Nos gustaría agradecer a los técnicos de laboratorio de la Clínica Mayo Micobacteriología laboratorio por su ayuda durante el estudio.

Cada laboratorio debe realizar una evaluación de riesgos en colaboración con su Oficial Institucional de Seguridad Biológica para determinar el nivel apropiado de bioseguridad para la preparación del inóculo y para pipetear del inóculo en la placa. En nuestro laboratorio, utilizamos el nivel 3 de bioseguridad de laboratorio (BSL3) las prácticas hasta que las placas de microtitulación se inoculan, y sellado con un sello adhesivo plástico. Estas placas se colocan dentro de una bolsa de plástico, que es el calor también está sellado. Lectura e interpretación de incubación de la placa de microtitulación se lleva a cabo en un laboratorio de bioseguridad nivel 2 (BSL2). 1. Etiquetado de los materiales <ol><li> Etiqueta de una placa de agar sangre, tres placas de Middlebrook 7H10, caldo de Middlebrook 7H9, una solución salina entre tubo de vidrio de cuentas y una placa de microtitulación AST con los identificadores adecuados (por ejemplo, el nombre del paciente, número de historia clínica) en el BSL2. </li><li> Etiqueta de la sangre y agar 7H10 plateado como &quot;comprobar la pureza&quot;. Recuentos periódicos colonia del inóculo se recomienda asegurarse de que una concentración adecuada de los organismos se utiliza en la prueba. El recuento de colonias cuando se realiza tendrá que contar con dos de las placas de 7H10 etiquetados con &quot;count 01.01 colonia&quot; y del &quot;número 1 / 50 colonias&quot;. </li></ol> 2. Preparación técnico para ir a la BSL3 <ol><li> El equipo de protección personal es necesario trabajar en la BSL3 e incluye sólido frente vestido, cubierta de la cabeza, guantes, cubiertas para los zapatos, y un respirador. Cada institución debe establecer su propio plan de protección respiratoria, en colaboración con el oficial de su institución de Higiene Industrial. En nuestro laboratorio, tenemos varios respiradores disponibles, incluida la alimentación respiradores purificadores de aire (PAPR) y equipado con filtro HEPA, los respiradores N95 mitad de la cara. </li></ol> 3. Preparación de la cabina de seguridad biológica <ol><li> En el BSL3, preparar la cabina de seguridad biológica (BSC) por la desinfección de la superficie y colocando una toalla de papel empapada con desinfectante de aproximadamente 6 pulgadas desde el panel de ventilación de aire. </li><li> Coloque un contenedor de residuos de la izquierda, y nephlometer pequeñas y vórtice de la derecha. </li><li> Bucles de inoculación, pipetors y consejos se colocan al lado de la toalla de papel. </li><li> Coloque una parrilla con el organismo (en caldo o en medio sólido) y el 0,5 McFarland estándar en la toalla de papel. </li></ol> 4. La calibración del nefelómetro <ol><li> Coloque un tubo de solución salina entre cuentas de vidrio en el nefelómetro y poner el instrumento a cero con la solución salina en blanco entre. </li><li> Coloque el estándar de 0,5 McFarland en el nefelómetro y calibrar según las instrucciones del fabricante. </li></ol> 5. Preparación del inóculo <ol><li> De trabajo en las colonias raspar BSC, de medio sólido con un asa estéril en el tubo debidamente etiquetado salinas entre cuentas de vidrio hasta un ≥ 0,5 McFarland estándar se obtiene. </li><li> Inspeccione visualmente la muestra, y comparar con el estándar de McFarland. </li><li> Vórtice de la muestra durante 30-60 segundos. </li><li> Deje que el inóculo que conformarse con 15 minutos (un cronómetro). </li></ol> 6. La inoculación de la placa de microtitulación <ol><li> Ajuste del inóculo con solución salina caldo entre usar el nefelómetro calibrado a la McFarland 0,5 en pasos de 4.1 y 4.2. Esto se debe completar en 30 minutos (según las especificaciones del fabricante). </li><li> Utilice una pipeta de 200 ly una punta de aerosol LARGO pipeta resistentes a eliminar 100 L de inóculo a partir de la tercera TOP 1 / 3 de la solución salina tubo de cuentas entre. Inocular el caldo Middlebrook 7H9. Poco a poco el caldo inoculado vórtice 7H9 durante 20 segundos. </li><li> Retire la placa de microtitulación AST del paquete del fabricante. No lo use si el desecante es de color azul o si el paquete de aluminio está dañado. </li><li> Preparar un tubo de dilución de 1:50 para su posterior uso en la preparación de la dilución 1:50 con la pipeta 50 l de agua estéril en un tubo estéril. </li><li> Con cuidado, vierta el caldo 7H9 inóculo en un canal. Evitar generar aerosoles. </li><li> Coloque la placa de microtitulación de AST con la etiqueta hacia usted. </li><li> Utilice una pipeta de 12 canales para añadir 100 ml de inóculo a cada pocillo. </li><li> Preparar las diluciones para el recuento de colonias: <ol type="a"><li> La dilución de 1 / 1 se prepara a partir del control positivo y (H12) por rayar un bucle l en la placa de 7H10 con la etiqueta &quot;1.1&quot;. </li><li> La dilución 1 / 50 se prepara mediante la inoculación de un circuito de 1 l de control positivo y (H12) y girando en el tubo con 50 l de tubo de agua preparada anteriormente. Una racha de L de esta dilución a la placa &quot;1 / 50&quot;. </li></ol></li><li> Inocular las placas de la pureza de la cubeta con la pipeta 50 L en un agar sangre debidamente etiquetados y la placa de 7H10 y raya para el aislamiento. </li><li> Lugarun sello adhesivo en la placa de microtitulación de AST. Presione con firmeza en cada pozo para asegurar estanqueidad adecuada mediante el uso de la parte posterior del blanco de la junta. Evitar las arrugas. </li><li> La desinfección de la placa de microtitulación frotándolo con una toalla de papel empapado con un desinfectante tuberculocida y placa de microtitulación en una bolsa de plástico y cierre con un sello de calor o de la cinta. <ol type="a"><li> La bolsa de plástico sirve como un contenedor secundario para asegurarse de que todos los caldos se encuentra debe haber una fuga o derrame de la placa principal. </li></ol></li></ol> 7. Desinfección de materiales y el gabinete de seguridad biológica <ol><li> Para evitar la producción de aerosoles, en otro lugar a través en la parte superior del canal inoculadas antes de colocarlo suavemente en el recipiente de descarte. </li><li> Desinfectar todos los materiales incluyendo nephlometer, vórtice, placas, tubos, etc al lavarlo con un desinfectante tuberculocida y esperar el momento adecuado para asegurar la desinfección de acuerdo a las instrucciones del fabricante antes de sacarlo del BSC. Por ejemplo, ProSpray, el desinfectante tuberculocida usamos, requiere 15 minutos para matar las micobacterias. </li><li> Coloque el recipiente de descarte en una bolsa de riesgo biológico y sellado antes de esterilizar. </li></ol> 8. Incubación <ol><li> Las placas deben ser incubadas con los medios de comunicación hacia abajo y se debe tener cuidado para evitar derrames o salpicaduras de cualquier líquido. </li><li> Incubar la placa de microtitulación AST placa, 7H10 y las placas de agar sangre en una incubadora de 35 a 37 ° C con 5-10% CO 2 con 5-10% CO 2 en el laboratorio BSL2. <ol class="lalpha"><li> En nuestro laboratorio, esto es aceptable, ya que la placa esté bien sellada y dentro de una bolsa de plástico sellada. </li></ol></li><li> No más de 2 placas de microtitulación AST se deben apilar en la incubadora de acuerdo a las instrucciones del fabricante. </li></ol> 9. Los resultados representativos Interpretación de los resultados: Sellado AST placas de microtitulación se pueden leer de forma manual usando un espejo como una ayuda o con un lector de placas automatizado como el Vizion (Diagnóstico TREK). Las placas pueden ser examinadas en cuanto a los 7 días después de la inoculación. Examinar los pozos de control de crecimiento en puestos de H11 y H12 para determinar si son positivos (es decir, tener un depósito de células en el fondo del pozo). Si los pozos de control de crecimiento no son positivas, reincubate la placa y volver a examinar de forma periódica (por ejemplo, el día 10, día 14) hasta los pozos de control de crecimiento son positivas. <ol type="a"><li> Espejo decía: <ol type="i"><li> Lugar cerrado AST placa de microtitulación en la plataforma; </li><li> Lea los pozos de control de crecimiento después de 7 días de incubación, cuando éste sea negativo, reincubate AST placa de microtitulación; </li><li> Si los controles de crecimiento son aceptables, a continuación, lea los pozos que contiene droga. Para cada medicamento, registrar la primera dilución que no tiene el crecimiento mediante la comparación de los pozos de drogas con el control del crecimiento. </li><li> El crecimiento se presenta como la turbidez o como un depósito de células en el fondo de un pozo. </li></ol></li><li> Instrumento de lectura: seguir las instrucciones del fabricante, las instrucciones a continuación son un ejemplo de cómo la lectura se hace con la plataforma Vizion: <ol type="i"><li> Log in </li><li> Use &quot;MYCOTB&quot; software placa de AST. </li><li> Coloque la placa de sellado con el código de barras frente al usuario. El crecimiento se presenta como la turbidez o como un depósito de células en el fondo de un pozo. </li><li> Lea controla el crecimiento, cuando éste sea negativo, reincubate AST placa de microtitulación, si es positivo leer la placa tocando la pantalla con el primer pozo de cada medicamento que no muestra crecimiento. </li></ol></li></ol> Leer las placas pureza. La placa de agar sangre no debe mostrar un crecimiento después de 48 horas y la placa de pureza 7H10 debe tener un crecimiento confluente del tipo de organismo una. Si está contaminado, la prueba de microtitulación AST debe repetirse a partir de un cultivo puro. Calcular el recuento de colonias del control positivo y utilizando la tabla siguiente: Key Colony Conde <table border="1"><tbody><tr><td> # Colonias contadas en la placa de la etiqueta &quot;1.1&quot; </td><td> # Colonias contadas en la placa &quot;1 / 50&quot; </td><td> Inóculo recuento de colonias (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 a 1 X 10 5 </td></tr><tr><td> &gt; 100 </td><td> ≤ 10 </td><td> 1 X 10 5 al 05 X 10 5 </td></tr><tr><td> &gt; 100 </td><td> &gt; 10 </td><td> &gt; 5 X 10 5 </td></tr></tbody></table> No puede ser posterior con ácido para-aminosalicílico en algunas cepas de Mycobacterium tuberculosissis. Esto puede ser interpretado por mirar a todos los pozos que tienen y el uso de pellets en el primer pozo que tiene el mismo tamaño de las pastillas como la dilución siguiente como el punto final. La resistencia es rara, con este medicamento. <img src="/files/ftp_upload/3094/3094fig1.jpg" alt="Figura 1"/> Figura 1. Los resultados representativos con la placa MYCOTB. <table border="1"><tbody><tr><td> Abreviatura de drogas </td><td> Droga </td><td> MIC μ / ml </td></tr><tr><td> OFL </td><td> ofloxacina </td><td> 16 </td></tr><tr><td> MXF </td><td> moxifloxacino </td><td> 8 </td></tr><tr><td> RIF </td><td> rifampicina </td><td> &gt; 16 </td></tr><tr><td> AMI </td><td> amikacina </td><td> 0.5 </td></tr><tr><td> STR </td><td> estreptomicina </td><td> 16 </td></tr><tr><td> RFB </td><td> rifabutina </td><td> 8 </td></tr><tr><td> PAS </td><td> p-aminosalicílico </td><td> ≤ 0,5 </td></tr><tr><td> ETH </td><td> etionamida </td><td> 5 </td></tr><tr><td> CYC </td><td> cicloserina </td><td> 4 </td></tr><tr><td> INH </td><td> isoniazida </td><td> 2 </td></tr><tr><td> KAN </td><td> kanamicina </td><td> 1.2 </td></tr><tr><td> EMB </td><td> etambutol </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 ejemplo de una placa de microtitulación AST que tiene controles de crecimiento positivo se muestra en la Figura 1 y el MIC final (g / ml) para cada droga es un círculo. Un estudio realizado en nuestro laboratorio indican que el método de la placa de microtitulación de AST es equivalente a la proporción de oro método estándar de agar para determinar la susceptibilidad de Mycobacterium tuberculosis. Más rápida presentación de los resultados se obtuvo con microtiter AST placa de microtitulación en comparación con el método de agar proporción tradicional con la interpretación final de AST microplaca y el método de proporción de agar a 14 y 21 días, respectivamente.El AST microplaca tiene la ventaja de la primera prueba y medicamentos de segunda línea al mismo tiempo que puede ayudar a prevenir demoras excesivas con cepas resistentes. La placa de microtitulación de AST es mucho más fácil de configurar y leer que la APM y los dos un espejo manual y un sistema semi-automático, como el Vizion se puede utilizar para leer las placas. La disponibilidad de un valor de MIC para probar la resistencia a los medicamentos antituberculosos pueden proporcionar información nueva y valiosa a los médicos en relación con el valor de la concentración crítica tradicional, especialmente para aquellos aislados con &quot;límite&quot; la susceptibilidad.

<table border="1"><tbody><tr><th> Nombre del reactivo </th><th> Empresa </th><th> Número de catálogo </th></tr><tr><td> AST placas de microtitulación y sello adhesivo </td><td> TREK Diagnóstico, Cleveland, Ohio </td><td> MYCOTB </td></tr><tr><td> McFarland estándar de 0,5 </td><td> TREK Diagnóstico, Cleveland, Ohio </td><td> E1041 </td></tr><tr><td> Middlebrook 7H10 placas </td><td> Pescador </td><td></td></tr><tr><td> Placas de agar sangre </td><td> Pescador </td><td></td></tr><tr><td> Estéril bucles l 1 </td><td> Pescador </td><td></td></tr><tr><td> Bucles estéril de 10 l </td><td> Pescador </td><td></td></tr><tr><td> Sensititre Saline-Tween caldo de bolas de cristal </td><td> TREK Diagnóstico, Cleveland, Ohio </td><td> 27143 </td></tr><tr><td> 7H9 caldo OADC </td><td> TREK Diagnóstico, Cleveland, Ohio </td><td> T3440 </td></tr><tr><td> Consejos largo Pipet </td><td> Pescador </td><td></td></tr><tr><td> Los comederos estéril </td><td> TREK Diagnóstico, Cleveland, Ohio </td><td></td></tr><tr><td> M. tuberculosis </td><td> ATCC </td><td> 27294 </td></tr><tr><td> Pipeters: 1000, 200, 20μL </td><td> Pescador </td><td></td></tr><tr><td> Consejos para 1000, 20, 20 l </td><td> Pescador </td><td></td></tr><tr><td> Pipeteador multicanal </td><td> Pescador </td><td></td></tr><tr><td> Incubadora de 5-10% de CO2 </td><td> Pescador </td><td></td></tr><tr><td> Las bolsas de plástico, pequeños, CO 2 permeable </td><td> Poli de plástico, Delano, PA </td><td></td></tr><tr><td> Desinfectante TB (tales como ProSpray C-60) </td><td> Certol Internacional, Commerce City, CO </td><td> PSC60/32 </td></tr><tr><td> Espejo Caja lector </td><td> Pescador </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 rápida detección de resistencia a los antimicrobianos es importante en el esfuerzo por controlar el aumento de la resistencia de Mycobacterium tuberculosis (Mtb). Pruebas de susceptibilidad antimicrobiana (AST) de Mtb tradicionalmente ha sido realizado por el método de la proporción de agar o las pruebas macrobroth en un instrumento como el BACTEC (Becton Dickinson, Sparks, MD), VersaTREK (Diagnóstico TREK, Cleveland, OH) o BacT / ALERT (bioMérieux, Hazelwood, MO). El método de proporción de agar, mientras que considera el &quot;gold&quot; estándar de AST, es un trabajo intensivo y requiere el cálculo de la resistencia mediante la realización de recuentos de colonias en agar que contiene el fármaco en comparación con el agar libre de drogas. Si hay crecimiento de ≥ 1% en el medio que contiene el fármaco en comparación con la droga libre de soporte, el microorganismo se considera resistente a ese medicamento. Los métodos macrobroth requieren instrumentación y el punto de prueba de ruptura (&quot;crítica&quot;), las concentraciones de drogas para los medicamentos de primera línea (isoniazida, etambutol, rifampicina y pirazinamida). El método descrito aquí está comercialmente disponible en un formato de 96 pocillos de microtitulación [MYCOTB (Diagnóstico TREK)] y contiene concentraciones crecientes de 12 de los antimicrobianos utilizados para el tratamiento de la tuberculosis entre los dos primeros (isoniacida, rifampicina, etambutol) y medicamentos de segunda línea (amikacina, cicloserina, etionamida, kanamicina, moxifloxacina, ofloxacino, ácido para-aminosalicílico, la rifabutina y estreptomicina). Pirazinamida, un fármaco de primera línea, no está incluido en la placa de microtitulación, debido a su necesidad de condiciones de la prueba ácida. Ventajas del sistema de microtitulación incluyen tanto la facilidad de instalar y más rápido tiempo de vuelta (14 días) en comparación con la proporción de agar tradicional (21 días). Además, la placa se puede configurar de inóculo preparado utilizando caldo o medio sólido. Dado que el formato de placa de microtitulación es nuevo y ya Mtb presenta retos únicos de seguridad en el laboratorio, este protocolo se describe cómo configurar de forma segura, incubar y leer la placa de microtitulación.

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

Pruebas de susceptibilidad antimicrobiana de Mycobacterium tuberculosis es difícil, pero fundamental para el cuidado del paciente. Este formato de placa de microtitulación ofrece la prueba de 12 medicamentos antimicobacterianos y proporciona la concentración inhibitoria mínima (CIM) los valores, que ayudará en el tratamiento adecuado.

Pruebas de susceptibilidad antimicrobiana de Mycobacterium tuberculosis complex para drogas de primera línea y la segunda por dilución en caldo en un formato de placa de microtitulación

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

38.0K vistas.  Mayo Clinic. Pruebas de susceptibilidad antimicrobiana de Mycobacterium tuberculosis es difícil, pero fundamental para el cuidado del paciente. Este formato de placa de microtitulación ofrece la prueba de 12 medicamentos antimicobacterianos y proporciona la concentración inhibitoria mínima (CIM) los valores, que ayudará en el tratamiento adecuado.

Video: 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.

A 96 de microtitulación y placa basada en el método para la formación de Control y pruebas de sensibilidad antifúngica Candida albicans Las biopelículas

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

Investigación

Inmunología e Infección

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.

Preparación de la muestra de<em&gt; Mycobacterium tuberculosis</em&gt; Extractos de Estudios de Resonancia Magnética Nuclear metabolómica

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.

Crecimiento de Mycobacterium tuberculosis Las biopelículas

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.

Demostración de un resistente a múltiples fármacos<em&gt; Mycobacterium tuberculosis</em&gt; Microarray Amplificación

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

Preparation of a Blood Culture Pellet for Rapid Bacterial Identification and Antibiotic Susceptibility Testing

Bloodstream infections and sepsis are a major cause of morbidity and mortality. The successful outcome of patients suffering from bacteremia depends on a rapid identification of the infectious agent to guide optimal antibiotic treatment. The analysis of Gram stains from positive blood culture can be rapidly conducted and already significantly impact the antibiotic regimen. However, the accurate identification of the infectious agent is still required to establish the optimal targeted treatment. We present here a simple and fast bacterial pellet preparation from a positive blood culture that can be used as a sample for several essential downstream applications such as identification by MALDI-TOF MS, antibiotic susceptibility testing (AST) by disc diffusion assay or automated AST systems and by automated PCR-based diagnostic testing. The performance of these different identification and AST systems applied directly on the blood culture bacterial pellets is very similar to the performance normally obtained from isolated colonies grown on agar plates. Compared to conventional approaches, the rapid acquisition of a bacterial pellet significantly reduces the time to report both identification and AST. Thus, following blood culture positivity, identification by MALDI-TOF can be reported within less than 1 hr whereas results of AST by automated AST systems or disc diffusion assays within 8 to 18 hr, respectively. Similarly, the results of a rapid PCR-based assay can be communicated to the clinicians less than 2 hr following the report of a bacteremia. Together, these results demonstrate that the rapid preparation of a blood culture bacterial pellet has a significant impact on the identification and AST turnaround time and thus on the successful outcome of patients suffering from bloodstream infections.

A rapid bacterial pellet preparation from a positive blood culture can be used as a sample for applications such as identification by MALDI-TOF, Gram staining, antibiotic susceptibility testing and PCR-based test. The results can be rapidly communicated to clinicians to improve the outcome of patients suffering from bloodstream infections.

Preparación de una Cultura de pellets de sangre para Rapid bacteriana Identificación y Prueba de Susceptibilidad a los antibióticos

Bloodstream infections and sepsis are a major cause of morbidity and mortality. The successful outcome of patients suffering from bacteremia depends on a ...

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.

Una referencia Caldo método de microdilución para Dalbavancina<em&gt; In Vitro</em&gt; Pruebas de Susceptibilidad de bacterias que crecen en aerobiosis

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 modelo de tejido 3D Pulmón humano de Estudios funcionales en<em&gt; Mycobacterium tuberculosis</em&gt; Infección

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.

Sistema para la eficacia y la citotoxicidad de detección de inhibidores de focalización intracelular<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.

Análisis de 18FDG PET/CT la proyección de imagen como una herramienta para estudiar la tuberculosis del Mycobacterium infección y tratamiento en Primates no humanos

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.

Proyección de imagen de tuberculosis del Mycobacterium en ratones con reportero enzima fluorescencia

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