Gostaríamos de agradecer aos técnicos de laboratório da Clínica Mayo Micobactérias laboratório por sua ajuda durante o estudo.

Cada laboratório deve realizar uma avaliação de risco, em colaboração com o seu Diretor de Segurança Institucional biológica para determinar o nível de biossegurança adequado para preparação do inóculo e para a pipetagem do inóculo na placa. Em nosso laboratório, utilizamos nível de biossegurança 3 de laboratório (BSL3) práticas até as placas de microtitulação são inoculados, e selada com um selo adesivo de plástico. Estas placas são então colocados dentro de um saco plástico, que é o calor, também lacrado. Incubação e interpretação da leitura da placa de microtitulação, é realizado em um laboratório de nível de segurança 2 (BSL2). 1. Rotulagem de materiais <ol><li> Rotular uma placa de ágar sangue, três Middlebrook 7H10 placas, Middlebrook 7H9 caldo, uma solução salina entre tubo de vidro e um talão de microtitulação placa AST com os identificadores apropriados (por exemplo, nome do paciente, número do prontuário) na BSL2. </li><li> Rótulo agar sangue e 7H10 banhado como &quot;check pureza&quot;. Contagem de colônias periódica do inóculo são recomendadas para garantir que uma concentração adequada de organismos é usado no teste. Contagem de colônias quando realizada precisará ter duas das placas 7H10 rotulada com &quot;contagem 01/01 colônia&quot; e &quot;Contagem de 1 / 50 colônia&quot;. </li></ol> 2. Preparações tecnólogo de ir para o BSL3 <ol><li> Equipamentos de proteção individual apropriado é necessário para trabalhar no BSL3 e inclui sólido frente vestido, cobrir a cabeça, luvas, propés, e um respirador. Cada instituição deverá estabelecer o seu plano de proteção respiratória própria em colaboração com a sua instituição oficial de Higiene Industrial. Em nosso laboratório, temos vários respiradores disponíveis, incluindo os respiradores purificadores de ar alimentado (PAPRs) e equipado com filtro HEPA, N95 meia-face respiradores. </li></ol> 3. Preparação da cabine de segurança biológica <ol><li> No BSL3, prepare o gabinete de segurança biológica (CSB) por desinfecção da superfície e colocando uma toalha de papel embebido com desinfetante aproximadamente 6 polegadas a partir do painel de ventilação de ar. </li><li> Coloque um recipiente do lado esquerdo, e nephlometer pequenas e vortexer à direita. </li><li> Loops inoculação, pipetors e dicas são colocados ao lado da toalha de papel. </li><li> Coloque um rack com o organismo (em caldo ou em meio sólido) e do padrão de McFarland 0,5 na toalha de papel. </li></ol> 4. Calibração do nefelômetro <ol><li> Coloque um tubo de vidro de soro fisiológico entre talão na nefelômetro e defina o instrumento para zero usando o espaço em branco entre salina. </li><li> Coloque o padrão de McFarland 0,5 na nefelômetro e calibrar de acordo com as instruções do fabricante. </li></ol> 5. Preparação do inóculo <ol><li> Trabalhando nas colônias BSC, raspe a partir de meio sólido de uma agulha esterilizada para a solução salina tween devidamente rotulados de vidro do tubo até que um talão ≥ 0,5 McFarland padrão é obtido. </li><li> Inspecionar visualmente a amostra, e comparar com o padrão McFarland. </li><li> Amostra Vortex por 30-60 segundos. </li><li> Permitir que o inóculo de se contentar com 15 minutos (definir um temporizador). </li></ol> 6. Inoculação da placa de microtitulação <ol><li> Ajustar o inóculo com caldo de solução salina entre usando o nefelômetro calibrado para o McFarland 0,5 em passos de 4.1 e 4.2. Este deve ser concluída dentro de 30 minutos (por especificações do fabricante). </li><li> Use uma pipeta 200 mL e uma longa aerosol ponta da pipeta resistente a remover 100 mL de inóculo do rd 03/01 TOP do tubo de soro fisiológico entre talão. Inocular o caldo Middlebrook 7H9. Lentamente vortex o caldo 7H9 inoculados por 20 segundos. </li><li> Remova a placa de microtitulação AST da embalagem do fabricante. Não use se o dessecante é azul ou se o pacote folha está danificado. </li><li> Prepare um tubo de diluição 1:50 para uso posterior na preparação da diluição 1:50 pipetando 50 mL de água estéril em um tubo estéril. </li><li> Com cuidado, despeje o caldo 7H9 inóculo em uma calha. Evitar a geração de aerossóis. </li><li> Coloque a placa de microtitulação AST com o rótulo voltado para você. </li><li> Use uma pipeta de 12 canais para adicionar 100 mL do inóculo em cada poço. </li><li> Preparar diluições para contagem de colônias: <ol type="a"><li> A diluição de 1 / 1 é preparado a partir do controlo positivo bem (H12) por listras 1 loop mL sobre a placa 7H10 rotulados como &quot;01/01&quot;. </li><li> A diluição de 1 / 50 é preparada através da inoculação de um loop 1 ml de controle positivo bem (H12) e rodando-o no tubo com 50 ul de tubo de água preparada anteriormente. ML raia 1 do presente de diluição para a chapa denominada &quot;1 / 50&quot;. </li></ol></li><li> Inocular as placas pureza da calha pipetando 50 mL em um ágar sangue devidamente rotulados e placa 7H10 e raia para o isolamento. </li><li> Lugarum selo adesivo na placa de microtitulação AST. Pressione firmemente cada poço para garantir vedação adequada usando o fundo branco do selo. Evitar vincos. </li><li> Desinfetar a placa de microtitulação esfregando-o com uma toalha de papel embebido com um desinfectante tuberculocida e placa de microtitulação lugar em um saco plástico e feche com um selo de calor ou fita. <ol type="a"><li> O saco de plástico serve como um recipiente secundário para garantir que todo o caldo está contido deve haver um vazamento ou derramamento do prato principal. </li></ol></li></ol> 7. Desinfecção de materiais e cabines de segurança biológica <ol><li> Para evitar a produção de aerossóis, coloque outra calha na parte superior da calha inoculados antes de colocar o suavemente para dentro do recipiente de descarte. </li><li> Desinfetar todos os materiais, incluindo nephlometer, vortexer, chapas, tubos, etc, limpando-se com um desinfectante tuberculocida e esperar o momento adequado para garantir a desinfecção de acordo com as instruções do fabricante antes de remover a partir do BSC. Por exemplo, Prospray, o desinfetante tuberculocida que usamos, requer 15 minutos para matar micobactérias. </li><li> Coloque o recipiente de descarte em um saco de risco biológico e vedação antes da autoclavagem. </li></ol> 8. Incubação <ol><li> As placas devem ser incubadas com meio para baixo e deve ser tomado cuidado para evitar derramamento ou respingos qualquer líquido. </li><li> Incubar a placa AST placa de microtitulação, 7H10 e placas de ágar sangue em uma incubadora de 35-37 ° C com 5-10% CO 2 com CO 2 em 5-10% do laboratório BSL2. <ol class="lalpha"><li> Em nosso laboratório, isso é aceitável, porque a placa é bem fechados e dentro de um saco plástico selado. </li></ol></li><li> Não mais do que duas placas de microtitulação AST devem ser empilhadas na incubadora de acordo com as instruções do fabricante. </li></ol> 9. Resultados representativos Interpretação dos resultados: Placas de microtitulação selada AST podem ser lidos manualmente usando um espelho como uma ajuda ou com um leitor automático de placas, como a Vizion (TREK Diagnostics). As placas podem ser examinados logo 7 dias após a inoculação. Examine os poços controlar o crescimento em posições H11 e H12 para determinar se eles são positivos (ou seja, têm um depósito de células no fundo do poço). Se a poços de controlo de crescimento não são positivas, reincubate a placa e re-examinar periodicamente (por exemplo, dia 10, dia 14) até a poços de controlo de crescimento são positivas. <ol type="a"><li> Espelho leia-se: <ol type="i"><li> Lugar selado AST placa de microtitulação para a plataforma; </li><li> Leia poços de controlo de crescimento após 7 dias de incubação, se este for negativo, reincubate AST placa de microtitulação; </li><li> Se os controles de crescimento são aceitáveis, em seguida, ler os poços de drogas que contêm. Para para cada droga, gravar primeira diluição que não tem crescimento, comparando os poços de drogas com o controle do crescimento. </li><li> Crescimento aparece como turbidez ou como um depósito de células no fundo de um poço. </li></ol></li><li> Instrumento de leitura: siga as instruções do fabricante; as instruções abaixo são um exemplo de como a leitura seria feita com a plataforma Vizion: <ol type="i"><li> Log in </li><li> Use &quot;MYCOTB&quot; software placa AST. </li><li> Coloque a placa selada com o código de barras voltado para o usuário. Crescimento aparece como turbidez ou como um depósito de células no fundo de um poço. </li><li> Leia controla o crescimento, se este for negativo, reincubate AST placa de microtitulação; se positivo ler a placa ao tocar a tela com o primeiro poço de cada medicamento que não apresenta crescimento. </li></ol></li></ol> Leia as placas pureza. A placa de ágar sangue não deve apresentar crescimento após 48 horas ea placa de pureza 7H10 deve ter crescimento confluente de um tipo de organismo. Se contaminados, o teste de microtitulação AST precisa ser repetido a partir de uma cultura pura. Calcule contagem de colônias a partir do controle positivo bem usando a tabela abaixo: Chave Contagem de Colônia <table border="1"><tbody><tr><td> # Colônias contou com placa rotulados como &quot;01/01&quot; </td><td> # Colônias contou com placa rotulado como &quot;1 / 50&quot; </td><td> Inóculo Contagem de Colônia (UFC / mL) </td></tr><tr><td> &lt;50 </td><td> 0 </td><td> &lt;5 X 10 4 </td></tr><tr><td> 5-10 </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 de 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> Pode haver fuga com para-aminossalicílico em algumas cepas de Mycobacterium tuberculosis. Isto pode ser interpretado, olhando para todos os poços que têm pellets e usando o primeiro poço que tem o mesmo tamanho da pelota como a diluição seguinte como o ponto final. A resistência é rara com este medicamento. <img src="/files/ftp_upload/3094/3094fig1.jpg" alt="Figura 1"/> Figura 1. Resultados representativos usando a placa MYCOTB. <table border="1"><tbody><tr><td> Abreviatura de drogas </td><td> Droga </td><td> Μ MIC / mL </td></tr><tr><td> OFL </td><td> ofloxacina </td><td> 16 </td></tr><tr><td> MXF </td><td> moxifloxacina </td><td> 8 </td></tr><tr><td> RIF </td><td> rifampicina </td><td> &gt; 16 </td></tr><tr><td> AMI </td><td> amicacina </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-aminossalicílico </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> canamicina </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>

Um exemplo de uma placa de microtitulação AST que controla o crescimento positivo é mostrado na Figura 1 e do MIC endpoint (mg / ml) para cada droga é um círculo. Um estudo realizado em nosso laboratório indicaram que a placa de microtitulação AST método é equivalente ao método de proporção padrão agar ouro para testes de susceptibilidade do Mycobacterium tuberculosis. Mais rápido relato dos resultados foi obtida com microtitulação placa de microtitulação AST quando comparado ao método tradicional agar proporção com a interpretação final para AST placa de microtitulação e método das proporções em ágar 14 e 21 dias, respectivamente.A AST placa de microtitulação tem a vantagem de testar primeiro e segunda linha de medicamentos simultaneamente, o que pode ajudar a prevenir morosidade excessiva com cepas resistentes. O AST placa de microtitulação também é muito mais fácil de configurar e ler que a APM e ambos espelho um manual e um sistema semi-automático como o Vizion pode ser usado para ler as placas. A disponibilidade de um valor de MIC para TB testes de resistência poderiam fornecer novas informações valiosas para os médicos em relação ao valor tradicional concentração crítica, especialmente para aqueles isolados com susceptibilidade &quot;borderline&quot;.

<table border="1"><tbody><tr><th> Nome do reagente </th><th> Companhia </th><th> Número de catálogo </th></tr><tr><td> Placas de microtitulação AST e selo adesivo </td><td> TREK Diagnostics, Cleveland Ohio </td><td> MYCOTB </td></tr><tr><td> Padrão de McFarland 0,5 </td><td> TREK Diagnostics, Cleveland, Ohio </td><td> E1041 </td></tr><tr><td> Middlebrook 7H10 placas </td><td> Pescador </td><td></td></tr><tr><td> Agar Plates sangue </td><td> Pescador </td><td></td></tr><tr><td> Estéril loops 1 mL </td><td> Pescador </td><td></td></tr><tr><td> Loops estéril 10 L </td><td> Pescador </td><td></td></tr><tr><td> Sensititre Saline-Tween caldo de contas de vidro </td><td> TREK Diagnostics, Cleveland, Ohio </td><td> 27143 </td></tr><tr><td> 7H9 OADC Caldo </td><td> TREK Diagnostics, Cleveland, Ohio </td><td> T3440 </td></tr><tr><td> Dicas longo Pipetar </td><td> Pescador </td><td></td></tr><tr><td> Cochos estéril </td><td> TREK Diagnostics, 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> Dicas de 1000, 20, 20 mL </td><td> Pescador </td><td></td></tr><tr><td> Pipeta multicanal </td><td> Pescador </td><td></td></tr><tr><td> Incubadora de CO% 05-10 fevereiro </td><td> Pescador </td><td></td></tr><tr><td> Sacos de plástico, pequena, CO 2 permeáveis </td><td> Poly Plásticos, Delano, PA </td><td></td></tr><tr><td> Desinfetante TB (Tais como ProSpray C-60) </td><td> Certol International, Commerce City, CO </td><td> PSC60/32 </td></tr><tr><td> Box espelho Leitor </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

A rápida detecção de resistência aos antimicrobianos é importante no esforço para controlar o aumento da resistência do Mycobacterium tuberculosis (Mtb). Testes de susceptibilidade antimicrobiana (AST) de Mtb tem sido tradicionalmente realizada pelo método de agar de proporção ou por meio de testes macrobroth em um instrumento como o BACTEC (Becton Dickinson, Sparks, MD), VersaTREK (TREK Diagnostics, Cleveland, OH) ou BacT / ALERT (bioMérieux, Hazelwood, MO). O método das proporções em ágar, enquanto considerado o padrão &quot;ouro&quot; da AST, é trabalhoso e requer o cálculo de resistência através da realização de contagem de colônias em ágar contendo drogas em relação ao livre de drogas agar. Se houver ≥ crescimento de 1% no meio de drogas que contêm, em comparação com drogas meio livre, o organismo é considerado resistente a esta droga. Os métodos macrobroth requerem instrumentação e ponto de teste de quebra (&quot;crítica&quot;), as concentrações da droga para os medicamentos de primeira linha (isoniazida, etambutol, rifampicina e pirazinamida). O método descrito aqui é comercialmente disponível em um formato de 96 poços de microtitulação placa [MYCOTB (TREK Diagnostics)] e contém concentrações crescentes de 12 antimicrobianos utilizados para o tratamento de tuberculose, incluindo ambos os primeiro (isoniazida, rifampicina, etambutol) e medicamentos de segunda linha (amicacina, cicloserina, etionamida, canamicina, a moxifloxacina, ofloxacina, para-aminossalicílico, rifabutina, e estreptomicina). Pirazinamida, uma droga de primeira linha, não está incluído na placa de microtitulação, devido à sua necessidade de condições de teste ácido. Vantagens do sistema de microtitulação incluem tanto a facilidade de configurar e mais rápido por sua vez em torno do tempo (14 dias) em comparação com proporção tradicionais com ágar (21 dias). Além disso, a placa pode ser configurada a partir de inóculo preparado usando o caldo de carne ou meio sólido. Desde o formato microplaca é novo e desde Mtb apresenta desafios únicos de segurança no laboratório, este protocolo irá descrever como a segurança de instalação, incubar e ler a placa de microtitulação.

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

Testes de susceptibilidade antimicrobiana de Mycobacterium tuberculosis é um desafio, mas fundamental para o atendimento ao paciente. Este formato de placa de microtitulação oferece testes de 12 drogas antimicobacterianos e fornece concentração inibitória mínima (MIC) valores, o que ajudará no tratamento adequado.

Teste de susceptibilidade antimicrobiana do complexo Mycobacterium tuberculosis da Droga primeira e segunda linha de diluição em caldo em um formato de placa de microtitulação

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 visualizações.  Mayo Clinic. Testes de susceptibilidade antimicrobiana de Mycobacterium tuberculosis é um desafio, mas fundamental para o atendimento ao paciente. Este formato de placa de microtitulação oferece testes de 12 drogas antimicobacterianos e fornece concentração inibitória mínima (MIC) valores, o que ajudará no tratamento adequado.

Vídeo: 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 Bem microtitulação Método da Placa com base para a Formação Monitoramento e Teste de susceptibilidade antifúngica de Candida albicans Biofilmes

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

Imunologia e Infecção

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.

Preparação de amostras de<em&gt; Mycobacterium tuberculosis</em&gt; Extractos para Estudos Nucleares metabolômica Ressonância Magnética

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.

Crescimento de Mycobacterium tuberculosis Biofilmes

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.

Demonstrando um multi-resistente<em&gt; Mycobacterium tuberculosis</em&gt; Ampliação Microarray

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.

Preparação de uma Cultura Pellet sangue para rápida identificação bacteriana e Antibiótico Teste à Susceptibilidade

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.

Um método de microdiluição em caldo de referência para Dalbavancin<em&gt; In Vitro</em&gt; Teste à Susceptibilidade de bactérias que crescem aeròbia

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.

Um modelo de tecido Pulmão humano 3D para estudos funcionais em<em&gt; Mycobacterium tuberculosis</em&gt; Infecção

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 de Eficácia e citotoxicidade Rastreio de inibidores Segmentação 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.

Análise de 18FDG PET-CT Imaging como uma ferramenta para estudar infecção por Mycobacterium tuberculosis e tratamento em primatas não-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.

Mycobacterium tuberculosis em camundongos com fluorescência de enzima do repórter de imagem

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