Los autores desean agradecer a la Fundación de Fibrosis Quística que proporcionó fondos para esta investigación y a MedImmune por su generosa donación de anticuerpos anti-Psl0096. Para este estudio, las imágenes se realizaron en el centro de imágenes CAMiLoD de la Universidad de Toronto.

Se requiere la aprobación de la Junta de Ética en Investigación (REB, por sus siglas en inglés) para recolectar y almacenar muestras de esputo de sujetos humanos. Los estudios presentados en este documento fueron aprobados por el Hospital para Niños Enfermos REB#1000058579.
1. Recolección de esputo
<ol><li>Almacenar el esputo expectorado en un recipiente de recogida estéril y conservar inmediatamente a 4 °C durante un máximo de 24 h antes de la fijación. NOTA: Dejar el esputo demasiado tiempo a 4 °C sin fijación puede provocar la degradación celular, en particular la degradación de los glóbulos blancos. Es preferible la fijación lo antes posible.</li>
	<li>Transfiera las muestras de esputo a un tubo estéril de 15 ml.</li>
	<li>Agregue un volumen igual de paraformaldehído (PFA) al 4% a las muestras de esputo. Por ejemplo, si la muestra de esputo es de 0,5 ml, agregue 0,5 ml de PFA al 4 %. Mezclar mediante una inversión suave.</li>
	<li>Incubar el esputo durante la noche a 4 °C.</li>
	<li>Lave la muestra añadiendo 5 ml de solución salina tamponada con fosfato (PBS) a cada 2 ml de esputo fijo. NOTA: No se requiere centrifugación para este paso de lavado.</li>
	<li>Retire con cuidado el sobrenadante con una pipeta. NOTA: Evite succionar el esputo con la pipeta apuntando la punta en dirección opuesta al esputo y aspire muy lentamente el líquido de la superficie. La etapa de lavado no implica centrifugación para no alterar la integridad estructural de los tapones de esputo.</li>
	<li>Repita los pasos 1.6 y 1.7 dos veces más.</li>
	<li>Vuelva a suspender el gránulo en 2 veces el volumen de PBS con azida sódica al 0,01 % (p/v).</li>
	<li>Almacenar el esputo a 4 °C.</li>
</ol>2. Procesamiento de esputo MiPACT (técnica de limpieza de tejidos)
<ol><li>Desgasificar los componentes del hidrogel (30% acrilamida 29:1:bis-acrilamida, endurecedor y PBS) en un recipiente sellado que contenga un paquete anaeróbico previamente durante 72 h. NOTA: El paquete anaeróbico y el recipiente sellado son necesarios porque el oxígeno inhibe la polimerización de la acrilamida. Alternativamente, el oxígeno se puede eliminar realizando este paso en una campana anaeróbica, aplicando un vacío a un recipiente sellado o burbujeando gas N2 a través de la mezcla de acrilamida.<ol><li>Agregue 2 ml de una solución de acrilamida:bis-acrilamida al 30% 29:1 a un tubo de 15 ml sin la tapa dentro del recipiente anaeróbico sellado.</li>
		<li>Haga una caldo concentrado de endurecedor al 10% (p/v) añadiendo 0,5 g de endurecedor a 5 ml de PBS en un tubo de 15 ml. Deje la tapa fuera del tubo y guárdela en el recipiente anaeróbico sellado.</li>
		<li>Deje unos pocos ml de PBS estéril en un tubo, sin tapa, dentro del recipiente anaeróbico.</li>
	</ol></li>
	<li>Preparar 5 ml de solución de hidrogel con una concentración final de endurecedor al 0,2 % y acrilamida 29:1 al 4 % en PBS en un tubo de 15 ml. Mezclar por inversión y esterilizar con filtro.</li>
	<li>Retire las muestras de esputo de la nevera y córtelas en pequeñas secciones (de aproximadamente 5 mm de diámetro) en condiciones estériles con un bisturí. NOTA: Si el esputo es bastante fluido, este paso aún se puede realizar, sin embargo, se necesita más paciencia y práctica para eliminar el esputo de la solución de almacenamiento con pinzas antes de usar el bisturí para separar las fracciones deseadas.</li>
	<li>Coloque las muestras de esputo cortadas dentro de un pocillo de un portaobjetos de cubreobjetos de 8 cámaras.</li>
	<li>Añadir 300 μL de solución de hidrogel esterilizada con filtro del paso 2.2 a cada uno de los pocillos que contengan esputo.</li>
	<li>Coloque el cubreobjetos de 8 cámaras dentro de un recipiente sellado que contenga un paquete anaeróbico durante 3 h a 37 °C. NOTA: Una vez polimerizado, el hidrogel con esputo incrustado debe tener la consistencia de un gel firme.</li>
	<li>Transfiera las muestras de esputo de hidrogel solidificado a un tubo de cultivo de 15 ml que contenga 5 ml de dodecil sulfato de sodio (SDS) al 8 %, pH 8, y deje que las muestras se aclaren durante 3-14 días a 37 °C (con o sin agitación), hasta que el esputo se vuelva transparente. NOTA: Los tiempos de aclaramiento dependen de la composición del esputo y, por lo general, pueden disminuirse con agitación. Sin embargo, tenga cuidado de no aumentar la velocidad de agitación hasta el punto de interrumpir la integridad de la muestra. Las muestras más ricas en ADN tardan más en aclararse en comparación con las muestras ricas en moco.</li>
	<li>Decantar la solución SDS al 8% en un contenedor de recogida de residuos. Con pinzas estériles, transfiera cada muestra incluida en hidrogel a un tubo cónico estéril de 50 ml. Agregue 10 ml de PBS a cada uno de los tubos cónicos de 50 ml para lavar los hidrogeles y deje reposar la solución durante 30 minutos a 1 hora antes de decantar. Repita esto 2 veces más. NOTA: Este paso de lavado no implica centrifugación. El exceso de líquido y sobrenadante se elimina cuidadosamente con una pipeta.</li>
	<li>Almacenar las muestras lavadas en PBS con azida sódica al 0,01% (p/v) y 1x inhibidor de la RNasa a 4 °C.</li>
</ol>3. Protocolo de hibridación in situ fluorescente de hidrogel (FISH)
<ol><li>Retire las muestras de hidrogel de su solución de almacenamiento con pinzas estériles y coloque las muestras sobre una superficie estéril (como un portaobjetos de vidrio o una placa de Petri).</li>
	<li>Con un bisturí estéril, corte los hidrogeles en rodajas de ~ 1 mm de grosor.</li>
	<li>Coloque las secciones de hidrogel de 1 mm dentro de tubos estériles de 1,5 ml.</li>
	<li>Preparar 1 mL del tampón de hibridación (25% formamida, 0,9 M NaCl, 20 mM Tris-HCl [pH 7,6], 0,01% SDS, H2O purificado y desionizado) en un tubo de 1,5 mL.</li>
	<li>Agregue la sonda PseaerA marcada con fluorescencia (150.7 nM12) al tampón de hibridación y mezcle por inversión. NOTA: La solución de formalina debe mantenerse alejada de una llama abierta. El tampón de hibridación puede prepararse en avanzado y almacenarse en alícuotas a -20 °C13.</li>
	<li>Agregue 200-500 μL de tampón de hibridación a cada sección de ~ 1 mm de hidrogel y asegúrese de que la totalidad de la muestra de hidrogel esté sumergida.</li>
	<li>Deje que la sonda PseaerA se hibrida con las muestras de hidrogel colocándolas en la oscuridad durante ~ 18-24 h, a 46 °C, sin agitar.</li>
	<li>Decantar el tampón de hibridación en un contenedor de recogida de residuos.</li>
	<li>Enjuague las muestras una vez con tampón de lavado esterilizado por filtro (337,5 mM de NaCl, 20 mM de Tris-HCl, 5 mM de EDTA (ácido etilendiaminotetraacético) [pH 7,2], 0,01 % de SDS y H2O purificado y desionizado) añadiendo 1 ml de tampón de lavado a cada uno de los tubos de 1,5 ml y luego retírelo. NOTA: El tampón de lavado se puede hacer con anticipación y almacenar a temperatura ambiente (RT).</li>
	<li>Añadir 1 ml de tampón de lavado fresco a los tubos, luego incubar las muestras en la oscuridad durante 6 h a 48 °C, sin agitar.</li>
</ol>4. Unión de hidrogel y anticuerpos Psl0096
<ol><li>Retire estérilmente el tampón de lavado con una pipeta de 1 ml.</li>
	<li>Enjuague las muestras con un BSA al 2% (p/v) en una solución de PBS agregando y quitando 1 ml de la solución de BSA al 2%.</li>
	<li>Añadir 500 μL de la solución BSA/PBS al 2% a las muestras de hidrogel para bloquear la unión a proteínas no específicas. A continuación, incubar las muestras durante la noche, en la oscuridad, a RT, sin agitar.</li>
	<li>Retire estérilmente la solución de bloqueo con una pipeta de 1 ml.</li>
	<li>Prepare la solución de anticuerpos Psl0096-Texas Red diluyendo el anticuerpo hasta una concentración final de 0,112 μg/ml en 500 μl de BSA/PBS fresco al 2%.</li>
	<li>Añadir la solución de anticuerpos de 500 μL a las muestras de hidrogel e incubarlas a RT durante 6 h, protegidas de la luz, sin agitar.</li>
</ol>5. Tinción con DAPI (4′,6′-diamidino-2-fenilindol)
<ol><li>Prepare la solución de adaptación del índice de refracción (RIMS) añadiendo 40 g de medio de gradiente de densidad no iónico, 30 μl de Tween20, 3 μg de azida sódica y 30 ml de PBS a un matraz que contenga una barra de agitación magnética. Revuelva la solución durante 15 minutos en un agitador magnético o hasta que se disuelva por completo.</li>
	<li>Filtrar: esterilizar la solución en un tubo cónico de 50 ml con una jeringa de 10 ml y un filtro estéril de 0,2 μm. NOTA: La solución puede almacenarse a 4 °C durante varios meses.</li>
	<li>Retire la solución de anticuerpos Psl0096-Texas Red con una pipeta estéril de 1 ml.</li>
	<li>Enjuague las muestras de hidrogel agregando y luego retirando 1 ml de PBS.</li>
	<li>Incubar las muestras de hidrogel con 250 μL de solución RIMS y 10 μg/mL de DAPI a RT con agitación suave, en la oscuridad, durante la noche.</li>
	<li>Antes de la obtención de imágenes confocal, monte las muestras en cámaras de perfusión de 0,9 mm o 1,7 mm y séllelas con un cubreobjetos de vidrio. NOTA: Después de la FISH y/o inmunohistoquímica y antes de la inmersión en RIMS, se pueden aplicar tinciones de lectina fluorescente si se desea la visualización de la mucosa del esputo12.</li>
</ol>6. Imágenes
<ol><li>Realice imágenes de microscopía de escaneo láser confocal utilizando técnicas estándar con aumentos de 25x, 40x, 63x o 100x.</li>
</ol>

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El propósito de este protocolo es permitir vislumbrar la organización in situ de las células de P. aeruginosa en el esputo de pacientes con FQ. Las muestras de esputo deben almacenarse a 4 °C hasta que se procesen si no pueden fijarse inmediatamente. Se ha demostrado que el número de células de P. aeruginosa en el esputo no cambia significativamente si se procesa a 1 h, 24 h o 48 h, cuando se almacena a 4 °C, aunque si se deja a 25 °C durante 24 o 48 h, los recuentos de células bacterianas aume...

En este estudio, se diseñaron experimentos para visualizar la estructura in vivo de Pseudomonas aeruginosa dentro del esputo de pacientes pediátricos con fibrosis quística (FQ). Las infecciones por P. aeruginosa se vuelven crónicas en el 30-40% de la población pediátrica con FQ; Una vez que las infecciones crónicas se establecen, son casi imposibles de eliminar1. Los aislados de P. aeruginosa de pacientes con infección temprana son generalmente más susceptibles a los antimicrobianos, por lo tanto, estos se tratan con antibióticos antipseudomonales para prevenir el establecimiento de infección crónica2. Desafortunadamente, no todos los aislados de P. aeruginosa se eliminan eficazmente del pulmón después de la terapia con antibióticos. Los mecanismos precisos asociados con el fracaso de los antibióticos no se han dilucidado por completo. Estudios previos han demostrado que las variaciones en la densidad celular de la biopelícula, la agregación y la producción de polisacáridos puedenafectar la eficacia de los antibióticos. P. aeruginosa produce tres polisacáridos extracelulares: Pel, Psl y alginato4. La mayoría de las cepas de P. aeruginosa tienen la capacidad genética de expresar cada uno de los exopolisacáridos, aunque a menudo un tipo de polisacárido se expresa predominantemente5. El alginato de exopolisacárido se asocia con infecciones crónicas en el pulmón de FQ, lo que resulta en un fenotipo mucoide 6,7. Los polisacáridos Pel y Psl tienen múltiples funciones, entre ellas ayudar a la unión inicial y al mantenimiento de la estructura de la biopelícula, y conferir resistencia a los antibióticos8.
Se han desarrollado métodos dirigidos a visualizar in vivo las estructuras de los tejidos para una variedad de tipos de muestras 9,10,11. Más recientemente, se han adaptado para visualizar las comunidades microbianas in vivo dentro del esputo de pacientes con FQ12. La optimización de un protocolo de limpieza de tejidos específicamente para la identificación de comunidades microbianas dentro del esputo fue desarrollada por DePas et al., 201612. El término MiPACT, que significa identificación microbial idespués de la tecnología de la LERA de Pa sive,se acuñó para la eliminación del esputo de FQ 11,12. En el caso de las técnicas de limpieza de tejidos, las muestras se fijan primero y luego se vuelven transparentes, dejando intacta su arquitectura inherente para la tinción y la visualización microscópica11. La fijación y eliminación de muestras de esputo de FQ permite a los investigadores responder preguntas relacionadas con la estructura de la biopelícula, la densidad celular bacteriana, las asociaciones polimicrobianas y las asociaciones entre patógenos y células huésped. La ventaja de examinar directamente las bacterias que se han conservado dentro del esputo es que pueden analizarse y visualizarse en un contexto específico del huésped. Aunque el crecimiento in vitro de aislados clínicos en el laboratorio para la experimentación puede ser muy informativo, estos métodos no pueden recrear completamente el entorno pulmonar de la FQ, lo que resulta en una desconexión entre los resultados de laboratorio y los resultados de los pacientes.
Los métodos presentados aquí se pueden utilizar para fijar y eliminar el esputo para visualizar las bacterias, ya sea de pacientes con FQ o pacientes con otras infecciones respiratorias. El tipo específico de tinción y análisis microscópico descrito en este documento es la hibridación fluorescente in situ (FISH), seguida de la unión anti-Psl-anticuerpo dentro del hidrogel y el análisis posterior mediante microscopía de barrido láser confocal (CLSM). Después de la limpieza de tejidos, también se pueden aplicar otros métodos de inmunohistoquímica y microscopía.

<table>
	<tbody>
		<tr>
			<td>29:1 acrylamide bisacrylamide, 30 % solution</td>
			<td>BioRad</td>
			<td>161-0146</td>
			<td></td>
		</tr>
		<tr>
			<td>8-Chambered Coverglass Nunc Lab-Tek</td>
			<td>ThermoFischer Scientific</td>
			<td>155411</td>
			<td></td>
		</tr>
		<tr>
			<td>Anaerogen2.5L</td>
			<td>Oxid Inc.</td>
			<td>35108</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverwell perfusion chambers</td>
			<td>Electron Microscopry Sciences</td>
			<td>70326 -12/-14</td>
			<td></td>
		</tr>
		<tr>
			<td>HistoDenz</td>
			<td>Sigma</td>
			<td>D2158</td>
			<td></td>
		</tr>
		<tr>
			<td>Protect RNA Rnase Inhibitor</td>
			<td>Sigma</td>
			<td>R7387</td>
			<td></td>
		</tr>
		<tr>
			<td>PseaerA - GGTAACCGTCCCCCTTGC</td>
			<td>Eurofins</td>
			<td></td>
			<td>Order Details: Product: Modified DNA Oligo; Name: PseaerA; Sequence: [Alexa488]GGTAACCGTCCCCCTTGC; Synthesis: 50 nmol; Purification: HPLC; Ship state: Full yield (dry)</td>
		</tr>
		<tr>
			<td>Psl0096-Texas Red</td>
			<td>Medimmune</td>
			<td></td>
			<td>The Psl0096-Texas red antibodies were a gift kindly provided by Medimmune and the company should be contacted for order inquiries.</td>
		</tr>
		<tr>
			<td>VA-044 Hardener</td>
			<td>Wako</td>
			<td>27776-21-21</td>
			<td></td>
		</tr>
	</tbody>
</table>

visualization of pseudomonas aeruginosa within the sputum of cystic fibrosis patients

La detección temprana y la erradicación de Pseudomonas aeruginosa en los pulmones de los pacientes con fibrosis quística pueden reducir la posibilidad de desarrollar una infección crónica. El desarrollo de infecciones crónicas por P. aeruginosa se asocia con una disminución de la función pulmonar y un aumento de la morbilidad. Por lo tanto, existe un gran interés en dilucidar las razones del fracaso en la erradicación de P. aeruginosa con terapia antibiótica que ocurre en aproximadamente el 10-40% de los pacientes pediátricos. Uno de los muchos factores que pueden afectar la eliminación del huésped de P. aeruginosa y la susceptibilidad a los antibióticos son las variaciones en la organización espacial (como la agregación o la formación de biopelículas) y la producción de polisacáridos. Por lo tanto, nos interesaba visualizar las características in situ de P. aeruginosa en el esputo de pacientes con FQ. Se aplicó una técnica de limpieza de tejidos a las muestras de esputo después de incrustar las muestras en una matriz de hidrogel para retener las estructuras 3D relativas a las células huésped. Después de la limpieza del tejido, se agregaron etiquetas fluorescentes y colorantes para permitir la visualización. Se realizó hibridación fluorescente in situ para la visualización de células bacterianas, unión de anticuerpos anti-Psl marcados con fluorescencia para la visualización del exopolisacárido y tinción de DAPI para teñir las células huésped para obtener información estructural. Estos métodos permitieron obtener imágenes de alta resolución de P. aeruginosa dentro del esputo de pacientes con FQ mediante microscopía de barrido láser confocal.

El diseño general del experimento se resume en la Figura 1 y la Figura 2. La Figura 1 proporciona un resumen de los protocolos de procesamiento y eliminación del esputo. El procesamiento y la eliminación del esputo pueden tardar hasta 17 días. Sin embargo, el protocolo puede detenerse y las muestras se pueden almacenar después de la fijación con PFA (día 2) o después de la limpieza del tejido (días 5-17 dependiendo del tiem...

Watch this Scientific Journal Video about Visualization of Pseudomonas aeruginosa within the Sputum of Cystic Fibrosis Patients at JoVE.com

Visualization of Pseudomonas aeruginosa within the Sputum of Cystic Fibrosis Patients

Este protocolo proporciona métodos para la visualización de células bacterianas y polisacáridos del locus de síntesis de polisacáridos (Psl) dentro del esputo de pacientes con fibrosis quística.

Visualización de Pseudomonas aeruginosa en el esputo de pacientes con fibrosis quística

Early detection and eradication of Pseudomonas aeruginosa within the lungs of cystic fibrosis patients can reduce the chance of developing chronic ...

visualization-pseudomonas-aeruginosa-within-sputum-cystic-fibrosis

Research

JoVE Journal

Immunology and Infection

Visualization of Pseudomonas aeruginosa within the Sputum of Cystic Fibrosis Patients

2.9K vistas.  Hospital for Sick Children. Este protocolo proporciona métodos para la visualización de células bacterianas y polisacáridos del locus de síntesis de polisacáridos (Psl) dentro del esputo de pacientes con fibrosis quística.

Video: Visualization of Pseudomonas aeruginosa within the Sputum of Cystic Fibrosis Patients

Co-culture Models of Pseudomonas aeruginosa Biofilms Grown on Live Human Airway Cells

Bacterial biofilms have been associated with a number of different human diseases, but biofilm development has generally been studied on non-living surfaces. In this paper, we describe protocols for forming Pseudomonas aeruginosa biofilms on human airway epithelial cells (CFBE cells) grown in culture. In the first method (termed the Static Co-culture Biofilm Model), P. aeruginosa is incubated with CFBE cells grown as confluent monolayers on standard tissue culture plates. Although the bacterium is quite toxic to epithelial cells, the addition of arginine delays the destruction of the monolayer long enough for biofilms to form on the CFBE cells. The second method (termed the Flow Cell Co-culture Biofilm Model), involves adaptation of a biofilm flow cell apparatus, which is often used in biofilm research, to accommodate a glass coverslip supporting a confluent monolayer of CFBE cells. This monolayer is inoculated with P. aeruginosa and a peristaltic pump then flows fresh medium across the cells. In both systems, bacterial biofilms form within 6-8 hours after inoculation. Visualization of the biofilm is enhanced by the use of P. aeruginosa strains constitutively expressing green fluorescent protein (GFP). The Static and Flow Cell Co-culture Biofilm assays are model systems for early P. aeruginosa infection of the Cystic Fibrosis (CF) lung, and these techniques allow different aspects of P. aeruginosa biofilm formation and virulence to be studied, including biofilm cytotoxicity, measurement of biofilm CFU, and staining and visualizing the biofilm.

Co-culture Models of Pseudomonas aeruginosa Biofilms Grown on Live Human Airway Cells

This paper describes different methods of growing Pseudomonas aeruginosa biofilms on cultured human airway epithelial cells. These protocols can be adapted to study different aspects of biofilm formation, including visualization of the biofilm, staining of the biofilm, measuring the colony forming units (CFU) of the biofilm, and studying biofilm cytotoxicity.

Los modelos de co-cultivo de Pseudomonas aeruginosa Biofilms Cultivado en las células vivas las vías respiratorias humanas

Bacterial biofilms have been associated with a number of different human diseases, but biofilm development has generally been studied on non-living ...

Investigación

Inmunología e Infección

Pseudomonas aeruginosa and Saccharomyces cerevisiae Biofilm in Flow Cells

Many microbial cells have the ability to form sessile microbial communities defined as biofilms that have altered physiological and pathological properties compared to free living microorganisms. Biofilms in nature are often difficult to investigate and reside under poorly defined conditions1. Using a transparent substratum it is possible to device a system where simple biofilms can be examined in a non-destructive way in real-time: here we demonstrate the assembly and operation of a flow cell model system, for in vitro 3D studies of microbial biofilms generating high reproducibility under well-defined conditions2,3.
The system consists of a flow cell that serves as growth chamber for the biofilm. The flow cell is supplied with nutrients and oxygen from a medium flask via a peristaltic pump and spent medium is collected in a waste container. This construction of the flow system allows a continuous supply of nutrients and administration of e.g. antibiotics with minimal disturbance of the cells grown in the flow chamber. Moreover, the flow conditions within the flow cell allow studies of biofilm exposed to shear stress. A bubble trapping device confines air bubbles from the tubing which otherwise could disrupt the biofilm structure in the flow cell.
The flow cell system is compatible with Confocal Laser Scanning Microscopy (CLSM) and can thereby provide highly detailed 3D information about developing microbial biofilms. Cells in the biofilm can be labeled with fluorescent probes or proteins compatible with CLSM analysis. This enables online visualization and allows investigation of niches in the developing biofilm. Microbial interrelationship, investigation of antimicrobial agents or the expression of specific genes, are of the many experimental setups that can be investigated in the flow cell system.

Pseudomonas aeruginosa and Saccharomyces cerevisiae Biofilm in Flow Cells

Protocol describing the application of a flow cell system for growing and analyzing microbial biofilms for Confocal Laser Scanning Microscopy (CLSM).

Pseudomonas aeruginosa y Biofilm Saccharomyces cerevisiae en las celdas de flujo

Many microbial cells have the ability to form sessile microbial communities defined as biofilms that have altered physiological and pathological ...

RNA Isolation of Pseudomonas aeruginosa Colonizing the Murine Gastrointestinal Tract

Pseudomonas aeruginosa (PA) infections result in significant morbidity and mortality in hosts with compromised immune systems, such as patients with leukemia, severe burn wounds, or organ transplants1. In patients at high-risk for developing PA bloodstream infections, the gastrointestinal (GI) tract is the main reservoir for colonization2, but the mechanisms by which PA transitions from an asymptomatic colonizing microbe to an invasive, and often deadly, pathogen are unclear. Previously, we performed in vivo transcription profiling experiments by recovering PA mRNA from bacterial cells residing in the cecums of colonized mice 3 in order to identify changes in bacterial gene expression during alterations to the host&#x2019;s immune status.
As with any transcription profiling experiment, the rate-limiting step is in the isolation of sufficient amounts of high quality mRNA. Given the abundance of enzymes, debris, food residues, and particulate matter in the GI tract, the challenge of RNA isolation is daunting. Here, we present a method for reliable and reproducible isolation of bacterial RNA recovered from the murine GI tract. This method utilizes a well-established murine model of PA GI colonization and neutropenia-induced dissemination4. Once GI colonization with PA is confirmed, mice are euthanized and cecal contents are recovered and flash frozen. RNA is then extracted using a combination of mechanical disruption, boiling, phenol/chloroform extractions, DNase treatment, and affinity chromatography. Quantity and purity are confirmed by spectrophotometry (Nanodrop Technologies) and bioanalyzer (Agilent Technologies) (Fig 1). This method of GI microbial RNA isolation can easily be adapted to other bacteria and fungi as well.

RNA Isolation of Pseudomonas aeruginosa Colonizing the Murine Gastrointestinal Tract

A reliable method for the RNA isolation of Pseudomonas aeruginosa recovered from murine cecums is described. The RNA recovered is of sufficient quantity and quality for subsequent qPCR, transcription profiling, and RNA Seq experiments. This technique can be adapted for RNA isolation of other intestinal microbes.

El aislamiento de ARN Pseudomonas aeruginosa Que colonizan el tracto gastrointestinal murino

Pseudomonas aeruginosa (PA) infections result in significant morbidity and mortality in hosts with compromised immune systems, such as patients with ...

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 ...

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model itself. Early phases of inflammation and infection have been widely studied by using the Pseudomonas aeruginosa agar-beads mouse model, while only few reports have focused on the long-term chronic infection in vivo. The main challenge for long term chronic infection remains the low bacterial burden by P. aeruginosa and the low percentage of infected mice weeks after challenge, indicating that bacterial cells are progressively cleared by the host.
This paper presents a method for obtaining efficient long-term chronic infection in mice. This method is based on the embedding of the P. aeruginosa clinical strains in the agar-beads in vitro, followed by intratracheal instillation in C57Bl/6NCrl mice. Bilateral lung infection is associated with several measurable read-outs including weight loss, mortality, chronic infection, and inflammatory response. The P. aeruginosa RP73 clinical strain was preferred over the PAO1 reference laboratory strain since it resulted in a comparatively lower mortality, more severe lesions, and higher chronic infection. P. aeruginosa colonization may persist in the lung for over three months. Murine lung pathology resembles that of CF patients with advanced chronic pulmonary disease.
This murine model most closely mimics the course of the human disease and can be used both for studies on the pathogenesis and for the evaluation of novel therapies.

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

We describe the agar-beads method to establish persistent long-term chronic Pseudomonas aeruginosa airway infection in the mouse model.

Crónica a largo plazo<em&gt; Pseudomonas aeruginosa</em&gt; Infección de las vías respiratorias en los ratones

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model ...

Pseudomonas aeruginosa Induced Lung Injury Model

In order to study human acute lung injury and pneumonia, it is important to develop animal models to mimic various pathological features of this disease. Here we have developed a mouse lung injury model by intra-tracheal injection of bacteria Pseudomonas aeruginosa (P. aeruginosa or PA). Using this model, we were able to show lung inflammation at the early phase of injury. In addition, alveolar epithelial barrier leakiness was observed by analyzing bronchoalveolar lavage (BAL); and alveolar cell death was observed by Tunel assay using tissue prepared from injured lungs. At a later phase following injury, we observed cell proliferation required for the repair process. The injury was resolved 7 days from the initiation of P. aeruginosa injection. This model mimics the sequential course of lung inflammation, injury and repair during pneumonia. This clinically relevant animal model is suitable for studying pathology, mechanism of repair, following acute lung injury, and also can be used to test potential therapeutic agents for this disease.

Pseudomonas aeruginosa Induced Lung Injury Model

We have developed a mouse lung injury model by intra-tracheal injection of bacteria Pseudomonas aeruginosa. This model mimics lung injury during pneumonia and is clinically relevant.

Pseudomonas aeruginosa inducida por la lesión pulmonar Modelo

In order to study human acute lung injury and pneumonia, it is important to develop animal models to mimic various pathological features of this disease. ...

An IL-8 Transiently Transgenized Mouse Model for the In Vivo Long-term Monitoring of Inflammatory Responses

Airway inflammation is often associated with bacterial infections and represents a major determinant of lung disease. The in vivo determination of the pro-inflammatory capabilities of various factors is challenging and requires terminal procedures, such as bronchoalveolar lavage and the removal of lungs for in situ analysis, precluding longitudinal visualization in the same mouse. Here, lung inflammation is induced through the intratracheal instillation of Pseudomonas aeruginosa culture supernatant (SN) in transiently transgenized mice expressing the luciferase reporter gene under the control of a heterologous IL-8 bovine promoter. Luciferase expression in the lung is monitored by in vivo bioluminescent image (BLI) analysis over a 2.5- to 48-h timeframe following the instillation. The procedure can be repeated multiple times within 2 - 3 months, thus permitting the evaluation of the inflammatory response in the same mice without the need to terminate the animals. This approach permits the monitoring of pro- and anti-inflammatory factors acting in the lung in real time and appears suitable for functional and pharmacological studies.

An IL-8 Transiently Transgenized Mouse Model for the In Vivo Long-term Monitoring of Inflammatory Responses

The method described here allows for the visualization of IL-8 promoter-dependent inflammation activation in the lungs of mice through non-invasive bioluminescence imaging (BLI). The same animal can be subjected to BLI multiple times for up to two months from the time of delivery of the luciferase reporter construct.

Un modelo de ratón IL-8 transgénicamente transgéni-<em&gt; In Vivo</em&gt; Monitoreo a Largo Plazo de las Respuestas Inflamatorias

Airway inflammation is often associated with bacterial infections and represents a major determinant of lung disease. The in vivo determination of the ...

Replication of the Ordered, Nonredundant Library of Pseudomonas aeruginosa strain PA14 Transposon Insertion Mutants

Pseudomonas aeruginosa is a phenotypically and genotypically diverse and adaptable Gram-negative bacterium ubiquitous in human environments. P. aeruginosa is able to form biofilms, develop antibiotic resistance, produce virulence factors, and rapidly evolve in the course of a chronic infection. Thus P. aeruginosa can cause both acute and chronic, difficult to treat infections, resulting in significant morbidity in certain patient populations. P. aeruginosa strain PA14 is a human clinical isolate with a conserved genome structure that infects a variety of mammalian and nonvertebrate hosts making PA14 an attractive strain for studying this pathogen. In 2006, a nonredundant transposon insertion mutant library containing 5,459 mutants corresponding to 4,596 predicted PA14 genes was generated. Since then, distribution of the PA14 library has allowed the research community to better understand the function of individual genes and complex pathways of P. aeruginosa. Maintenance of library integrity through the replication process requires proper handling and precise techniques. To that end, this manuscript presents protocols that describe in detail the steps involved in library replication, library quality control and proper storage of individual mutants.

Replication of the Ordered, Nonredundant Library of Pseudomonas aeruginosa strain PA14 Transposon Insertion Mutants

Pseudomonas aeruginosa infection causes significant morbidity in vulnerable hosts. The nonredundant transposon insertion mutant library of P. aeruginosa strain PA14, designated as PA14NR Set, facilitates analysis of gene functionality in numerous processes. Presented here is a protocol to generate high-quality copies of the PA14NR Set mutant library.

Replicación de la ordena, no redundante biblioteca de Pseudomonas aeruginosa cepa PA14 mutantes de inserción de transposones

Pseudomonas aeruginosa is a phenotypically and genotypically diverse and adaptable Gram-negative bacterium ubiquitous in human environments. P. aeruginosa ...

Culture of Small Colony Variant of Pseudomonas aeruginosa and Quantitation of its Alginate

Pseudomonas aeruginosa, an opportunistic Gram-negative bacterial pathogen, can overproduce an exopolysaccharide alginate resulting in a unique phenotype called mucoidy. Alginate is linked to chronic lung infections resulting in poor prognosis in patients with cystic fibrosis (CF). Understanding the pathways that regulate the production of alginate can aid in the development of novel therapeutic strategies targeting the alginate formation. Another disease-related phenotype is the small colony variant (SCV). SCV is due to the slow growth of bacteria and often associated with increased resistance to antimicrobials. In this paper, we first show a method of culturing a genetically defined form of P. aeruginosa SCV due to pyrimidine biosynthesis mutations. Supplementation of nitrogenous bases, uracil or cytosine, returns the normal growth to these mutants, demonstrating the presence of a salvage pathway that scavenges free bases from the environment. Next, we discuss two methods for the measurement of bacterial alginate. The first method relies on the hydrolysis of the polysaccharide to its uronic acid monomer followed by derivatization with a chromogenic reagent, carbazole, while the second method uses an ELISA based on a commercially available, alginate-specific mAb. Both methods require a standard curve for quantitation. We also show that the immunological method is specific for alginate quantification and may be used for the measurement of alginate in the clinical specimens.

Culture of Small Colony Variant of Pseudomonas aeruginosa and Quantitation of its Alginate

Here, we describe a growth condition to culture the small colony variant of Pseudomonas aeruginosa. We also describe two separate methods for the detection and quantitation of the exopolysaccharide alginate produced by P. aeruginosa using a traditional uronic acid carbazole assay and an alginate-specific monoclonal antibody (mAb) based ELISA.

Cultivo de la Pequeña Colonia Variante de Pseudomonas aeruginosa y Cantidad de su Alginato

Pseudomonas aeruginosa, an opportunistic Gram-negative bacterial pathogen, can overproduce an exopolysaccharide alginate resulting in a unique phenotype ...

A Delayed Inoculation Model of Chronic Pseudomonas aeruginosa Wound Infection

Pseudomonas aeruginosa (P. aeruginosa) is a major nosocomial pathogen of increasing relevance to human health and disease, particularly in the setting of chronic wound infections in diabetic and hospitalized patients. There is an urgent need for chronic infection models to aid in the investigation of wound pathogenesis and the development of new therapies against this pathogen. Here, we describe a protocol that uses delayed inoculation 24 hours after full-thickness excisional wounding. The infection of the provisional wound matrix present at this time forestalls either rapid clearance or dissemination of infection and instead establishes chronic infection lasting 7&#8211;10 days without the need for implantation of foreign materials or immune suppression. This protocol mimics a typical temporal course of post-operative infection in humans. The use of a luminescent P. aeruginosa strain (PAO1:lux) allows for quantitative daily assessment of bacterial burden for P. aeruginosa wound infections. This novel model may be a useful tool in the investigation of bacterial pathogenesis and the development of new therapies for chronic P. aeruginosa wound infections.

A Delayed Inoculation Model of Chronic Pseudomonas aeruginosa Wound Infection

We describe a delayed inoculation protocol for generating chronic wound infections in immunocompetent mice.

Un modelo de inoculación tardía de la infección crónica por la herida de Pseudomonas aeruginosa

Pseudomonas aeruginosa (P. aeruginosa) is a major nosocomial pathogen of increasing relevance to human health and disease, particularly in the setting of ...