We thank Saran Kupul for technical assistance. This work was supported in part by NIH grant R01-AI104952 to FW. Further support was provided by the University of Alabama at Birmingham (UAB) Center for AIDS Research (CFAR), an NIH funded program (P30 AI027767) that was made possible by the following NIH Institutes: NIAID, NIMH, NIDA, NICHD, NHLDI, NIA.

1. Biofilm Initiation <ol><li> Wachsen einer Kultur des Biofilms produzierenden Organismus in einem nährstoffreichen Medium. Impfen Staphylococcus aureus - Stamm Newman in 10 ml Mueller-Hinton - Medium aus einem Glycerin Lager. Führen Sie alle Arbeiten , die mit der Handhabung von S. aureus mit Handschuhen und in einem Biosicherheitsschrank. </li><li> bei 37 ° C für 16 Stunden inkubieren auf einem Rotationsschüttler (100-200 rpm). </li><li> Bestimmen Sie OD 600 der Kultur eines Spektrometers und eine Standardküvette mit (Weglänge: 1cm). </li><li> Berechne das Volumen (V) der Kultur benötigt 20 herzustellen ml des Inokulums mit OD 600 (Inokulum) von 0,1 in chelexed Roswell Park Memorial Institute Medium (cRPMI) 9 mit der Formel: [V = 20 ml * 0,1 / OD 600 (Kultur )] Hinweis: Referenz 9 enthält Einzelheiten über cRPMI Vorbereitung. <ol><li> Die berechnete Kulturvolumen (von oben) in ein Zentrifugenröhrchen und Pelletzellen durch bei 10.000 x g für 1 min dreht. Um prepadas verbrauchte Nährmedium und resuspendieren Zellpellet in 20 ml cRPMI re Biofilm Inokulum absaugen. </li></ol></li><li> Gießen Inokulum in einen 25-ml-Reservoir. </li><li> Entfernen Sie vorsichtig den Deckel aus einer sterilen 96-Well-Platte peg diese gerade nach oben ziehen. Hinweis: Bitte achten Sie dürfen nicht die Heringe zu verunreinigen nach unten aus dem Deckel hängen. Der Deckel kann in einem Biosicherheitsschrank auf eine saubere Oberfläche den Kopf gestellt werden. </li><li> Verwendung eines 200 &amp; mgr; l Mehrkanalpipette, fügen 125 ul Impfgut zu jeder Vertiefung der Reihen A bis G der Bodenplatte. </li><li> Füllen Sie 125 ul sterile cRPMI Medium in jeder Vertiefung der Reihe H als Sterilitätskontrolle. </li><li> Nehmen Sie die peg-Deckel und halten Sie ihn mit den Zapfen über dem Plattenboden nach unten. sorgfältig ausrichten, um die einzelnen Stifte mit ihren jeweiligen Brunnen. Vermeiden Sie Körperkontakt zwischen der Platte und den Zapfen. Nach der Ausrichtung senken vorsichtig nach unten den Deckel. Vermeiden Sie Fehlstellungen wie ausgeführt Nachjustierungen nach den Zapfen der Bodenplatte berührt haben cont kannaminate Sterilität Kontrollen in Reihe H. </li><li> Verschließen Sie den Deckel auf die Platte mit Paraffin Filmverdampfung und in einen verschließbaren Plastikbeutel zu verhindern, Abdichtung fest. Halten Sie das Luftvolumen in der Tasche so gering wie möglich Verdunstung zu minimieren. </li><li> Inkubieren ohne für 48 Stunden in einem 5% CO 2 Inkubator bei 37 ° C schüttelnd. Hinweis: 48 Stunden des Wachstums von S. optimal gewesen aureus, sondern wird auf den Organismus verwendet , variieren. Hinweis: Obwohl statische Inkubation ein guter Ausgangspunkt ist der Test zu optimieren, sanft bei 150 Umdrehungen pro Minute auf einem Schüttler Schütteln ist eine mögliche Variante , die die Biofilmbildung von einigen S. verbessern kann aureus - Isolaten. </li></ol> 2. Herstellung der Biofilm Challenge-Platte <ol><li> Am Tag des Biofilms Herausforderung nehmen eine frische 96-Well-Platte und fügen 100 ul cRPMI, auf RT ins Gleichgewicht gebracht, in jede Vertiefung der Zeilen B bis H. inkonsistente Ergebnisse zu vermeiden, verwenden eine 96-Well-Platte, die 200 &amp; mgr; l aufnehmen kanninnerhalb der Medien, wenn die Heringe, und verfügt über Vertiefungen mit Abmessungen identisch mit denen des Biofilms Einleitung Platte. </li><li> Verdünne bis zu 4 Testverbindungen getrennt in 1,0 ml cRPMI die gewünschte Endkonzentration, um zu 2x für eine Endverdünnung in Schritt 2.7 zu berücksichtigen. Hinweis: Ein guter Ausgangspunkt ist 8-fach über die hemmende Konzentration von planktonischen Zellen. </li><li> In 200 ul der Verbindung 1 in den Vertiefungen A1 bis A3, die Verbindung 2 in Brunnen A4 bis A6, Verbindung 3 in Brunnen A7 bis A9 und Verbindung 4 in Brunnen A10 bis A12. </li><li> Abwechselnd verdünnen die Testverbindungen mit einer Mehrkanalpipette mit und 100 &amp; mgr; l aus Reihe A übertragen B und mischen zu rudern. </li><li> Auf diese Weise weiterhin 100 ul Vertiefung zu Vertiefung zu übertragen. Mischen Sie nach jeder Übertragung und stoppen in der Reihe F. </li><li> Nach dem Mischen vertreiben 100 &amp; mgr; l aus Reihe F in einen Abfallbehälter. Verwenden Sie keine Flüssigkeit in Reihen G und H. übertragen </li><li> 100 l cRPMI zu jeder Vertiefung der Platte mit einer Mehrkanalpipettebringen das Volumen auf 200 ul. Siehe Abbildung 1 für die endgültige Plattenlayout. </li></ol> 3. Biofilm Herausforderung <ol><li> Mit 200 &amp; mgr; l phosphatgepufferter Salzlösung (PBS) auf eine frische sterile 96-Well-Platte, die Vertiefungen mit Abmessungen identisch mit denen des Biofilms Initiationsplatte verfügt. </li><li> Entfernen Sie den Plastikbeutel und Paraffinfilm aus inkubiert Biofilm Initiationsplatte. </li><li> Heben Sie den Deckel gerade nach oben. Vermeiden Sie den Kontakt zwischen den Zapfen und dem Plattenboden, da dies den Biofilm beschädigen können. Halten Sie Unterteil für die nachfolgende OD 600 - Analyse (siehe 3.8). </li><li> Wegspülen planktonischen Zellen durch den Stift-Deckel auf die Platte legen enthält PBS (aus Schritt 3.1). Versenken Pflöcke für 5 Sekunden, heben Sie aus PBS, und tauchen noch einmal, man aufpassen, nicht die Zapfen gegen die Seiten gut zu treffen. </li><li> Setzen Sie den abgespült peg-Deckel in die Herausforderung Platte. Vermeiden Sie unnötigen Kontakt zwischen Zapfen und Bodenplatte, da dies der Biofilm führt zu Schäden aninkonsistente Ergebnisse. </li><li> Die Dichtungsplatte mit Paraffinfilm und in einen verschließbaren Beutel aus Kunststoff, wie in Schritt 1.10 beschrieben. </li><li> Inkubiere die Platte für 24 Stunden bei 37 ° C in einem 5% CO 2 Inkubator ohne Schütteln. </li><li> Nehmen Unterseite des Biofilms Initiationsplatte von 3,3. Resuspendieren der Bakterien in jeder Vertiefung einer Mehrkanalpipette mit und die OD 600 mit einem geeigneten Mikroplatten - Reader messen Wachstum zu bestätigen in allen Vertiefungen , aber die Sterilität Kontrollen in Reihe H. vorkommenden </li></ol> 4. Biofilm Bestimmung <ol><li> Verwenden Leser eine Platte mit einer Inkubationskabine ausgestattet und in der Lage nehmen kinetische Fluoreszenzmesswerte zur Erfassung von Resazurin Umwandlung. Legen Sie eine 24-Stunden-kinetische Fluoreszenzmessung unter Verwendung eines 530 nm Anregung und 590 nm Emission auf. </li><li> Stellen Sie die Raumtemperatur auf 37 ° C und haben die Platte alle 20 Minuten lesen. Stellen Sie den Leser Platte von der Unterseite der Platte zu messen, nach Herstelleranweisunggen. </li><li> Bereiten Waschplatte durch Zugabe von 200 ul PBS mit einer Mehrkanalpipette in eine sterile 96-Well-Platte. </li><li> Bereiten Biofilm Wiederherstellungsmedium durch Zugabe von 400 &amp; mgr; l von 0,8 mg / ml Stammlösung zu 20 ml cRPMI Medium auf eine Endkonzentration von 16 &amp; mgr; g / ml Resazurin Resazurin. Hinweis: Resazurin ist eine bekannte hautreizend. Verwenden Sie einen Laborkittel, Schutzbrille und Handschuhe bei der Handhabung. CRPMI als Biofilm Recovery-Medien wurde verwendet, da es den niedrigsten Hintergrundsignal liefert, aber es kann von Mueller-Hinton-Medien gegebenenfalls ersetzt werden. </li><li> In 150 ul Biofilm Recovery Medium mit einer Mehrkanalpipette in jede Vertiefung einer frischen 96-Well-Platte. </li><li> Transfer Herausforderung Platte aus Inkubator in einem Biosicherheitsschrank und sorgfältig Plastiktüte und Siegelfolie entfernen. </li><li> Entfernen Sie die peg-Deckel von der Herausforderung Platte und abzuspülen planktonischen Zellen in PBS, wie in 3.4 beschrieben. Halten Sie den Boden der Herausforderung Platte für nachfolgende OD 600 anaLyse (siehe 4.10). </li><li> Nach dem Spülen, legen Sie den zapfen Deckel in den Plattenboden des Biofilms Recovery-Medium enthält. </li><li> Wickeln Sie die Seite der Platte mit Paraffinfilm, wobei darauf geachtet, nicht den Boden der Umfang Brunnen zu behindern. Unmittelbar nach der Platte Verpackung, legen Sie sie auf dem Plattenleser und eine kinetische Lese über einen Zeitraum von 24 h beginnen, indem die bisherigen Resazurin kinetische Protokoll von Schritt 4.1 zu initiieren. </li><li> Um das Wachstum von planktonischen Zellen zu quantifizieren , die während der Herausforderung auftreten, lesen Sie die OD 600 der gesamten Herausforderung Plattenboden kann oder auch nicht ein Mikroplattenleser. Folgen Sie die Anweisungen in Schritt 3.8. Hinweis: vorsichtig resuspendieren den Inhalt jeder Vertiefung als Shedding Zellen aus den Biofilm neigen dazu, in Klumpen am Boden der Vertiefung zu wachsen. Alle Blasen innerhalb des Schachtes wird stark mit OD 600 Lesungen stören und müssen vor dem Lese vermieden oder entfernt werden. </li></ol>

<ol>
	<li>Bjarnsholt, T., Ciofu, O., Molin, S., Givskov, M., Hoiby, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applying+insights+from+biofilm+biology+to+drug+development+-+can+a+new+approach+be+developed.">Applying insights from biofilm biology to drug development - can a new approach be developed.</a> Nat Rev Drug Discov. 12, 791-808 (2013).</li><li>Song, Z., 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=Prosthesis+infections+after+orthopedic+joint+replacement:+the+possible+role+of+bacterial+biofilms.">Prosthesis infections after orthopedic joint replacement: the possible role of bacterial biofilms.</a> Orthop Rev (Pavia). 5, 65-71 (2013).</li><li>Hall-Stoodley, L., Costerton, J. W., Stoodley, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+biofilms:+from+the+natural+environment+to+infectious+diseases).">Bacterial biofilms: from the natural environment to infectious diseases).</a> Nat Rev Microbiol. 2, 95-108 (2004).</li><li>Bordi, C., de Bentzmann, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hacking+into+bacterial+biofilms:+a+new+therapeutic+challenge.">Hacking into bacterial biofilms: a new therapeutic challenge.</a> Ann Intensive Care. 1, 19 (2011).</li><li>Fux, C. A., Shirtliff, M., Stoodley, P., Costerton, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Can+laboratory+reference+strains+mirror+'real-world'+pathogenesis?.">Can laboratory reference strains mirror 'real-world' pathogenesis?.</a> Trends Microbiol. 13, 58-63 (2005).</li><li>Kwasny, S. M., Opperman, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Static+biofilm+cultures+of+Gram-positive+pathogens+grown+in+a+microtiter+format+used+for+anti-biofilm+drug+discovery.">Static biofilm cultures of Gram-positive pathogens grown in a microtiter format used for anti-biofilm drug discovery.</a> Curr Protoc Pharmacol. Chapter 13, Unit 13A 18 (2010).</li><li>Ceri, H., 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=The+MBEC+Assay+System:+multiple+equivalent+biofilms+for+antibiotic+and+biocide+susceptibility+testing.">The MBEC Assay System: multiple equivalent biofilms for antibiotic and biocide susceptibility testing.</a> Methods Enzymol. 337, 377-385 (2001).</li><li>Ceri, H., 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=The+Calgary+Biofilm+Device:+new+technology+for+rapid+determination+of+antibiotic+susceptibilities+of+bacterial+biofilms.">The Calgary Biofilm Device: new technology for rapid determination of antibiotic susceptibilities of bacterial biofilms.</a> J Clinical Microbiol. 37, 1771-1776 (1999).</li><li>Baker, J., 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=Copper+stress+induces+a+global+stress+response+in+Staphylococcus+aureus+and+represses+sae+and+agr+expression+and+biofilm+formation.">Copper stress induces a global stress response in Staphylococcus aureus and represses sae and agr expression and biofilm formation.</a> Appl Environ Microbiol. 76, 150-160 (2010).</li><li>Haeili, M., 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=Copper+complexation+screen+reveals+compounds+with+potent+antibiotic+properties+against+methicillin-resistant+Staphylococcus+aureus.">Copper complexation screen reveals compounds with potent antibiotic properties against methicillin-resistant Staphylococcus aureus.</a> Antimicrob Agents Chemother. 58, 3727-3736 (2014).</li><li>O'Brien, J., Wilson, I., Orton, T., Pognan, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigation+of+the+Alamar+Blue+(resazurin)+fluorescent+dye+for+the+assessment+of+mammalian+cell+cytotoxicity.">Investigation of the Alamar Blue (resazurin) fluorescent dye for the assessment of mammalian cell cytotoxicity.</a> Eur J Biochem. 267, 5421-5426 (2000).</li><li>Mah, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishing+the+minimal+bactericidal+concentration+of+an+antimicrobial+agent+for+planktonic+cells+(MBC-P)+and+biofilm+cells+(MBC-B).">Establishing the minimal bactericidal concentration of an antimicrobial agent for planktonic cells (MBC-P) and biofilm cells (MBC-B).</a> J Vis Exp. (83), e50854 (2014).</li><li>Junker, L. M., Clardy, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+screens+for+small-molecule+inhibitors+of+Pseudomonas+aeruginosa+biofilm+development.">High-throughput screens for small-molecule inhibitors of Pseudomonas aeruginosa biofilm development.</a> Antimicrob Agents Chemother. 51, 3582-3590 (2007).</li><li>Shah, D., 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=Persisters:+a+distinct+physiological+state+of+E.+coli.">Persisters: a distinct physiological state of E. coli.</a> BMC Microbiol. 6, 53 (2006).</li><li>Lewis, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Riddle+of+biofilm+resistance.">Riddle of biofilm resistance.</a> Antimicrob Agents Chemother. 45, 999-1007 (2001).</li></ol>

The authors have nothing to disclose.

Hier haben wir ein abgewandeltes Biofilm - Assay zur Bestimmung der Aktivität der getesteten Inhibitoren auf S. beschrieben aureus Biofilm auf den Stoffwechselzustand von Biofilm assoziierten Zellen konzentriert. Während die beschriebene Biofilm Initiierung und Widerspruchsverfahren meist nachgeahmten Herstellerempfehlungen, die Verwendung von Resazurin Farbstoff Biofilm-assoziierte Zellen, die eine 24-Stunden-Inhibitor Herausforderung überleben dramatisch vereinfacht die empfohlene Verfahren der Ze...

Pathogene Keime können 1 Biofilmen in vivo führt zu nicht akuten chronischen Infektionen bilden. Biofilm-assoziierte Infektion ist eine ernste Risikofaktor für medizinische Verfahren , die Implantation von Fremdkörpern (zB künstliche Knochenersatz, Brustimplantate) oder die Installation von Trachealtuben oder Urinkatheter 2 beinhalten. In diesen Zusammenhängen ist eine antiinfektive Therapie fast immer notwendig , als Biofilm-basierte Infektionen auf ihren eigenen selten gelöscht werden, selbst in immunkompetenten Individuen. Staphylococcus aureus eine der am häufigsten beobachteten Krankheitserreger in Biofilm-Komplikationen beteiligt ist , während der Verwendung auftreten , von invasive medizinische Geräte 3. Sie mehr resistent gegen die Behandlung als planktonischen Zellen 1,4, und die Bewertung der vorhergesagten klinische Wirksamkeit Leider macht das Wesen von Biofilmen als Schutzbarriere ist ein wichtiger Bestandteil der beiden AnfangsArzneimittelentwicklung sowie Arzneimittelresistenzüberwachung. Es gibt immer mehr Anerkennung , dass Laborbedingungen auf planktonischen Kulturen konzentriert, kann nicht treu reale Krankheit 5 darstellen. Weiterhin das Problem Compoundierung, ist die Replikation von Biofilm - Phänotypen schwierig, und vorhandene Biofilm - Modelle sind langwierig und leiden unter hohen inter- und intra Assay Variabilität 6. viele Forscher, durch die Notwendigkeit, Standard zu planktonischen Zellassays von Medikamentenempfindlichkeit, wodurch möglicherweise ein wichtiger Aspekt der bakteriellen Virulenz zu vernachlässigen und Krankheit So. Hier beschreiben wir das Protokoll für Bakterien Testen, insbesondere S. aureus, in pre-grown Biofilmen eine 96-Well - basierten Biofilm System 7,8 verwendet. Während das Protokoll für die Biofilmbildung und die Herausforderung im Wesentlichen die Empfehlungen des Herstellers folgt präsentieren wir eine alternative informationsreiche Methode, um die Lebensfähigkeit des bioF zur Quantifizierungilm nach Herausforderung. Kurz gesagt, sind Bakterien in der Stange Platte kultiviert, wo Biofilmen an der Platte des Deckels angebracht an den vorstehenden Zapfen bilden. Nach der Biofilmbildung sind die Zapfen in die Vertiefungen einer frischen Platte mit PBS gefüllt zu entfernen planktonischen Zellen vorsichtig getaucht. Der PEG-Deckel mit Biofilmen angebracht ist, wird auf eine neue Herausforderung Platte übertragen, verschiedene Konzentrationen der Antibiotika enthält, untersucht werden. Nach einer zweiten Inkubation werden Deckel wieder entfernt, gewaschen und in ein Rückgewinnungsplatte Resazurin Farbstoff enthält, überführt, wo sie einer abschließenden Inkubation unterzogen werden. Resazurin Umwandlung kann nach einer definierten Erholungszeit als Endpunkt Lesen kinetisch oder verbucht. Dieser Farbstoff-basierte Methode , um die Lebensfähigkeit von Biofilmen zu quantifizieren , unterscheidet sich erheblich von den langweiligen KBE (Kolonie bildende Einheiten) zählen basierte Methodik im ursprünglichen Protokoll 7 beschrieben. OD 600 Messungen der Droge Herausforderung Platte und Resazurin Umwandlungskinetik dienen als Lebensfähigkeit Leseouts von planktonischen und Biofilm-Zellen, die jeweils eine schnelle, zuverlässige, informationsreiche und technisch einfachen Test zur Biofilm Überleben anbietet.

<table border="0" cellpadding="0" cellspacing="0" width="612">
	<tbody>
		<tr height="61">
			<td height="61" style="height:61px;width:227px;">&#160;Mueller-Hinton Medium</td>
			<td style="width:89px;">Oxoid</td>
			<td style="width:103px;">CM0405</td>
			<td style="width:195px;">Follow recommendations of manufacturer</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:227px;">RPMI-medium</td>
			<td style="width:89px;">Corning&#160;</td>
			<td style="width:103px;">17-105-CV</td>
			<td style="width:195px;"></td>
		</tr>
		<tr height="101">
			<td height="101" style="height:101px;width:227px;">CRPMI</td>
			<td style="width:89px;">Ref 9</td>
			<td style="width:103px;"></td>
			<td style="width:195px;">RPMI-1640 medium chelexed for 1h with Chelex 100 resin and then supplemented with 10% unchelexed RPMI-1640</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:227px;">Chelex 100 Resin</td>
			<td style="width:89px;">Bio Rad</td>
			<td style="width:103px;">142-2822</td>
			<td style="width:195px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:227px;">MBEC-plates</td>
			<td style="width:89px;">Innovotech</td>
			<td style="width:103px;">19111</td>
			<td style="width:195px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:227px;">Resazurin Sodium Salt</td>
			<td style="width:89px;">Sigma</td>
			<td style="width:103px;">R7017</td>
			<td style="width:195px;">800&#181;g/ml in DI water&#160;&#160;&#160;&#160; Filter sterile</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:227px;">Micro Plate Shaking Platform&#160;</td>
			<td style="width:89px;">Heidolph Titramax 1000</td>
			<td style="width:103px;"></td>
			<td style="width:195px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:227px;">Cytation 3 Plate Reader</td>
			<td style="width:89px;">Biotek</td>
			<td style="width:103px;"></td>
			<td style="width:195px;"></td>
		</tr>
		<tr height="57">
			<td height="57" style="height:58px;width:227px;">Gen5 software</td>
			<td style="width:89px;">Biotek</td>
			<td style="width:103px;"></td>
			<td style="width:195px;">Recording and analysis of resazurin conversion</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:227px;">Neocuproine</td>
			<td style="width:89px;">Sigma</td>
			<td style="width:103px;">N1501&#160;</td>
			<td style="width:195px;">prepare 10 mM stock in 100% Ethanol, store at -80&#186;C</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:227px;">Copper sulfate</td>
			<td style="width:89px;">Acros Organics</td>
			<td style="width:103px;">7758-99-8</td>
			<td style="width:195px;">prepare a 100 mM stock solution in water, store at 4&#186;C</td>
		</tr>
		<tr height="121">
			<td height="121" style="height:121px;width:227px;">Cu-Neocuproine</td>
			<td style="width:89px;">Self-Made</td>
			<td style="width:103px;"></td>
			<td style="width:195px;">Generated by mixing equal molarities of neocuproine and copper sulfate. Mix was diluted in CRPMI medium to desired concentration.</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:227px;">Gentamicin</td>
			<td style="width:89px;">Sigma</td>
			<td style="width:103px;">G3632-1G</td>
			<td style="width:195px;"></td>
		</tr>
	</tbody>
</table>

targeting biofilm associated staphylococcus aureus using resazurin based drug-susceptibility assay

Most pathogenic bacteria are able to form biofilms during infection, but due to the difficulty of manipulating and assessing biofilms, the vast majority of laboratory work is conducted with planktonic cells. Here, we describe a peg plate biofilm assay as performed with Staphylococcus aureus. Bacterial biofilms are grown on pegs attached to a 96-well microtiter plate lid, washed through gentle submersion in buffer, and placed in a drug challenge plate. After subsequent incubation they are again washed and moved to a final recovery plate, in which the fluorescent dye resazurin serves as a viability indicator. This assay offers greatly increased ease-of-use, reliability, and reproducibility, as well as a wealth of data when conducted as a kinetic read. Moreover, this assay can be adapted to a medium-throughput drug screening approach by which an endpoint fluorescent readout is taken instead, offering a path for drug discovery efforts.

Der Resazurin-Assay ist empfindlich genug, um zuverlässig sehr wenige lebensfähige Zellen replizieren in drogenfreien Medium nach der Herausforderung der Lage zu erkennen. In dieser Methode definieren wir die minimale Biofilm auszurotten Konzentration als die niedrigste Konzentration, bei der keine Resazurin Umwandlung innerhalb von 24 h zu sehen ist. Der Resazurin-Assay beruht auf oxidativen Moleküle in metabolisch aktiven Zellen gefunden, die den Farbstoff Farbe von blau zu rosa kon...

Watch this Scientific Journal Video about Targeting Biofilm Associated Staphylococcus aureus Using Resazurin Based Drug-susceptibility Assay at JoVE.com

Targeting Biofilm Associated Staphylococcus aureus Using Resazurin Based Drug-susceptibility Assay

Targeting Biofilm Assoziierte<em&gt; Staphylococcus aureus</em&gt; Verwendung von Resazurin Based Drug-Anfälligkeit Assay

Most bacterial infections produce a biofilm. By virtue of their environment, biofilm associated bacteria are often phenotypically drug resistant. Novel antibacterial molecules that kill bacteria in biofilms are thus a high priority. We establish an assay to quickly screen for antimicrobial compounds that are effective at eradicating biofilms.

Most pathogenic bacteria are able to form biofilms during infection, but due to the difficulty of manipulating and assessing biofilms, the vast majority ...

targeting-biofilm-associated-staphylococcus-aureus-using-resazurin

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

Targeting Biofilm Associated Staphylococcus aureus Using Resazurin Based Drug-susceptibility Assay

13.0K Aufrufe.  University of Alabama at Birmingham. Most bacterial infections produce a biofilm. By virtue of their environment, biofilm associated bacteria are often phenotypically drug resistant. Novel antibacterial molecules that kill bacteria in biofilms are thus a high priority. We establish an assay to quickly screen for antimicrobial compounds that are effective at eradicating biofilms. 

Serie: Targeting Biofilm Associated Staphylococcus aureus Using Resazurin Based Drug-susceptibility Assay

Microtiter Dish Biofilm Formation Assay

Biofilms are communities of microbes attached to surfaces, which can be found in medical, industrial and natural settings. In fact, life in a biofilm probably represents the predominate mode of growth for microbes in most environments. Mature biofilms have a few distinct characteristics. Biofilm microbes are typically surrounded by an extracellular matrix that provides structure and protection to the community. Microbes growing in a biofilm also have a characteristic architecture generally comprised of macrocolonies (containing thousands of cells) surrounded by fluid-filled channels. Biofilm-grown microbes are also notorious for their resistance to a range of antimicrobial agents including clinically relevant antibiotics.
The microtiter dish assay is an important tool for the study of the early stages in biofilm formation, and has been applied primarily for the study of bacterial biofilms, although this assay has also been used to study fungal biofilm formation. Because this assay uses static, batch-growth conditions, it does not allow for the formation of the mature biofilms typically associated with flow cell systems. However, the assay has been effective at identifying many factors required for initiation of biofilm formation (i.e, flagella, pili, adhesins, enzymes involved in cyclic-di-GMP binding and metabolism) and well as genes involved in extracellular polysaccharide production. Furthermore, published work indicates that biofilms grown in microtiter dishes do develop some properties of mature biofilms, such a antibiotic tolerance and resistance to immune system effectors.
This simple microtiter dish assay allows for the formation of a biofilm on the wall and/or bottom of a microtiter dish. The high throughput nature of the assay makes it useful for genetic screens, as well as testing biofilm formation by multiple strains under various growth conditions. Variants of this assay have been used to assess early biofilm formation for a wide variety of microbes, including but not limited to, pseudomonads, Vibrio cholerae, Escherichia coli, staphylocci, enterococci, mycobacteria and fungi.
In the protocol described here, we will focus on the use of this assay to study biofilm formation by the model organism Pseudomonas aeruginosa. In this assay, the extent of biofilm formation is measured using the dye crystal violet (CV). However, a number of other colorimetric and metabolic stains have been reported for the quantification of biofilm formation using the microtiter plate assay. The ease, low cost and flexibility of the microtiter plate assay has made it a critical tool for the study of biofilms.

The assay describes a rapid means to measure early biofilm formation in bacteria and fungi. This method uses a microtiter plate as the substratum for microbial biofilm formation, and the biofilm is visualized using crystal violet strain. The assay provides either a qualitative or quantitative assay for early biofilm formation.

Mikrotiterschale Biofilmbildung Assay

Biofilms are communities of microbes attached to surfaces, which can be found in medical, industrial and natural settings.  In fact, life in a biofilm ...

Forschung

Immunologie und Infektion

The Use of Drip Flow and Rotating Disk Reactors for Staphylococcus aureus Biofilm Analysis

Most microbes in nature are thought to exist as surface-associated communities in biofilms.1 Bacterial biofilms are encased within a matrix and attached to a surface.2 Biofilm formation and development are commonly studied in the laboratory using batch systems such as microtiter plates or flow systems, such as flow-cells. These methodologies are useful for screening mutant and chemical libraries (microtiter plates)3 or growing biofilms for visualization (flow cells)4. Here we present detailed protocols for growing Staphylococcus aureus in two additional types of flow system biofilms: the drip flow biofilm reactor and the rotating disk biofilm reactor.
Drip flow biofilm reactors are designed for the study of biofilms grown under low shear conditions.5 The drip flow reactor consists of four parallel test channels, each capable of holding one standard glass microscope slide sized coupon, or a length of catheter or stint. The drip flow reactor is ideal for microsensor monitoring, general biofilm studies, biofilm cryosectioning samples, high biomass production, medical material evaluations, and indwelling medical device testing.6,7,8,9
The rotating disk reactor consists of a teflon disk containing recesses for removable coupons.10 The removable coupons can by made from any machinable material. The bottom of the rotating disk contains a bar magnet to allow disk rotation to create liquid surface shear across surface-flush coupons. The entire disk containing 18 coupons is placed in a 1000 mL glass side-arm reactor vessel. A liquid growth media is circulated through the vessel while the disk is rotated by a magnetic stirrer. The coupons are removed from the reactor vessel and then scraped to collect the biofilm sample for further study or microscopy imaging. Rotating disc reactors are designed for laboratory evaluations of biocide efficacy, biofilm removal, and performance of anti-fouling materials.9,11,12,13

The Use of Drip Flow and Rotating Disk Reactors for Staphylococcus aureus Biofilm Analysis

Protocols for utilizing open system flow biofilms with drip flow reactors and rotating disk reactors are presented in detail.

Die Verwendung von Drip Flow und Rotating Disk-Reaktoren für Staphylococcus aureus Biofilm Analysis

Most microbes in nature are thought to exist as surface-associated communities in biofilms.1  Bacterial biofilms are encased within a matrix and attached ...

Subcutaneous Infection of Methicillin Resistant Staphylococcus Aureus (MRSA)

MRSA is a worldwide threat to public health, and MRSA skin and soft-tissue infections now account for more than half of all soft-tissue infections in the United States. Among soft-tissue infections, myositis, pyomyositis, and necrotizing fasciitis have been increasingly reported in association with MRSA arising from the community. To understand the interplay between MRSA and host immunity leading to more severe infection, the availability of animal models is critical, permitting the study of host and bacterial factors. Several infection models have been introduced to assess the pathogenesis of S. aureus during superficial skin infection. Here, we describe a subcutaneous infection model that examines the skin, subcutaneous, and muscle pathologies.

Subcutaneous Infection of Methicillin Resistant Staphylococcus Aureus MRSA

Murine skin and soft tissue infection model is utilized for assessing the virulence function of methicillin resistant Staphylococcus aureus (MRSA) and the host immunological responses. Here, we presented a subcutaneous infection model for skin and soft tissue infection.

Subkutane Infektion von Methicillin-resistenten Staphylococcus Aureus (MRSA)

MRSA is a worldwide threat to public health, and MRSA skin and soft-tissue infections now account for more than half of all soft-tissue infections in the ...

Experimental Endocarditis Model of Methicillin Resistant Staphylococcus aureus (MRSA) in Rat

Endovascular infections, including endocarditis, are life-threatening infectious syndromes1-3. Staphylococcus aureus is the most common world-wide cause of such syndromes with unacceptably high morbidity and mortality even with appropriate antimicrobial agent treatments4-6. The increase in infections due to methicillin-resistant S. aureus (MRSA), the high rates of vancomycin clinical treatment failures and growing problems of linezolid and daptomycin resistance have all further complicated the management of patients with such infections, and led to high healthcare costs7, 8. In addition, it should be emphasized that most recent studies with antibiotic treatment outcomes have been based in clinical settings, and thus might well be influenced by host factors varying from patient-to-patient. Therefore, a relevant animal model of endovascular infection in which host factors are similar from animal-to-animal is more crucial to investigate microbial pathogenesis, as well as the efficacy of novel antimicrobial agents. Endocarditis in rat is a well-established experimental animal model that closely approximates human native valve endocarditis. This model has been used to examine the role of particular staphylococcal virulence factors and the efficacy of antibiotic treatment regimens for staphylococcal endocarditis. In this report, we describe the experimental endocarditis model due to MRSA that could be used to investigate bacterial pathogenesis and response to antibiotic treatment.

Experimental Endocarditis Model of Methicillin Resistant Staphylococcus aureus MRSA in Rat

Experimental rat endocarditis model due to methicillin-resistant S. aureus.

Experimentelle Endokarditis Modell von Methicillin-resistenten<em&gt; Staphylococcus aureus</em&gt; (MRSA) in Ratten

Endovascular infections, including endocarditis, are life-threatening infectious syndromes1-3. Staphylococcus aureus is the most common world-wide cause ...

Staphylococcus aureus Growth using Human Hemoglobin as an Iron Source

S. aureus is a pathogenic bacterium that requires iron to carry out vital metabolic functions and cause disease. The most abundant reservoir of iron inside the human host is heme, which is the cofactor of hemoglobin. To acquire iron from hemoglobin, S. aureus utilizes an elaborate system known as the iron-regulated surface determinant (Isd) system1. Components of the Isd system first bind host hemoglobin, then extract and import heme, and finally liberate iron from heme in the bacterial cytoplasm2,3. This pathway has been dissected through numerous in vitro studies4-9. Further, the contribution of the Isd system to infection has been repeatedly demonstrated in mouse models8,10-14. Establishing the contribution of the Isd system to hemoglobin-derived iron acquisition and growth has proven to be more challenging. Growth assays using hemoglobin as a sole iron source are complicated by the instability of commercially available hemoglobin, contaminating free iron in the growth medium, and toxicity associated with iron chelators. Here we present a method that overcomes these limitations. High quality hemoglobin is prepared from fresh blood and is stored in liquid nitrogen. Purified hemoglobin is supplemented into iron-deplete medium mimicking the iron-poor environment encountered by pathogens inside the vertebrate host. By starving S. aureus of free iron and supplementing with a minimally manipulated form of hemoglobin we induce growth in a manner that is entirely dependent on the ability to bind hemoglobin, extract heme, pass heme through the bacterial cell envelope and degrade heme in the cytoplasm. This assay will be useful for researchers seeking to elucidate the mechanisms of hemoglobin-/heme-derived iron acquisition in S. aureus and possibly other bacterial pathogens.

Staphylococcus aureus Growth using Human Hemoglobin as an Iron Source

Here we describe a growth assay for Staphylococcus aureus using hemoglobin as the sole source of available nutrient iron. This assay establishes the role of bacterial factors involved in hemoglobin-derived iron acquisition.

Staphylococcus aureus Wachstum mit menschlichen Hämoglobins als Eisenquelle

S. aureus is a pathogenic bacterium that requires iron to  carry out vital metabolic functions and cause disease. The most abundant  reservoir of iron ...

Multiplex PCR Assay for Typing of Staphylococcal Cassette Chromosome Mec Types I to V in Methicillin-resistant Staphylococcus aureus

Staphylococcal Cassette Chromosome mec (SCCmec) typing is a very important molecular tool for understanding the epidemiology and clonal strain relatedness of methicillin-resistant Staphylococcus aureus (MRSA), particularly with the emerging outbreaks of community-associated MRSA (CA-MRSA) occurring on a worldwide basis. Traditional PCR typing schemes classify SCCmec by targeting and identifying the individual mec and ccr gene complex types, but require the use of many primer sets and multiple individual PCR experiments. We designed and published a simple multiplex PCR assay for quick-screening of major SCCmec types and subtypes I to V, and later updated it as new sequence information became available. This simple assay targets individual SCCmec types in a single reaction, is easy to interpret and has been extensively used worldwide. However, due to the sophisticated nature of the assay and the large number of primers present in the reaction, there is the potential for difficulties while adapting this assay to individual laboratories. To facilitate the process of establishing a MRSA SCCmec assay, here we demonstrate how to set up our multiplex PCR assay, and discuss some of the vital steps and procedural nuances that make it successful.

Multiplex PCR Assay for Typing of Staphylococcal Cassette Chromosome Mec Types I to V in Methicillin-resistant Staphylococcus aureus

We demonstrate a simple multiplex PCR assay for quick-screening and typing of Staphylococcal Cassette Chromosome mec (SCCmec) types I-V for methicillin-resistant Staphylococcus aureus, and provide some of the vital steps and procedural nuances that make it successful for adapting this assay to individual laboratories.

Multiplex PCR Assay f

Staphylococcal Cassette Chromosome mec (SCCmec) typing  is a very important molecular tool for understanding the epidemiology and  clonal strain ...

Studying Interactions of Staphylococcus aureus with Neutrophils by Flow Cytometry and Time Lapse Microscopy

We present methods to study the effect of phenol soluble modulins (PSMs) and other toxins produced and secreted by Staphylococcus aureus on neutrophils. To study the effects of the PSMs on neutrophils we isolate fresh neutrophils using density gradient centrifugation. These neutrophils are loaded with a dye that fluoresces upon calcium mobilization. The activation of neutrophils by PSMs initiates a rapid and transient increase in the free intracellular calcium concentration. In a flow cytometry experiment this rapid mobilization can be measured by monitoring the fluorescence of a pre-loaded dye that reacts to the increased concentration of free Ca2+. Using this method we can determine the PSM concentration necessary to activate the neutrophil, and measure the effects of specific and general inhibitors of the neutrophil activation.	
	To investigate the expression of the PSMs in the intracellular space, we have constructed reporter fusions of the promoter of the PSM&alpha; operon to GFP. When these reporter strains of S. aureus are phagocytosed by neutrophils, the induction of expression can be observed using fluorescence microscopy.

Studying Interactions of Staphylococcus aureus with Neutrophils by Flow Cytometry and Time Lapse Microscopy

We present methods to study the effect of PSMs and other toxins secreted by Staphylococcus aureus on neutrophils using flow cytometry and fluorescence microscopy.

Studieren Wechselwirkungen von<em&gt; Staphylococcus aureus</em&gt; Mit Neutrophilen durch Durchflusszytometrie und Mikroskopie Zeitraffer

We present methods to study the effect of phenol soluble modulins (PSMs)  and other toxins produced and secreted by Staphylococcus  aureus on neutrophils. ...

Genotyping of Staphylococcus aureus by Ribosomal Spacer PCR (RS-PCR)

The ribosomal spacer PCR (RS-PCR) is a highly resolving and robust genotyping method for S. aureus that allows a high throughput at moderate costs and is, therefore, suitable to be used for routine purposes. For best resolution, data evaluation and data management, a miniaturized electrophoresis system is required. Together with such an electrophoresis system and the in-house developed software (freely available here) assignment of the pattern of bands to a genotype is standardized and straight forward. DNA extraction is simple (boiling prep), setting-up of the reactions is easy and they can be run on any standard PCR machine. PCR cycling is common except prolonged ramping and elongation times. Compared to spa typing and Multi Locus Sequence Typing (MLST), RS-PCR does not require DNA sequencing what simplifies the analysis considerably and allows a high throughput. Furthermore, the resolution for bovine strains of S. aureus is at least as good as spa typing and better than MLST or pulsed-field gel electrophoresis (PFGE). The RS-PCR data base includes presently a total of 141 genotypes and variants. The method is highly associated with the virulence gene pattern, contagiosity and pathogenicity of S. aureus strains involved in bovine mastitis. S. aureus genotype B (GTB) is contagious and causes herds problems causing large costs in the Switzerland and other European countries. All the other genotypes observed in Switzerland infect individual cows and quarters. Genotyping by RS-PCR allows the reliable prediction of the epidemiological and the pathogenic potential of S. aureus involved in bovine intramammary infection (IMI), two key factors for clinical veterinary medicine. Because of these beneficial properties together with moderate costs and a high sample throughput the goal of this publication is to give a detailed, step-by-step protocol for easily establishing and running RS-PCR for genotyping S. aureus in other laboratories.

Genotyping of Staphylococcus aureus by Ribosomal Spacer PCR RS-PCR

Here, ribosomal spacer PCR (RS-PCR) is used together with a miniaturized electrophoresis system as a fast and high resolution method for genotyping S. aureus at moderate costs allowing a high throughput.

Genotypisierung<em&gt; Staphylococcus aureus</em&gt; Durch ribosomalen Spacer PCR (RS-PCR)

The ribosomal spacer PCR (RS-PCR) is a highly resolving and robust genotyping method for S. aureus that allows a high throughput at moderate costs and is, ...

Methodology for the Study of Horizontal Gene Transfer in Staphylococcus aureus

One important feature of the major opportunistic human pathogen Staphylococcus aureus is its extraordinary ability to rapidly acquire resistance to antibiotics. Genomic studies reveal that S. aureus carries many virulence and resistance genes located in mobile genetic elements, suggesting that horizontal gene transfer (HGT) plays a critical role in S. aureus evolution. However, a full and detailed description of the methodology used to study HGT in S. aureus is still lacking, especially regarding natural transformation, which has been recently reported in this bacterium. This work describes three protocols that are useful for the in vitro investigation of HGT in S. aureus: conjugation, phage transduction, and natural transformation. To this aim, the cfr gene (chloramphenicol/florfenicol resistance), which confers the Phenicols, Lincosamides, Oxazolidinones, Pleuromutilins, and Streptogramin A (PhLOPSA)-resistance phenotype, was used. Understanding the mechanisms through which S. aureus transfers genetic materials to other strains is essential to comprehending the rapid acquisition of resistance and helps to clarify the modes of dissemination reported in surveillance programs or to further predict the spreading mode in the future.

Methodology for the Study of Horizontal Gene Transfer in Staphylococcus aureus

We describe here three different protocols for the in vitro investigation of conjugation, transduction, and natural transformation in Staphylococcus aureus.

Methodik für die Studie des horizontalen Gentransfers in<em&gt; Staphylococcus aureus</em

One important feature of the major opportunistic human pathogen Staphylococcus aureus is its extraordinary ability to rapidly acquire resistance to ...

A Mouse Model to Assess Innate Immune Response to Staphylococcus aureus Infection

Staphylococcus aureus (S. aureus)&#160;infections, including methicillin resistant stains, are an enormous burden on the healthcare system. With incidence rates of S. aureus infection climbing annually, there is a demand for additional research in its&#160;pathogenicity. Animal models of infectious disease advance our understanding of the host-pathogen response and lead to the development of effective therapeutics. Neutrophils play a primary role in the innate immune response that controls S. aureus infections by forming an abscess to wall off the infection and facilitate bacterial clearance; the number of neutrophils that infiltrate an S. aureus skin infection often correlates with disease outcome. LysM-EGFP mice, which possess the enhanced green fluorescent protein (EGFP) inserted in the Lysozyme M (LysM) promoter region (expressed primarily by neutrophils), when used in conjunction with in vivo whole animal fluorescence imaging (FLI) provide&#160;a means of quantifying neutrophil emigration noninvasively and longitudinally into wounded skin. When combined with a bioluminescent S. aureus strain and sequential in vivo whole animal bioluminescent imaging (BLI), it is possible to longitudinally monitor both the neutrophil recruitment dynamics and in vivo bacterial burden at the site of infection in anesthetized mice from onset of infection to resolution or death. Mice are more resistant to a number of virulence factors produced by S. aureus that facilitate effective colonization and infection in humans. Immunodeficient mice provide a more sensitive animal model to examine persistent S. aureus infections and the ability of therapeutics to boost innate immune responses. Herein, we characterize responses in LysM-EGFP mice that have been bred to MyD88-deficient mice (LysM-EGFP&#215;MyD88-/- mice) along with wild-type LysM-EGFP mice to investigate S. aureus skin wound infection. Multispectral simultaneous detection enabled study of neutrophil recruitment dynamics by using in vivo FLI, bacterial burden by using in vivo BLI, and wound healing longitudinally and noninvasively over time.

A Mouse Model to Assess Innate Immune Response to Staphylococcus aureus Infection

An approach is described for real-time detection of the innate immune response to cutaneous wounding and Staphylococcus aureus infection of mice. By comparing LysM-EGFP mice (which possess fluorescent neutrophils) with a LysM-EGFP crossbred immunodeficient mouse strain, we advance our understanding of infection and the development of approaches to combat infection.

Ein Mausmodell zu beurteilen, angeborene Immunantwort auf Staphylococcus Aureus -Infektion

Staphylococcus aureus (S. aureus)&#160;infections, including methicillin resistant stains, are an enormous burden on the healthcare system. With incidence ...

Targeting Biofilm Assoziierte

Zusammenfassung

Weitere Videos entdecken

Kapitel in diesem Video

Mikrotiterschale Biofilmbildung Assay

Die Verwendung von Drip Flow und Rotating Disk-Reaktoren für Staphylococcus aureus Biofilm Analysis

Subkutane Infektion von Methicillin-resistenten Staphylococcus Aureus (MRSA)

Experimentelle Endokarditis Modell von Methicillin-resistenten

Staphylococcus aureus Wachstum mit menschlichen Hämoglobins als Eisenquelle

Multiplex PCR Assay f

Studieren Wechselwirkungen von

Genotypisierung

Methodik für die Studie des horizontalen Gentransfers in

Ein Mausmodell zu beurteilen, angeborene Immunantwort auf Staphylococcus Aureus -Infektion