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

Die Tierversuche wurden von der Tierpflege Ausschuss und Institutional Biosafety Ausschüsse der Universität von Illinois in Chicago genehmigt. HINWEIS: Maske, Augenschutz, Kleid oder Overall und Doppelhandschuhe: Alle Verfahren, die Pseudomonas sollte mit biologischen Sicherheitsstufe 2 (BSL2) Praktiken, die umfassen, sind aber nicht beschränkt durchgeführt werden. Arbeiten Sie in zertifizierten Sicherheitswerkbank. Gönnen Instrumente in Kontakt mit Bakterien mit Bleichmittel oder Chlordioxid Desinfektionsmittel. Verwenden Sie eine versiegelte Verpackung für den Transport Proben. 1. P. aeruginosa Kultur und Wachstum <ol><li> Shop P. aeruginosa PA103 als Bakterienbestand in einer Schraubkappe Kryoröhrchen bei -80 ° C. </li><li> Legen Sie das Fläschchen in einem Rack in einem Kasten mit 70% Ethanol benetzt Papierhandtücher und zu ergreifen, um die biologischen Sicherheitsstufe 2 (BSL2) Labor. </li><li> Streak die Bakterien auf Schafblut-Agar-Platten wachsen bei 37 ° C für ~ 15 Stunden in einem Inkubator. </li&gt; <li> In einer BSL2- Haube, kratzen die Bakterien von der Platte mit Bakterien Schleife und resuspendieren in 5 ml PBS. </li><li> Bewahren Sie die Lager bei 4 ° C für bis zu 3 Monate. Allerdings bestimmen die Titer alle 2 Wochen. </li><li> Seriell zu verdünnen, die Bakterien in PBS (in der Regel von 1:10 4 bis 1:10 7) und die Platte des bekannten Verdünnung auf Schafblut-Agar-Platten. </li><li> Inkubieren Sie die Platten für ~ 15 Std. Graf Kolonien und berechnen Colony Forming Unit (CFU) nach Bakterienkonzentration zu bestimmen. </li><li> Resuspendieren der entsprechenden Konzentrationen (~ 5 x 10 3 KbE / ul) in 0,5 ml PBS in 1,5 ml steriler Schraubkappe Kryoröhrchen. Dichten Sie die Kryoröhrchen und legen Sie sie in einem Kryoröhrchen Rack auf Desinfektionsmittel beladene Papierhandtücher in einem Schnappdeckel-Box. </li><li> Transportieren Sie das Kästchen, um BSL2- Tieranlage. Dort angekommen, öffnen Sie die Box in der BSL2- Schrank. </li></ol> 2. P. aeruginosa Instillation <ol><li> Führen Überleben Chirurgie aseptisch (sterile Handschuhe, sterile InstrumenteUnd aseptischer Techniken). Verwenden Sie eine sterile Abdeckung, eine Arbeitsfläche für sterile (autoklaviert) chirurgische Instrumente bereitzustellen. Sterilisieren Instrumente mit einer Heiß Perle Sterilisator zwischen jeder Instillation Verfahren. </li><li> Wiegen Mäusen vor Betäubung. Mäuse anästhesiert mit Ketamin (100 mg / kg), Xylazin (5 mg / kg) in 0.1 - 0.2 ml PBS intraperitoneal (ip). Bestimmung der Wirksamkeit der Betäubung durch Nichtreaktionsfähigkeit auf Zehenklemm. Verwenden Sie einen Tierarzt Salbe auf die Augen bis zur Trockene unter Narkose zu verhindern. </li><li> Rain Mäuse auf einer chirurgischen Bord in BSL2- Schrank. </li><li> Identifizieren Sie den Bereich für den Schnitt nach unten. Rasur dieses Gebiet und bereiten die Haut mit Wechselstrom Alkohol und Povidoniod Tupfer 3 mal. </li><li> Behandlung der Schnittbereich mit einem Lokalanästhetikum (Lidocain), da dies der Anästhesie ist ausreichend für eine kleinere chirurgische Eingriffe, wie einen Hautschnitt, um die Luftröhre gelangen. </li><li> Machen Sie einen kleinen Schnitt (ca. 5 mm) an der Mittellinie des Halses, und benutzen stumpfe forceps sanft bewegen die Muskeln für den Zugriff auf die Luftröhre. Setzen Sie die Luftröhre durch chirurgische Dissektion. </li><li> Zeichnen Bakterienlösung in eine 1 ml Einwegspritze mit 27 G-Nadel. Für jede Maus, verabreichen 20-30 ul Bakterien bei der entsprechenden Konzentration (bis zu 10 5 CFU je Maus). </li><li> Setzen Sie die Nadel in die Luftröhre. Injizieren Lösung langsam in den Luftröhren. </li><li> Stellen Sie sicher, dass die Tier keucht, die normalerweise an, dass Lösung in die Lunge erreicht. </li><li> Schließen Sie die Wunde mit sterilem Nahtmaterial (6-0 Monofilament) unter aseptischen Bedingungen. </li><li> Verwenden Sie 0,1 mg / kg Buprenorphin durch subkutane Injektion als post-op Analgesie zu postoperativen Schmerzen zu kontrollieren. </li><li> Stellen Sie sicher, dass die Zeit von der ersten Narkoseeinleitung erforderlichen Schließung Schnitt weniger als 15 min. </li><li> Nach der Injektion entsorgen Spritzen und Nadeln in einem geeigneten Biohazard Behälter für scharfe Gegenstände. </li><li> Behandeln Sie die chirurgischen Instrumente mit Chlordioxid basierend DISInfectant für 15 min, spülen und zum Labor für die Sterilisation und Wiederverwendung wie nötig. </li><li> Haus die Mäuse einzeln in saubere Käfige bei warmen Umgebung. </li><li> Überprüfen Sie auf dem Tier alle 30 Minuten, bis er das Bewusstsein wiedererlangt und beginnt sich zu bewegen; und später in 12 Stunden Intervallen für die ersten 3 Tage nach der Operation. </li><li> Halten die Mäuse in der Tier BSL2 Einrichtung während des Experiments. Stellen Sie sicher, dass nur in versiegelten Kiste enthaltene Gewebe verlässt das Tier BSL2- Möglichkeiten. Wenn die Mäuse exprimieren einem der moribund Verhaltensweisen (definiert als Atemnot, Lethargie, Versagen in Reaktion auf sanfte Stimulierung ambulate) an einem Punkt in der Studie euthanize die Mäuse durch CO 2 Inhalation aus einem Flaschenquelle gefolgt durch zervikale Dislokation. </li><li> Euthanize die Versuchspersonen durch Ausbluten unter Narkose. <ol><li> Sammeln Lungengewebe von Mäusen getötet. Führen Sie diese Verfahren in einem BSL2 Kapuze. Falls erforderlich, überweisen Sie den Proben mit verschlossenen Röhrchen in einem rac platziertk on Desinfektions beladene Papierhandtücher in einem Schnappdeckel-Box für weitere Prozess. </li></ol></li></ol>

<ol>
	<li>Ware, L. B., Matthay, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+acute+respiratory+distress+syndrome.">The acute respiratory distress syndrome.</a> N Engl J Med. 342, 1334-1349 (2000).</li><li>Matthay, M. A., Ware, L. B., Zimmerman, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+acute+respiratory+distress+syndrome.">The acute respiratory distress syndrome.</a> J Clin Invest. 122, 2731-2740 (2012).</li><li>Shimabukuro, D. W., Sawa, T., Gropper, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Injury+and+repair+in+lung+and+airways.">Injury and repair in lung and airways.</a> Crit Care Med. 31 (8), 524-531 (2003).</li><li>Wansleeben, C., Barkauskas, C. E., Rock, J. R., Hogan, B. 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T., Blackwell, T. S., Christman, J. W., Prince, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogen-host+interactions+in+pseudomonas+aeruginosa+pneumonia.">Pathogen-host interactions in pseudomonas aeruginosa pneumonia.</a> Am J Respir Crit Care Med. 171, 1209-1223 (2005).</li><li>Liu, Y., 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=Foxm1+mediates+the+progenitor+function+of+type+ii+epithelial+cells+in+repairing+alveolar+injury+induced+by+pseudomonas+aeruginosa.">Foxm1 mediates the progenitor function of type ii epithelial cells in repairing alveolar injury induced by pseudomonas aeruginosa.</a> J Exp Med. 208, 1473-1484 (2011).</li><li>Kurahashi, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogenesis+of+septic+shock+in+pseudomonas+aeruginosa+pneumonia.">Pathogenesis of septic shock in pseudomonas aeruginosa pneumonia.</a> J Clin Invest. 104, 743-750 (1999).</li><li>Kumar, P. 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The authors have nothing to disclose.

Die Pseudomonas-Maus Lungenverletzung Modell, das wir beschreiben imitiert den gesamten Prozess der Entzündung, Lungenverletzung, Reparatur und Auflösung, die nach einer akuten Lungenschädigung oder Lungenentzündung auftreten. Es hat einzigartige Vorteile im Vergleich mit einigen anderen Verletzungen Modelle, dass es klinisch relevanten und relativ sicher und einfach zu handhaben. Der kritische Schritt in dem Verfahren ist, dass die Injektion von Bakterienlösung muss sehr langsam. Wenn ...

Lungen Umweltkrankheitserreger ausgesetzt und sind anfällig für Entzündungen und Verletzungen 1-3. Während pathologischen Zuständen, wie Lungenentzündung oder Adult Respiratory Distress Syndrome (ARDS), Krankheitserreger sowie Entzündungsfaktoren von Leukozyten freigesetzt induzieren Verletzungen und Tod von Alveolarzellen 1-3. Es ist wichtig zu Tiermodellen von akutem Lungenversagen entwickeln, um die Untersuchung der Pathologie von Verletzungen sowie Mechanismus der Reparatur zu erleichtern. Gegenwärtig sind die meisten Leute benutzen Hyperoxie und Bleomycin induzierten Mauslungenverletzung Modelle 4. Jedoch sind die Mechanismen der Hyperoxie Schädigung verursacht sind nicht die gleichen wie die häufigste Lungenverletzungen, die bei Lungenentzündung oder ARDS 5 auftreten. Bleomycin induzierte akute Verletzung ist selten im klinischen Kontext 4. Eine Maus Lungenverletzung Modell mit intratracheale Injektion von P. Hier berichten wir aeruginosa 6,7. Dieses Modell ist klinisch relevant, und ahmt die prozesse, die folgende Lungenentzündung 8 geschehen. Als opportunistische, nosokomiale Erreger der immungeschwächten Personen, P. aeruginosa infiziert in der Regel die Lungenwege, der Harnwege, Verbrennungen, Wunden, und verursacht auch andere Blutinfektionen 6. Die Bakterien setzen Virulenzfaktor Exotoxin A, multiplizieren und lösen Immunreaktionen 6. Intra-trachael Verabreichung von P. aeruginosa spiegelt die Situation in der menschlichen Exposition gegenüber den Bakterien, die Lungenentzündung verursachen und die Pathologie ist wahrscheinlich anders als die vor kurzem berichtet, Influenza-Virus H1N1 induzierten Lungenschädigung Modell 9 zu sein. Da P. aeruginosa ist ein opportunistisches Pathogen, das relativ sicher im Vergleich zu einigen der virulenten Pathogenen handhaben ist. Hier verwendeten wir intratracheale Injektion, um die Bakterien zu verabreichen, weil wir festgestellt, dass dieses Verfahren eingeführt mehr Bakterien in den distalen Bereich der Alveolen der Lunge im Vergleich mitEinige andere Verfahren wie die Verwendung eines Katheters über den Mund. Verglichen mit anderen akuten Lungenverletzung Modelle, die P. hier beschriebenen aeruginosa Modell eignet sich für das Studium Lungenschädigung durch Bakterien und durch übermäßige Entzündung induziert. Im Gegensatz zu anderen Tiermodellen, die P. verwenden aeruginosa Sepsis 10,11 induzieren, hier verwenden wir intratracheale Injektion dieser Bakterien, um lokalisierte akute Lungenschädigung zu induzieren.

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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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			<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>
		</tr>
		<tr height="26">
			<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>
		</tr>
		<tr height="26">
			<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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</table>

pseudomonas aeruginosa induced lung injury model

Um die menschliche akute Lungenschädigung oder Lungenentzündung zu untersuchen, ist es wichtig, Tiermodelle zu entwickeln, um verschiedene pathologische Merkmale dieser Krankheit zu imitieren. Hier haben wir eine Maus Lungenverletzung Modell durch intratracheale Injektion von Bakterien Pseudomonas aeruginosa (P. aeruginosa oder PA) entwickelt. Mit diesem Modell konnten wir Lungenentzündung in der frühen Phase der Verletzung zeigen. Zusätzlich wurde Alveolarepithel- Sperr Undichtigkeit durch Analysieren bronchoalveoläre Lavage (BAL) beobachtet; und alveoläre Zelltod wurde durch TUNEL-Assay mit Gewebe aus verletzten Lunge vorbereitet beobachtet. Zu einem späteren Phase nach der Verletzung, beobachteten wir die Zellproliferation für den Reparaturvorgang erforderlich. Die Verletzung wurde von 7 Tagen ab der Einleitung des P. gelöst aeruginosa Injektion. Dieses Modell imitiert die sequentielle Verlauf Lungenentzündung, Verletzungen und Reparatur während einer Lungenentzündung. Diese klinisch relevanten Tiermodell eignet sich für das Studium der Pathologie, Mechanismus der Reparatur, fach akuter Lungenverletzung, und kann auch verwendet werden, um potentielle therapeutische Mittel für diese Krankheit zu testen.

Ab 24 bis 72 Stunden nach P. aeruginosa Injektion erhöhte Zellularität wurde in Lungenschnitten beobachtet (1A-D). Die Lunge begonnen, von 96 Stunden nach der Verletzung (1E) erholen. 7 Tage nach P. aeruginosa, normalen Alveolen Morphologie wurde weitgehend wiederhergestellt (1F). Tunnel Färbung mit Lungenabschnitte bei 24 h nach P. vorbereitet aeruginosa zeigte Zelltod in Alveolen Zellen (1G-I). Um den Reparaturpr...

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 induzierte Lungenschädigung-Modell

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

Research

JoVE Journal

Immunology and Infection

Pseudomonas aeruginosa Induced Lung Injury Model

18.4K Aufrufe.  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. 

Serie: 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-Kultur Modelle Pseudomonas aeruginosa Biofilme auf lebenden menschlichen Zellen gezüchtet Airway

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

Forschung

Immunologie und Infektion

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 und Saccharomyces cerevisiae Biofilm im Flow Cells

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.

RNA Isolierung von Pseudomonas aeruginosa Kolonisierung der Murine Gastrointestinaltrakt

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.

Einsatz von künstlichen Sputum Mittlere bis antibiotische Wirkung gegen die getestet<em&gt; Pseudomonas aeruginosa</em&gt; AGB in einen engeren Bezug zu den Mukoviszidose-Lunge

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.

Langzeitchronik<em&gt; Pseudomonas aeruginosa</em&gt; Airway-Infektion bei Mäusen

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.

Replikation von bestellt, Nonredundant Bibliothek von Pseudomonas Aeruginosa Stamm PA14 Transposon Insertion Mutanten

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.

Methoden zur Erkennung von zytotoxischen amyloiden folgende Infektion der pulmonalen Endothelzellen durch 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.

Kultur der kleinen Kolonie Variante von Pseudomonas aeruginosa und Quantitation seines Alginats

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.

Ein verzögertes Impfmodell der chronischen Pseudomonas aeruginosa Wundinfektion

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.

Studium der Auswirkungen von Zigarettenrauch auf Pseudomonas-Infektion in Lungenepithelzellen

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