TB erkennt ein Forschungsstipendium von Cystic Fibrosis Kanada.

Beachten Sie, dass Forschungsethik Board (REB) erforderlich zu sammeln und zu speichern, Sputum-Proben von menschlichen Probanden. Diese Studien wurden von der Klinik zugelassen für kranke Kinder REB # 1000019444. 1. Vorbereiten CF Sputum-Proben <ol><li> Sammeln Sie Sputum Probe von Patienten während der Routine Abfragen der Mukoviszidose-Klinik und auf Eis halten. </li><li> Transport Sputumprobe auf Eis innerhalb der ersten Stunde nach der Entnahme, zum Forschungslabor, die Verarbeitung zu unterziehen. </li></ol> 2. Sputum Verarbeitung <ol><li> Aufzeichnen des Volumens des Sputum-Probe erhalten. Fügen Phosphat - gepufferter Salzlösung (PBS) , um das Volumen der Probe auf 2x (dh 2 Teile PBS, 1 Teil Probe). </li><li> Mischen Sie die Probe gut mit einer Transferpipette. Vortex die Probe auf die höchste Einstellung für 1 min vollständig zu mischen. </li><li> Aliquot 1 ml der obigen Mischung in die entsprechende Anzahl von 1,5 ml Mikrozentrifugenröhrchen und Spin bei 5000 × g nach unten für20 min bei 4 ° C. </li><li> Nach der Zentrifugation den Überstand und das Pellet verwerfen. </li><li> Filter sterilisieren den Überstand durch ein 0,22 um Filter und sammeln sich in einem sauberen Reaktionsgefäß. HINWEIS: Sterility des Filtrats durch Ausplattieren auf LB-Agar getestet und flüssigen Medien Animpfen. </li><li> Speichern von Sputum Überstand bei -80 ° C für zukünftige Verwendung. HINWEIS: Sputum von mehreren Patienten können auch nach der Filtration gesammelt werden. </li><li> Vor der Verwendung verdünnt Sputum Filtrat 1/10 v / v (100 &amp; mgr; l Sputum, 900 ul Medium) in gewünschte Medien. HINWEIS: Hier Standard LB-Medium (LB) Medien verwendet wurde. </li></ol> 3. Kammerdeckgläser Verfahren zur Bildung von Biofilmen <ol><li> Wachsen Bakteriumisolat von Interesse über Nacht in das gewünschte Medium bei 37 ° C unter Schütteln (200 rpm). Hinweis: Eine Anzahl verschiedener Bakterien verwendet wurden, einschließlich der klinischen Isolate von Pseudomonas aeruginosa, Staphylococcus aureus,Burkholderia cepacia und Achromobacter xylosoxidans. Wahl der Medien hängt von Stämmen und Bedingungen von Interesse kann jedoch LB-Medien für erste Experimente verwendet werden. </li><li> Von Übernachtkultur, legen 40 &amp; mgr; l der Kultur in die 4 ml frisches Medium wachsen und für 3-4 h bei 37 ° C unter Schütteln (200 Upm) , um eine Kultur mit einer optischen Dichte bei 600 nm (OD 600) von ungefähr 0,5 zu erhalten , -0.6. </li><li> Verdünnen Sie die Kultur aus Schritt 3,2 bis 1/5 in das gewünschte Medium mit 10% Sputum Filtrat oder ohne Auswurf Filtrat (als Kontrolle). Andere Konzentration von Sputum Filtrat kann (dh 50% oder 100%) getestet werden. </li><li> Verwenden Sie 200 ul der Verdünnung Vertiefungen Schiebekammern auf Saatgut. </li><li> Lassen Bakterien für 4 Stunden bei 37 ° C zu befestigen ohne Schütteln. </li><li> Nach 4 Stunden, um die Medien zu entfernen und sanft den Biofilm mit 1x frisches Medium waschen. Ersetzen mit 200 &amp; mgr; l frisches Medium. Hinweis: Um die Auswirkungen von Sputum auf dem Biofilm studieren, die fresh Medien sollten Sputum Überstand enthalten. </li><li> Erlauben Biofilmen für gewünschte Zeitdauer bei 37 ° C zu wachsen, ohne Schütteln, Medien alle 12 Stunden ersetzt, ohne Waschen, bis die Zeit für die Mikroskopie. ANMERKUNG: Um die Wirkung von Sputum Überstände auf Biofilm antibiotischen Empfänglichkeit untersuchen, werden Antibiotika zum Medium hinzugefügt folgenden 24 h Biofilmwachstum und werden in den Medien bis zum Anfärben und Abbildung von Biofilmen gehalten. </li></ol> 4. Die Färbung Biofilme und konfokale Mikroskopie <ol><li> gewünschte Wachstumszeit verfolgt (24-48 h funktioniert am besten), entfernen Sie Medien aus der Kammer Brunnen und sanft jede Kammer zweimal mit 300 ul sterilem PBS waschen. </li><li> Bereiten Sie Färbemischung für Biofilm durch Mischen von 1 ul jeder Farbstoff (sofern in der Lebensfähigkeit Kit) für jeden ml Lösung benötigt. Machen Farbstoff in Wasser oder Media-Lösung. HINWEIS: Das Wasser wird vom Hersteller empfohlen. </li><li> Mit 200 &amp; mgr; l der Farbstoffmischung in jede Vertiefung chambered coverglass und für 45 min bei Raumtemperatur im Dunkeln inkubieren. </li><li> Entfernen Färbung Mischung aus Kammern und waschen Sie jede Vertiefung mit 300 ul sterilem PBS. Entfernen PBS und ersetzen mit frischem Wasser oder Medien. </li><li> Fahren Sie mit der Visualisierung von Biofilmen durch konfokale Mikroskopie. </li></ol> 5. Visualizing Biofilme mit konfokale Mikroskopie <ol><li> Lesen gefärbten Biofilmen in Kammern unmittelbar nach dem Färben (innerhalb von 1 h). Minimieren Verzögerung in Visualisierung der Objektträger durch Anfärben August 1-02-well Kammern zu einer Zeit. </li><li> Führen Bildgebung mit konfokalen Mikroskop mit Lasern für die Anregung und Filtersätze für die Erfassung. HINWEIS: Hier wird die sich drehende Scheibe konfokalen System mit spektraler borealis Laser (Grün: 491nm, Rot: 561 nm) zur Anregung verwendet wurden. Emissionsfiltersätze von 515/40 und 624/40 wurden verwendet, um die Flecken aus dem Biofilm Lebensfähigkeit Kit zu visualisieren. </li><li> Nehmen Sie Bilder mit einem 25X Wasser Ziel auf konfokalen Mikroskop mit Kamera. </li><li&gt; Verwenden Z-Stacks des Biofilms zu modellieren. Nehmen Sie Bilder jede 0,5-1 &amp; mgr; m ausgehend von der ersten in-Fokusebene auf den letzten im Fokus befindlichen Rahmen des Biofilms (typischerweise Spanning 30-80 &amp; mgr; m für 48 Stunden Biofilme) </li><li> Nehmen Sie 3-5 Bilder von jedem gut. HINWEIS: So für einen 8 gut chambered Abdeckglas, 24-40 Bilder erzeugt werden. </li><li> Speichern Sie die Bilder für die Analyse. HINWEIS: Bilder gespeichert werden sollen als OME-TIFF - Dateien mit COMSTAT 18,19 analysiert werden. Hinweise zur Biofilmbildanalyse kann bei http://www.comstat.dk/ finden. Sobald Bilder importiert werden, können Parameter wie die durchschnittliche Dicke, Biomasse und Flächendeckung für jeden Kanal (rot und grün) analysiert werden. </li></ol>

<ol><li>Beaudoin, T., Waters, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Infections+with+biofilm+formation%3A+selection+of+antimicrobials+and+role+of+prolonged+antibiotic+therapy%5BTitle%5D)+AND+%22Pediatr.Infect.Dis.J%22%5BJournal%5D)">Infections with biofilm formation: selection of antimicrobials and role of prolonged antibiotic therapy.</a> Pediatr.Infect.Dis.J. , (2016).</li>
<li>Donlan, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Biofilms%3A+microbial+life+on+surfaces%5BTitle%5D)+AND+%22Emerg.Infect.Dis%22%5BJournal%5D)">Biofilms: microbial life on surfaces.</a> Emerg.Infect.Dis. 8 (9), 881-890 (2002).</li>
<li>Hobley, L., Harkins, C., MacPhee, C. E., Stanley-Wall, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Giving+structure+to+the+biofilm+matrix%3A+an+overview+of+individual+strategies+and+emerging+common+themes%5BTitle%5D)+AND+%22FEMS+Microbiol.Rev%22%5BJournal%5D)">Giving structure to the biofilm matrix: an overview of individual strategies and emerging common themes.</a> FEMS Microbiol.Rev. 39 (5), 649-669 (2015).</li>
<li>Katharios-Lanwermeyer, S., Xi, C., Jakubovics, N. S., Rickard, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mini-review%3A+Microbial+coaggregation%3A+ubiquity+and+implications+for+biofilm+development%5BTitle%5D)+AND+%22Biofouling%22%5BJournal%5D)">Mini-review: Microbial coaggregation: ubiquity and implications for biofilm development.</a> Biofouling. 30 (10), 1235-1251 (2014).</li>
<li>Donlan, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Biofilm+formation%3A+a+clinically+relevant+microbiological+process%5BTitle%5D)+AND+%22Clin.Infect.Dis%22%5BJournal%5D)">Biofilm formation: a clinically relevant microbiological process.</a> Clin.Infect.Dis. 33 (8), 1387-1392 (2001).</li>
<li>Bjarnsholt, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+in+vivo+biofilm%5BTitle%5D)+AND+%22Trends+Microbiol%22%5BJournal%5D)">The in vivo biofilm.</a> Trends Microbiol. 21 (9), 466-474 (2013).</li>
<li>Mah, T. F., Pitts, B., Pellock, B., Walker, G. C., Stewart, P. S., O'Toole, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+genetic+basis+for+Pseudomonas+aeruginosa+biofilm+antibiotic+resistance%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">A genetic basis for Pseudomonas aeruginosa biofilm antibiotic resistance.</a> Nature. 426 (6964), 306-310 (2003).</li>
<li>Mah, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Biofilm-specific+antibiotic+resistance%5BTitle%5D)+AND+%22Future+Microbiol%22%5BJournal%5D)">Biofilm-specific antibiotic resistance.</a> Future Microbiol. 7 (9), 1061-1072 (2012).</li>
<li>Beaudoin, T., Zhang, L., Hinz, A. J., Parr, C. J., Mah, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+biofilm-specific+antibiotic+resistance+gene+ndvB+is+important+for+expression+of+ethanol+oxidation+genes+in+Pseudomonas+aeruginosa+biofilms%5BTitle%5D)+AND+%22J.+Bacteriol%22%5BJournal%5D)">The biofilm-specific antibiotic resistance gene ndvB is important for expression of ethanol oxidation genes in Pseudomonas aeruginosa biofilms.</a> J. Bacteriol. 194 (12), 3128-3136 (2012).</li>
<li>Beaudoin, T., Aaron, S. D., Giesbrecht-Lewis, T., Vandemheen, K., Mah, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Characterization+of+clonal+strains+of+Pseudomonas+aeruginosa+isolated+from+cystic+fibrosis+patients+in+Ontario%2C+Canada%5BTitle%5D)+AND+%22Can.+J.+Microbiol%22%5BJournal%5D)">Characterization of clonal strains of Pseudomonas aeruginosa isolated from cystic fibrosis patients in Ontario, Canada.</a> Can. J. Microbiol. 56 (7), 548-557 (2010).</li>
<li>Vidya, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chronic+infection+phenotypes+of+Pseudomonas+aeruginosa+are+associated+with+failure+of+eradication+in+children+with+cystic+fibrosis%5BTitle%5D)+AND+%22Eur.J.Clin.Microbiol.Infect.Dis%22%5BJournal%5D)">Chronic infection phenotypes of Pseudomonas aeruginosa are associated with failure of eradication in children with cystic fibrosis.</a> Eur.J.Clin.Microbiol.Infect.Dis. , (2015).</li>
<li>Beaudoin, T., Lafayette, S., Nguyen, D., Rousseau, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mucoid+Pseudomonas+aeruginosa+caused+by+mucA+mutations+result+in+activation+of+TLR2+in+addition+to+TLR5+in+airway+epithelial+cells%5BTitle%5D)+AND+%22Biochem.Biophys.Res.Commun%22%5BJournal%5D)">Mucoid Pseudomonas aeruginosa caused by mucA mutations result in activation of TLR2 in addition to TLR5 in airway epithelial cells.</a> Biochem.Biophys.Res.Commun. 428 (1), 150-154 (2012).</li>
<li>Beaudoin, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+level+of+p38alpha+mitogen-activated+protein+kinase+activation+in+airway+epithelial+cells+determines+the+onset+of+innate+immune+responses+to+planktonic+and+biofilm+Pseudomonas+aeruginosa%5BTitle%5D)+AND+%22J.Infect.Dis%22%5BJournal%5D)">The level of p38alpha mitogen-activated protein kinase activation in airway epithelial cells determines the onset of innate immune responses to planktonic and biofilm Pseudomonas aeruginosa.</a> J.Infect.Dis. 207 (10), 1544-1555 (2013).</li>
<li>LaFayette, S. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cystic+fibrosis-adapted+quorum+sensing+mutants+cause+hyperinflammatory+responses%5BTitle%5D)+AND+%22Sci.Adv%22%5BJournal%5D)">Cystic fibrosis-adapted quorum sensing mutants cause hyperinflammatory responses.</a> Sci.Adv. 1 (6), e1500199(2015).</li>
<li>Staudinger, B. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Conditions+associated+with+the+cystic+fibrosis+defect+promote+chronic+Pseudomonas+aeruginosa+infection%5BTitle%5D)+AND+%22Am.J.Respir.Crit.Care+Med%22%5BJournal%5D)">Conditions associated with the cystic fibrosis defect promote chronic Pseudomonas aeruginosa infection.</a> Am.J.Respir.Crit.Care Med. 189 (7), 812-824 (2014).</li>
<li>Kennedy, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Activity+of+Tobramycin+against+Cystic+Fibrosis+Isolates+of+Burkholderia+cepacia+Complex+Grown+as+Biofilms%5BTitle%5D)+AND+%22Antimicrob.Agents+Chemother%22%5BJournal%5D)">Activity of Tobramycin against Cystic Fibrosis Isolates of Burkholderia cepacia Complex Grown as Biofilms.</a> Antimicrob.Agents Chemother. 60 (1), 348-355 (2015).</li>
<li>Tom, S. K., Yau, Y. C., Beaudoin, T., LiPuma, J. J., Waters, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effect+of+High-Dose+Antimicrobials+on+Biofilm+Growth+of+Achromobacter+Species+Isolated+from+Cystic+Fibrosis+Patients%5BTitle%5D)+AND+%22Antimicrob.Agents+Chemother%22%5BJournal%5D)">Effect of High-Dose Antimicrobials on Biofilm Growth of Achromobacter Species Isolated from Cystic Fibrosis Patients.</a> Antimicrob.Agents Chemother. 60 (1), 650-652 (2015).</li>
<li>Heydorn, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quantification+of+biofilm+structures+by+the+novel+computer+program+COMSTAT%5BTitle%5D)+AND+%22Microbiology%22%5BJournal%5D)">Quantification of biofilm structures by the novel computer program COMSTAT.</a> Microbiology. 146 (Pt 10), 2395-2407 (2000).</li>
<li>Vorregaard, M. Comstat2 - a modern 3D image analysis environment for biofilms
 . <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aVorregaard%2C+M+%22Informatics+and+Mathematical+Modelling%22">Informatics and Mathematical Modelling</a>. , Technical University of Denmark. Kongens Lyngby, Denmark. (2008).</li>
<li>Jurcisek, J. A., Dickson, A. C., Bruggeman, M. E., Bakaletz, L. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vitro+Biofilm+Formation+in+an+8-well+Chamber+Slide%5BTitle%5D)+AND+%22J.+Vis.+Exp%22%5BJournal%5D)">In vitro Biofilm Formation in an 8-well Chamber Slide.</a> J. Vis. Exp. (47), e2481(2011).</li>
</ol>

Die hierin beschriebenen Verfahren ermöglichen die Visualisierung von bakteriellen Biofilmen in Gegenwart von exogenen Produkten gewachsen. Es überrascht nicht, die Herstellung der exoproducts ist von Bedeutung, wenn diese Art von System. Zum Beispiel Dithiothreitol (DTT), wird häufig auf die menschliche Sputum-Proben verwendet, um die Proben helfen verflüssigen. Jedoch allein der Wirkung von DTT Biofilmentwicklung verringern kann und die Lebensfähigkeit (Daten nicht gezeigt). Somit sind geeignete Kontrollen für a...

Bakterielle Biofilme sind Gruppen von Mikroorganismen, die miteinander und umhüllt in einer selbst sezerniert Matrix gebunden sind. 1,2 Classically stellen sie Bakterien physisch an einen abiotischen oder biotischen Oberfläche unter den Bedingungen der Strömung gebildet angebracht. Biofilme sind auch gezeigt worden, unter statischen Bedingungen (keine Strömung) und distal von Oberflächen, beispielsweise an der Luft-Flüssigkeit-Grenzfläche der thermischen Pools oder Häutchen gebildet in Reagenzgläsern zu wachsen. Diese Biofilme sind seit langem in der Umwelt erkannt und sind eine große Nachteil für industrielle Prozesse, wie sie in Wasserbehältern oder in Rohren bilden können, was zu Biofouling, Korrosion und Verstopfung. 3,4 Biofilme sind auch im Gesundheitsbereich kritisch, da sie in Katheter bedingten Infektionen, Lungeninfektionen bei Mukoviszidose-Patienten, sowie in zahlreichen anderen Infektionen beteiligt haben gezeigt werden. 5,6 Eines der Markenzeichen von Biofilm - Infektionen ist die deerhöhte Empfindlichkeit von Bakterien gegen Antibiotika und die Beeinträchtigung der Clearance durch das angeborene Immunsystem. 7-9 Die gut untersucht, klinisch relevante Szenarien Biofilm-basierten Infektion beteiligt tritt bei Patienten mit zystischer Fibrose (CF), die chronisch infiziert sind , mit Pseudomonas aeruginosa Biofilme. P. aeruginosa kann eine Reihe von Änderungen bei Etablierung einer chronischen Infektion zu unterziehen , die es sehr schwierig zu behandeln , machen. 10,11 Biofilme können unterschiedlich angeborenen Immunität aktivieren und Entzündung fahren. 12-14 Da diese Infektionen zu einer erhöhten Morbidität und Mortalität bei CF - Patienten führen, ist es wichtig , Faktoren zu verstehen , die Biofilmbildung in diesem Zusammenhang beeinflussen können. Eine neue Studie schlägt vor , dass Host-Faktoren bei der Bildung von P. aeruginosa Biofilm Aggregate kritisch sind. 15 Diese Biofilme tragen zu einer reduzierten Empfindlichkeit gegenüber Antibiotika und Abwehrmechanismen des Wirts. Die Presenz von Wirt-abgeleiteten Faktoren, wie Neutrophilen-Elastase, sowie sekretierte Produkte von Mikroorganismen, die in der CF-Lunge, haben das Potential stark zu Biofilmbildung und Entwicklung modulieren. 16 Darüber hinaus interagieren Biofilmen mit dem Host - Expression zahlreicher Wege zu modulieren und Entzündungen auslösen. Während Hochdurchsatzverfahren, wie zum Beispiel die Standardkristallviolett-Assay, einige Informationen in Bezug auf die Biofilmverfahren zur Verfügung stellen kann, die Visualisierung des Biofilms in Reaktion auf diese Faktoren liefern weitergehende Informationen. In diesem Manuskript beschreiben wir ein Verfahren zur Faktoren aus dem Sputum von Patienten mit CF mit der Entwicklung von Biofilmen in vitro zu studieren. Dieses Verfahren ermöglicht eine schnelle Visualisierung von auf Sputum enthaltenden Wirtsfaktoren ausgesetzt Biofilmen Kommerzielle Biofilm Lebensfähigkeit Kit. Diese Technik kann verwendet werden, um visuell Veränderungen zu identifizieren, die während der Biofilmwachstum in Gegenwart von exogeno auftretenuns Produkte und stellt ein verbessertes Verfahren der Änderungen in Biofilmentwicklung unter verschiedenen Bedingungen zu untersuchen.

<table><tbody><tr><td>Lab-Tek II Chambered coverglass, #1.5 borosilicate, 8-well</td><td>Thermo Sicher Scientific</td><td>155409</td><td></td></tr><tr><td>Filmtracer Live/Dead Biofilm Viabilty Kit</td><td>Thermo Fisher Scientific</td><td>L10316</td><td></td></tr><tr><td>Blood agar plates</td><td>Thermo Fisher Scientific</td><td>R10215</td><td>Confirming viability via CFU counts or selecting colonies for innoculation</td></tr><tr><td>COMSTAT</td><td>Availble software online</td><td></td><td>COMSTAT is software to analyze biofilm images. Available www.comstat.dk&amp;nbsp;</td></tr><tr><td>Millers LB Broth</td><td>Thermo Fisher Scientific</td><td>12780-052</td><td>Standard media for overnight gowth/biofilm growth</td></tr><tr><td>Millex-GV Syringe Filters</td><td>Millipore</td><td>SLGV013SL</td><td>Filtering of sputum supernants</td></tr><tr><td>Phosphate Buffered Saline (Dulbecco A)</td><td>Oxoid</td><td>BR0014G</td><td>Washing of biofilm chambers after media removal</td></tr><tr><td>Zeiss AxioVert 200M</td><td>Carl Zeiss</td><td></td><td></td></tr><tr><td>Hamamatsu C9100-13 EM-CCD</td><td>QS Technologies Inc.</td><td></td><td></td></tr><tr><td>Spectral Borealis</td><td>Qs Technologies Inc.</td><td></td><td></td></tr><tr><td>Perkin Elmer Volocity</td><td>QS Technologies Inc.</td><td></td><td>Instructions for this software can be found at: http://cellularimaging.perkinelmer.com/pdfs/manuals/VolocityuserGuide.pdf</td></tr></tbody></table>

visualizing the effects of sputum on biofilm development using a chambered coverglass model

Biofilme bestehen aus Gruppen von in einem selbst sezerniert Matrix eingeschlossen Bakterien. Sie spielen eine wichtige Rolle in der industriellen Verschmutzung sowie in der Entwicklung und Persistenz von vielen gesundheitsbezogenen Infektionen. Eines der am besten beschrieben und untersucht Biofilmen in der menschlichen Erkrankung tritt bei chronischen Lungeninfektion von Mukoviszidose-Patienten. Wenn Biofilme im Rahmen des Host studieren, können viele Faktoren die Biofilmbildung und Entwicklung beeinflussen. Um zu ermitteln, wie Wirtsfaktoren die Bildung von Biofilmen und Entwicklung beeinflussen können, haben wir eine statische chambered Abdeckglas Methode Biofilmen in Gegenwart von Wirt abgeleiteten Faktoren in Form von Sputum Stände zu wachsen. Die Bakterien werden in die Kammern ausgesät und Sputum Filtrate ausgesetzt. mit einem handelsüblichen Biofilm Lebensfähigkeit Kit vor der konfokalen Mikroskopie und Analyse folgenden 48 h des Wachstums werden Biofilme gefärbt. Nach der Bildaufnahme können Biofilm Eigenschaften mit Hilfe Plattformen unterschiedlicher Software bewertet werden.Dieses Verfahren ermöglicht es uns, Schlüsseleigenschaften von Biofilmwachstum in Gegenwart verschiedener Substanzen einschließlich Antibiotika zu visualisieren.

Das Gesamtdesign des Experiments ist in Abbildung 1 dargestellt. Die Verwendung dieses Protokoll bietet eine bequeme Methode , die Veränderungen in Biofilmen für verschiedene Zeiträume (beispielsweise 24, 48 oder 72 Stunden) gezüchtet zu visualisieren. Wichtig ist, dass exogene Signale, wie Sputum Filtrate können hinzugefügt werden, um die Veränderungen in der Biofilmentwicklung zu visualisieren. Wie in Figur 2 zu sehen ist , kann die Geg...

Watch this Scientific Journal Video about Visualizing the Effects of Sputum on Biofilm Development Using a Chambered Coverglass Model at JoVE.com

Visualizing the Effects of Sputum on Biofilm Development Using a Chambered Coverglass Model

This protocol describes the visualization of biofilm development following exposure to host-factors using a slide chamber model. This model allows for direct visualization of biofilm development as well as analysis of biofilm parameters using computer software programs.

Visualisierung der Auswirkungen von Sputum auf Biofilm-Entwicklung eines Modells mit Kammerdeckgläser

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the ...

visualizing-effects-sputum-on-biofilm-development-using-chambered

Research

JoVE Journal

Immunology and Infection

8.3K Aufrufe.  Hospital for Sick Children. This protocol describes the visualization of biofilm development following exposure to host-factors using a slide chamber model. This model allows for direct visualization of biofilm development as well as analysis of biofilm parameters using computer software programs.

Serie: Visualizing the Effects of Sputum on Biofilm Development Using a Chambered Coverglass 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

In vitro Biofilm Formation in an 8-well Chamber Slide

The chronic nature of many diseases is attributed to the formation of bacterial biofilms which are recalcitrant to traditional antibiotic therapy. Biofilms are community-associated bacteria attached to a surface and encased in a matrix. The role of the extracellular matrix is multifaceted, including facilitating nutrient acquisition, and offers significant protection against environmental stresses (e.g. host immune responses). In an effort to acquire a better understanding as to how the bacteria within a biofilm respond to environmental stresses we have used a protocol wherein we visualize bacterial biofilms which have formed in an 8-well chamber slide. The biofilms were stained with the BacLight Live/Dead stain and examined using a confocal microscope to characterize the relative biofilm size, and structure under varying incubation conditions. Z-stack images were collected via confocal microscopy and analyzed by COMSTAT. This protocol can be used to help elucidate the mechanism and kinetics by which biofilms form, as well as identify components that are important to biofilm structure and stability.

In vitro Biofilm Formation in an 8-well Chamber Slide

This article describes the procedure for the formation and visualization of a bacterial biofilm grown within an 8-well chamber slide

In-vitro-Bildung von Biofilmen in einem 8-well Chamber Slide

The chronic nature of many diseases is attributed to the formation of bacterial biofilms which are recalcitrant to traditional antibiotic therapy. ...

Growth of Mycobacterium tuberculosis Biofilms

Mycobacterium tuberculosis, the etiologic agent of human tuberculosis, has an extraordinary ability to survive against environmental stresses including antibiotics. Although stress tolerance of M. tuberculosis is one of the likely contributors to the 6-month long chemotherapy of tuberculosis 1, the molecular mechanisms underlying this characteristic phenotype of the pathogen remain unclear. Many microbial species have evolved to survive in stressful environments by self-assembling in highly organized, surface attached, and matrix encapsulated structures called biofilms 2-4. Growth in communities appears to be a preferred survival strategy of microbes, and is achieved through genetic components that regulate surface attachment, intercellular communications, and synthesis of extracellular polymeric substances (EPS) 5,6. The tolerance to environmental stress is likely facilitated by EPS, and perhaps by the physiological adaptation of individual bacilli to heterogeneous microenvironments within the complex architecture of biofilms 7.
In a series of recent papers we established that M. tuberculosis and Mycobacterium smegmatis have a strong propensity to grow in organized multicellular structures, called biofilms, which can tolerate more than 50 times the minimal inhibitory concentrations of the anti-tuberculosis drugs isoniazid and rifampicin 8-10. M. tuberculosis, however, intriguingly requires specific conditions to form mature biofilms, in particular 9:1 ratio of headspace: media as well as limited exchange of air with the atmosphere 9. Requirements of specialized environmental conditions could possibly be linked to the fact that M. tuberculosis is an obligate human pathogen and thus has adapted to tissue environments. In this publication we demonstrate methods for culturing M. tuberculosis biofilms in a bottle and a 12-well plate format, which is convenient for bacteriological as well as genetic studies. We have described the protocol for an attenuated strain of M. tuberculosis, mc27000, with deletion in the two loci, panCD and RD1, that are critical for in vivo growth of the pathogen 9. This strain can be safely used in a BSL-2 containment for understanding the basic biology of the tuberculosis pathogen thus avoiding the requirement of an expensive BSL-3 facility. The method can be extended, with appropriate modification in media, to grow biofilm of other culturable mycobacterial species.
Overall, a uniform protocol of culturing mycobacterial biofilms will help the investigators interested in studying the basic resilient characteristics of mycobacteria. In addition, a clear and concise method of growing mycobacterial biofilms will also help the clinical and pharmaceutical investigators to test the efficacy of a potential drug.

Growth of Mycobacterium tuberculosis Biofilms

Mycobacterium tuberculosis forms drug tolerant biofilms when cultured in certain conditions. Here we describe methods for culturing M. tuberculosis biofilms and determining the frequency of drug tolerant persisters. These protocols will be useful for further studies into the mechanisms of drug tolerance in M. tuberculosis.

Wachstum von Mycobacterium tuberculosis Biofilme

Mycobacterium tuberculosis, the etiologic agent of human tuberculosis, has an extraordinary ability to survive against environmental stresses including ...

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 ...

Methodologies for Studying B. subtilis Biofilms as a Model for Characterizing Small Molecule Biofilm Inhibitors

This work assesses different methodologies to study the impact of small molecule biofilm inhibitors, such as D-amino acids, on the development and resilience of Bacillus subtilis biofilms. First, methods are presented that select for small molecule inhibitors with biofilm-specific targets in order to separate the effect of the small molecule inhibitors on planktonic growth from their effect on biofilm formation. Next, we focus on how inoculation conditions affect the sensitivity of multicellular, floating B. subtilis cultures to small molecule inhibitors. The results suggest that discrepancies in the reported effects of such inhibitors such as D-amino acids are due to inconsistent pre-culture conditions. Furthermore, a recently developed protocol is described for evaluating the contribution of small molecule treatments towards biofilm resistance to antibacterial substances. Lastly, scanning electron microscopy (SEM) techniques are presented to analyze the three-dimensional spatial arrangement of cells and their surrounding extracellular matrix in a B. subtilis biofilm. SEM facilitates insight into the three-dimensional biofilm architecture and the matrix texture. A combination of the methods described here can greatly assist the study of biofilm development in the presence and absence of biofilm inhibitors, and shed light on the mechanism of action of these inhibitors.

Methodologies for Studying B. subtilis Biofilms as a Model for Characterizing Small Molecule Biofilm Inhibitors

This study presents the development of reproducible methodologies to study biofilm inhibitors and their effects on Bacillus subtilis multicellularity.

Methodologien für das Studium<em&gt; B. subtilis</em&gt; Biofilme als Modell für kleine Molekül-Inhibitoren Biofilm Charakterisieren

This work assesses different methodologies to study the impact of small molecule biofilm inhibitors, such as D-amino acids, on the development and ...

The WinCF Model - An Inexpensive and Tractable Microcosm of a Mucus Plugged Bronchiole to Study the Microbiology of Lung Infections

Many chronic airway diseases result in mucus plugging of the airways. Lungs of an individual with cystic fibrosis are an exemplary case where their mucus-plugged bronchioles create a favorable habitat for microbial colonization. Various pathogens thrive in this environment interacting with each other and driving many of the symptoms associated with CF disease. Like any microbial community, the chemical conditions of their habitat have a significant impact on the community structure and dynamics. For example, different microorganisms thrive in differing levels of oxygen or other solute concentrations. This is also true in the CF lung, where oxygen concentrations are believed to drive community physiology and structure. The methods described here are designed to mimic the lung environment and grow pathogens in a manner more similar to that from which they cause disease. Manipulation of the chemical surroundings of these microbes is then used to study how the chemistry of lung infections governs its microbial ecology. The method, called the WinCF system, is based on artificial sputum medium and narrow capillary tubes meant to provide an oxygen gradient similar to that which exists in mucus-plugged bronchioles. Manipulating chemical conditions, such as the media pH of the sputum or antibiotics pressure, allows for visualization of the microbiological differences in those samples using colored indicators, watching for gas or biofilm production, or extracting and sequencing the nucleic acid contents of each sample.

The mucus plugged airways of cystic fibrosis (CF) patients are an ideal environment for microbial pathogens to thrive. The manuscript describes a novel method for studying the CF lung microbiome in an environment that mimics where they cause disease and how alterations of chemical conditions can drive microbial dynamics.

Das WinCF-Modell - Ein preiswerter und tragbarer Mikrokosmos eines Schleims verstopfte Bronchiole zur Untersuchung der Mikrobiologie von Lungeninfektionen

Many chronic airway diseases result in mucus plugging of the airways. Lungs of an individual with cystic fibrosis are an exemplary case where their ...

Quantifying the Effects of Antimicrobials on In vitro Biofilm Architecture using COMSTAT Software

Biofilms are aggregates of microorganisms that rely on a self-produced matrix of extracellular polymeric substance for protection and structural integrity. The nosocomial pathogen, Pseudomonas aeruginosa, is known to adopt a biofilm mode of growth, causing chronic pulmonary infection in patients with cystic fibrosis (CF). The computer program, COMSTAT, is a useful tool for quantifying antimicrobial-induced changes in P. aeruginosa biofilm architecture by extracting data from three-dimensional confocal images. However, standardized operation of the software is less commonly addressed, which is important for optimal reporting of biofilm behavior and cross-center comparison. Thus, the aim of this protocol is to provide a simple and reproducible framework for quantifying in vitro biofilm structures under varying antimicrobial conditions via COMSTAT. The technique is modeled using a CF P. aeruginosa isolate, grown in the form of biofilm replicates, and exposed to tobramycin and the anti-Psl monoclonal antibody, Psl0096. The step-by-step approach aims to reduce user ambiguity and minimize the chance of overlooking crucial image-processing steps. Specifically, the protocol emphasizes the elimination of subjective variations associated with the manual operation of COMSTAT, including image segmentation and the selection of appropriate quantitative analysis functions. Although this method requires users to spend additional time processing confocal images prior to running COMSTAT, it helps minimize misrepresented biofilm heterogenicity in automated outputs.

Quantifying the Effects of Antimicrobials on In vitro Biofilm Architecture using COMSTAT Software

Antimicrobial-induced changes to Pseudomonas aeruginosa biofilm architecture differ among clinical isolates cultured from patients with cystic fibrosis and chronic pulmonary infection. Following confocal microscopy, COMSTAT software can be utilized to quantify variations in biofilm architecture (e.g., surface area, thickness, biomass) for individual isolates to assess the efficacy of anti-infective agents.

Quantifizierung der Auswirkungen von Antibiotika auf die In-vitro-Biofilmarchitektur mit der COMSTAT-Software

Biofilms are aggregates of microorganisms that rely on a self-produced matrix of extracellular polymeric substance for protection and structural ...

Antibiotic Efficacy Testing in an Ex vivo Model of Pseudomonas aeruginosa and Staphylococcus aureus Biofilms in the Cystic Fibrosis Lung

The effective prescription of antibiotics for the bacterial biofilms present within the lungs of individuals with cystic fibrosis (CF) is limited by a poor correlation between antibiotic susceptibility testing (AST) results using standard diagnostic methods (e.g., broth microdilution, disk diffusion, or Etest) and clinical outcomes after antibiotic treatment. Attempts to improve AST by the use of off-the-shelf biofilm growth platforms show little improvement in results. The limited ability of in vitro biofilm systems to mimic the physicochemical environment of the CF lung and, therefore bacterial physiology and biofilm architecture, also acts as a brake on the discovery of novel therapies for CF infection. Here, we present a protocol to perform AST of CF pathogens grown as mature, in vivo-like biofilms in an ex vivo CF lung model comprised of pig bronchiolar tissue and synthetic CF sputum (ex vivo&#160;pig lung, EVPL).
Several in vitro&#160;assays exist for biofilm susceptibility testing, using either standard laboratory medium or various formulations of synthetic CF sputum in microtiter plates. Both growth medium and biofilm substrate (polystyrene plate vs. bronchiolar tissue) are likely to affect biofilm antibiotic tolerance. We show enhanced tolerance of clinical Pseudomonas aeruginosa and Staphylococcus aureus isolates in the ex vivo model; the effects of antibiotic treatment of biofilms is not correlated with the minimum inhibitory concentration (MIC) in standard microdilution assays or a sensitive/resistant classification in disk diffusion assays.
The ex vivo platform could be used for bespoke biofilm AST of patient samples and as an enhanced testing platform for potential antibiofilm agents during pharmaceutical research and development. Improving the prescription or acceleration of antibiofilm drug discovery through the use of more in vivo-like testing platforms could drastically improve health outcomes for individuals with CF, as well as reduce the costs of clinical treatment and discovery research.

Antibiotic Efficacy Testing in an Ex vivo Model of Pseudomonas aeruginosa and Staphylococcus aureus Biofilms in the Cystic Fibrosis Lung

This workflow can be used to perform antibiotic susceptibility testing using an established&#160;ex vivo model of bacterial biofilm in the lungs of individuals with cystic fibrosis. Use of this model could enhance the clinical validity of MBEC (minimal biofilm eradication concentration) assays.

Antibiotika-Wirksamkeitstests in einem Ex-vivo-Modell von Pseudomonas aeruginosa und Staphylococcus aureus Biofilmen in der Mukoviszidose-Lunge

The effective prescription of antibiotics for the bacterial biofilms present within the lungs of individuals with cystic fibrosis (CF) is limited by a ...

<meta charset="utf8"></head><body>Anpassbares CF-Modell für antimikrobielle Tests und mikrobielle Dynamikanalysen

Development of a Polymicrobial Colony Biofilm Model to Test Antimicrobials in Cystic Fibrosis

A range of bacteria biofilm models exist for the testing of antibiotics. However, many of these are limited to a single experimental output, such as colony-forming units or metabolic activity. Furthermore, many biofilm models do not reflect the biological and physiochemical properties of the human host environment. This is an important issue in many conditions, but most noticeably in cystic fibrosis (CF). A large proportion of people with CF suffer from both chronic and intermittent infections, and in vitro, antibiotic susceptibility testing poorly correlates with patient treatment outcomes. Some biofilm models incorporate CF lung-relevant media, including synthetic sputum mimics, but do not consider the polymicrobial nature of the environment, which alters biofilm architecture, physiology, and the way microbes respond to treatment. The solid-air interface colony biofilm model described here is highly adaptable and incorporates both CF-relevant media and a polymicrobial context. This model can also be used for mid-throughput screening of antimicrobials and to study their effect on polymicrobial dynamics. Output measurements from the model can be colony-forming units, metabolic activity, and confocal microscopy analysis. The model can easily be adapted to different microorganisms, media, temperatures, and variable oxygen conditions and can be used to test a wide range of chemical, biological, and physical treatments.

<meta charset="utf8"></head><body>Entwicklung eines polymikrobiellen Kolonie-Biofilmmodells zur Erprobung von Antibiotika bei Mukoviszidose

This methodology allows for the establishment of a polymicrobial biofilm model in cystic fibrosis for antimicrobial sensitivity testing in research and clinical laboratories. This model provides accurate and reliable results over a range of outputs.

Entwicklung eines polymikrobiellen Kolonie-Biofilmmodells zur Erprobung von Antibiotika bei Mukoviszidose

A range of bacteria biofilm models exist for the testing of antibiotics. However, many of these are limited to a single experimental output, such as ...

Visualisierung der Auswirkungen von Sputum auf Biofilm-Entwicklung eines Modells mit Kammerdeckgläser

Zusammenfassung

Weitere Videos entdecken

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

In-vitro-Bildung von Biofilmen in einem 8-well Chamber Slide

Wachstum von Mycobacterium tuberculosis Biofilme

Einsatz von künstlichen Sputum Mittlere bis antibiotische Wirkung gegen die getestet

Methodologien für das Studium

Das WinCF-Modell - Ein preiswerter und tragbarer Mikrokosmos eines Schleims verstopfte Bronchiole zur Untersuchung der Mikrobiologie von Lungeninfektionen

Quantifizierung der Auswirkungen von Antibiotika auf die In-vitro-Biofilmarchitektur mit der COMSTAT-Software

Antibiotika-Wirksamkeitstests in einem Ex-vivo-Modell von Pseudomonas aeruginosa und Staphylococcus aureus Biofilmen in der Mukoviszidose-Lunge

Entwicklung eines polymikrobiellen Kolonie-Biofilmmodells zur Erprobung von Antibiotika bei Mukoviszidose

Entwicklung eines polymikrobiellen Kolonie-Biofilmmodells zur Erprobung von Antibiotika bei Mukoviszidose