This work was supported by National Institutes of Health grants HL105947-01 (YL), HL07829-16 (AM), HL090152 (AM).

Les expériences sur les animaux ont été approuvés par le Comité de protection des animaux et des comités institutionnels sur la biosécurité de l&#39;Université de l&#39;Illinois à Chicago. NOTE: Toutes les procédures impliquant des pseudomonas doivent être effectuées avec le niveau de biosécurité 2 (BSL2) pratiques, qui comprennent, mais ne sont pas limités à: masque, protection oculaire, blouse ou combinaison, gants et doubles. Travailler dans le cabinet de biosécurité certifié. Traiter les instruments en contact avec des bactéries avec l&#39;eau de Javel ou de dioxyde de chlore désinfectant à base. Utilisez une boîte étanche pour transporter les échantillons. 1. P. aeruginosa Culture et de croissance <ol><li> Magasin P. aeruginosa PA103 comme un stock de bactéries dans un bouchon à vis cryovial à -80 ° C. </li><li> Placer le flacon dans un rack dans une boîte avec 70% d&#39;éthanol mouillé des serviettes en papier et prendre au laboratoire de biosécurité de niveau 2 (niveau L2). </li><li> Streak les bactéries sur des plaques de gélose au sang de mouton et se développent à 37 ° C pendant environ 15 heures dans un incubateur. </li&gt; <li> Dans une hotte BSL2, rayer les bactéries de la plaque avec des bactéries boucle et remettre en suspension dans 5 ml de PBS. </li><li> Conservez le stock à 4 ° C pendant 3 mois. Toutefois, déterminer le titre toutes les 2 semaines. </li><li> Diluer en série les bactéries dans du PBS (en général de 01:10 à 01:10 4 7) et la plaque à la dilution connue sur des plaques de gélose au sang de mouton. </li><li> Incuber les plaques pour ~ 15 heures. Compter les colonies et calculer unités formant colonie (UFC) pour déterminer la concentration de bactéries. </li><li> Remettre en suspension les concentrations appropriées (~ 5 x 10 3 UFC / ul) dans 0,5 ml de PBS dans des cryotubes de 1,5 ml à bouchon à vis stériles. Sceller les cryotubes et les placer dans un rack cryovial sur des serviettes en papier chargés désinfectant dans une boîte de couvercle à pression. </li><li> Transporter la boîte à l&#39;animalerie BSL2. Une fois là, ouvrir la boîte dans l&#39;armoire de BSL2. </li></ol> 2. P. aeruginosa instillation <ol><li> Effectuer la chirurgie de survie de façon aseptique (gants stériles, des instruments stérileset les techniques aseptiques). Utilisez un champ stérile pour fournir une surface de travail pour (autoclave) instruments chirurgicaux stériles. Stériliser les instruments à l&#39;aide d&#39;un stérilisateur de perles chaud entre chaque instillation trachéale procédure. </li><li> Peser souris avant l&#39;anesthésie. Anesthésier les souris avec de la kétamine (100 mg / kg), de xylazine (5 mg / kg), en 0.1 - 0.2 ml de PBS par voie intrapéritonéale (ip). Déterminer l&#39;efficacité de l&#39;anesthésique par des non-réactivité de pincement de l&#39;orteil. Utilisez une pommade vétérinaire sur les yeux pour prévenir la sécheresse sous anesthésie. </li><li> Retenez souris sur un bord chirurgicale dans BSL2 armoire. </li><li> Identifier la zone de la coupe vers le bas. Rasez ce domaine et préparer la peau à l&#39;aide alternance alcool et povidone prélèvements d&#39;iode 3 fois. </li><li> Traiter la zone d&#39;incision avec un anesthésique local (lidocaïne) que cette anesthésie est suffisante pour une chirurgie mineure comme une incision de la peau pour accéder à la trachée. </li><li> Faire une petite incision (environ 5 mm) sur la ligne médiane du cou, et utiliser émoussé forceps de se déplacer doucement sur le muscle pour l&#39;accès à la trachée. Exposer la trachée par dissection chirurgicale. </li><li> Dessinez solution de bactéries dans une seringue de 1 ml à usage unique avec aiguille 27 G. Pour chaque souris, l&#39;administration de 20 à 30 pl de bactéries à la concentration appropriée (jusqu&#39;à 10 5 CFU chaque souris). </li><li> Insérer l&#39;aiguille dans la trachée. Injecter la solution lentement dans la trachée. </li><li> Veiller à ce que les halètements des animaux, ce qui indique généralement que la solution a atteint dans les poumons. </li><li> Fermer la plaie avec des sutures stériles (6-0 monofilament) dans des conditions aseptiques. </li><li> Utilisez 0,1 mg / kg de buprénorphine par injection sous-cutanée post-op analgésie pour contrôler la douleur post-chirurgicale. </li><li> Assurez-vous que le temps nécessaire à l&#39;induction de l&#39;anesthésie initiale de fermeture de l&#39;incision est inférieure à 15 min. </li><li> Après l&#39;injection, jeter les seringues et aiguilles dans un container approprié approprié. </li><li> Traiter les instruments chirurgicaux avec Disi base de dioxyde de chlorenfectant pendant 15 minutes, rincer et retourner au laboratoire pour la stérilisation et de réutiliser au besoin. </li><li> Maison des souris individuellement dans des cages propres au cadre chaleureux. </li><li> Vérifiez sur l&#39;animal toutes les 30 min jusqu&#39;à ce qu&#39;elle reprenne conscience et commence à se déplacer; et plus tard, à des intervalles de 12 h pour les 3 premiers jours après la chirurgie. </li><li> Conserver les souris dans le centre de BSL2 animal tout au long de l&#39;expérience. Assurez-vous que seul tissu contenu dans la boîte scellée de quitter l&#39;établissement BSL2 animale. Si les souris expriment l&#39;un des comportements moribonds (définie comme la détresse respiratoire, léthargie, absence de déambuler en réponse à une stimulation douce) à tout moment de l&#39;étude, euthanasier les souris par inhalation de CO 2 provenant d&#39;une source en bouteille suivie par dislocation cervicale. </li><li> Euthanasier le sujet expérimental par exsanguination sous anesthésie. <ol><li> Recueillir les tissus pulmonaires de souris euthanasiées. Effectuer ces procédures dans une hotte de BSL2. Si nécessaire, transférer l&#39;échantillon en utilisant des tubes étanches placées dans un rack sur des serviettes en papier chargés désinfectant dans une boîte de couvercle à pression pour plus de processus. </li></ol></li></ol>

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The authors have nothing to disclose.

Le poumon de la souris modèle de lésion Pseudomonas que nous décrivons ici imite l&#39;ensemble du processus de l&#39;inflammation, des lésions pulmonaires, la réparation, et la résolution qui se produisent suite à une lésion pulmonaire aiguë ou la pneumonie. Il a des avantages uniques comparaison avec plusieurs autres modèles de lésion en ce qu&#39;il est cliniquement pertinent et relativement sûr et facile à manipuler. L&#39;étape critique dans la procédure est que l&#39;inj...

Poumons sont exposés à des agents pathogènes environnementaux et sont sensibles à l&#39;inflammation et les lésions 1-3. Dans des conditions pathologiques telles que la pneumonie ou syndrome de détresse respiratoire adulte (SDRA), les agents pathogènes ainsi que les facteurs inflammatoires libérés par les leucocytes provoquent des blessures et la mort des cellules alvéolaires 1-3. Il est important de développer des modèles animaux de lésion pulmonaire aiguë de faciliter l&#39;étude de la pathologie de la blessure ainsi que le mécanisme de réparation. Actuellement, la plupart des gens utilisent des modèles de lésions pulmonaires hyperoxie et la bléomycine souris induit 4. Cependant, les mécanismes d&#39;hyperoxie blessures causées ne sont pas les mêmes que la plupart des lésions pulmonaires courantes qui se produisent au cours de la pneumonie ou de l&#39;ARDS 5. La bléomycine blessure aiguë induite est rare dans un contexte clinique 4. Nous rapportons ici un modèle de lésion pulmonaire de la souris par injection intra-trachéale de P. aeruginosa 6,7. Ce modèle est cliniquement pertinente, et imite la processus qui se produisent suite à une pneumonie 8. Comme, un pathogène nosocomial opportuniste des individus immunodéprimés, P. aeruginosa infecte habituellement les voies pulmonaires, des voies urinaires, des brûlures, des plaies, et provoque également d&#39;autres infections du sang 6. La bactérie produit virulence facteur exotoxine A, se multiplient et déclenchent des réactions immunitaires 6. L&#39;administration intra-trachael de P. aeruginosa correspond à la situation dans l&#39;exposition des humains à des bactéries qui provoquent la pneumonie et à la pathologie est susceptible d&#39;être différente de la grippe H1N1 du virus de la grippe pulmonaire induite modèle de lésion récemment rapporté 9. Depuis P. aeruginosa est un agent pathogène opportuniste, il est relativement sûr à manipuler par rapport à certains des agents pathogènes les plus virulentes. Ici, nous avons utilisé l&#39;injection intra-trachéale à administrer les bactéries, car nous avons constaté que cette méthode introduit plus de bactéries dans la région distale alvéoles du poumon par rapport àd&#39;autres procédures telles que l&#39;utilisation d&#39;un cathéter par la bouche. Comparé à d&#39;autres modèles de lésion pulmonaire aiguë, le P. aeruginosa modèle décrit ici est adapté à l&#39;étude des lésions pulmonaires induites par les bactéries et par une inflammation excessive. Contrairement à d&#39;autres modèles animaux qui utilisent P. aeruginosa pour induire une septicémie 10,11, ici nous utilisons injection intra-trachéale de ces bactéries à induire des lésions pulmonaires aiguës localisées.

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			<td height="26" style="height:27px;width:215px;">Name</td>
			<td style="width:145px;">Company</td>
			<td style="width:145px;">Catalog number&#160;</td>
			<td style="width:145px;">Comments</td>
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		<tr height="38">
			<td height="38" style="height:38px;width:215px;">Anesthetic: Ketamin, xylazine, lidocaine, buprenorphine</td>
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			<td style="width:145px;">pharmaceutical grade</td>
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			<td height="26" style="height:27px;width:215px;">27g needle</td>
			<td style="width:145px;">Fisher</td>
			<td style="width:145px;">1482648</td>
			<td style="width:145px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:215px;">syringe</td>
			<td style="width:145px;">Fisher</td>
			<td style="width:145px;">14823434</td>
			<td style="width:145px;">1 ml</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:215px;">scissors</td>
			<td style="width:145px;">Fine Science tools</td>
			<td style="width:145px;"></td>
			<td style="width:145px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:215px;">forceps</td>
			<td style="width:145px;">Fine Science tools</td>
			<td style="width:145px;"></td>
			<td style="width:145px;"></td>
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			<td height="26" style="height:27px;width:215px;">suture&#160;</td>
			<td style="width:145px;">Fisher</td>
			<td style="width:145px;">NC0147607</td>
			<td style="width:145px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;">Eye gauge, glove, gown</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;">Biosafety Cabinet</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;">chlorine dioxide based sterilant&#160;</td>
			<td></td>
			<td></td>
			<td>Clidox</td>
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			<td height="26" style="height:27px;">sheep blood agar plates&#160;</td>
			<td>Medex supply</td>
			<td>HL-1160</td>
			<td></td>
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pseudomonas aeruginosa induced lung injury model

Afin d&#39;étudier les lésions pulmonaires aiguës humaine et la pneumonie, il est important de développer des modèles animaux pour imiter différentes caractéristiques pathologiques de cette maladie. Ici, nous avons développé un modèle de lésion pulmonaire de la souris par injection intra-trachéale de bactéries Pseudomonas aeruginosa (P. aeruginosa ou PA). En utilisant ce modèle, nous avons pu montrer une inflammation des poumons à la phase précoce de la lésion. En outre, alvéolaire perméabilité de la barrière epitheliale a été observée par analyse de lavage broncho-alvéolaire (BAL); et la mort cellulaire alvéolaire a été observé par essai TUNEL en utilisant un tissu préparé à partir de poumons blessés. Lors d&#39;une étape ultérieure après la lésion, on a observé une prolifération cellulaire requis pour le processus de réparation. La blessure a été résolue 7 jours à compter de l&#39;ouverture de P. injection aeruginosa. Ce modèle imite le parcours séquentiel de l&#39;inflammation pulmonaire, une lésion et de réparation pendant une pneumonie. Ce modèle animal cliniquement pertinente est adapté pour l&#39;étude de la pathologie, le mécanisme de réparation, fuite une lésion pulmonaire aiguë, et peut également être utilisé pour tester des agents thérapeutiques potentiels pour cette maladie.

A partir de 24 à 72 heures après P. injection aeruginosa, cellularité accrue a été observée dans les coupes pulmonaires (figure 1A-D). Le poumon a commencé à récupérer à partir de 96 h après la lésion (figure 1E). 7 jours après P. aeruginosa, morphologie alvéoles normale a été en grande partie restaurée (figure 1F). Sections à l&#39;aide du poumon tunnel de coloration préparés à 24 heures après P. aeruginosa a m...

Watch this Scientific Journal Video about Pseudomonas aeruginosa Induced Lung Injury Model at JoVE.com

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 lésion pulmonaire induite par modèle

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. ...

pseudomonas-aeruginosa-induced-lung-injury-model

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JoVE Journal

Immunology and Infection

Pseudomonas aeruginosa Induced Lung Injury Model

18.4K vues.  University of Illinois at Chicago. 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. 

Vidéo: Pseudomonas aeruginosa Induced Lung Injury Model

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.

Co-culture des modèles Pseudomonas aeruginosa Les biofilms Cultivé sur le Live cellules des voies respiratoires de l&#39;homme

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

Recherche

Immunologie et infection

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 et le biofilm Saccharomyces cerevisiae en cellules à flux

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.

Isolement des ARN Pseudomonas aeruginosa La colonisation du tractus gastro-intestinal murin

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.

L&#39;utilisation de moyen d&#39;expectoration artificielle pour tester l&#39;efficacité des antibiotiques contre<em&gt; Pseudomonas aeruginosa</em&gt; Dans des conditions plus pertinents à la fibrose kystique du poumon

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.

Chronique à long terme<em&gt; Pseudomonas aeruginosa</em&gt; Infection des voies respiratoires chez les souris

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 ...

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.

Réplication de l’ordonné, bibliothèque non redondante de la souche Pseudomonas aeruginosa PA14 Transposon Mutants d’Insertion

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

Methods for Detecting Cytotoxic Amyloids Following Infection of Pulmonary Endothelial Cells by Pseudomonas aeruginosa

Patients who survive pneumonia have elevated death rates in the months following hospital discharge. It has been hypothesized that infection of pulmonary tissue during pneumonia results in the production of long-lived cytotoxins that can lead to subsequent end organ failure. We have developed in vitro assays to test the hypothesis that cytotoxins are produced during pulmonary infection. Isolated rat pulmonary endothelial cells and the bacterium Pseudomonas aeruginosa are used as model systems, and the production of cytoxins following infection of the endothelial cells by the bacteria is demonstrated using cell culture followed by direct quantitation using lactate dehydrogenase assays and a novel microscopic method utilizing ImageJ technology. The amyloid nature of these cytotoxins was demonstrated by thioflavin T binding assays and by immunoblotting and immunodepletion using A11 anti-amyloid antibody. Further analyses using immunoblotting demonstrated that oligomeric tau and A&#946; were produced and released by endothelial cells following infection by P. aeruginosa. These methods should be readily adaptable to analyses of human clinical samples.

Methods for Detecting Cytotoxic Amyloids Following Infection of Pulmonary Endothelial Cells by Pseudomonas aeruginosa

Simple methods are described for demonstrating the production of cytotoxic amyloids following infection of pulmonary endothelium by Pseudomonas aeruginosa.

Méthodes pour détecter les cytotoxiques amyloïdes suivant Infection de pulmonaire cellules endothéliales par Pseudomonas aeruginosa

Patients who survive pneumonia have elevated death rates in the months following hospital discharge. It has been hypothesized that infection of pulmonary ...

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.

Culture de la petite colonie Variante de Pseudomonas aeruginosa et Quantitation de son Alginate

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 modèle d’inoculation retardée de pseudomonas chronique infection de blessure d’aeruginosa

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

Studying Effects of Cigarette Smoke on Pseudomonas Infection in Lung Epithelial Cells

Cigarette smoking is the major etiological cause for lung emphysema and chronic obstructive pulmonary disease (COPD). Cigarette smoking also promotes susceptibility to bacterial infections in the respiratory system. However, the effects of cigarette smoking on bacterial infections in human lung epithelial cells have yet to be thoroughly studied. Described here is a detailed protocol for the preparation of cigarette smoking extracts (CSE), treatment of human lung epithelial cells with CSE, and bacterial infection and infection determination. CSE was prepared with a conventional method. Lung epithelial cells were treated with 4% CSE for 3 h. CSE-treated cells were, then, infected with Pseudomonas at a multiplicity of infection (MOI) of 10. Bacterial loads of the cells were determined by three different methods. The results showed that CSE increased Pseudomonas load in lung epithelial cells. This protocol, therefore, provides a simple and reproducible approach to study the effect of cigarette smoke on bacterial infections in lung epithelial cells.

Studying Effects of Cigarette Smoke on Pseudomonas Infection in Lung Epithelial Cells

Described here is a protocol to study how cigarette smoke extract affects bacterial colonization in lung epithelial cells.

Étude des effets de la fumée de cigarette sur l’infection de Pseudomonas dans les cellules épithéliales pulmonaires

Cigarette smoking is the major etiological cause for lung emphysema and chronic obstructive pulmonary disease (COPD). Cigarette smoking also promotes ...