Le travail a été réalisé avec le soutien financier de l&#39;Institut national de la Santé et l&#39;American Lung Association.

1. Biofilms plus en plus de M. la tuberculose dans une bouteille de 250 mL vis plafonné <ol><li> La préparation des médias: Dissoudre 0,5 g de KH 2 PO 4, 0,5 g de MgSO 4, 4 g de L-asparagine, 2g d&#39;acide citrique, 0,05 g de citrate d&#39;ammonium ferrique, 60mL de glycérol dans 900 ml d&#39;eau. Ajuster le pH à 7,0 avec NaOH. Autoclave, frais et juste avant de commencer l&#39;expérience, ajouter stérile ZnSO 4 à une concentration finale de 0,1% p / v Depuis mc 2 7000 est un auxotrophe pantothénate cette souche nécessite également de l&#39;acide pantothénique à 10μg/mL de concentration finale. </li></ol> Note: Ceci est une composition standard des médias Sauton utilisés pour M. tuberculosis. Toutefois, si nécessaire d&#39;autres médias spécialisés peuvent également être utilisés pour d&#39;autres espèces de mycobactéries. <ol start="2"><li> La préparation des inoculums: Grow M. la tuberculose dans 7H9OADC avec 0,05% de Tween-80 pendant une semaine, ou DO 600 de 0,7 à 1,0. Le culture peut être directement utilisée comme inoculum à une dilution de 1:100. </li><li> Distribuer 25 ml de Sauton médias à une bouteille de 250 ml en polystyrène vis plafonné (Corning). Ajouter 250μl de l&#39;inoculum dans le milieu, bouchon de la bouteille très serré et le placer tranquillement dans un incubateur humidifié 37 ° C pendant 3 semaines. Observez la bouteille une fois par jour pour s&#39;assurer qu&#39;il n&#39;y a pas de contamination. </li><li> A la fin de la troisième semaine, dévisser le bouchon de la bouteille afin de permettre une nouvelle croissance de M. la tuberculose à l&#39;interface. Si le bouchon n&#39;est pas desserré, à ce stade, puis la concentration en oxygène insuffisante dans le conteneur va retarder la croissance des bactéries encore. </li></ol> 2. La croissance de M. biofilms tuberculose dans des plaques 12 puits <ol><li> Préparer les médias et l&#39;inoculum de mc 2 7000 tel que décrit dans les sections A1 et A2. </li><li> Mélanger 60 ml de médias avec 600μl d&#39;inoculum. Distribuer 4,5 millions deL du mélange dans chaque puits de la plaque. Recouvrir la plaque avec un couvercle. Envelopper la plaque à plusieurs reprises avec du parafilm. Incuber la plaque tranquillement dans incubateur humidifié à 37 ° C pendant 5 semaines. </li></ol> 3. Détermination de la fréquence des persistants de drogues tolérantes en M. la tuberculose biofilms <ol><li> Grow M. biofilms tuberculose dans un format de 12 puits comme décrit dans la section B. Cela va prendre un total d&#39;environ 5 semaines de. </li><li> Une fois que les biofilms sont élevés (après 5 semaines d&#39;incubation) d&#39;injecter votre choix de l&#39;antibiotique à une concentration désirée dans les médias sous les biofilms en utilisant une microsonde dans une pipette. </li></ol> Remarque: Le volume des médias sous les pellicules réduit à environ 3.0mL. Donc, les enquêteurs devraient donc calculer la quantité de médicament. <ol start="3"><li> Swirl doucement la plaque de sorte que l&#39;antibiotique est bien diffusé dans le milieu. Pour obtenir des résultats statistiquement significatifs, injecter le antibiotiquesotique dans quatre puits. En parallèle, injecter le même volume de solvant dans lequel l&#39;antibiotique a été dissous dans quatre autres puits, et de laisser les quatre derniers puits de la plaque vierge. Recouvrir la plaque avec un couvercle et de mettre de nouvelles couches de parafilm autour de la plaque. Placez-le revenir dans l&#39;incubateur pour la période désirée. </li><li> À la fin de l&#39;incubation, ouvrir la plaque et ajouter le Tween-80 à la concentration finale de 0,1% (volume / volume) dans chacun des puits. Swirl toute la plaque en douceur pour la dispersion uniforme du détergent. Incuber la plaque à température ambiante pendant 15 minutes. Mélanger le contenu de chaque puits avec la pipette plusieurs fois afin que le contenu entier peut être uniformément transférés à un tube de 15 ml conique. </li><li> Centrifuger le contenu du tube à 4000rpm pendant 10 minutes à température ambiante. Reprendre le culot dans 5 ml de tampon de lavage fraîche (PBS avec 10% de glycérol et 0,05% de Tween-80). Répétez les trois temps de lavage. Reprendre le culot dans 5 ml de tampon de lavage. Gardez-le sur la bascule pour overnight à 4 ° C. </li></ol> Remarque: Bien que basse température a été initialement développé pour M. smegmatis (afin de minimiser sa croissance lors de la dispersion) et utilisé pour M. tuberculosis ainsi, les espèces à croissance lente peut très probablement être secoué à la température ambiante sans aucun impact sur ​​le résultat. Rocking à la température ambiante pourrait être nécessaire si vous travaillez dans BSL-3 installation. <ol start="6"><li> Préparer une seringue stérile équipée micropointe (2-200 pl), en coupant son extrémité la plus large à la taille appropriée, en l&#39;adaptant à la seringue et l&#39;envelopper avec du parafilm. Passez l&#39;ensemble du contenu du tube à travers la seringue à embout-équipée et recueillir dans un des tubes de 15 ml frais. Répétez cette étape 5 à 6 fois jusqu&#39;à ce que vous observez une suspension assez homogène. </li><li> Préparer des dilutions en série de la suspension et la plaque des dilutions sur la plaque 7H11OADC pour déterminer le nombre de colonies viables dans chaque puits. Incuber les boîtes pendant trois semaines à 37 ° C incubateur. Déterminer la fréCY d&#39;objets persistants dans la population biofilm en calculant le rapport du nombre de colonies obtenues dans antibiotique traitée à celles obtenues lors de solvant plaques traitées. </li></ol> 4. Les résultats représentatifs Lorsqu&#39;elle est cultivée dans une bouteille, la croissance de M. la tuberculose peut être vu à la base de la bouteille à la fin de la première semaine. À la fin de la deuxième semaine, la croissance inégale des bactéries sur l&#39;interface air-support peut être vu, même si la croissance à l&#39;interface air-media est toujours visible à la fin de la troisième semaine (fig. 1A). À ce moment de la fixation des bactéries à la paroi du récipient est également observée. A partir de ce point de la croissance de la culture se fait principalement sur l&#39;interface air-médias. Le liquide sous la surface de la croissance est clair. En règle générale, la structure évolue d&#39;ici la fin de la cinquième semaine (figure 1B). Si l&#39;incubation est prolongée, les structures de va commencer à couler au fond du récipient. Curieusement,serrage du bouchon jusqu&#39;à la fin de la troisième semaine est une étape importante dans le processus, et pour des raisons inconnues une bouteille lâche-capped retarde significativement l&#39;initiation de la croissance sur l&#39;interface 9. Dans le format de 12 puits, un biofilm robuste à l&#39;interface air-médiatique est perçue dans chacun des puits à la fin de cinq semaines (figure 2A). Si les plaques ne sont pas enveloppés complètement, puis la croissance du biofilm différentiel est observée. Dans le pire des cas, l&#39;évaporation importante des médias peuvent retarder la croissance des bactéries (figure 2B). Ainsi, l&#39;emballage de la plaque est nécessaire à la fois pour empêcher l&#39;évaporation ainsi que de fournir un environnement pour la formation de biofilm (voir paragraphe précédent). Le nombre de bacilles viables dans les biofilms déterminés avec cette technique est tout à fait reproductible. Réponse de M. tuberculose biofilms varie avec la nature des antibiotiques. Pour un antibiotique bactéricide tels que la rifampicine, la perte de la viabilité suit une bipha<html> tendance sic 9. Un déclin rapide de la viabilité au cours des trois à quatre premiers jours, suivie d&#39;une phase persistante dans laquelle un petit pourcentage de la population restent totalement récalcitrant aux antibiotiques, quelle que soit la concentration de l&#39;antibiotique ou le temps d&#39;exposition. La figure 3 montre le nombre de bacilles viables dans les biofilms matures après une exposition de 7 jours à 50μg/mL (50 fois plus élevé que le MIC) de la rifampicine. <img alt="Figure 1A." src="/files/ftp_upload/3820/3820fig1A.jpg" /> Figure 1A. Apparition précoce de M. bacilles de la tuberculose sur l&#39;interface air-media de bactéries après 3 semaines d&#39;incubation. <img alt="Figure 1B." src="/files/ftp_upload/3820/3820fig1B.jpg" /> La figure 1B. Elevage biofilms de M. la tuberculose sur l&#39;interface air-médias après cinq semaines d&#39;incubation. 820/3820fig2A.jpg &quot;/&gt; Figure 2A. 5-semaine biofilms vieux de M. la tuberculose cultivé dans 12 puits de format. <img alt="La figure 2B." src="/files/ftp_upload/3820/3820fig2B.jpg" /> La figure 2B. Une tentative a échoué à croître M. biofilms tuberculose dans 12 puits de plaque sans parafilm. <img alt="Figure 3." src="/files/ftp_upload/3820/3820fig3.jpg" /> Figure 3. Un graphique représentatif sur la fréquence des persistants de drogues tolérantes en M. la tuberculose biofilms cultivés dans 12 puits format et exposées à 50μg/mL de rifampicine pendant sept jours.

<ol>
	<li>Saltini, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemotherapy+and+diagnosis+of+tuberculosis.">Chemotherapy and diagnosis of tuberculosis.</a> Respir. Med. 100, 2085-2097 (2006).</li><li>Hall-Stoodley, L., 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=Biofilm+formation+and+dispersal+and+the+transmission+of+human+pathogens.">Biofilm formation and dispersal and the transmission of human pathogens.</a> Trends Microbiol. 13, 7-10 (2005).</li><li>Costerton, J. W., Stewart, P. S., Greenberg, E. 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:+a+common+cause+of+persistent+infections.">Bacterial biofilms: a common cause of persistent infections.</a> Science. 284, 1318-1322 (1999).</li><li>Blankenship, J. R., Mitchell, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+to+build+a+biofilm:+a+fungal+perspective.">How to build a biofilm: a fungal perspective.</a> Curr Opin Microbiol. 9, 588-594 (2006).</li><li>Henke, J. M., Bassler, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+social+engagements.">Bacterial social engagements.</a> Trends Cell Biol. 14, 648-656 (2004).</li><li>Kolter, R., Losick, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=One+for+all+and+all+for+one.">One for all and all for one.</a> Science. 280, 226-227 (1998).</li><li>Branda, S. 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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+of+Mycobacterium+tuberculosis+biofilms+containing+free+mycolic+acids+and+harbouring+drug-tolerant+bacteria.">Growth of Mycobacterium tuberculosis biofilms containing free mycolic acids and harbouring drug-tolerant bacteria.</a> Mol. Microbiol. 69, 164-174 (2008).</li><li>Ojha, A. K., Trivelli, X., Guerardel, Y., Kremer, L., Hatfull, G. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzymatic+hydrolysis+of+trehalose+dimycolate+releases+free+mycolic+acids+during+mycobacterial+growth+in+biofilms.">Enzymatic hydrolysis of trehalose dimycolate releases free mycolic acids during mycobacterial growth in biofilms.</a> J. Biol. Chem. 285, 17380-17389 (2010).</li><li>Dye, C., Lonnroth, K., Jaramillo, E., Williams, B. G., Raviglione, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trends+in+tuberculosis+incidence+and+their+determinants+in+134+countries.">Trends in tuberculosis incidence and their determinants in 134 countries.</a> Bull World Health Organ. 87, 683-691 (2009).</li><li>Jindani, A., Dore, C. J., Mitchison, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bactericidal+and+sterilizing+activities+of+antituberculosis+drugs+during+the+first+14+days.">Bactericidal and sterilizing activities of antituberculosis drugs during the first 14 days.</a> Am. J. Respir. Crit. Care Med. 167, 1348-1354 (2003).</li><li>Carter, G., Wu, M., Drummond, D. C., Bermudez, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+biofilm+formation+by+clinical+isolates+of+Mycobacterium+avium.">Characterization of biofilm formation by clinical isolates of Mycobacterium avium.</a> J. Med. Microbiol. 52, 747-752 (2003).</li><li>Hall-Stoodley, L., Lappin-Scott, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biofilm+formation+by+the+rapidly+growing+mycobacterial+species+Mycobacterium+fortuitum.">Biofilm formation by the rapidly growing mycobacterial species Mycobacterium fortuitum.</a> FEMS Microbiol. Lett. 168, 77-84 (1998).</li><li>Alibaud, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temperature-dependent+regulation+of+mycolic+acid+cyclopropanation+in+saprophytic+mycobacteria:+role+of+the+Mycobacterium+smegmatis+1351+gene+(MSMEG_1351)+in+CIS-cyclopropanation+of+alpha-mycolates.">Temperature-dependent regulation of mycolic acid cyclopropanation in saprophytic mycobacteria: role of the Mycobacterium smegmatis 1351 gene (MSMEG_1351) in CIS-cyclopropanation of alpha-mycolates.</a> J. Biol. Chem. 285, 21698-21707 (2010).</li></ol>

Nous n&#39;avons rien à communiquer.

La tuberculose (TB), causée par l&#39;infection de Mycobacterium tuberculosis, demeure une menace majeure pour la santé publique mondiale. Près d&#39;un tiers de la population mondiale est estimée à asymptomatique infecté par l&#39;agent pathogène, environ 9 millions de nouveaux cas apparaissent chaque année dans la clinique avec des symptômes de tuberculose évolutive et environ 1,7 million de personnes meurent de l&#39;infection chaque année 11. Le lourd fardeau de la maladie est principa...

<table border="1">
	<tbody>
		<tr>
			<td>Equipment and supplies</td>
			<td>SUPPLIER</td>
			<td>CATALOG NUMBER</td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>VWR</td>
			<td>Model # 1923/25</td>
		</tr>
		<tr>
			<td>Polystyrene culture bottles</td>
			<td>Fisher Scientific</td>
			<td>03-374-300</td>
		</tr>
		<tr>
			<td>12-well tissue culture plate</td>
			<td>VWR</td>
			<td>62406-165</td>
		</tr>
		<tr>
			<td>50-mL conical tubes</td>
			<td>VWR</td>
			<td>89039-660</td>
		</tr>
		<tr>
			<td>Rocker</td>
			<td>Thermo Scientific</td>
			<td>57019-662</td>
		</tr>
		<tr>
			<td>Chromatographic refrigerator</td>
			<td>VWR</td>
			<td>55702-520</td>
		</tr>
		<tr>
			<td>petri dish</td>
			<td>VWR</td>
			<td>25384-342</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>REAGENT</td>
			<td>SUPPLIER</td>
			<td>CATALOG NUMBER</td>
		</tr>
		<tr>
			<td>KH2PO4 (monobasic)</td>
			<td>EMD</td>
			<td>PX1565-1</td>
		</tr>
		<tr>
			<td>MgSO4</td>
			<td>Fisher</td>
			<td>M65-500</td>
		</tr>
		<tr>
			<td>L-asparagine</td>
			<td>Sigma</td>
			<td>A4284-100G</td>
		</tr>
		<tr>
			<td>citric acid</td>
			<td>Sigma</td>
			<td>C1857-100G</td>
		</tr>
		<tr>
			<td>ferric ammonium citrate</td>
			<td>Sigma</td>
			<td>F5879-100G</td>
		</tr>
		<tr>
			<td>glycerol</td>
			<td>EMD</td>
			<td>GX0185-5</td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Sigma</td>
			<td>S8045-500G</td>
		</tr>
		<tr>
			<td>ZnSO4</td>
			<td>Sigma</td>
			<td>Z4750-500G</td>
		</tr>
		<tr>
			<td>D-pantothenic acid</td>
			<td>Sigma</td>
			<td>P2250-25G</td>
		</tr>
		<tr>
			<td>Difco Middlebrook 7H9 Broth</td>
			<td>Becton Dickinson</td>
			<td>271310</td>
		</tr>
		<tr>
			<td>Middlebrook OADC Enrichment</td>
			<td>BBL</td>
			<td>212351</td>
		</tr>
		<tr>
			<td>Tween-80</td>
			<td>Fisher</td>
			<td>T164-500</td>
		</tr>
		<tr>
			<td>250mL storage bottle</td>
			<td>Corning</td>
			<td>430281</td>
		</tr>
		<tr>
			<td>12 well plates</td>
			<td>Falcon (BD)</td>
			<td>353043</td>
		</tr>
		<tr>
			<td>rifampicin</td>
			<td>Sigma</td>
			<td>R3501-1G</td>
		</tr>
		<tr>
			<td>methanol</td>
			<td>J.T. Baker</td>
			<td>9070-05</td>
		</tr>
		<tr>
			<td>10mlLsyringe</td>
			<td>Becton Dickinson</td>
			<td>301604</td>
		</tr>
		<tr>
			<td>1-200&mu;L pipet tips</td>
			<td>VWR</td>
			<td>89079-458</td>
		</tr>
		<tr>
			<td>parafilm M</td>
			<td>VWR</td>
			<td>PM-996</td>
		</tr>
		<tr>
			<td>15mL centrifuge tube</td>
			<td>Greiner Bio-One</td>
			<td>188-285</td>
		</tr>
		<tr>
			<td>Difco Mycobacteria 7H11 Agar</td>
			<td>Becton Dickinson</td>
			<td>283810</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Fisher</td>
			<td>BP358-1</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma</td>
			<td>P9333-500G</td>
		</tr>
		<tr>
			<td>Na2HPO4 (dibasic)</td>
			<td>Sigma</td>
			<td>S0876-500G</td>
		</tr>
	</tbody>
</table>

growth of mycobacterium tuberculosis biofilms

Mycobacterium tuberculosis, l&#39;agent étiologique de la tuberculose humaine, a une capacité extraordinaire de survivre contre les stress environnementaux, notamment des antibiotiques. Bien que la tolérance au stress de M. la tuberculose est l&#39;une des contributeurs susceptibles à la chimiothérapie de 6 mois à long de la tuberculose 1, les mécanismes moléculaires qui sous-tendent ce phénotype caractéristique de l&#39;agent pathogène restent floues. Beaucoup d&#39;espèces microbiennes ont évolué pour survivre dans des environnements stressants par auto-assemblage en très organisée, la surface fixée, et la matrice des structures encapsulées appelé biofilms 2-4. La croissance dans les collectivités qui semble être une stratégie de survie préféré des microbes, et est réalisé grâce à des composants génétiques qui régulent la surface de fixation, les communications intercellulaires, et la synthèse de substances polymériques extracellulaires (EPS) 5,6. La tolérance au stress environnemental est probablement facilité par EPS, et peut-être par le physiol&#39;adaptation logique de bacilles individu à microenvironnements hétérogènes au sein de l&#39;architecture complexe des biofilms 7. Dans une série d&#39;articles récents, nous avons établi que M. la tuberculose et le Mycobacterium smegmatis ont une forte propension à se développer dans des structures multicellulaires organisés, appelés biofilms, qui peuvent tolérer plus de 50 fois les concentrations minimales inhibitrices des médicaments anti-tuberculeux isoniazide et 8-10 rifampicine. M. la tuberculose, cependant, nécessite curieusement conditions spécifiques à former des biofilms matures, en particulier de rapport de 9:01 espace de tête: les médias ainsi que l&#39;échange limité de l&#39;air avec l&#39;atmosphère 9. Exigences de conditions environnementales spécialisées pourrait être lié au fait que M. la tuberculose est un agent pathogène obligatoire humaine et a donc adapté aux environnements de tissus. Dans cette publication, nous la démonstration de méthodes de culture de M. tuberculosebiofilms dans une bouteille et un format de plaque à 12 puits, ce qui est pratique pour les études bactériologiques ainsi que génétiques. Nous avons décrit le protocole d&#39;une souche atténuée de M. la tuberculose, mc 2 7000, avec la suppression dans les deux locus, panCD et RD1, qui sont cruciaux pour la croissance in vivo de la 9 pathogène. Cette souche peut être utilisé sans danger dans une enceinte BSL-2 pour la compréhension de la biologie de base de l&#39;agent pathogène tuberculose évitant ainsi l&#39;exigence d&#39;un coûteux BSL-3 installation. La méthode peut être étendue, moyennant des modifications appropriées dans les médias, à se développer un biofilm d&#39;autres espèces de mycobactéries cultivables. Dans l&#39;ensemble, un protocole uniforme de biofilms culture mycobactérienne aidera les enquêteurs s&#39;intéressent à l&#39;étude des caractéristiques de base élastiques de mycobactéries. En outre, une méthode claire et concise de la croissance des biofilms mycobactériens aidera également le inv clinique et pharmaceutiqueestigators pour tester l&#39;efficacité d&#39;un médicament potentiel.

Watch this Scientific Journal Video about Growth of Mycobacterium tuberculosis Biofilms at JoVE.com

Growth of Mycobacterium tuberculosis Biofilms

Mycobacterium tuberculosis forme des biofilms de drogues tolérantes quand elles sont cultivées dans certaines conditions. Nous décrivons ici les méthodes de culture, M. biofilms tuberculose et la détermination de la fréquence des persistants de drogue tolérante. Ces protocoles seront utiles pour d&#39;autres études sur les mécanismes de la tolérance au médicament chez M. la tuberculose.

La croissance de la Mycobacterium tuberculosis Les biofilms

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

growth-of-mycobacterium-tuberculosis-biofilms

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Immunology and Infection

Growth of Mycobacterium tuberculosis Biofilms

23.6K vues.  University of Pittsburgh. Mycobacterium tuberculosis forme des biofilms de drogues tolérantes quand elles sont cultivées dans certaines conditions. Nous décrivons ici les méthodes de culture, M. biofilms tuberculose et la détermination de la fréquence des persistants de drogue tolérante. Ces protocoles seront utiles pour d'autres études sur les mécanismes de la tolérance au médicament chez M. la tuberculose.

Vidéo: Growth of Mycobacterium tuberculosis Biofilms

Antimicrobial Susceptibility Testing of Mycobacterium Tuberculosis Complex for First and Second Line Drugs by Broth Dilution in a Microtiter Plate Format

The rapid detection of antimicrobial resistance is important in the effort to control the increase in resistant Mycobacterium 
tuberculosis (Mtb). Antimicrobial susceptibility testing (AST) of Mtb has traditionally been performed by the agar method of proportion or by 
macrobroth testing on an instrument such as the BACTEC (Becton Dickinson, Sparks, MD), VersaTREK (TREK Diagnostics, Cleveland, OH) or BacT/ALERT (bioM&eacute;rieux, Hazelwood, MO). The agar proportion method, while considered the &#x201C;gold&#x201D; standard of AST, is labor intensive and requires calculation of resistance by performing colony counts on drug-containing agar as compared to drug-free agar. If there is &ge;1% growth on the drug-containing medium as compared to drug-free medium, the organism is considered resistant to that drug. The macrobroth methods require instrumentation and test break point ("critical") drug concentrations for the first line drugs (isoniazid, ethambutol, rifampin, and pyrazinamide). The method described here is commercially available in a 96 well microtiter plate format [MYCOTB (TREK Diagnostics)] and contains increasing concentrations of 12 antimicrobials used for treatment of tuberculosis including both first (isoniazid, rifampin, ethambutol) and second line drugs (amikacin, cycloserine, ethionamide, kanamycin, moxifloxacin, ofloxacin, para-aminosalicylic acid, rifabutin, and streptomycin). Pyrazinamide, a first line drug, is not included in the microtiter plate due to its need for acidic test conditions. Advantages of the microtiter system include both ease of set up and faster turn around time (14 days) compared with traditional agar proportion (21 days). In addition, the plate can be set up from inoculum prepared using either broth or solid medium. Since the microtiter plate format is new and since Mtb presents unique safety challenges in the laboratory, this protocol will describe how to safely setup, incubate and read the microtiter plate.

Antimicrobial susceptibility testing of Mycobacterium tuberculosis is challenging but critical for patient care. This microtiter plate format offers testing of 12 antimycobacterial drugs and provides minimum inhibitory concentration (MIC) values, which will aid in appropriate treatment.

Test de sensibilité aux antimicrobiens du complexe Mycobacterium tuberculosis des drogues de première et deuxième par dilution en bouillon dans un format microplaque

The rapid detection of antimicrobial resistance is important in the effort to control the increase in resistant Mycobacterium 
tuberculosis (Mtb). ...

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Immunologie et infection

Sample Preparation of Mycobacterium tuberculosis Extracts for Nuclear Magnetic Resonance Metabolomic Studies

Mycobacterium tuberculosis is a major cause of mortality in human beings on a global scale. The emergence of both multi- (MDR) and extensively-(XDR) drug-resistant strains threatens to derail current disease control efforts. Thus, there is an urgent need to develop drugs and vaccines that are more effective than those currently available. The genome of M. tuberculosis has been known for more than 10 years, yet there are important gaps in our knowledge of gene function and essentiality. Many studies have since used gene expression analysis at both the transcriptomic and proteomic levels to determine the effects of drugs, oxidants, and growth conditions on the global patterns of gene expression. Ultimately, the final response of these changes is reflected in the metabolic composition of the bacterium including a few thousand small molecular weight chemicals. Comparing the metabolic profiles of wild type and mutant strains, either untreated or treated with a particular drug, can effectively allow target identification and may lead to the development of novel inhibitors with anti-tubercular activity. Likewise, the effects of two or more conditions on the metabolome can also be assessed. Nuclear magnetic resonance (NMR) is a powerful technology that is used to identify and quantify metabolic intermediates. In this protocol, procedures for the preparation of M. tuberculosis cell extracts for NMR metabolomic analysis are described. Cell cultures are grown under appropriate conditions and required Biosafety Level 3 containment,1 harvested, and subjected to mechanical lysis while maintaining cold temperatures to maximize preservation of metabolites. Cell lysates are recovered, filtered sterilized, and stored at ultra-low temperatures. Aliquots from these cell extracts are plated on Middlebrook 7H9 agar for colony-forming units to verify absence of viable cells. Upon two months of incubation at 37 &deg;C, if no viable colonies are observed, samples are removed from the containment facility for downstream processing. Extracts are lyophilized, resuspended in deuterated buffer and injected in the NMR instrument, capturing spectroscopic data that is then subjected to statistical analysis. The procedures described can be applied for both one-dimensional (1D) 1H NMR and two-dimensional (2D) 1H-13C NMR analyses. This methodology provides more reliable small molecular weight metabolite identification and more reliable and sensitive quantitative analyses of cell extract metabolic compositions than chromatographic methods. Variations of the procedure described following the cell lysis step can also be adapted for parallel proteomic analysis.

Sample Preparation of Mycobacterium tuberculosis Extracts for Nuclear Magnetic Resonance Metabolomic Studies

The metabolomic profile of Mycobacterium tuberculosis is determined after growth in broth cultures. Conditions can be varied to test the effects of nutritional supplements, oxidants, and anti-tuberculosis agents on the metabolic profile of this microorganism. Procedure for extract preparation is applicable for both 1D 1H and 2D 1H-13C NMR analyses.

Préparation de l&#39;échantillon de<em&gt; Mycobacterium tuberculosis</em&gt; Extraits pour Magnétique Nucléaire études métabolomiques résonance

Mycobacterium tuberculosis is a major cause of mortality in human beings on a global scale. The emergence of both multi- (MDR) and extensively-(XDR) ...

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.

Un modèle expérimental pour étudier la tuberculose-paludisme co-infection à transmission naturelle de Mycobacterium tuberculosis Et 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.

Faisant preuve d&#39;une multi-résistante<em&gt; Mycobacterium tuberculosis</em&gt; Amplification de puces à ADN

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

A Novel Microdissection Approach to Recovering Mycobacterium tuberculosis Specific Transcripts from Formalin Fixed Paraffin Embedded Lung Granulomas

Microdissection has been used for the examination of tissues at DNA, RNA, and protein levels for over a decade. Laser capture microscopy (LCM) is the most common microdissection technique used today. In this technique, a laser is used to focally melt a thermoplastic membrane that overlies a dehydrated tissue section1. The tissue section composite is then lifted and separated from the membrane. Although this technique can be used successfully for tissue examination, it is time consuming and expensive. Furthermore, the successful completion of procedures using this technique requires the use of a laser, thus limiting its use. A new more affordable and practical microdissection approach called mesodissection is a possible solution to the pitfalls of LCM. This technique employs the MESO-1/MeSectr system to mill the desired tissue from a slide mounted tissue sample while concurrently dispensing and aspirating fluid to recover the desired tissue sample into a consumable mill bit. Before the dissection process begins, the user aligns the formalin fixed paraffin embedded (FFPE) slide with a hematoxylin and eosin stained (H&#38;E) reference slide. Thereafter, the operator annotates the desired dissection area and proceeds to dissect the appropriate segment. The program generates an archived image of the dissection. The main advantage of mesodissection is the short duration needed to dissect a slide, taking an average of ten minutes from set up to sample generation in this experiment. Additionally, the system is significantly more cost effective and user friendly. A slight disadvantage is that it is not as precise as laser capture microscopy. In this article we demonstrate how mesodissection can be used to extract RNA from slides from FFPE granulomas caused by Mycobacterium tuberculosis (Mtb).

A Novel Microdissection Approach to Recovering Mycobacterium tuberculosis Specific Transcripts from Formalin Fixed Paraffin Embedded Lung Granulomas

Microdissection has been extensively employed for the examination of DNA, RNA, and protein within tissue. Laser capture microscopy (LCM) is the most commonly used method, but a new milling technique, mesodissection, is recently available. We demonstrate RNA extraction from mesodissected formalin fixed paraffin embedded tissue slides of Mycobacterium tuberculosis granulomas.

Une nouvelle approche microdissection de la récupération<em&gt; Mycobacterium tuberculosis</em&gt; Transcriptions spécifiques de formol paraffine fixe embarqué poumon granulomes

Microdissection has been used for the examination of tissues at DNA, RNA, and protein levels for over a decade. Laser capture microscopy (LCM) is the most ...

A 3D Human Lung Tissue Model for Functional Studies on Mycobacterium tuberculosis Infection

Tuberculosis (TB) still holds a major threat to the health of people worldwide, and there is a need for cost-efficient but reliable models to help us understand the disease mechanisms and advance the discoveries of new treatment options. In vitro cell cultures of monolayers or co-cultures lack the three-dimensional (3D) environment and tissue responses. Herein, we describe an innovative in vitro model of a human lung tissue, which holds promise to be an effective tool for studying the complex events that occur during infection with Mycobacterium tuberculosis (M. tuberculosis). The 3D tissue model consists of tissue-specific epithelial cells and fibroblasts, which are cultured in a matrix of collagen on top of a porous membrane. Upon air exposure, the epithelial cells stratify and secrete mucus at the apical side. By introducing human primary macrophages infected with M. tuberculosis to the tissue model, we have shown that immune cells migrate into the infected-tissue and form early stages of TB granuloma. These structures recapitulate the distinct feature of human TB, the granuloma, which is fundamentally different or not commonly observed in widely used experimental animal models. This organotypic culture method enables the 3D visualization and robust quantitative analysis that provides pivotal information on spatial and temporal features of host cell-pathogen interactions. Taken together, the lung tissue model provides a physiologically relevant tissue micro-environment for studies on TB. Thus, the lung tissue model has potential implications for both basic mechanistic and applied studies. Importantly, the model allows addition or manipulation of individual cell types, which thereby widens its use for modelling a variety of infectious diseases that affect the lungs.

A 3D Human Lung Tissue Model for Functional Studies on Mycobacterium tuberculosis Infection

Human tuberculosis infection is a complex process, which is difficult to model in vitro. Here we describe a novel 3D human lung tissue model that recapitulates the dynamics that occur during infection, including the migration of immune cells and early granuloma formation in a physiological environment.

Un modèle de tissu 3D Poumon humain pour les études fonctionnelles sur<em&gt; Mycobacterium tuberculosis</em&gt; Infection

Tuberculosis (TB) still holds a major threat to the health of people worldwide, and there is a need for cost-efficient but reliable models to help us ...

Ratiometric Imaging of Extracellular pH in Dental Biofilms

The pH in bacterial biofilms on teeth is of central importance for dental caries, a disease with a high worldwide prevalence. Nutrients and metabolites are not distributed evenly in dental biofilms. A complex interplay of sorption to and reaction with organic matter in the biofilm reduces the diffusion paths of solutes and creates steep gradients of reactive molecules, including organic acids, across the biofilm. Quantitative fluorescent microscopic methods, such as fluorescence life time imaging or pH ratiometry, can be employed to visualize pH in different microenvironments of dental biofilms. pH ratiometry exploits a pH-dependent shift in the fluorescent emission of pH-sensitive dyes. Calculation of the emission ratio at two different wavelengths allows determining local pH in microscopic images, irrespective of the concentration of the dye. Contrary to microelectrodes the technique allows monitoring both vertical and horizontal pH gradients in real-time without mechanically disturbing the biofilm. However, care must be taken to differentiate accurately between extra- and intracellular compartments of the biofilm. Here, the ratiometric dye, seminaphthorhodafluor-4F 5-(and-6) carboxylic acid (C-SNARF-4) is employed to monitor extracellular pH in in vivo grown dental biofilms of unknown species composition. Upon exposure to glucose the dye is up-concentrated inside all bacterial cells in the biofilms; it is thus used both as a universal bacterial stain and as a marker of extracellular pH. After confocal microscopic image acquisition, the bacterial biomass is removed from all pictures using digital image analysis software, which permits to exclusively calculate extracellular pH. pH ratiometry with the ratiometric dye is well-suited to study extracellular pH in thin biofilms of up to 75 &#181;m thickness, but is limited to the pH range between 4.5 and 7.0.

A pH-sensitive ratiometric dye is used in combination with confocal laser scanning microscopy and digital image analysis to monitor extracellular pH in dental biofilms in real-time.

Imagerie Ratiométrique du pH extracellulaire dans biofilms dentaires

The pH in bacterial biofilms on teeth is of central importance for dental caries, a disease with a high worldwide prevalence. Nutrients and metabolites ...

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.

Système d&#39;efficacité et de dépistage des inhibiteurs Cytotoxicité ciblage intracellulaire<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 ...

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.

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.

Un haut-débit Assay Compatible pour évaluer l&#39;efficacité des médicaments contre Macrophage repiquées<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 ...

Analysis of 18FDG PET/CT Imaging as a Tool for Studying Mycobacterium tuberculosis Infection and Treatment in Non-human Primates

Mycobacterium tuberculosis remains the number one infectious agent in the world today. With the emergence of antibiotic resistant strains, new clinically relevant methods are needed that evaluate the disease process and screen for potential antibiotic and vaccine treatments. Positron Emission Tomography/Computed Tomography (PET/CT) has been established as a valuable tool for studying a number of afflictions such as cancer, Alzheimer&#39;s disease, and inflammation/infection. Outlined here are a number of strategies that have been employed to evaluate PET/CT images in cynomolgus macaques that are infected intrabronchially with low doses of M. tuberculosis. Through evaluation of lesion size on CT and uptake of 18F-fluorodeoxyglucose (FDG) in lesions and lymph nodes in PET images, these described methods show that PET/CT imaging can predict future development of active versus latent disease and the propensity for reactivation from a latent state of infection. Additionally, by analyzing the overall level of lung inflammation, these methods determine antibiotic efficacy of drugs against M. tuberculosis in the most clinically relevant existing animal model. These image analysis methods are some of the most powerful tools in the arsenal against this disease as not only can they evaluate a number of characteristics of infection and drug treatment, but they are also directly translatable to a clinical setting for use in human studies.

Analysis of 18FDG PET/CT Imaging as a Tool for Studying Mycobacterium tuberculosis Infection and Treatment in Non-human Primates

Here, we present a protocol to describe the analysis of 18F-FDG PET/CT imaging in non-human primates that have been infected with M. tuberculosis to study disease process, drug treatment, and disease reactivation.

Analyse de 18FDG TEP/CT d’imagerie comme un outil pour étudier Mycobacterium tuberculosis Infection et traitement chez les Primates Non humains

Mycobacterium tuberculosis remains the number one infectious agent in the world today. With the emergence of antibiotic resistant strains, new clinically ...