Nous tenons à remercier les technologues de laboratoire du Laboratoire de la Clinique Mayo Mycobactériologie pour leur aide pendant l&#39;étude.

Chaque laboratoire doit effectuer une évaluation des risques en collaboration avec leur agent de sécurité institutionnel biologique pour déterminer le niveau approprié de biosécurité pour la préparation de l&#39;inoculum et pour le pipetage de l&#39;inoculum dans la plaque. Dans notre laboratoire, nous utilisons de biosécurité de niveau 3 en laboratoire (BSL3) jusqu&#39;à ce que les pratiques des plaques de microtitration sont inoculés, et scellé avec un sceau plastique adhésif. Ces plaques sont ensuite placés dans un sac en plastique, qui est également scellé à la chaleur. La lecture et l&#39;interprétation d&#39;incubation de la microplaque est menée dans un laboratoire de biosécurité de niveau 2 (BSL2). 1. L&#39;étiquetage des matériaux <ol><li> Etiquette une gélose au sang, trois plaques 7H10, le bouillon Middlebrook 7H9, une solution saline entre billes de verre du tube et une plaque de microtitration AST avec les identifiants appropriés (par exemple, le nom du patient, numéro de dossier médical) dans la BSL2. </li><li> Étiquette de la gélose sang et 7H10 plaqués comme «vérification de la pureté&quot;. Nombre de colonies périodique de l&#39;inoculum est recommandé de s&#39;assurer que la concentration appropriée des organismes est utilisé dans le test. Nombre de colonies lorsqu&#39;elles sont effectuées aurez besoin d&#39;avoir deux des plaques 7H10 étiquetés avec &quot;compter 1 / 1 colonie» et «compter 1 / 50 colonie». </li></ol> 2. Technologue des préparations d&#39;aller dans le BSL3 <ol><li> Équipement protecteur personnel approprié est nécessaire pour travailler dans le BSL3 et comprend solide devant robe, se couvrir la tête, des gants, des couvre-chaussures, et un respirateur. Chaque établissement devrait établir son propre plan de protection respiratoire, en collaboration avec l&#39;agent de leur institution d&#39;hygiène industrielle. Dans notre laboratoire, nous avons plusieurs respirateurs disponibles, y compris appareils respiratoires d&#39;épuration d&#39;air (PAPR) et équipé filtre HEPA, N95 demi-masque respiratoire. </li></ol> 3. Préparation de l&#39;enceinte de sécurité biologique <ol><li> Dans le BSL3, préparer l&#39;enceinte de sécurité biologique (BSC) par la désinfection de la surface et placer une serviette en papier imbibée de désinfectant environ 6 pouces à partir du panneau de ventilation d&#39;air. </li><li> Placer un conteneur à déchets sur la gauche, et nephlometer petites et Vortexer sur la droite. </li><li> Boucles d&#39;inoculation, pipetors et des conseils sont placés à côté de la serviette en papier. </li><li> Placer une grille avec l&#39;organisme (en bouillon ou sur milieu solide) et le standard McFarland 0,5 sur la serviette en papier. </li></ol> 4. Calibrage de la néphélomètre <ol><li> Placer un tube de verre entre salins billes dans le néphélomètre et régler l&#39;instrument à zéro en utilisant le blanc salines interpolation. </li><li> Placez le standard McFarland 0,5 dans le néphélomètre et calibrer selon les instructions du fabricant. </li></ol> 5. Préparation de l&#39;inoculum <ol><li> Travailler dans des colonies de la BSC gratter, de milieu solide à l&#39;aide d&#39;une anse stérile dans le tube entre correctement étiquetés salines de billes de verre jusqu&#39;à ce qu&#39;un ≥ 0,5 McFarland standard est obtenue. </li><li> Inspecter visuellement l&#39;échantillon, et de comparer à la norme McFarland. </li><li> Échantillons Vortex pendant 30-60 secondes. </li><li> Laisser l&#39;inoculum se contenter de 15 minutes (régler une minuterie). </li></ol> 6. L&#39;inoculation de la plaque de microtitration <ol><li> Ajustez l&#39;inoculum avec le bouillon entre saline à l&#39;aide du néphélomètre calibré pour le McFarland 0,5 par pas de 4.1 et 4.2. Ceci doit être complété dans les 30 minutes (les spécifications du fabricant par personne). </li><li> Utiliser une pipette 200 ul et une pointe d&#39;aérosol LONG pipette résistant à enlever 100 ul d&#39;inoculum de la RD TOP 1 / 3 du tube salines perles interpolation. Inoculer le bouillon Middlebrook 7H9. Lentement vortex le bouillon inoculé 7H9 pendant 20 secondes. </li><li> Retirer la plaque de microtitration AST de l&#39;emballage du fabricant. Ne pas utiliser si le déshydratant est le bleu ou si l&#39;emballage d&#39;aluminium est endommagé. </li><li> Préparer un tube dilution 1:50 pour une utilisation ultérieure dans la préparation de la dilution 1:50 par pipetage 50 pl d&#39;eau stérile dans un tube stérile. </li><li> Versez délicatement l&#39;inoculum bouillon 7H9 dans une auge. Éviter de générer des aérosols. </li><li> Placer la plaque de microtitration AST avec l&#39;étiquette face à vous. </li><li> Utiliser une pipette à 12 canaux d&#39;ajouter 100 ml de l&#39;inoculum à chaque puits. </li><li> Préparer des dilutions pour les décomptes de colonies: <ol type="a"><li> La dilution 1 / 1 est préparé à partir du contrôle positif et (H12) en stries une boucle ul sur la plaque de 7H10 étiqueté &quot;1 / 1&quot;. </li><li> La dilution au 1 / 50 est préparé par inoculation d&#39;une boucle de 1 pl du contrôle positif et (H12) et il tourbillonnant dans le tube avec 50 ul de tubes d&#39;eau préparé ci-dessus. Streak 1 ul de cette dilution à la plaque portant la mention «1 / 50&quot;. </li></ol></li><li> Inoculer les plaques pureté de l&#39;auge par pipetage 50 ul sur une gélose au sang convenablement étiquetés et la plaque de 7H10 et à balayage pour l&#39;isolement. </li><li> Placerun joint d&#39;adhésif sur la plaque de microtitration AST. Appuyez fermement sur chaque puits afin d&#39;assurer une étanchéité adéquate en utilisant le dos blanc du phoque. Éviter les plis. </li><li> Désinfectez la plaque de microtitration en l&#39;essuyant avec une serviette en papier imbibée d&#39;un désinfectant et tuberculocide microplaque placer dans un sac en plastique et fermer avec un sceau de la chaleur ou du ruban adhésif. <ol type="a"><li> Le sac en plastique sert de récipient secondaire pour s&#39;assurer que tous bouillon est contenue devrait y avoir une fuite ou un déversement de la plaque principale. </li></ol></li></ol> 7. Désinfection des matériaux et des enceintes de sécurité biologique <ol><li> Pour éviter de produire des aérosols, placez un autre bac sur le dessus de la cuvette inoculé avant de le placer délicatement dans le récipient jeter. </li><li> Désinfecter tout le matériel dont nephlometer, Vortexer, plaques, tubes, etc par les essuyer avec un désinfectant tuberculocide et attendre le moment approprié pour assurer la désinfection conformément aux instructions du fabricant avant de retirer du BSC. Par exemple, Prospray, le désinfectant tuberculocide que nous utilisons, il faut 15 minutes pour tuer les mycobactéries. </li><li> Placez le récipient jeter dans un sac de Biohazard et sceller avant l&#39;autoclavage. </li></ol> 8. D&#39;incubation <ol><li> Les plaques doivent être incubés avec les médias vers le bas et les soins devraient être prises pour éviter de renverser ou à des éclaboussures de liquide. </li><li> Incuber la plaque AST plaque de microtitration, 7H10 et une gélose au sang dans un incubateur à 35-37 ° C avec 5-10% de CO 2 avec 5-10% de CO 2 dans le laboratoire de BSL2. <ol class="lalpha"><li> Dans notre laboratoire, ce qui est acceptable parce que la plaque est bien fermé et à l&#39;intérieur d&#39;un sac plastique scellé. </li></ol></li><li> Pas plus de 2 plaques de microtitration AST doivent être empilés dans l&#39;incubateur selon les instructions du fabricant. </li></ol> 9. Les résultats représentatifs Interprétation des résultats: Scellé AST microplaques peuvent être lus manuellement à l&#39;aide d&#39;un miroir comme une aide ou avec un lecteur de plaque automatisés tels que l&#39;Vizion (Diagnostics TREK). Les plaques peuvent être examinés dès que 7 jours après l&#39;inoculation. Examiner les puits de contrôle de croissance dans des postes H11 et H12 pour déterminer si elles sont positives (ie., ont un dépôt de cellules sur le fond du puits). Si les puits de contrôle de croissance ne sont pas positifs, réincuber la plaque et réexaminer périodiquement (par exemple, le jour 10, jour 14) jusqu&#39;à ce que le puits de contrôle de croissance sont positifs. <ol type="a"><li> Miroir lire: <ol type="i"><li> Placez scellés AST plaque de microtitration sur la plateforme; </li><li> Lire puits contrôle de la croissance après 7 jours d&#39;incubation; si cela est négatif, réincuber AST plaque de microtitration; </li><li> Si les contrôles de croissance sont acceptables, alors lisez les puits contenant un médicament. Pour chaque médicament, d&#39;enregistrer la première dilution qui n&#39;a pas de croissance en comparant les puits de drogue avec le contrôle de la croissance. </li><li> La croissance apparaît comme la turbidité ou comme un dépôt de cellules au fond d&#39;un puits. </li></ol></li><li> Instrument Lire: suivez les instructions des fabricants; les instructions ci-dessous sont un exemple de comment la lecture serait faite avec la plate-forme Vizion: <ol type="i"><li> Log in </li><li> Utilisez &quot;MYCOTB&quot; logiciel plaque AST. </li><li> Placer la plaque scellée avec le code-barres face à l&#39;utilisateur. La croissance apparaît comme la turbidité ou comme un dépôt de cellules au fond d&#39;un puits. </li><li> Lire contrôle la croissance, si ce n&#39;est négatif, réincuber AST plaque de microtitration; si elle est positive lire la plaque en touchant l&#39;écran avec le premier puits de chaque médicament qui ne montrent pas de croissance. </li></ol></li></ol> Lire les plaques pureté. La plaque de gélose au sang devrait montrer une croissance nulle après 48 heures et la plaque de la pureté 7H10 devrait avoir une croissance confluente d&#39;un type d&#39;organisme. S&#39;il est contaminé, le test AST microtitration doit être répété à partir d&#39;une culture pure. Calculer le nombre de colonies à partir du contrôle positif et en utilisant le tableau ci-dessous: Principales nombre de colonies <table border="1"><tbody><tr><td> # Colonies compté sur la plaque portant la mention «1 / 1&quot; </td><td> # Colonies compté sur la plaque portant la mention «1 / 50&quot; </td><td> Le nombre de colonies d&#39;inoculum (UFC / ml) </td></tr><tr><td> &lt;50 </td><td> 0 </td><td> &lt;5 x 10 4 </td></tr><tr><td> 50-100 </td><td> 0-2 </td><td> 5 x 10 4 à 1 X 10 5 </td></tr><tr><td> &gt; 100 </td><td> ≤ 10 </td><td> 1 x 10 5 à 5 X 10 5 </td></tr><tr><td> &gt; 100 </td><td> &gt; 10 </td><td> &gt; 5 x 10 5 </td></tr></tbody></table> Il peut y avoir de fuite d&#39;acide para-aminosalicylique chez certaines souches de Mycobacterium tuberculoseSIS. Cela peut être interprété en regardant tous les puits qui ont granulés et en utilisant le premier puits qui a la même taille que le pellet dilution suivante comme point de fin. La résistance est rare avec ce médicament. <img src="/files/ftp_upload/3094/3094fig1.jpg" alt="Figure 1"/> Figure 1. Les résultats représentatifs en utilisant la plaque MYCOTB. <table border="1"><tbody><tr><td> Abréviation de drogue </td><td> Drogues </td><td> Μ MIC / ml </td></tr><tr><td> OFL </td><td> ofloxacine </td><td> 16 </td></tr><tr><td> MXF </td><td> moxifloxacine </td><td> 8 </td></tr><tr><td> FRR </td><td> rifampine </td><td> &gt; 16 </td></tr><tr><td> IAM </td><td> amikacine </td><td> 0.5 </td></tr><tr><td> STR </td><td> streptomycine </td><td> 16 </td></tr><tr><td> RFB </td><td> rifabutine </td><td> 8 </td></tr><tr><td> PAS </td><td> acide p-aminosalicylique </td><td> ≤ 0,5 </td></tr><tr><td> EPF </td><td> éthionamide </td><td> 5 </td></tr><tr><td> CYC </td><td> cyclosérine </td><td> 4 </td></tr><tr><td> INH </td><td> isoniazide </td><td> 2 </td></tr><tr><td> KAN </td><td> kanamycine </td><td> 1.2 </td></tr><tr><td> EMB </td><td> éthambutol </td><td> 4,0 </td></tr></tbody></table>

<ol>
	<li>Wengenack, N., Hall, L., Labombardi, V., Parrish, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+the+New+Sensititre+(MYCOTB)+MIC+Plate+for+the+Susceptibility+Testing+of+Mycobacterium+tuberculosis+(Mtb)+to+First+and+Second+Line+Drugs.">Evaluation of the New Sensititre (MYCOTB) MIC Plate for the Susceptibility Testing of Mycobacterium tuberculosis (Mtb) to First and Second Line Drugs.</a> , (2010).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HHS+Publication+No.+(CDC)+21-112:+Biosafety+in+Microbiological+and+Biomedical+Laboratories+(BMBL).">HHS Publication No. (CDC) 21-112: Biosafety in Microbiological and Biomedical Laboratories (BMBL).</a> U.S. Department of Health and Human Services Centers for Disease and Preventions. , (2009).</li><li>Morcillo, N., Imperiale, B., Digiulio, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+MGIT+960+and+the+colorimetric-based+method+for+tuberculosis+drug+susceptibility+testing.">Evaluation of MGIT 960 and the colorimetric-based method for tuberculosis drug susceptibility testing.</a> Int J Tuberc Lung Dis. 14, 1169-1175 (2010).</li><li>Bemer, P., Bodmer, T., Munzinger, J., Perrin, M., Vincent, V., Drugeon, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multicenter+Evaluation+of+the+MB/BACT+System+for+Susceptibility+Testing+of+Mycobacterium+tuberculosis.">Multicenter Evaluation of the MB/BACT System for Susceptibility Testing of Mycobacterium tuberculosis.</a> J Clin Microbiol. 42, 1030-1034 (2004).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SENSITITRE+MYCOTB+Susceptibility+Plate:+For+Mycobacterium+Susceptibility+Testing.">SENSITITRE MYCOTB Susceptibility Plate: For Mycobacterium Susceptibility Testing.</a> Product Insert. , (2010).</li><li>CLSI. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Susceptiblity Testing of Mycobacteria, Nocardia, and Other Aerobic Actinomycetes; Approved Standard. Document M24-A. , (2003).</li><li>Sullivan, N., Sotos, J., Anhalt, K., Allen, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preliminary+results+from+a+new+TREK+Sensititre+Mycobacterium+tuberculosis+MIC+plate+(MYCOTB).">Preliminary results from a new TREK Sensititre Mycobacterium tuberculosis MIC plate (MYCOTB).</a> , (2010).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VersaTREK+MYCO+SUSCEPTIBLITY+KIT.">VersaTREK MYCO SUSCEPTIBLITY KIT.</a> Product insert. , 7115-7160 (2008).</li><li>Parrish, N. M., Carroll, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Role+of+the+Clinical+Mycobacteriology+Laboratory+in+the+Diagnosis+and+Management+of+Tuberculosis+in+Low+Prevalance+Settings.">The Role of the Clinical Mycobacteriology Laboratory in the Diagnosis and Management of Tuberculosis in Low Prevalance Settings.</a> J Clin Microbiol. , (2010).</li><li>Kam, K. M., Sloutsky, A., Yip, C. W., Bulled, N., Zingol, M., Espinal, M., Kim, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+critical+concentrations+of+second-line+anti-tuberculosis+drugs+with+clinical+and+microbiological+relevance.">Determination of critical concentrations of second-line anti-tuberculosis drugs with clinical and microbiological relevance.</a> Int J Tubercl Lung Dis. 14, 282-288 (2010).</li><li>Woods, G. L., Warren, N. G., Inderlied, C. B., Murray, P. R., Barron, E. J., Jorgensen, J. H., Landry, M. L., Pfaller, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Susceptibility+Test+Methods:+Mycobacteria,+Nocardia,+and+Other+Actinomycetes.">Susceptibility Test Methods: Mycobacteria, Nocardia, and Other Actinomycetes.</a> Manual of Clinical Microbiology. , 1223-1247 (2007).</li><li>Bergmann, J. S., Woods, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+the+ESP+Culture+System+II+for+Testing+Susceptibilities+of+Mycobacterium+tuberculosis+Isolates+to+Four+Primary+Antituberculosis+Drugs.">Evaluation of the ESP Culture System II for Testing Susceptibilities of Mycobacterium tuberculosis Isolates to Four Primary Antituberculosis Drugs.</a> J Clin Microbiol. 36, 2940-2943 (1998).</li></ol>

Un exemple d&#39;une plaque de microtitration AST qui a des contrôles de croissance positif est montré dans la figure 1 et le MIC critère (pg / ml) pour chaque médicament est encerclé. Une étude menée dans notre laboratoire ont indiqué que la méthode AST plaque de microtitration est équivalente à la méthode standard de l&#39;or agar proportion pour les tests de sensibilité de Mycobacterium tuberculosis. Plus rapide présentation des résultats a été obtenue avec microtitration AST plaque de microtitration par rapport à la méthode des proportions agar traditionnelle avec l&#39;interprétation finale pour AST plaque de microtitration et la méthode de la proportion de gélose à 14 et 21 jours, respectivement à AST plaque de microtitration a l&#39;avantage de tester d&#39;abord et médicaments de seconde ligne simultanément ce qui peut aider à prévenir les retards excessifs avec des souches résistantes. La plaque de microtitration AST est aussi beaucoup plus facile à configurer et à lire que l&#39;APM et la fois un miroir manuelle et d&#39;un système semi-automatisé comme la VIZION peut être utilisé pour lire les plaques. La disponibilité d&#39;une valeur de CMI pour les tests résistance aux antituberculeux peuvent fournir de précieuses informations aux médecins de nouvelles par rapport à la valeur critique de concentration traditionnels, en particulier pour ceux des isolats avec «limite» de susceptibilité.

<table border="1"><tbody><tr><th> Nom du réactif </th><th> Société </th><th> Numéro de catalogue </th></tr><tr><td> AST plaques de microtitration et joint adhésif </td><td> Diagnostics TREK, Cleveland, Ohio </td><td> MYCOTB </td></tr><tr><td> Standard McFarland 0,5 </td><td> Diagnostics TREK, Cleveland, Ohio </td><td> E1041 </td></tr><tr><td> Middlebrook 7H10 plaques </td><td> Fisher </td><td></td></tr><tr><td> Gélose au sang </td><td> Fisher </td><td></td></tr><tr><td> Stérile boucles 1 uL </td><td> Fisher </td><td></td></tr><tr><td> Boucles stériles 10 uL </td><td> Fisher </td><td></td></tr><tr><td> Sensititre Saline-Tween billes de verre de bouillon </td><td> Diagnostics TREK, Cleveland, Ohio </td><td> 27143 </td></tr><tr><td> 7H9 OADC Bouillon </td><td> Diagnostics TREK, Cleveland, Ohio </td><td> T3440 </td></tr><tr><td> Conseils Pipet longue </td><td> Fisher </td><td></td></tr><tr><td> Auges stérile </td><td> Diagnostics TREK, Cleveland, Ohio </td><td></td></tr><tr><td> M. tuberculosis </td><td> ATCC </td><td> 27294 </td></tr><tr><td> Pipeters: 1000, 200, 20 pi </td><td> Fisher </td><td></td></tr><tr><td> Conseils 1000, 20, 20 pl </td><td> Fisher </td><td></td></tr><tr><td> Pipetteur multicanaux </td><td> Fisher </td><td></td></tr><tr><td> Incubateur CO% 5 au 10 fév </td><td> Fisher </td><td></td></tr><tr><td> Les sacs en plastique, les petits, le CO 2 perméables </td><td> Poly Plastic, Delano, PA </td><td></td></tr><tr><td> Désinfectants tuberculose (tels que ProSpray C-60) </td><td> Certol international, Commerce City, CO </td><td> PSC60/32 </td></tr><tr><td> Boîte Miroir lecteur </td><td> Fisher </td><td></td></tr></tbody></table>

antimicrobial susceptibility testing of mycobacterium tuberculosis complex for first and second line drugs by broth dilution in a microtiter plate format

La détection rapide de la résistance antimicrobienne est important dans l&#39;effort pour contrôler l&#39;augmentation de la résistance de Mycobacterium tuberculosis (MTB). Tests de sensibilité aux antimicrobiens (AST) de Vtt a traditionnellement été effectuée par la méthode de la proportion d&#39;agar ou par des essais macrobroth sur un instrument comme le BACTEC (société Becton Dickinson, Sparks, MD), VersaTREK (Diagnostics TREK, Cleveland, OH) ou BacT / ALERTE (bioMérieux, Hazelwood, MO). La méthode des proportions agar-agar, bien que considérées comme &quot;l&#39;or&quot; standard de l&#39;AST, est laborieuse et nécessite le calcul de la résistance en effectuant nombre de colonies sur les médicaments contenant de l&#39;agar par rapport à la drogue sans agar. Si la croissance est ≥ 1% sur le support contenant un médicament par rapport à la drogue sans support, l&#39;organisme est considéré comme résistant à ce médicament. Les méthodes macrobroth besoin d&#39;instrumentation et de test de point de rupture («critique») des concentrations de médicament pour les médicaments de première ligne (isoniazide, éthambutol, la rifampicine, pyrazinamide et l&#39;). La méthode décrite ici est disponible commercialement dans un format 96 plaques de microtitration ainsi [MYCOTB (Diagnostics TREK)] et contient des concentrations croissantes de 12 antimicrobiens utilisés pour le traitement de la tuberculose, y compris à la fois première (isoniazide, rifampicine, éthambutol) et des médicaments de deuxième ligne (amikacine, cyclosérine, éthionamide, kanamycine, la moxifloxacine, ofloxacine, acide para-aminosalicylique, la rifabutine et streptomycine). Pyrazinamide, un médicament de première ligne, n&#39;est pas inclus dans la plaque de microtitration en raison de son besoin de conditions d&#39;essai acide. Avantages du système de microtitration comprennent à la fois facilité d&#39;installation et plus rapidement tourner autour du temps (14 jours) par rapport à la proportion d&#39;agar traditionnelle (21 jours). En outre, la plaque peut être mis en place à partir inoculum préparé en utilisant soit un bouillon ou un milieu solide. Comme le format microplaque est nouveau et depuis Vtt présente des défis uniques de la sécurité dans le laboratoire, ce protocole va décrire de façon sécuritaire l&#39;installation, incuber et de lire la plaque de microtitration.

Watch this Scientific Journal Video about Antimicrobial Susceptibility Testing of Mycobacterium Tuberculosis Complex for First and Second Line Drugs by Broth Dilution in a Microtiter Plate Format at J

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

Antibiogramme de Mycobacterium tuberculosis est difficile mais essentiel pour les soins aux patients. Ce format microplaque offre des tests de 12 antimycobactériens et fournit la concentration minimale inhibitrice (CMI), qui aidera à un traitement approprié.

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). ...

antimicrobial-susceptibility-testing-mycobacterium-tuberculosis

Research

JoVE Journal

Immunology and Infection

38.1K vues.  Mayo Clinic. Antibiogramme de Mycobacterium tuberculosis est difficile mais essentiel pour les soins aux patients. Ce format microplaque offre des tests de 12 antimycobactériens et fournit la concentration minimale inhibitrice (CMI), qui aidera à un traitement approprié.

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

A 96 Well Microtiter Plate-based Method for Monitoring Formation and Antifungal Susceptibility Testing of Candida albicans Biofilms

Candida albicans remains the most frequent cause of fungal infections in an expanding population of compromised patients and candidiasis is now the third most common infection in US hospitals. Different manifestations of candidiasis are associated with biofilm formation, both on host tissues and/or medical devices (i.e. catheters). Biofilm formation carries negative clinical implications, as cells within the biofilms are protected from host immune responses and from the action of antifungals. We have developed a simple, fast and robust in vitro model for the formation of C. albicans biofilms using 96 well microtiter-plates, which can also be used for biofilm antifungal susceptibility testing. The readout of this assay is colorimetric, based on the reduction of XTT (a tetrazolium salt) by metabolically active fungal biofilm cells. A typical experiment takes approximately 24 h for biofilm formation, with an additional 24 h for antifungal susceptibility testing. Because of its simplicity and the use of commonly available laboratory materials and equipment, this technique democratizes biofilm research and represents an important step towards the standardization of antifungal susceptibility testing of fungal biofilms.

A 96 Well Microtiter Plate-based Method for Monitoring Formation and Antifungal Susceptibility Testing of Candida albicans Biofilms

We describe a simple, rapid and robust method for the formation of Candida albicans biofilms using 96 well microtiter plates and its utility in antifungal susceptibility testing of cells within biofilms.

Une plaque de microtitrage à 96 puits à base Méthode de formation suivi et les tests de sensibilité antifongique des Candida albicans Les biofilms

Candida albicans remains the most frequent cause of fungal infections in an expanding population of compromised patients and candidiasis is now the third ...

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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) ...

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.

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 ...

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 Reference Broth Microdilution Method for Dalbavancin In Vitro Susceptibility Testing of Bacteria that Grow Aerobically

Antimicrobial susceptibility testing (AST) is performed to assess the in vitro activity of antimicrobial agents against various bacteria. The AST results, which are expressed as minimum inhibitory concentrations (MICs) are used in research for antimicrobial development and monitoring of resistance development and in the clinical setting for antimicrobial therapy guidance. Dalbavancin is a semi-synthetic lipoglycopeptide antimicrobial agent that was approved in May 2014 by the Food and Drug Administration (FDA) for the treatment of acute bacterial skin and skin structure infections caused by Gram-positive organisms. The advantage of dalbavancin over current anti-staphylococcal therapies is its long half-life, which allows for once-weekly dosing. Dalbavancin has activity against Staphylococcus aureus (including both methicillin-susceptible S. aureus [MSSA] and methicillin-resistant S. aureus [MRSA]), coagulase-negative staphylococci, Streptococcus pneumoniae, Streptococcus anginosus group, &#946;-hemolytic streptococci and vancomycin susceptible enterococci. Similar to other recent lipoglycopeptide agents, optimization of CLSI and ISO broth susceptibility test methods includes the use of dimethyl sulfoxide (DMSO) as a solvent when preparing stock solutions and polysorbate 80 (P80) to alleviate adherence of the agent to plastic. Prior to the clinical studies and during the initial development of dalbavancin, susceptibility studies were not performed with the use of P-80 and MIC results tended to be 2-4 fold higher and similarly higher MIC results were obtained with the agar dilution susceptibility method. Dalbavancin was first included in CLSI broth microdilution methodology tables in 2005 and amended in 2006 to clarify use of DMSO and P-80. The broth microdilution (BMD) procedure shown here is specific to dalbavancin and is in accordance with the CLSI and ISO methods, with step-by-step detail and focus on the critical steps added for clarity.

A Reference Broth Microdilution Method for Dalbavancin In Vitro Susceptibility Testing of Bacteria that Grow Aerobically

Here, we present a protocol for determination of dalbavancin susceptibility of clinically relevant Gram-positive bacteria using a broth microdilution reference methodology.

Une méthode de référence de microdilution de bouillon pour dalbavancine<em&gt; In Vitro</em&gt; Tests de sensibilité des bactéries qui se développent Aerobically

Antimicrobial susceptibility testing (AST) is performed to assess the in vitro activity of antimicrobial agents against various bacteria. The AST results, ...

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 ...

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 ...

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 ...

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.

Imagerie de Mycobacterium tuberculosis chez la souris avec la journaliste Enzyme Fluorescence

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.

Modélisation de la tuberculose chez Mycobacterium marinum adultes infectés poisson zèbre

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