We thank Dr. Frank Wolschendorf for access to the Cytation 3 automated imaging plate reader. This work was funded in part by NIH grant R01-AI104499 to OK. Parts of the work were performed in the UAB CFAR facilities and by the UAB CFAR Flow Cytometry Core/Joint UAB Flow Cytometry Core, which are funded by NIH/NIAID P30 AI027767 and by NIH 5P30 AR048311.

HINWEIS: M. tuberculosis mc 2 6206 ist ein avirulenten Stamm 17, 18, alle Arbeiten in diesem Protokoll kann in einem Biosafety Level 2 - Anlage (BSL-2) durchgeführt werden. 1. Kulturbedingungen für Green Fluorescent Protein exprimieren M. tuberculosis mc 2 6206 (Mtb -GFP) HINWEIS: Die M. tuberculosis H37Rv - auxotrophe Stamm mc 2 6206 abgeleitet (Δ panCD, Δ leuCD) , transformiert mit dem Plasmid gfp pMN437 exprimierenden 16 in diesem Protokoll verwendet wird. Es ist möglich , die Mtb -GFP Stamm mit nicht - GFP - exprimierenden Wildtyp - Stamm zu ersetzen für Forscher leichter Zugänglichkeit des Stammes zu ermöglichen. Allerdings ist die GFP - Expression wünschenswert visuelle Bestätigung der Phagozytose und die Bildung von Mtb / Makrophagen - Aggregate zu ermöglichen. Für long Zeitlagerung, Mtb -GFP wurden ergänzt mit 20% Glycerin bei -80 ° C in vollständiger 7H9 Medium (in Schritt 1.1 beschrieben) eingefroren. <ol><li> Bereiten vollständige Mtb -GFP Medien (7H9-C) durch Ergänzung 7H9 - Medium mit 10% OADC Middlebrook, 0,02% Tyloxapol, 24 ug / ml D-Pantothensäure, 50 ug / ml L-Leucin und 50 ug / ml Hygromycin B. </li><li> Ein Fläschchen mit Mtb -GFP auftauen und in einem 30 ml quadratischem Boden PETG Kolben auf 10 ml 7H9-C hinzufügen. </li><li> Bei 37 ° C auf einem Rotationsschüttler (50 rpm). Nehmen Sie Messungen der optischen Dichte (OD 600) alle paar Tage , bis die OD 600 erreicht 1,0 (ca. 5-8 Tage). </li><li> Verdünnen und Passage Kultur nach Bedarf eine OD 600 zwischen 0,2 bis 1,0 zu halten. </li><li> Zur Infektion eine etablierte Kultur der Mtb -GFP innerhalb der logarithmischen Wachstumsphase (OD 600 von 0,6-1) verwenden. Um klumpig Kulturen zu vermeiden, beschallen die Mtb Kulturflasche in einem Wasserbad (130 W, 3 x 5 s Pulse) ONCe oder zweimal pro Woche </li></ol> 2. Kulturbedingungen für THP-1-Zellen <ol><li> Bereiten vollständige THP-1-Zellmedium (RPMI-C) von RPMI 1640 Medium mit 10% hitzeinaktiviertem fötalem Rinderserum, 2 mM L-Glutamin und 10 mM HEPES ergänzt. </li><li> Inkubiere Zellen in einem T-75 - Kolben bei 37 ° C in einer befeuchteten Atmosphäre von 5% CO 2. </li><li> Zählen von Zellen unter Verwendung eines Hämozytometers oder Durchflusszytometrie jeden zweiten Tag und aufrechtzuerhalten THP-1-Zellen in einer Dichte zwischen 0,1 bis 0.600.000 pro mL. </li></ol> 3. Infektion Protokoll zum Generieren Mtb / Makrophagen - Aggregatstrukturen <ol><li> THP-1 Zellpräparation (pro 96-well - Platte) <ol><li> Zentrifuge 7 x 10 6 THP-1 - Zellen bei 250 xg für 5 min. Resuspendieren in 7 ml RPMI-C. </li></ol></li><li> Mtb -GFP Vorbereitung <ol><li> Zentrifuge 2,8 x 10 8 Mtb -GFP bei 3.200 xg für 5 min in einem Schwenkbecherzentrifuge ueine Umwandlung von 1,0 OD 600 = 3 x 10 8 Bakterien / ml singen. </li><li> Wasche einmal mit RPMI-C und zentrifugiert wie in Schritt 3.2.1. </li><li> Resuspendieren in 7 ml RPMI-C und 10 s vortexen. </li></ol></li><li> Infektion HINWEIS: Siehe Abbildung 1 für eine Vorlage. <ol><li> Vorbereiten eine Platte mit 96 Vertiefungen durch Zugabe von 200 &amp; mgr; l sterilem Wasser in den Reihen A und H und Spalt 1 und 12 für eine Wasserverdampfung von Felgenkulturmedium zu verhindern. </li><li> Füge 200 &amp; mgr; l RPMI-C, bis Spalte 2 (B2 bis G2) für die Hintergrundkontrolle (Blank). </li><li> Mtb -GFP Suspension (Stufe 3.2) zu THP-1 - Zellsuspension (Schritt 3.1) und gut mischen durch Pipettieren zu infizieren, hinzuzufügen. Die endgültige THP-1 Zelldichte beträgt 5 x 10 5 pro ml , und die entsprechende Vielzahl der Infektion ist 40. </li><li> Gießen Sie die THP-1 / Mtb -GFP Suspension in einem 25 - ml - Reservoir. </li><li> In 200 ul THP-1 / Mtb -GFP Suspension auf die restlichen 96 Vertiefungen (B3 bis G11) usinga Mehrkanalpipette. HINWEIS: Wenn mit mehreren 96-Well - Platten arbeiten, resuspendieren regelmäßig die restlichen THP-1 / Mtb Suspension in dem Vorratsbehälter eine gleichmäßige Mischung zu gewährleisten , zu jeder Vertiefung zugegeben. </li><li> Inkubieren bei 37 ° C mit 5% CO 2 für 7-10 Tage. </li><li> Änderungsmedien alle 2 Tage durch langsames Entfernen 100 ul verbrauchtes Medium von der Oberseite jeder Vertiefung und sanft Zugabe von 100 &amp; mgr; l vorgewärmtes RPMI-C einen Mehrkanal-Pipette. Resuspendieren Sie nicht Brunnen und stören die Mtb / Makrophagen - Aggregate auf dem Boden der Wells. </li><li> Sichtprüfung der Vertiefungen durch Fluoreszenzmikroskopie (4-10 x Objektiv) täglich, Kenntnis von der Größe von Mtb / Makrophagen - Aggregate nehmen. Am Tag 7-10, sollte Mtb / Makrophagen - Aggregate ausreichend groß sein (siehe 2 für die Bezugnahme auf Abbildung) für die Arzneimittelwirksamkeitsprüfung (Abschnitt 4) , um fortzufahren. </li><li> Falls gewünscht, können die Aufnahmen der 96-Mulden eine automatisierte Cell Imaging System ausgestattetmit Hellfeld und GFP - Filter setzt Mtb / Makrophagen - Aggregatbildung zu dokumentieren. HINWEIS: Wenn die Benutzer keinen Zugriff auf ein automatisiertes Imaging-System haben, Bilder von repräsentativen Brunnen mit GFP und Hellfeld-Funktionen unter Verwendung eines beliebigen geeigneten Mikroskop aufgenommen werden. </li></ol></li></ol> 4. Growth Inhibition Assay zur Wirksamkeit von Medikamenten gegen Mtb Beurteilen von Mtb / Makrophagen - Aggregates Abgeleitet <ol><li> Arzneimittelzubereitung (dreifacher Ausfertigung Bedingungen für zwei Medikamente) HINWEIS: Siehe Abbildung 1A für Vorlage. <ol><li> In einem separaten 96-Well-Platte, fügen Sie 125 ul 7H9-C Medien B2 bis zu G10. </li><li> Bereiten zwei Medikamente bei der doppelten höchsten gewünschte Endkonzentration in 1 ml 7H9-C für die Verdünnung zu berücksichtigen, in Schritt 4.2.4. </li><li> In 250 ul jeder Droge in die Vertiefungen B11-C11-D11 und E11-F11-G11, die jeweils für drei Exemplaren Behandlungen. </li><li> Verwendung einer Mehrkanalpipette, seriell dilute die Testmedikamente zweifach um 125 &amp; mgr; l von B11-G11 in B10-G10 bewegt. Mischen durch Pipettieren 5 mal bei jedem Schritt. </li><li> Weiter 125 &amp; mgr; l von einer Spalte zu bewegen (von rechts nach links) über die Platte, und nicht mehr, nachdem Spalte 4. </li><li> Nach dem Mischen Spalte 4, verwerfen 125 &amp; mgr; l in einen Abfallbehälter. Die Spalten 2 und 3 sollten keine Medikamente enthalten, die für die Verwendung als Hintergrund (Blank) und positive Wachstumskontrollen zu ermöglichen. Hinweis: Alternativ folgen Vorlage in 1B zum Testen Droge Bibliotheken. Jede 96-Well-Platte 58 Medikamente in einer einzigen Konzentration aufnehmen kann. Bereiten Sie dieses Medikament Platte doppelt um die gewünschte endgültige Konzentration verwendet wird, da es in der Mitte in Schritt 4.2.4 verdünnt werden. </li></ol></li><li> Medikamentöse Behandlung: <ol><li> Rufen Sie die 96-Well - Platte der Mtb - infizierten Makrophagen (Mtb / Makrophagen - Aggregate) enthält. </li><li> dekantieren sorgfältig alle relevanten Vertiefungen (B2 bis G11) mit einer Mehrkanalpipette.Führen Sie diese in einem zweistufigen Weise: zunächst 150 ul entfernen, ohne die Platte kippen, und entfernen Sie dann verbleibenden Medien (~ 50 &amp; mgr; l) durch die Platte Kippen und die Pipettenspitze an den unteren Rand des Bohrlochs eingesetzt wird. Als Mtb / Makrophagen - Aggregate an der Unterseite des Schachts adhärenten sind, sollten kein Material verloren. </li><li> Fügen Sie vorsichtig 100 ul 7H9-C Medien auf alle relevanten Brunnen (B2 durch G11) der Platte die Mtb / Makrophagen - Aggregate enthält. </li><li> Verwendung einer Mehrkanalpipette, dann 100 &amp; mgr; l aus der Arzneimittel enthaltenden 96-Well-Platte (Schritt 4.1) in die entsprechenden Vertiefungen der Platte Infektion. </li><li> Platz in versiegelten Beutel und Inkubation für 3 Tage bei 37 ° C. </li></ol></li></ol> 5. Quantifizierung der Wirksamkeit von Medikamenten den Assay Resazurin Mikrotitter Verwendung <ol><li> Bereiten Resazurin Stammlösung in einer Endkonzentration von 0,8 mg / ml in H 2 O. Man filtriert durch 0,22 um Porengrße PVDF - Membran für Sterilisierung.</li><li> Bereiten Resazurin - Lösung von Resazurin Arbeitsstammlösung Mischen, H 2 O und Tween-80 (20% ige Lösung in H 2 O) in einem 2: 1: 1 - Verhältnis. Endkonzentrationen 0,4 mg / ml Resazurin und 5% Tween-80. </li><li> Unter Verwendung eines Plattenlesegerät, ein Setup-Programm, um die Fluoreszenz bei 530 nm Anregung und 590 nm Emission alle 30 min für 24 h bei 37 ° C gelesen. Vorwärmen Plattenleser auf 37 ° C. </li><li> Fügen Sie 20 &amp; mgr; l Resazurin Arbeitslösung zu allen relevanten Vertiefungen (B2 bis G11) der arzneimittelbehandelten Platte (Schritt 4.11) eine Mehrkanal-Pipette. </li><li> Legen Sie die Platte auf dem Plattenleser und starten Sie das Programm einrichten in Schritt 5.3. ANMERKUNG: Um die Verarbeitung von großen Mengen von Assayplatten zu erleichtern, ist es ausreichend, den Test in einem 37 ° C Inkubator zu entwickeln und auszuführen einzelne Fluoreszenz all 24 h liest. Dies gilt auch für Benutzer, den Zugriff auf einen Plattenleser nicht mit kinetischen und Inkubation Fähigkeiten haben. </li></ol>

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The authors have nothing to disclose.

Hier haben wir im Detail eine alternative Mtb - Infektionsmodell geeignet für die Arzneimittelwirksamkeitstests beschrieben. Dieses Modell berücksichtigt zwei wichtige Faktoren , die eine stärkere Berücksichtigung in der frühen TB Arzneimittelentwicklung gegeben werden sollte: das Vorhandensein von physiologisch relevanten Hindernisse für Arzneimittelpenetration und metabolischen Veränderungen von Mtb während der Infektion. Während wir früher die Vorteile unserer Infektionsmodell gezeigt habe...

<html> Tuberkulose (TB) bleibt eine ernsthafte globale Bedrohung für die Gesundheit trotz der Verfügbarkeit von Anti-TB - Chemotherapien seit über 40 Jahren ein. Dies ist zum Teil auf die Forderung nach langen Behandlungszeiten von mehr als 6 Monaten mehrere Arzneimittelkombinationen, die die Nichteinhaltung 2 zu Patient führt. Die Entstehung von arzneimittelresistenten TB in den letzten Jahren hat weitere Probleme in einem Bereich verstärkt , in der erfolgreiche Entwicklung klinisch zugelassenen Medikamente 3 praktisch nicht existent ist. Denn trotz erschöpfende Anti-TB - Medikamentenentwicklung, nur ein einziges Medikament für die klinische Anwendung FDA gewesen 4 in den letzten 40 Jahren zugelassen. So werden neue Generationen von Anti-TB-Medikamente sind dringend erforderlich, um dieses Problem zu lösen. Ein wesentliches Problem bei der TB Drug Discovery ist der Mangel an erfolgreichen Transfer von Verbindungen mit in - vitro - Aktivität zur Wirksamkeit in der klinischen Einstellung= &quot;Xref&quot;&gt; 5, 6, 7. Zunächst wurden Zielbasierte Ansätze verwendet 5 für Anti- Mtb Drogen zu screenen, die in ganze Bakterienzellen zu übersetzen ist fehlgeschlagen. Selbst wenn Mtb - Zellen verwendet werden, wird es oft durchgeführt Brühe gewachsenen Kulturen verwendet, die nicht genau Arzneimittelwirksamkeit in vivo 8, 9 vorherzusagen. Diese Probleme wurden erkannt und Wirkstoff - Screening - Assays gegen Makrophagen enthalten , Mtb oder latent Mtb wurden erfolgreich etabliert 8, 10, 11, 12. Aber auch diese erweiterte Tests geben keine ausreichende Berücksichtigung der Durchdringung Barrieren, die Drogen in den nicht-gefäßLungenLäsionen auftreten, und in der Nekrosen an der Stelle der Infektion. Tatsächlich, Auch für die First-Line - TB - Medikamente Rifampicin, suboptimale Dosierung wurde 13, 14, 15 als auch in vivo Gewebe und Zerebrospinalflüssigkeit (CSF) Penetration aufgrund unzureichender in Frage gestellt als Wirksamkeit gegen intrazelluläre Mtb 8 verringert, 9. Als solche neue Modelle und Tests, die Berücksichtigung dieser Parameter während der frühen Führung Entwicklungsprozess zweifellos TB Drug Discovery nehmen würde Anstrengungen verbessern würde. Um diesem Bedarf zu begegnen, wir eine kostengünstige, schnelle und BSL-2 - kompatible Alternative Infektionsmodell für Mtb Arzneimittelwirksamkeitsprüfung 16 vor kurzem etabliert. Diese Infektionsmodell produziert dicht Mtb verpackt in großen Makrophagen Aggregatstrukturen, die physiologisch relevante zelluläre Penetrationsbarrieren rekapituliert und erzeugt Makrophagen-agierten <em&gt; Mtb mit einem veränderten physiologischen Zustand latenten Mtb ähnelt. Mtb von dieser Infektion Modell abgeleitet wurde mit dem Resazurin Mikrotitertest (REMA) kombiniert Wirksamkeit von Medikamenten zu bewerten, die mit der berichteten Fähigkeit von gemeinsamen TB - Medikamente Ergebnisse im Einklang mit anderen intrazellulären Infektionsmodellen und korreliert gut produziert hohen CSF - Konzentrationen in Bezug auf Serumkonzentrationen erzielen 16. Hier beschreiben wir ausführlich in der Erzeugung von Mtb / Makrophagen - Aggregatstrukturen Makrophagen-agierten Mtb geeignet für die Arzneimittelempfindlichkeitstests unter Verwendung von REMA herzustellen. Insbesondere zeigen wir, wie diese Infektion System auf eine 96-Well-Format für die Kompatibilität mit Durchsatz-Screening von Kandidaten-Anti-TB-Medikamente angepasst werden könnte.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>7H9</td>
			<td>BD Difco</td>
			<td>271310</td>
			<td>Follow manufacturer&#39;s recommendations</td>
		</tr>
		<tr>
			<td>Middlebrook OADC</td>
			<td>BD Biosciences</td>
			<td>212351</td>
			<td></td>
		</tr>
		<tr>
			<td>Tyloxapol</td>
			<td>Sigma</td>
			<td>T8761</td>
			<td>Prepare 20% stock solution in H2O; filter sterilize</td>
		</tr>
		<tr>
			<td>D-Pantothenic acid hemicalcium salt</td>
			<td>Sigma</td>
			<td>P5710</td>
			<td>Prepare 24 mg/ml&#160; stock solution in H2O; filter sterilize</td>
		</tr>
		<tr>
			<td>L-leucine</td>
			<td>MP Biomedicals</td>
			<td>194694</td>
			<td>Prepare 50 mg/ml&#160; stock solution in H2O; filter sterilize</td>
		</tr>
		<tr>
			<td>Hygromycin B</td>
			<td>EMD Millipore</td>
			<td>400051</td>
			<td>Prepare 200 mg/ml&#160; stock solution in H2O</td>
		</tr>
		<tr>
			<td>Nalgene Square PETG media bottle</td>
			<td>Thermo Fisher</td>
			<td>2019-0030</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640 media</td>
			<td>Hyclone</td>
			<td>SH30027.01</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Atlanta Biologicals</td>
			<td>S12450H</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Corning</td>
			<td>MT25005CI</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Hyclone</td>
			<td>SH30237.01</td>
			<td></td>
		</tr>
		<tr>
			<td>Cytation 3 plate reader</td>
			<td>Biotek</td>
			<td></td>
			<td>Interchangable with any fluorescent plate reader and microscope</td>
		</tr>
		<tr>
			<td>Gen5 Software</td>
			<td>Biotek</td>
			<td></td>
			<td>Recording and analysis of rezasurin coversion</td>
		</tr>
		<tr>
			<td>Rifampicin&#160;</td>
			<td>Fisher Scientific</td>
			<td>BP2679250</td>
			<td>Prepare 10 mg/ml stock solution in H2O</td>
		</tr>
		<tr>
			<td>Moxifloxacin Hydrochloride</td>
			<td>Acros Organics</td>
			<td>457960010</td>
			<td>Prepare 10 mg/ml stock solution in H2O</td>
		</tr>
		<tr>
			<td>Resazurin Sodium Salt</td>
			<td>Sigma</td>
			<td>R7017</td>
			<td>Prepare 800 &#956;g/mL stock solution in H2O; filter sterilize</td>
		</tr>
		<tr>
			<td>Tween-80</td>
			<td>Fisher Scientific</td>
			<td>T164500</td>
			<td>Prepare 20% stock solution in H2O; filter sterilize</td>
		</tr>
	</tbody>
</table>

a high-throughput compatible assay to evaluate drug efficacy against macrophage passaged mycobacterium tuberculosis

The early drug development process for anti-tuberculosis drugs is hindered by the inefficient translation of compounds with in vitro activity to effectiveness in the clinical setting. This is likely due to a lack of consideration for the physiologically relevant cellular penetration barriers that exist in the infected host. We recently established an alternative infection model that generates large macrophage aggregate structures containing densely packed M. tuberculosis (Mtb) at its core, which was suitable for drug susceptibility testing. This infection model is inexpensive, rapid, and most importantly BSL-2 compatible. Here, we describe the experimental procedures to generate Mtb/macrophage aggregate structures that would produce macrophage-passaged Mtb for drug susceptibility testing. In particular, we demonstrate how this infection system could be directly adapted to the 96-well plate format showing throughput capability for the screening of compound libraries against Mtb. Overall, this assay is a valuable addition to the currently available Mtb drug discovery toolbox due to its simplicity, cost effectiveness, and scalability.

Um die Robustheit der Anpassung dieser Infektionsmodell auf 96-Well - Plattenformat bestätigen, untersuchten wir hier die Wirkstoffansprechverhaltens von Mtb abgeleitet aus unserer 96-Well angepasst Infektionsmodell Rifampicin (RIF) und Moxifloxacin (Moxi) nach der Vorlage in Abbildung 1A. Wir zeigen , dass die Erzeugung von Mtb / Makrophagen Aggregatstrukturen Schlüssel zu diesem Assay kann zuverlässig in einer 96-Well - Plattenformat (Figur...

Watch this Scientific Journal Video about A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis at JoVE.com

A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis

New models and assays that would improve the early drug development process for next-generation anti-tuberculosis drugs are highly desirable. Here, we describe a quick, inexpensive, and BSL-2 compatible assay to evaluate drug efficacy against Mycobacterium tuberculosis that can be easily adapted for high-throughput screening.

Ein Hochdurchsatz-Assay unterstützte Wirksamkeit von Medikamenten gegen Makrophagen agierten zur Bewertung<em&gt; Mycobacterium tuberculosis</em

The early drug development process for anti-tuberculosis drugs is hindered by the inefficient translation of compounds with in vitro activity to ...

a-high-throughput-compatible-assay-to-evaluate-drug-efficacy-against

Research

JoVE Journal

Immunology and Infection

A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis

7.7K Aufrufe.  University of Alabama at Birmingham. New models and assays that would improve the early drug development process for next-generation anti-tuberculosis drugs are highly desirable. Here, we describe a quick, inexpensive, and BSL-2 compatible assay to evaluate drug efficacy against Mycobacterium tuberculosis that can be easily adapted for high-throughput screening. 

Serie: A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis

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 ...

Forschung

Immunologie und Infektion

An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei

Coinfections naturally occur due to the geographic overlap of distinct types of pathogenic organisms. Concurrent infections most likely modulate the respective immune response to each single pathogen and may thereby affect pathogenesis and disease outcome. Coinfected patients may also respond differentially to anti-infective interventions. Coinfection between tuberculosis as caused by mycobacteria and the malaria parasite Plasmodium, both of which are coendemic in many parts of sub-Saharan Africa, has not been studied in detail. In order to approach the challenging but scientifically and clinically highly relevant question how malaria-tuberculosis coinfection modulate host immunity and the course of each disease, we established an experimental mouse model that allows us to dissect the elicited immune responses to both pathogens in the coinfected host. Of note, in order to most precisely mimic naturally acquired human infections, we perform experimental infections of mice with both pathogens by their natural routes of infection, i.e. aerosol and mosquito bite, respectively.

An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei

Tuberculosis and malaria are two of the most prevalent infections in humans and major causes of morbidity and mortality in impoverished populations in the tropics. We established an experimental model system to study outcome of malaria-tuberculosis coinfection in mice after challenge with both pathogens via their natural route of infection.

Eine experimentelle Modell zum Studium Tuberkulose-Koinfektion auf Natur Malaria Übertragung von Mycobacterium tuberculosis Und Plasmodium berghei</i

Coinfections naturally occur due to the geographic overlap of distinct types of pathogenic organisms. Concurrent infections most likely modulate the ...

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

Simplifying microarray workflow is a necessary first step for creating MDR-TB microarray-based diagnostics that can be routinely used in lower-resource environments. An amplification microarray combines asymmetric PCR amplification, target size selection, target labeling, and microarray hybridization within a single solution and into a single microfluidic chamber. A batch processing method is demonstrated with a 9-plex asymmetric master mix and low-density gel element microarray for genotyping multi-drug resistant Mycobacterium tuberculosis (MDR-TB). The protocol described here can be completed in 6 hr and provide correct genotyping with at least 1,000 cell equivalents of genomic DNA. Incorporating on-chip wash steps is feasible, which will result in an entirely closed amplicon method and system. The extent of multiplexing with an amplification microarray is ultimately constrained by the number of primer pairs that can be combined into a single master mix and still achieve desired sensitivity and specificity performance metrics, rather than the number of probes that are immobilized on the array. Likewise, the total analysis time can be shortened or lengthened depending on the specific intended use, research question, and desired limits of detection. Nevertheless, the general approach significantly streamlines microarray workflow for the end user by reducing the number of manually intensive and time-consuming processing steps, and provides a simplified biochemical and microfluidic path for translating microarray-based diagnostics into routine clinical practice.

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

An amplification microarray combines asymmetric PCR amplification and microarray hybridization into a single chamber, which significantly streamlines microarray workflow for the end user. Simplifying microarray workflow is a necessary first step for creating microarray-based diagnostics that can be routinely used in lower-resource environments.

Der Nachweis eines multiresistenten<em&gt; Mycobacterium tuberculosis</em&gt; Verstärkung Microarray

Simplifying microarray workflow is a necessary first step for creating MDR-TB microarray-based diagnostics that can be routinely used in lower-resource ...

Establishment and Optimization of a High Throughput Setup to Study Staphylococcus epidermidis and Mycobacterium marinum Infection as a Model for Drug Discovery

Zebrafish are becoming a valuable tool in the preclinical phase of drug discovery screenings as a whole animal model with high throughput screening possibilities. They can be used to bridge the gap between cell based assays at earlier stages and in vivo validation in mammalian models, reducing, in this way, the number of compounds passing through to testing on the much more expensive rodent models. In this light, in the present manuscript is described a new high throughput pipeline using zebrafish as in vivo model system for the study of Staphylococcus epidermidis and Mycobacterium marinum infection. This setup allows the generation and analysis of large number of synchronous embryos homogenously infected. Moreover the flexibility of the pipeline allows the user to easily implement other platforms to improve the resolution of the analysis when needed. The combination of the zebrafish together with innovative high throughput technologies opens the field of drug testing and discovery to new possibilities not only because of the strength of using a whole animal model but also because of the large number of transgenic lines available that can be used to decipher the mode of action of new compounds.

Establishment and Optimization of a High Throughput Setup to Study Staphylococcus epidermidis and Mycobacterium marinum Infection as a Model for Drug Discovery

This video article describes the high throughput pipeline that has been successfully established to infect and analyze large numbers of zebrafish embryos providing a new powerful tool for compound testing and drug discovery using a whole animal vertebrate organism.

Aufbau und Optimierung eines Hochdurchsatz-Setup zum Studium<em&gt; Staphylococcus epidermidis</em&gt; Und<em&gt; Mycobacterium marinum</em&gt; Infektion als Modell für Drug Discovery

Zebrafish are becoming a valuable tool in the preclinical phase of drug discovery screenings as a whole animal model with high throughput screening ...

High-throughput Quantitative Real-time RT-PCR Assay for Determining Expression Profiles of Types I and III Interferon Subtypes

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific locked nucleic acid (LNA) and molecular beacon (MB) probes, transcript standards, automated multichannel pipetting, and plate drying. Determining expression among the type I interferons (IFN), especially the twelve IFN-&#945; subtypes, is limited by their shared sequence identity; likewise, the sequences of the type III IFN, especially IFN-&#955;2 and -&#955;3, are highly similar. This assay provides a reliable, reproducible, and relatively inexpensive means to analyze the expression of the seventeen interferon subtype transcripts.

This protocol describes a high-throughput qRT-PCR assay for the analysis of type I and III IFN expression signatures. The assay discriminates single base pair differences between the highly similar transcripts of these genes. Through batch assembly and robotic pipetting, the assays are consistent and reproducible.

Hochdurchsatz-quantitative Echtzeit-RT-PCR-Assay zur Bestimmung Expressionsprofile der Typen I und III Interferon-Formationsglieder

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific ...

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

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

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

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

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

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

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread of multi drug-resistant TB (MDR-TB), there is a real urgency to develop new therapeutic strategies against M. tuberculosis infections. Traditionally, compounds are evaluated based on their antibacterial activity under in vitro growth conditions in broth; however, results are often misleading for intracellular pathogens like M. tuberculosis since in-broth phenotypic screening conditions are significantly different from the actual disease conditions within the human body. Screening for inhibitors that work inside macrophages has been traditionally difficult due to the complexity, variability in infection, and slow replication rate of M. tuberculosis. In this study, we report a new approach to rapidly assess the effectiveness of compounds on the viability of M. tuberculosis in a macrophage infection model. Using a combination of a cytotoxicity assay and an in-broth M. tuberculosis viability assay, we were able to create a screening system that generates a comprehensive analysis of compounds of interest. This system is capable of producing quantitative data at a low cost that is within reach of most labs and yet is highly scalable to fit large industrial settings.

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

We have developed a modular high-throughput screening system for discovering novel compounds against Mycobacterium tuberculosis, targeting intracellular and in-broth growing bacteria as well as cytotoxicity against the mammalian host cell.

System zur Wirksamkeit und Zytotoxizität Screening von Inhibitoren Targeting intrazellulärer<em&gt; Mycobacterium tuberculosis</em

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread ...

An In Vitro Caseum Binding Assay that Predicts Drug Penetration in Tuberculosis Lesions

The eradication of tuberculosis disease requires drug regimens that can penetrate the multiple layers of complex pulmonary lesions. Drug distribution in the caseous cores of cavities and lesions is especially crucial because they harbor subpopulations of drug-tolerant bacteria also commonly referred to as persisters. Existing methods for the measurement of drug penetration in tuberculosis lesions involve costly and time-consuming in vivo pharmacokinetic studies coupled to bioanalytical or imaging techniques. The in vitro measurement of drug binding to caseum macromolecules was proposed as an alternative to such techniques since this binding hinders the passive diffusion of drug molecules through caseum. Rapid equilibrium dialysis is a fast and reliable system for performing plasma protein and tissue binding studies. In this protocol, we used a rapid equilibrium dialysis (RED) device to measure drug binding to homogenates of caseum that is excised from the lesions and cavities of tuberculosis-infected rabbits. The protocol also describes how to generate a surrogate matrix from lipid loaded THP-1 macrophages to use in place of caseum. This caseum/surrogate binding assay is an important tool in tuberculosis drug discovery and can be adapted to help study drug distribution in lesions or abscesses caused by other diseases.

An In Vitro Caseum Binding Assay that Predicts Drug Penetration in Tuberculosis Lesions

Here we describe a rapid equilibrium dialysis (RED) method to measure drug binding to caseum from pulmonary tuberculosis lesions and cavities. The protocol is also used with a foamy macrophage-derived matrix that is an effective surrogate to caseum.

Ein<em&gt; In Vitro</em&gt; Caseum-Bindungsassay, der die Drug Penetration in Tuberkulose-Läsionen vorhersagt

The eradication of tuberculosis disease requires drug regimens that can penetrate the multiple layers of complex pulmonary lesions. Drug distribution in ...

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

Reporter enzyme fluorescence (REF) utilizes substrates that are specific for enzymes present in target organisms of interest for imaging or detection by fluorescence or bioluminescence. We utilize BlaC, an enzyme expressed constitutively by all M. tuberculosis strains. REF allows rapid quantification of bacteria in lungs of infected mice. The same group of mice can be imaged at many time points, greatly reducing costs, enumerating bacteria more quickly, allowing novel observations in host-pathogen interactions, and increasing statistical power, since more animals per group are readily maintained. REF is extremely sensitive due to the catalytic nature of the BlaC enzymatic reporter and specific due to the custom flourescence resonance energy transfer (FRET) or fluorogenic substrates used. REF does not require recombinant strains, ensuring normal host-pathogen interactions. We describe the imaging of M. tuberculosis infection using a FRET substrate with maximal emission at 800 nm. The wavelength of the substrate allows sensitive deep tissue imaging in mammals. We will outline aerosol infection of mice with M. tuberculosis, anesthesia of mice, administration of the REF substrate, and optical imaging. This method has been successfully applied to evaluating host-pathogen interactions and efficacy of antibiotics targeting M. tuberculosis.

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

We describe the optical imaging of mice infected with Mycobacterium tuberculosis (M. tuberculosis) using reporter enzyme fluorescence (REF). This protocol facilitates the sensitive and specific detection of M. tuberculosis in pre-clinical animal models for pathogenesis, therapeutics and vaccine research.

Mycobacterium Tuberculosis in Mäusen mit Reporter Enzym Fluoreszenz Imaging

Reporter enzyme fluorescence (REF) utilizes substrates that are specific for enzymes present in target organisms of interest for imaging or detection by ...

Modeling Tuberculosis in Mycobacterium marinum Infected Adult Zebrafish

Mycobacterium tuberculosis is currently the deadliest human pathogen causing 1.7 million deaths and 10.4 million infections every year. Exposure to this bacterium causes a wide disease spectrum in humans ranging from a sterilized infection to an actively progressing deadly disease. The most common form is the latent tuberculosis, which is asymptomatic, but has the potential to reactivate into a fulminant disease. Adult zebrafish and its natural pathogen Mycobacterium marinum have recently proven to be an applicable model to study the wide disease spectrum of tuberculosis. Importantly, spontaneous latency and reactivation as well as adaptive immune responses in the context of mycobacterial infection can be studied in this model. In this article, we describe methods for the experimental infection of adult zebrafish, the collection of internal organs for the extraction of nucleic acids for the measurement of mycobacterial loads and host immune responses by quantitative PCR. The in-house-developed, M. marinum-specific qPCR assay is more sensitive than the traditional plating methods as it also detects DNA from non-dividing, dormant or recently dead mycobacteria. As both DNA and RNA are extracted from the same individual, it is possible to study the relationships between the diseased state, and the host and pathogen gene-expression. The adult zebrafish model for tuberculosis thus presents itself as a highly applicable, non-mammalian in vivo system to study host-pathogen interactions.

Modeling Tuberculosis in Mycobacterium marinum Infected Adult Zebrafish

Here, we present a protocol to model human tuberculosis in an adult zebrafish using its natural pathogen Mycobacterium marinum. Extracted DNA and RNA from the internal organs of infected zebrafish can be used to reveal the total mycobacterial loads in the fish and the host&#39;s immune responses with qPCR.

Modellierung von Tuberkulose Mycobacterium Marinum infizierten Erwachsenen Zebrafisch

Mycobacterium tuberculosis is currently the deadliest human pathogen causing 1.7 million deaths and 10.4 million infections every year. Exposure to this ...