Wir erkennen die Unterstützung des Vereinigten Königreichs National Institute for Health Research, die Dr Hadwen Trust for Humane Research, der führende britische medizinischen Forschung Nächstenliebe Finanzierung ausschließlich tierversuchsfreie Forschungsmethoden zu Tierversuchen ersetzen, und dem Wellcome Trust (Grant 089215). Wir erkennen auch Novartis Pharmaceuticals UK Ltd (unrestricted educational grant).

<ol>
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Microbiol. 43, 5085-5090 (2005).</li><li>Fung, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+expression+of+Pseudomonas+aeruginosa+in+a+mucin-containing+synthetic+growth+medium+mimicking+cystic+fibrosis+lung+sputum.">Gene expression of Pseudomonas aeruginosa in a mucin-containing synthetic growth medium mimicking cystic fibrosis lung sputum.</a> J. Med. Microbiol. 59, 1089-1100 (2010).</li><li>Andrews, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BSAC+standardized+disc+susceptibility+testing+method+(version+8).">BSAC standardized disc susceptibility testing method (version 8).</a> The Journal of antimicrobial chemotherapy. 64, 454-489 (2009).</li><li>Rybtke, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+implication+of+Pseudomonas+aeruginosa+biofilms+in+infections.">The implication of Pseudomonas aeruginosa biofilms in infections.</a> Inflamm Allergy Drug Targets. 10, 141-157 (2011).</li><li>Moreau-Marquis, S., Stanton, B. A., O'Toole, 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=Pseudomonas+aeruginosa+biofilm+formation+in+the+cystic+fibrosis+airway.">Pseudomonas aeruginosa biofilm formation in the cystic fibrosis airway.</a> Pulm. Pharmacol. Ther. 21, 595-599 (2008).</li><li>Ahmed, S. A., Gogal, R. M., Walsh, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+rapid+and+simple+non-radioactive+assay+to+monitor+and+determine+the+proliferation+of+lymphocytes:+an+alternative+to+[3H]thymidine+incorporation+assay.">A new rapid and simple non-radioactive assay to monitor and determine the proliferation of lymphocytes: an alternative to [3H]thymidine incorporation assay.</a> J. Immunol. Methods. 170, 211-224 (1994).</li><li>Fothergill, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+antibiotic+treatment+on+bacteriophage+production+by+a+cystic+fibrosis+epidemic+strain+of+Pseudomonas+aeruginosa.">Effect of antibiotic treatment on bacteriophage production by a cystic fibrosis epidemic strain of Pseudomonas aeruginosa.</a> Antimicrob. Agents Chemother. 55, 426-428 (2011).</li><li>Singh, P. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quorum-sensing+signals+indicate+that+cystic+fibrosis+lungs+are+infected+with+bacterial+biofilms.">Quorum-sensing signals indicate that cystic fibrosis lungs are infected with bacterial biofilms.</a> Nature. 407, 762-764 (2000).</li><li>Mowat, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pseudomonas+Aeruginosa+Population+Diversity+and+Turnover+in+Cystic+Fibrosis+Chronic+Infections.">Pseudomonas Aeruginosa Population Diversity and Turnover in Cystic Fibrosis Chronic Infections.</a> Am. J. Respir. Crit. Care. Med. , (2011).</li><li>Fothergill, J. L., Mowat, E., Ledson, M. J., Walshaw, M. J., Winstanley, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluctuations+in+phenotypes+and+genotypes+within+populations+of+Pseudomonas+aeruginosa+in+the+cystic+fibrosis+lung+during+pulmonary+exacerbations.">Fluctuations in phenotypes and genotypes within populations of Pseudomonas aeruginosa in the cystic fibrosis lung during pulmonary exacerbations.</a> J. Med. Microbiol. 59, 472-481 (2010).</li><li>Fouhy, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diffusible+signal+factor-dependent+cell-cell+signaling+and+virulence+in+the+nosocomial+pathogen+Stenotrophomonas+maltophilia.">Diffusible signal factor-dependent cell-cell signaling and virulence in the nosocomial pathogen Stenotrophomonas maltophilia.</a> J. Bacteriol. 189, 4964-4968 (2007).</li><li>Winstanley, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Newly+introduced+genomic+prophage+islands+are+critical+determinants+of+in+vivo+competitiveness+in+the+Liverpool+Epidemic+Strain+of+Pseudomonas+aeruginosa.">Newly introduced genomic prophage islands are critical determinants of in vivo competitiveness in the Liverpool Epidemic Strain of Pseudomonas aeruginosa.</a> Genome Res. 19, 12-23 (2009).</li><li>Carter, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+subtype+of+a+Pseudomonas+aeruginosa+cystic+fibrosis+epidemic+strain+exhibits+enhanced+virulence+in+a+murine+model+of+acute+respiratory+infection.">A subtype of a Pseudomonas aeruginosa cystic fibrosis epidemic strain exhibits enhanced virulence in a murine model of acute respiratory infection.</a> J. Infect. Dis. 202, 935-942 (2010).</li></ol>

Keine Interessenskonflikte erklärt.

In dieser Studie verwendeten wir ein neues in vitro Modell basierend auf ASM zu replizieren P. aeruginosa Biofilm Bedingungen innerhalb der CF Lunge 4. Das Modell wurde erfolgreich für die kleinräumige, Hochdurchsatz-Screening von antimikrobiellen Wirkstoffen modifiziert. Die kritischen Schritte dieses Tests sind: <ol><li> Konsequente Vorbereitung der ASM Medien und Aufrechterhaltung der Sterilität. Wir haben viele Stunden, um die Optimierung des in der jede Komponente hinzugefügt, um reproduzierbare Ergebnisse jedes Mal gewidmet ist. Die Filtration des ASM ist langsam, ist aber vorzuziehen, Autoklavieren, die die Muzin-Komponente beschädigt werden kann. Wir raten den Zusatz von Antibiotika, wie von anderen vorgeschlagen 11, weil diese erheblichen Selektionsdruck, Antriebs-Mutationen auferlegen können, induzieren Prophagen Lyse 16 und signifikante Veränderung der Expression von mehreren bakteriellen Genen. </li><li> Der Test sollte nach dem Volumen o optimiert werdenf ASM verwendet. Shaking Geschwindigkeiten sind für kleinere Volumina erhöht und einen kürzeren Lebenszyklus Biofilm beobachtet wird. </li></ol> Eine naheliegende Anwendung der kleinräumigen ASM Biofilm-Modell ist die realistischere Bestimmung der Biofilm antimikrobiellen Empfindlichkeiten (BSMIC 90). Anaerobe und mikroaerophilen Nischen in der CF-Lunge und es gibt Belege dafür, dass Sauerstoff tief innerhalb reife Biofilme 2, 17 begrenzt. Hier zeigen wir, dass 10/14 klinischen P. aeruginosa-Isolaten von CF-Patienten weisen eine erhebliche Sputum (4 - ≥ 128-fach) Abnahme der Empfindlichkeit auf Tobramycin unter mikroaerophilen Bedingungen in ASM. Die Ergebnisse dieser Studie legen nahe, dass Antibiotika wie Tobramycin, möglicherweise weniger wirksam gegen P. aeruginosa-Infektionen in der Lunge CF als durch herkömmliche Methoden Empfindlichkeitsprüfung angegeben. Diese Ergebnisse spiegeln früheren Studien über die antimikrobielle Empfindlichkeit von Biofilmen 10. Klein-Maßstab ASM-Assays somit eine einfache Plattform mit hohem Durchsatz zur Erzeugung aussagekräftiger Antibiotikaempfindlichkeit Daten besser zu informieren therapeutische Entscheidungen. Der Test wird in der gleichen Weise wie herkömmliche Antibiotika Empfindlichkeitsprüfung, daß einzelne Kolonien für das Screening, die nicht repräsentativ für die gesamte Bevölkerung können aufgenommen beschränkt. Wir glauben jedoch, dass ein Ansatz, (i) Verwendung von nicht-Oberfläche befestigt Biofilmwachstum und (ii) für mikroaerophilen Bedingungen, eine klare Alternative und eine mögliche Verbesserung bestehender Methoden darstellt. Wir schließen daraus, dass dieser Test ein geeignetes Modell, um zu studieren, ist P. aeruginosa Biofilm Populationen. Weitere Tests im klinischen Umfeld würde prüfen, ob Antibiotika-Empfindlichkeiten auf Biofilm-Basis gewachsen P. aeruginosa könnte auf unterschiedliche Antibiotika-Entscheidungen mit möglicherweise verbessert mikrobiologischen und klinischen Ergebnissen führen. Ähnliche Untersuchungen mit klassischen Biofilm-Modelle haben gezeigt, dass BSMIC Werten führenverschiedene Empfehlungen zur antibiotischen Behandlung 5,17. Zusätzlich zur Prüfung der Wirksamkeit von Antiinfektiva, stellt der ASM-System eine billige, einfache und reproduzierbare Alternative zu Tiermodellen für Studien wie die im Verständnis der Diversifizierung der P. richtet aeruginosa Bevölkerungen. Wir haben umfangreiche Heterogenität in natürlichen Populationen von P. beobachtet aeruginosa aus CF-Patienten Sputum 18, 19 erholt. Ähnliche phänotypische und genotypische Diversifizierung kann während des Wachstums in ASM-4 (und unsere unveröffentlichte Daten) beobachtet werden, so dass es ein attraktives In-vitro-Modell der CF Lungenerkrankungen. Die relative Einfachheit des ASM-Modell macht es einfach zu entwerfen langfristige Anpassung Experimente sollen, zum Beispiel bei der Überwachung der Auswirkungen von Antibiotika oder anderen Belastungen auf P. aeruginosa Bevölkerung Divergenz. Darüber hinaus können andere bakterielle Erreger in angebaut werdenASM. Zum Beispiel, Fouhy et al. ASM 2007 verwendet haben, um die Biofilmbildung von S. studieren maltophillia 20.

<table border="1"><tbody><tr><td> Name des Reagenz </td><td> Firma </td><td> Katalog-Nummer </td></tr><tr><td> DNA aus Fischsperma </td><td> Sigma-Aldrich </td><td> 74782 </td></tr><tr><td> Mucin aus Schweine-Magen-, Typ-II- </td><td> Sigma-Aldrich </td><td> M2378 </td></tr><tr><td> L-Alanin </td><td> Acros Organics </td><td> 102830250 </td></tr><tr><td> L-Arginin </td><td> Sigma-Aldrich </td><td> A5006 </td></tr><tr><td> L (+)-Asparagin-Monohydrat </td><td> Acros Organics </td><td> 175271000 </td></tr><tr><td> L (+)-Asparaginsäure </td><td> Acros Organics </td><td> 105041000 </td></tr><tr><td> L-Cystein </td><td> Sigma-Adrich </td><td> 168149 </td></tr&gt; <tr><td> L (+)-Glutaminsäure </td><td> Acros Organics </td><td> 156211000 </td></tr><tr><td> L-Glutamin </td><td> Sigma-Aldrich </td><td> G3126 </td></tr><tr><td> Glycine </td><td> Acros Organics </td><td> 220911000 </td></tr><tr><td> L-Histidin </td><td> Sigma-Adrich </td><td> H8000 </td></tr><tr><td> L-Isoleucin </td><td> Sigma-Aldrich </td><td> I2752 </td></tr><tr><td> L-Leucin </td><td> Sigma-Aldrich </td><td> L8000 </td></tr><tr><td> L (+)-Lysin-Monohydrochlorid </td><td> Acros Organics </td><td> 125222500 </td></tr><tr><td> L-Methionin </td><td> Sigma-Aldrich </td><td> M9625 </td></tr><tr><td> L-Phenylalanin </td><td> Acros Organics </td><td> 130310250 </td></tr><tr><td> L-Prolin </td><td> Sigma-Aldrich </td><td> P0380 </td></tr><tr><td> L-Serin </td><td> Acros Organics </td><td> 132660250 </td></tr><tr><td> L-Threonin </td><td> Acros Organics </td><td> 138930250 </td></tr><tr><td> L (-)-Tryptophan </td><td> Acros Organics </td><td> 140590250 </td></tr><tr><td> L-Tyrosin </td><td> Acros Organics </td><td> 140641000 </td></tr><tr><td> L-Valin </td><td> Sigma-Aldrich </td><td> V0500 </td></tr><tr><td> Diethylentriaminpentaessigsäure </td><td> Sigma-Aldrich </td><td> 32318 </td></tr><tr><td> NaCl </td><td> Fisher Scientific </td><td> S/3160/60 </td></tr><tr><td> KCl </td><td> BDH </td><td> BDH0258 </td></tr><tr><td&gt; KOH </td><td> BDH </td><td> BDH0262 </td></tr><tr><td> Eigelb-Emulsion </td><td> Sigma-Aldrich </td><td> 17148 </td></tr><tr><td> ME 2 Membran-Vakuumpumpe </td><td> Vacuubrand </td><td> 696126 </td></tr><tr><td> Steritop Filter (Porengröße: 0,22 um, Hals-Größe: 45 mm) </td><td> Millipore </td><td> SCGPT10RE </td></tr><tr><td> Luria-Bertani-Medium </td><td> Sigma </td><td> L2897 </td></tr><tr><td> 96-Well-Mikrotiterplatten </td><td> Sarstedt </td><td> 82,1581 </td></tr><tr><td> 24-Well-Gewebekultur-behandelten Platten </td><td> Iwaki </td><td> 3820-024 </td></tr><tr><td> CampyGen Gaserzeugung Packs </td><td> Oxoid </td><td> CN0025 </td></tr><tr><td> Microaerophilic Kammer </td><td> Oxoid </td><td> HP0011 </td></tr><tr><td> Tobramycin Sulfat </td><td> Sigma-Aldrich </td><td> T1783 </td></tr><tr><td> Cellulase von Aspergillus niger </td><td> Sigma-Aldrich </td><td> 22178 </td></tr><tr><td> Resazurin </td><td> Sigma-Aldrich </td><td> 199303 </td></tr><tr><td> Citrate.H 2 0 </td><td> BDH </td><td> BDH0288 </td></tr><tr><td> Fluostar Omega Mikroplatten-Reader </td><td> BMG-Labtech </td><td> SPECTROstar Omega </td></tr></tbody></table>

use of artificial sputum medium to test antibiotic efficacy against pseudomonas aeruginosa in conditions more relevant to the cystic fibrosis lung

Es wächst die Besorgnis über die Relevanz der in vitro antimikrobielle Empfindlichkeitstests wann Isolate von P. angewendet aeruginosa von zystischer Fibrose (CF)-Patienten. Bestehende Methoden beruhen auf einzelne oder wenige Isolate wachsen aerob und planktonically. Vorgegebenen cut-offs werden verwendet, um festzulegen, ob die Bakterien empfindlich oder resistent gegenüber einem bestimmten Antibiotikum 1 sind. Doch während chronische Lungeninfektionen bei CF, P. aeruginosa in Biofilmen Populationen existieren und es gibt Hinweise, dass die Umwelt weitgehend mikroaerophile 2 ist. Der extreme Unterschied zwischen den Bedingungen in Bakterien in der Lunge und denen während der diagnostischen Tests haben Zweifel an der Zuverlässigkeit und sogar Relevanz dieser Tests 3 genannt. Künstliche Sputum Medium (ASM) ein Kulturmedium, das die Komponenten des CF-Patienten Sputum, einschließlich Aminosäuren, Mucin und freie DNA. S. aeruginosa &lt;/ Em&gt; Wachstum im ASM Mimik Wachstum während der CF-Infektionen, mit der Bildung von Selbst-Aggregation Biofilmstruktur und Bevölkerung Divergenz 4,5,6. Das Ziel dieser Studie war es, eine Mikrotiterplatte Assay gegen antimikrobielle Empfindlichkeit von P. studieren entwickeln aeruginosa auf das Wachstum in ASM, die für beide mikroaerophile und aeroben Bedingungen beruht. Ein ASM-Assay wurde in einem Mikrotiterplatten-Format entwickelt. P. aeruginosa Biofilme wurden für 3 Tage vor der Inkubation mit antimikrobieller Mittel zu entwickeln in verschiedenen Konzentrationen für 24 Stunden. Nach Biofilm Störung, die Lebensfähigkeit der Zellen wurde durch Färbung mit Resazurin gemessen. Dieser Assay wurde verwendet, um die sessile Zelle minimale Hemmkonzentration (SMIC) von Tobramycin für 15 verschiedene P. ermitteln aeruginosa-Isolaten unter aeroben und mikroaerophilen Bedingungen und SMIC-Werte wurden mit denen mit Standard-Brühe Wachstum verglichen. Zwar gab eseinige Hinweise für eine erhöhte MHK-Werte für Isolate in ASM gewachsen, wenn ihre planktonischen Gegenstücken verglichen wurden die grössten Unterschiede zu Bakterien in mikroaerophilen Bedingungen, die eine wesentlich erhöhte Resistenz zeigte, bis zu einer&gt; 128 fache gegenüber Tobramycin in der ASM-System getestet gefunden, wenn gegenüber Assays unter aeroben Bedingungen durchgeführt. Der fehlende Zusammenhang zwischen Anfälligkeit aktuellen Prüfmethoden und dem klinischen Ergebnis der Gültigkeit der aktuellen Methoden 3 in Frage gestellt. Mehrere in vitro-Modelle wurden bereits auf S. Studie verwendet aeruginosa Biofilm 7, 8. Allerdings beruhen diese Methoden auf Oberfläche befestigt Biofilmen, während die ASM-Biofilme, die auf der CF-Lunge 9 beobachtet ähneln. Darüber hinaus hat verringert Sauerstoffkonzentration im Schleim wurde gezeigt, dass das Verhalten von P. verändern aeruginosa 2 und beeinflussen Antibiotikaempfindlichkeit 10. Deshalb using ASM unter mikroaerophilen Bedingungen können vorsehen, eine realistischere Umgebung, in der Empfindlichkeit gegenüber antimikrobiellen Mitteln zu studieren.

Watch this Scientific Journal Video about Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung at JoVE.com

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

Aktuelle Diagnostik antimikrobiellen Empfindlichkeitsprüfung beruht auf der Plankton-Wachstum von Isolaten in nährstoffreichen, aeroben Bedingungen. Hier beschäftigen wir eine Alternative künstliche Sputum mittel-bis Antimikrobielle Empfindlichkeit von Pseudomonas aeruginosa Biofilmen unter aeroben und mikroaerophilen Bedingungen mehr repräsentativ für die Mukoviszidose-Lunge zu untersuchen.

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 ...

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Research

JoVE Journal

Immunology and Infection

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

32.5K Aufrufe.  University of Liverpool. Aktuelle Diagnostik antimikrobiellen Empfindlichkeitsprüfung beruht auf der Plankton-Wachstum von Isolaten in nährstoffreichen, aeroben Bedingungen. Hier beschäftigen wir eine Alternative künstliche Sputum mittel-bis Antimikrobielle Empfindlichkeit von Pseudomonas aeruginosa Biofilmen unter aeroben und mikroaerophilen Bedingungen mehr repräsentativ für die Mukoviszidose-Lunge zu untersuchen.

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

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 ...

Forschung

Immunologie und Infektion

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 ...

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 ...

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 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. ...

A Robust Pneumonia Model in Immunocompetent Rodents to Evaluate Antibacterial Efficacy against S. pneumoniae, H. influenzae, K. pneumoniae, P. aeruginosa or A. baumannii

Efficacy of candidate antibacterial treatments must be demonstrated in animal models of infection as part of the discovery and development process, preferably in models which mimic the intended clinical indication. A method for inducing robust lung infections in immunocompetent rats and mice is described which allows for the assessment of treatments in a model of serious pneumonia caused by S. pneumoniae, H. influenzae, P. aeruginosa, K. pneumoniae or A. baumannii. Animals are anesthetized, and an agar-based inoculum is deposited deep into the lung via nonsurgical intratracheal intubation. The resulting infection is consistent, reproducible, and stable for at least 48 h and up to 96 h for most isolates. Studies with marketed antibacterials have demonstrated good correlation between in vivo efficacy and in vitro susceptibility, and concordance between pharmacokinetic/pharmacodynamic targets determined in this model and clinically accepted targets has been observed. Although there is an initial time investment when learning the technique, it can be performed quickly and efficiently once proficiency is achieved. Benefits of the model include elimination of the neutropenic requirement, increased robustness and reproducibility, ability to study more pathogens and isolates, improved flexibility in study design and establishment of a challenging infection in an immunocompetent host.

A Robust Pneumonia Model in Immunocompetent Rodents to Evaluate Antibacterial Efficacy against S. pneumoniae, H. influenzae, K. pneumoniae, P. aeruginosa or  A. baumannii

A method for establishing lung infections in immunocompetent rodents is described. With proficiency, this method can be performed quickly and easily to induce stable infection with many isolates of S. pneumoniae, H. influenzae, P. aeruginosa, K. pneumoniae and A. baumannii.

A Robust Pneumonia Modell in intaktem Immunsystem Nagetiere zur Bewertung antibakterieller Wirksamkeit gegen<i&gt; S. pneumoniae</i&gt;,<i&gt; H. influenzae</i&gt;,<i&gt; K. pneumoniae</i&gt;,<em&gt; P. aeruginosa</em&gt; oder<em&gt; A. baumannii</em

Efficacy of candidate antibacterial treatments must be demonstrated in animal models of infection as part of the discovery and development process, ...

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.

Ein IL-8 transientes transgenisiertes Mausmodell für die<em&gt; In vivo</em&gt; Langzeitüberwachung von entzündlichen Reaktionen

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

A Method to Test the Efficacy of Handwashing for the Removal of Emerging Infectious Pathogens

Handwashing is widely recommended to prevent infectious disease transmission. However, little comparable evidence exists on the efficacy of handwashing methods in general. Additionally, little evidence exists comparing handwashing methods to determine which are most efficacious at removing infectious pathogens. Research is needed to provide evidence for the different approaches to handwashing that may be employed during infectious disease outbreaks. Here, a laboratory method to assess the efficacy of handwashing methods at removing microorganisms from hands and their persistence in rinse water is described. Volunteers' hands are first spiked with the test organism and then washed with each handwashing method of interest. Generally, surrogate microorganisms are used to protect human subjects from disease. The number of organisms remaining on volunteers' hands after washing is tested using a modified "glove juice" method: the hands are placed in gloves with an eluent and are scrubbed to suspend the microorganisms and make them available for analysis by membrane filtration (bacteria) or plaque assay (viruses/bacteriophages). Rinse water produced from the handwashing is directly collected for analysis. Handwashing efficacy is quantified by comparing the log reduction value between samples taken after handwashing to samples with no handwashing. Rinse water persistence is quantified by comparing rinse water samples from various handwashing methods to samples collected after handwashing with just water. While this method is limited by the need to use surrogate organisms to preserve the safety of human volunteers, it captures aspects of handwashing that are difficult to replicate in an in vitro study and fills research gaps on handwashing efficacy and the persistence of infectious organisms in rinse water.

Handwashing is widely recommended to prevent infectious disease transmission. However, there is little evidence on which handwashing methods are most efficacious at removing infectious disease pathogens. We developed a method to assess the efficacy of handwashing methods at removing microorganisms.

Eine Methode, um die Wirksamkeit der Handwäsche für die Entfernung von auftauchenden infektiösen Pathogenen zu testen

Handwashing is widely recommended to prevent infectious disease transmission. However, little comparable evidence exists on the efficacy of handwashing ...

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 ...

Phage Therapy Application to Counteract Pseudomonas aeruginosa Infection in Cystic Fibrosis Zebrafish Embryos

Antimicrobial resistance, a major consequence of diagnostic uncertainty and antimicrobial overprescription, is an increasingly recognized cause of severe infections, complications, and mortality worldwide with a huge impact on our society and on the health system. In particular, patients with compromised immune systems or pre-existing and chronic pathologies, such as cystic fibrosis (CF), are subjected to frequent antibiotic treatments to control the infections with the appearance and diffusion of multidrug resistant isolates. Therefore, there is an urgent need to address alternative therapies to counteract bacterial infections. Use of bacteriophages, the natural enemies of bacteria, can be a possible solution. The protocol detailed in this work describes the application of phage therapy against Pseudomonas aeruginosa infection in CF zebrafish embryos. Zebrafish embryos were infected with P. aeruginosa to demonstrate that phage therapy is effective against P. aeruginosa infections as it reduces lethality, bacterial burden and pro-inflammatory immune response in CF embryos.

Phage Therapy Application to Counteract Pseudomonas aeruginosa Infection in Cystic Fibrosis Zebrafish Embryos

Presented here is a protocol for Pseudomonas aeruginosa infection and phage therapy application in cystic fibrosis (CF) zebrafish embryos.

Phage Therapy Anwendung gegen Pseudomonas aeruginosa Infektion bei Mukoviszidose Zebrafisch Embryonen

Antimicrobial resistance, a major consequence of diagnostic uncertainty and antimicrobial overprescription, is an increasingly recognized cause of severe ...

Visualization of Pseudomonas aeruginosa within the Sputum of Cystic Fibrosis Patients

Early detection and eradication of Pseudomonas aeruginosa within the lungs of cystic fibrosis patients can reduce the chance of developing chronic infection. The development of chronic P. aeruginosa infections is associated with a decline in lung function and increased morbidity. Therefore, there is a great interest in elucidating the reasons for the failure to eradicate P. aeruginosa with antibiotic therapy which occurs in approximately 10-40% of pediatric patients. One of many factors that can affect host clearance of P. aeruginosa and antibiotic susceptibility is variations in spatial organization (such as aggregation or biofilm formation) and polysaccharide production. Therefore, we were interested in visualizing the in situ characteristics of P. aeruginosa within the sputum of CF patients. A tissue clearing technique was applied to sputum samples after embedding the samples into a hydrogel matrix to retain the 3D structures relative to host cells. After tissue clearing, fluorescent labels and dyes were added to allow visualization. Fluorescence in situ hybridization was performed for the visualization of bacterial cells, binding of fluorescently labeled anti-Psl-antibodies for the visualization of the exopolysaccharide and DAPI staining to stain host cells to obtain structural insight. These methods allowed for the high-resolution imaging of P. aeruginosa within the sputum of CF patients via confocal laser scanning microscopy.

Visualization of Pseudomonas aeruginosa within the Sputum of Cystic Fibrosis Patients

This protocol provides methods for visualization of bacterial cells and polysaccharide synthesis locus (Psl) polysaccharide within the sputum of cystic fibrosis patients.