible, FIJI (http://fiji.sc), en la computadora. </li><li> Abra el archivo de imagen en FIJI. </li><li> Utilizando la función de línea, trace con precisión la barra de escala. </li><li> Haga clic en la pestaña Analizar en el programa FIJI y finalmente seleccione la función Seleccionar Escala. Aparecerá una ventana que requerirá el ajuste de la distancia conocida basada en la barra de escala. Cambie también la unidad de longitud y haga clic en Aceptar. </li><li> Ahora que el programa está calibrado para la distancia, utilice la función de línea para medir la longitud de la celda. Para obtener la longitud, utilice la función de línea para rastrear la célula en su totalidad. Seleccione de nuevo la pestaña Analizar y, a continuación, haga clic en Medir. La longitud debe aplicarOído en las unidades indicadas anteriormente. </li></ol>

<ol>
	<li>Lessa, F. C., 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=Burden+of+Clostridium+difficile+infection+in+the+United+States.">Burden of Clostridium difficile infection in the United States.</a> N Engl J Med. 372 (9), 825-834 (2015).</li><li>Vickers, R. 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=Ridinilazole:+a+novel+therapy+for+Clostridium+difficile+infection.">Ridinilazole: a novel therapy for Clostridium difficile infection.</a> Int J Antimicrob Agents. 48 (2), 137-143 (2016).</li><li>Shah, D., 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=Clostridium+difficile+infection:+update+on+emerging+antibiotic+treatment+options+and+antibiotic+resistance.">Clostridium difficile infection: update on emerging antibiotic treatment options and antibiotic resistance.</a> Expert Rev Anti Infect Ther. 8 (5), 555-564 (2010).</li><li>Ambrose, P. 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=New+EMA+guideline+for+antimicrobial+development.">New EMA guideline for antimicrobial development.</a> Lancet Infect Dis. 12 (4), 265-266 (2012).</li><li>Bass&#232;res, E., 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=Impact+on+toxin+production+and+cell+morphology+in+Clostridium+difficile+by+ridinilazole+(SMT19969),+a+novel+treatment+for+C.+difficile+infection.">Impact on toxin production and cell morphology in Clostridium difficile by ridinilazole (SMT19969), a novel treatment for C. difficile infection.</a> J Antimicrob Chemother. 71 (5), 1245-1251 (2016).</li><li>Endres, B. T., 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=A+novel+method+for+imaging+the+pharmacological+effects+of+antibiotic+treatment+on+Clostridium+difficile.">A novel method for imaging the pharmacological effects of antibiotic treatment on Clostridium difficile.</a> Anaerobe. 40, 10-14 (2016).</li><li>Endres, B. T., 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=Evaluating+the+Effects+of+Surotomycin+Treatment+on+Clostridium+difficile+Toxin+A+and+B+Production,+Immune+Response,+and+Morphological+Changes.">Evaluating the Effects of Surotomycin Treatment on Clostridium difficile Toxin A and B Production, Immune Response, and Morphological Changes.</a> Antimicrob Agents Chemother. 60 (6), 3519-3523 (2016).</li><li>Alam, M. J., Anu, A., Walk, S. T., Garey, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigation+of+potentially+pathogenic+Clostridium+difficile+contamination+in+household+environs.">Investigation of potentially pathogenic Clostridium difficile contamination in household environs.</a> Anaerobe. 27, 31-33 (2014).</li><li>Aitken, S. L., 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=In+the+Endemic+Setting,+Clostridium+difficile+Ribotype+027+Is+Virulent+But+Not+Hypervirulent.">In the Endemic Setting, Clostridium difficile Ribotype 027 Is Virulent But Not Hypervirulent.</a> Infect Control Hosp Epidemiol. , 1-6 (2015).</li><li>Basseres, E., 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=Impact+on+toxin+production+and+cell+morphology+in+Clostridium+difficile+by+ridinilazole+(SMT19969),+a+novel+treatment+for+C.+difficile+infection.">Impact on toxin production and cell morphology in Clostridium difficile by ridinilazole (SMT19969), a novel treatment for C. difficile infection.</a> J Antimicrob Chemother. 71 (5), 1245-1251 (2016).</li><li>Walters, B. A., Roberts, R., Stafford, R., Seneviratne, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relapse+of+antibiotic+associated+colitis:+endogenous+persistence+of+Clostridium+difficile+during+vancomycin+therapy.">Relapse of antibiotic associated colitis: endogenous persistence of Clostridium difficile during vancomycin therapy.</a> Gut. 24 (3), 206-212 (1983).</li><li>Chilton, C. 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C., 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=An+epidemic,+toxin+gene-variant+strain+of+Clostridium+difficile.">An epidemic, toxin gene-variant strain of Clostridium difficile.</a> New Eng J Med. 353 (23), 2433-2441 (2005).</li></ol>

KWG has received past and current research support from Merck &amp; Co. and Summit, PLC.

El objetivo del presente estudio fue crear un método de alto rendimiento para aislar C. difficile y probar la susceptibilidad a los antibióticos mediante microscopía electrónica de barrido (SEM) como medio para una caracterización más completa de la acción farmacológica del antibiótico. Usando los protocolos esbozados aquí, hemos demostrado que la obtención de imágenes de la respuesta fenotípica de la célula al tratamiento con antibióticos puede revelar una visión de la acción farmacológica de...

Clostridium difficile es una bacteria gram-positiva, formadora de esporas, causando aproximadamente 500,000 infecciones anualmente en los Estados Unidos y considerada por los Centros para el Control y la Prevención de Enfermedades (CDC) como el nivel más alto de riesgo. 1 En la última década se ha observado un considerable desarrollo de fármacos en antimicrobianos con actividad contra C. difficile . 2 , 3 Los estudios in vitro son un componente necesario del proceso de desarrollo del fármaco. Tradicionalmente, se usan estudios de susceptibilidad in vitro y matanza para validar futuros estudios en animales y otros in vivo . Aunque estos métodos cumplen un papel importante para evaluar la acción de matar, no capturan la respuesta fenotípica de las células al tratamiento farmacológico. Mediante la incorporación de microscopía electrónica de barrido (SEM) conD matando los estudios cinéticos, una caracterización más completa de los efectos directos antibióticos es posible. 5 , 6 , 7 Aquí, se presenta un método en el que SEM se utiliza como un medio para perfilar la eficacia del tratamiento con antibióticos.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>cotton gauze&#160;</td>
			<td>Caring</td>
			<td>PRM21408C</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Macron</td>
			<td>7532</td>
			<td></td>
		</tr>
		<tr>
			<td>50mL tubes</td>
			<td>Falcon</td>
			<td>352098</td>
			<td></td>
		</tr>
		<tr>
			<td>Brain Heart Infusion (BHI)&#160;</td>
			<td>Criterion</td>
			<td>C5141</td>
			<td></td>
		</tr>
		<tr>
			<td>L-cysteine</td>
			<td>Alfa Aesar</td>
			<td>A10389</td>
			<td></td>
		</tr>
		<tr>
			<td>yeast extract</td>
			<td>Criterion</td>
			<td>C741</td>
			<td></td>
		</tr>
		<tr>
			<td>sodium taurocholate</td>
			<td>Alfa Aesar</td>
			<td>A18346</td>
			<td></td>
		</tr>
		<tr>
			<td>anaerobic chamber</td>
			<td>Coy</td>
			<td></td>
			<td>vinyl anaerobic chamber</td>
		</tr>
		<tr>
			<td>cycloserinecefoxitin fructose agar (CCFA) plates</td>
			<td>Anaerobe systems</td>
			<td>AS-213</td>
			<td></td>
		</tr>
		<tr>
			<td>blood agar plates</td>
			<td>Hardy diagnostics</td>
			<td>A-10</td>
			<td></td>
		</tr>
		<tr>
			<td>latex agglutination reagent</td>
			<td>Oxoid</td>
			<td>DR1107A</td>
			<td>C. diff test kit</td>
		</tr>
		<tr>
			<td>microcentrifuge tubes</td>
			<td>Eppendorf</td>
			<td>222363204</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Gibco</td>
			<td>10010-031</td>
			<td></td>
		</tr>
		<tr>
			<td>4% paraformaldehyde</td>
			<td>Fisher Scientific</td>
			<td>50-259-98</td>
			<td></td>
		</tr>
		<tr>
			<td>microscope slides</td>
			<td>J. Melvin freed brand</td>
			<td>7525M</td>
			<td>75x25mm</td>
		</tr>
		<tr>
			<td>flow hood</td>
			<td>Labconco</td>
			<td>Class II type A2&#160;</td>
			<td>biosafety cabinet</td>
		</tr>
		<tr>
			<td>desk sputtering machine</td>
			<td>Denton Vacuum</td>
			<td>Desk II</td>
			<td></td>
		</tr>
		<tr>
			<td>tape</td>
			<td>Plastic Core</td>
			<td>05072-AB</td>
			<td>SPI Double Sided Adhesive Carbon Tape</td>
		</tr>
		<tr>
			<td>gold</td>
			<td>Denton Vacuum</td>
			<td>TAR001-0158</td>
			<td>2.375&#8221; Diameter x .002&#8221; Thick Gold foil</td>
		</tr>
		<tr>
			<td>scanning electron microscope</td>
			<td>FEI</td>
			<td>XL-30</td>
			<td></td>
		</tr>
	</tbody>
</table>

a protocol to characterize the morphological changes of clostridium difficile in response to antibiotic treatment

La evaluación de la acción antibiótica con el desarrollo de nuevos fármacos dirigidos hacia las bacterias anaerobias es difícil y técnicamente exigente. Para obtener información sobre el posible MOA, los cambios morfológicos asociados con la exposición a los antibióticos se pueden visualizar mediante microscopía electrónica de barrido (SEM). La integración de la imagen de SEM con las curvas de muerte tradicionales puede mejorar nuestra comprensión de la acción de la droga y avanzar en el proceso de desarrollo de fármacos. Para probar esta premisa, las curvas de muerte y los estudios de SEM se llevaron a cabo utilizando fármacos con MOA conocido pero diferente (vancomicina y metronidazol). Las células de C. difficile (R20291) se cultivaron con o sin la presencia de antibiótico hasta 48 h. Durante todo el intervalo de 48 h, las células se recogieron en múltiples puntos de tiempo para determinar la eficacia de los antibióticos y de imágenes en el SEM. De acuerdo con los informes anteriores, vancomicina y metronidazol tuvo una actividad bactericida significativa después de 24 h de tratamiento, medida por la unidad formadora de colonias (UFC)Ting Usando la imagen de SEM se determinó que el metronidazol tuvo efectos significativos sobre la longitud celular (&gt; 50% de reducción en la longitud de la célula para cada antibiótico, P &lt;0,05) en comparación con los controles y la vancomicina. Aunque la respuesta fenotípica al tratamiento farmacológico no se ha documentado previamente de esta manera, son consistentes con el MOA del fármaco demostrando la versatilidad y fiabilidad de la formación de imágenes y las mediciones y la aplicación de esta técnica para otros compuestos experimentales.

Clostridium difficile es una bacteria formadora de esporas y por lo tanto es esencial para determinar las diferencias morfológicas entre las células vegetativas y esporas antes de cualquier análisis funcional. La Figura 1 muestra imágenes representativas de células vegetativas que fueron capturadas durante la fase exponencial de la curva de crecimiento y las células de esporas. Como se muestra, las células vegetativas son largas, lisas, estructur...

Watch this Scientific Journal Video about A Protocol to Characterize the Morphological Changes of Clostridium difficile in Response to Antibiotic Treatment at JoVE.com

A Protocol to Characterize the Morphological Changes of Clostridium difficile in Response to Antibiotic Treatment

Antibiotic efficacy is most commonly determined by conducting killing kinetic studies and measuring colony forming units (CFUs). By integrating scanning electron microscopy (SEM) with these standard methods, we can distinguish the pharmacological effects of treatment between different antibiotics.

Protocolo para Caracterizar los Cambios Morfológicos de<em&gt; Clostridium difficile</em&gt; En respuesta al tratamiento antibiótico

Assessment of antibiotic action with new drug development directed towards anaerobic bacteria is difficult and technically demanding. To gain insight into ...

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Immunology and Infection

A Protocol to Characterize the Morphological Changes of Clostridium difficile in Response to Antibiotic Treatment

8.9K vistas.  University of Houston College of Pharmacy. Antibiotic efficacy is most commonly determined by conducting killing kinetic studies and measuring colony forming units (CFUs). By integrating scanning electron microscopy (SEM) with these standard methods, we can distinguish the pharmacological effects of treatment between different antibiotics.

Video: A Protocol to Characterize the Morphological Changes of Clostridium difficile in Response to Antibiotic Treatment

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 ...

Investigación

Inmunología e Infección

A Comparative Approach to Characterize the Landscape of Host-Pathogen Protein-Protein Interactions

Significant efforts were gathered to generate large-scale comprehensive protein-protein interaction network maps. This is instrumental to understand the pathogen-host relationships and was essentially performed by genetic screenings in yeast two-hybrid systems. The recent improvement of protein-protein interaction detection by a Gaussia luciferase-based fragment complementation assay now offers the opportunity to develop integrative comparative interactomic approaches necessary to rigorously compare interaction profiles of proteins from different pathogen strain variants against a common set of cellular factors.
This paper specifically focuses on the utility of combining two orthogonal methods to generate protein-protein interaction datasets: yeast two-hybrid (Y2H) and a new assay, high-throughput Gaussia princeps protein complementation assay (HT-GPCA) performed in mammalian cells.
A large-scale identification of cellular partners of a pathogen protein is performed by mating-based yeast two-hybrid screenings of cDNA libraries using multiple pathogen strain variants. A subset of interacting partners selected on a high-confidence statistical scoring is further validated in mammalian cells for pair-wise interactions with the whole set of pathogen variants proteins using HT-GPCA. This combination of two complementary methods improves the robustness of the interaction dataset, and allows the performance of a stringent comparative interaction analysis. Such comparative interactomics constitute a reliable and powerful strategy to decipher any pathogen-host interplays.

This article focuses on the identification of high-confident interaction datasets between host and pathogen proteins using a combination of two orthogonal methods: yeast two-hybrid followed by a high-throughput interaction assay in mammalian cells called HT-GPCA.

Un enfoque comparativo para caracterizar el paisaje del Huésped-Patógeno interacciones proteína-proteína

Significant efforts were gathered to generate  large-scale comprehensive protein-protein interaction network maps. This is  instrumental to understand the ...

Culturing and Maintaining Clostridium difficile in an Anaerobic Environment

Clostridium difficile is a Gram-positive, anaerobic, sporogenic bacterium that is primarily responsible for antibiotic associated diarrhea (AAD) and is a significant nosocomial pathogen. C. difficile is notoriously difficult to isolate and cultivate and is extremely sensitive to even low levels of oxygen in the environment. Here, methods for isolating C. difficile from fecal samples and subsequently culturing C. difficile for preparation of glycerol stocks for long-term storage are presented. Techniques for preparing and enumerating spore stocks in the laboratory for a variety of downstream applications including microscopy and animal studies are also described. These techniques necessitate an anaerobic chamber, which maintains a consistent anaerobic environment to ensure proper conditions for optimal C. difficile growth. We provide protocols for transferring materials in and out of the chamber without causing significant oxygen contamination along with suggestions for regular maintenance required to sustain the appropriate anaerobic environment for efficient and consistent C. difficile cultivation.

Culturing and Maintaining Clostridium difficile in an Anaerobic Environment

Clostridium difficile is a pathogenic bacterium that is a strict anaerobe and causes antibiotic associated diarrhea (AAD). Here, methods for isolating, culturing and maintaining C. difficile vegetative cells and spores are described. These techniques necessitate an anaerobic chamber, which requires regular maintenance to ensure proper conditions for optimal C. difficile cultivation.

El cultivo y mantenimiento<em&gt; Clostridium difficile</em&gt; En un ambiente anaeróbico

Clostridium  difficile is a Gram-positive, anaerobic, sporogenic bacterium  that is primarily responsible for antibiotic associated diarrhea (AAD) and is ...

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in mice. Recently, myeloid differentiation factor 88 (MyD88) signaling was shown to be important for initial T cell priming and memory T cell development during WNV NS4B-P38G mutant infection. In this study, two flow cytometry-based methods &#8211; an in vitro T cell priming assay and an intracellular cytokine staining (ICS) &#8211; were utilized to assess dendritic cells (DCs) and T cell functions. In the T cell priming assay, cell proliferation was analyzed by flow cytometry following co-culture of DCs from both groups of mice with carboxyfluorescein succinimidyl ester (CFSE) - labeled CD4+ T cells of OTII transgenic mice. This approach provided an accurate determination of the percentage of proliferating CD4+ T cells with significantly improved overall sensitivity than the traditional assays with radioactive reagents. A microcentrifuge tube system was used in both cell culture and cytokine staining procedures of the ICS protocol. Compared to the traditional tissue culture plate-based system, this modified procedure was easier to perform at biosafety level (BL) 3 facilities. Moreover, WNV- infected cells were treated with paraformaldehyde in both assays, which enabled further analysis outside BL3 facilities. Overall, these in vitro immunological assays can be used to efficiently assess cell-mediated immune responses during WNV infection.

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

Two flow cytometry-based methods &#8211; an in vitro T cell priming assay and intracellular cytokine staining were utilized to measure antigen presenting capacity of dendritic cells and antigen-specific T cell responses to a West Nile virus mutant infection in mice.

El análisis in vitro de la respuesta inmune celular Myd88 mediada por el Virus del Nilo Occidental cepa mutante Infección

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in ...

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 ...

Biomimetic Materials to Characterize Bacteria-host Interactions

Bacterial attachment to host cells is one of the earliest events during bacterial colonization of host tissues and thus a key step during infection. The biochemical and functional characterization of adhesins mediating these initial bacteria-host interactions is often compromised by the presence of other bacterial factors, such as cell wall components or secreted molecules, which interfere with the analysis. This protocol describes the production and use of biomimetic materials, consisting of pure recombinant adhesins chemically coupled to commercially available, functionalized polystyrene beads, which have been used successfully to dissect the biochemical and functional interactions between individual bacterial adhesins and host cell receptors. Protocols for different coupling chemistries, allowing directional immobilization of recombinant adhesins on polymer scaffolds, and for assessment of the coupling efficiency of the resulting &#8220;bacteriomimetic&#8221; materials are also discussed. We further describe how these materials can be used as a tool to inhibit pathogen mediated cytotoxicity and discuss scope, limitations and further applications of this approach in studying bacterial - host interactions.

Bacterial attachment to host cells is a key step during host colonization and infection. This protocol describes the generation of polymer-coupled recombinant adhesins as biomimetic materials which allow analysis of the contribution of individual adhesins to these processes, independent of other bacterial factors.

Materiales biomiméticos para caracterizar las interacciones bacteria-huésped

Bacterial attachment to host cells is one of the earliest events during bacterial colonization of host tissues and thus a key step during infection. The ...

Cefoperazone-treated Mouse Model of Clinically-relevant Clostridium difficile Strain R20291

Clostridium difficile is an anaerobic, gram-positive, spore-forming enteric pathogen that is associated with increasing morbidity and mortality and consequently poses an urgent threat to public health. Recurrence of a C. difficile infection (CDI) after successful treatment with antibiotics is high, occurring in 20-30% of patients, thus necessitating the discovery of novel therapeutics against this pathogen. Current animal models of CDI result in high mortality rates and thus do not approximate the chronic, insidious disease manifestations seen in humans with CDI. To evaluate therapeutics against C. difficile, a mouse model approximating human disease utilizing a clinically-relevant strain is needed. This protocol outlines the cefoperazone mouse model of CDI using a clinically-relevant and genetically-tractable strain, R20291. Techniques for clinical disease monitoring, C. difficile bacterial enumeration, toxin cytotoxicity, and histopathological changes throughout CDI in a mouse model are detailed in the protocol. Compared to other mouse models of CDI, this model is not uniformly lethal at the dose administered, allowing for the observation of a prolonged clinical course of infection concordant with the human disease. Therefore, this cefoperazone mouse model of CDI proves a valuable experimental platform to assess the effects of novel therapeutics on the amelioration of clinical disease and on the restoration of colonization resistance against C. difficile.

Cefoperazone-treated Mouse Model of Clinically-relevant Clostridium difficile Strain R20291

This protocol outlines the cefoperazone mouse model of Clostridium difficile infection (CDI) using a clinically-relevant and genetically-tractable strain, R20291. Emphasis on clinical disease monitoring, C. difficile bacterial enumeration, toxin cytotoxicity, and histopathological changes throughout CDI in a mouse model are detailed in the protocol.

Cefoperazona tratados modelo de ratón clínicamente relevante<em&gt; Clostridium difficile</em&gt; R20291 Strain

Clostridium difficile is an anaerobic, gram-positive, spore-forming enteric pathogen that is associated with increasing morbidity and mortality and ...

Examination of Host Phenotypes in Gambusia affinis Following Antibiotic Treatment

The commonality of antibiotic usage in medicine means that understanding the resulting consequences to the host is vital. Antibiotics often decrease host microbiome community diversity and alter the microbial community composition. Many diseases such as antibiotic-associated enterocolitis, inflammatory bowel disease, and metabolic disorders have been linked to a disrupted microbiota. The complex interplay between host, microbiome, and antibiotics needs a tractable model for studying host-microbiome interactions. Our freshwater vertebrate fish serves as a useful model for investigating the universal aspects of mucosal microbiome structure and function as well as analyzing consequential host effects from altering the microbial community. Methods include host challenges such as infection by a known fish pathogen, exposure to fecal or soil microbes, osmotic stress, nitrate toxicity, growth analysis, and measurement of gut motility. These techniques demonstrate a flexible and useful model system for rapid determination of host phenotypes.

Examination of Host Phenotypes in Gambusia affinis Following Antibiotic Treatment

This study involves methods to reveal effects on a model fish host following alteration of the skin and gut microbiome communities&#160;composition by an antibiotic.

El examen de los fenotipos de acogida en<em&gt; Gambusia affinis</em&gt; Tratamiento antibiótico Siguiendo

The commonality of antibiotic usage in medicine means that understanding the resulting consequences to the host is vital. Antibiotics often decrease host ...

Screening Assays to Characterize Novel Endothelial Regulators Involved in the Inflammatory Response

The endothelial layer is essential for maintaining homeostasis in the body by controlling many different functions. Regulation of the inflammatory response by the endothelial layer is crucial to efficiently fight against harmful inputs and aid in the recovery of damaged areas. When the endothelial cells are exposed to an inflammatory environment, such as the outer component of gram-negative bacteria membrane, lipopolysaccharide (LPS), they express soluble pro-inflammatory cytokines, such as Ccl5, Cxcl1 and Cxcl10, and trigger the activation of circulating leukocytes. In addition, the expression of adhesion molecules E-selectin, VCAM-1 and ICAM-1 on the endothelial surface enables the interaction and adhesion of the activated leukocytes to the endothelial layer, and eventually the extravasation towards the inflamed tissue. In this scenario, the endothelial function must be tightly regulated because excessive or defective activation in the leukocyte recruitment could lead to inflammatory-related disorders. Since many of these disorders do not have an effective treatment, novel strategies with a focus on the vascular layer must be investigated. We propose comprehensive assays that are useful to the search of novel endothelial regulators that modify leukocyte function. We analyze endothelial activation by using specific expression targets involved in leukocyte recruitment (such as, cytokines, chemokines, and adhesion molecules) with several techniques, including: real-time quantitative polymerase chain reaction (RT-qPCR), western-blot, flow cytometry and adhesion assays. These approaches determine endothelial function in the inflammatory context and are very useful to perform screening assays to characterize novel endothelial inflammatory regulators that are potentially valuable for designing new therapeutic strategies.

Vascular endothelium tightly controls leukocyte recruitment. Inadequate leukocyte extravasation contributes to human inflammatory diseases. Therefore, searching for novel regulatory elements of endothelial activation is necessary to design improved therapies for inflammatory disorders. Here, we describe a comprehensive methodology to characterize novel endothelial regulators that can modify leukocyte trafficking during inflammation.

Selección de ensayos para caracterizar nuevos reguladores endoteliales implicados en la respuesta inflamatoria

The endothelial layer is essential for maintaining homeostasis in the body by controlling many different functions. Regulation of the inflammatory ...

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple condensation reactions. DHDDS (dehydrodolichyl diphosphate synthase) is a eukaryotic long-chain cis-PT (forming cis double bonds from the condensation reaction) that catalyzes chain elongation of farnesyl diphosphate (FPP, an allylic diphosphate) via multiple condensations with isopentenyl diphosphate (IPP). DHDDS is of biomedical importance, as a non-conservative mutation (K42E) in the enzyme results in retinitis pigmentosa, ultimately leading to blindness. Therefore, the present protocol was developed in order to acquire large quantities of purified DHDDS, suitable for mechanistic studies. Here, the usage of protein fusion, optimized culture conditions and codon-optimization were used to allow the overexpression and purification of functionally active human DHDDS in E. coli. The described protocol is simple, cost-effective and time sparing. The homology of cis-PT among different species suggests that this protocol may be applied for other eukaryotic cis-PT as well, such as those involved in natural rubber synthesis.

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

A simple protocol for overexpression and purification of codon-optimized, human cis-prenyltransferase, under non-denaturing conditions, from Escherichia coli, is described, along with an enzymatic activity assay. This protocol can be generalized for production of other cis- prenyltransferase proteins in quantity and quality suitable for mechanistic studies.

Sobreexpresión y Purificación de Humanos<em&gt; Cis</em&gt; -preniltransferasa en<em&gt; Escherichia coli</em

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple ...

Protocolo para Caracterizar los Cambios Morfológicos de

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Capítulos en este video

Uso de un medio artificial de esputo para probar la eficacia de antibióticos contra la

Un enfoque comparativo para caracterizar el paisaje del Huésped-Patógeno interacciones proteína-proteína

El cultivo y mantenimiento

El análisis in vitro de la respuesta inmune celular Myd88 mediada por el Virus del Nilo Occidental cepa mutante Infección

Fenómica fagos: enfoques fisiológicos para caracterizar Novela Virales Proteínas

Materiales biomiméticos para caracterizar las interacciones bacteria-huésped

Cefoperazona tratados modelo de ratón clínicamente relevante

El examen de los fenotipos de acogida en

Selección de ensayos para caracterizar nuevos reguladores endoteliales implicados en la respuesta inflamatoria

Sobreexpresión y Purificación de Humanos