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

NOTA: Como M. tuberculosis mc 2 6206 é uma estirpe avirulenta 17, 18, todo o trabalho neste protocolo pode ser realizada em uma instalação de segurança biológica 2 Nível (BSL-2). 1. As condições de cultura para Green Fluorescent Protein Expressando M. tuberculosis mc 2 6206 (Mtb GFP) NOTA: O M. tuberculosis H37Rv derivado auxotróf ica estirpe MC 6206 2 (Δ PANCD, Δ leuCD) transformada com o plasmídeo que expressa GFP pMN437 é utilizado ao longo deste protocolo 16. É possível substituir a tensão Mtb-GFP com não expressando GFP do tipo selvagem tensão para permitir mais fácil acessibilidade da estirpe para os investigadores. No entanto, a expressão da GFP é desejável para permitir a confirmação visual da fagocitose e formação de agregados de M. tuberculosis / macrófagos. para lonarmazenagem a longo prazo g, Mtb -GFP foram congeladas a -80 ° C em meio completo (7H9 descritos no passo 1.1) suplementado com 20% de glicerol. <ol><li> Prepare completa meios de Mtb -GFP (7H9-C), completando 7H9 meio com 10% de Middlebrook OADC, 0,02% de tiloxapol, 24 ug / mL de ácido D-pantoténico, 50 ug / ml de L-leucina e 50 ug / ml de higromicina B. </li><li> Descongelar um frasco de Mtb -GFP e adicionar a 10 ml de 7H9-C num frasco de PETG fundo rectangular 30 mL. </li><li> Incubar a 37 ° C num agitador rotativo (50 rpm). Tome as medições de densidade óptica (DO 600) todos os dias até DO600 atinge 1.0 (aproximadamente 5-8 dias). </li><li> Diluir e da cultura passagem como necessário para manter um OD 600 entre 0,2-1,0. </li><li> Para a infecção, utilizar uma cultura estabelecida de Mtb -GFP dentro da fase de crescimento logarítmico (DO600 de 0,6-1). Para evitar culturas aglomerada, sonicar o frasco de cultura de M. tuberculosis num banho de água (130 W; 3 x 5 s pulsos) ONCe ou duas vezes por semana </li></ol> 2. Condições de cultura para As células THP-1 <ol><li> Prepare completa meios de células THP-1 (meio RPMI-C), completando meio RPMI 1640 com 10% de soro inactivado por calor fetal de bovino, 2 mM de L-glutamina, e 10 mM de HEPES. </li><li> Incubar as células em um frasco T-75 a 37 ° C numa atmosfera humidificada de 5% de CO 2. </li><li> Contagem de células utilizando um hemocitómetro ou por citometria de fluxo em dias alternados e manter as células THP-1 a uma densidade entre 0,1-0600000 por mL. </li></ol> 3. Infecção protocolo para gerar estruturas Mtb / macrófagos agregadas <ol><li> THP-1 preparação de células (por placa de 96 poços) <ol><li> Centrífuga 7 X 10 6 células THP-1 a 250 xg durante 5 min. Ressuspender em 7 mL de meio RPMI-C. </li></ol></li><li> Preparação Mtb -GFP <ol><li> Centrifugar 2,8 x 10 8 Mtb -GFP a 3200 xg durante 5 minutos numa centrifuga-L baldecantar uma conversão de OD 600 1.0 = 3 x 10 8 bactérias / mL. </li><li> Lavar uma vez com RPMI-C e centrifuga-se tal como no passo 3.2.1. </li><li> Ressuspender em 7 mL de meio RPMI-C e agitar com vortex durante 10 s. </li></ol></li><li> Infecção Nota: Consulte a Figura 1 para modelo. <ol><li> Prepara-se uma placa de 96 cavidades por adição de 200 uL de água estéril em filas A e H e as colunas 1 e 12 para um aro de água para evitar a evaporação do meio de cultura. </li><li> Adicionar 200 uL de RPMI-C para a coluna 2 (B2 para G2) para o controlo de fundo (em branco). </li><li> Para infectar, adicione suspensão Mtb -GFP (passo 3.2) para suspensão de células THP-1 (passo 3.1) e misture bem por pipetagem. A densidade de células THP-1 final é de 5 x 10 5 por ml e a multiplicidade correspondente de infecção é de 40. </li><li> Verter a suspensão de células THP-1 / Mtb -GFP em um reservatório de 25 ml. </li><li> Adicionar 200 mL de THP-1 / suspensão Mtb -GFP a todos os 96 poços restantes (B3 através G11) usinga pipeta multi-canal. NOTA: Se múltiplos que trabalha com placas de 96 poços, ressuspender regularmente a restante suspensão de células THP-1 / Mtb no reservatório para assegurar uma mistura uniforme é adicionado a cada poço. </li><li> Incubar a 37 ° C com 5% de CO 2 durante 7-10 dias. </li><li> Mudar meios a cada 2 dias, removendo-se lentamente 100 ul meios gasto a partir do topo de cada poço e suavemente adicionando 100 ul de RPMI pré-aquecido-C utilizando uma pipeta multi-canal. Não ressuspender poços e perturbar os agregados de Mtb / macrófagos no fundo dos poços. </li><li> Examine visualmente os poços por microscopia de fluorescência (objetiva 4-10x) diariamente, tomando nota do tamanho do Mtb agregados / macrófagos. Por dia 10/07, Mtb agregados / macrófagos deve ser suficientemente grande (consulte a Figura 2 para referência) para prosseguir para testes de eficácia da droga (Seção 4). </li><li> Se desejado, capturar imagens dos 96 poços utilizando um sistema de imagem de células automatizado equipadocom o campo luminoso e filtro GFP define para documentar a formação de agregados Mtb / macrófagos. NOTA: Se os usuários não têm acesso a um sistema de imagem automatizado, imagens de poços representativos podem ser tomadas utilizando qualquer microscópio apropriado com capacidades de GFP e de campo brilhante. </li></ol></li></ol> 4. Crescimento Ensaio de Inibição de Avaliação de Drogas eficácia contra Mtb Derivado do Mtb / macrófagos Agregados <ol><li> Preparação de drogas (em triplicado condições para duas drogas) NOTA: Ver Figura 1A para o modelo. <ol><li> Numa placa de 96 poços separada, adicionar 125 uL de meios de 7H9-C para B2 através de G10. </li><li> Preparar dois fármacos com o dobro da maior concentração final desejada em 1 ml de 7H9-C para dar conta da diluição no passo 4.2.4. </li><li> Adicionar 250 uL de cada fármaco aos poços B11-C11-D11 e E11-F11-G11, respectivamente, para tratamentos em triplicado. </li><li> Usando uma pipeta multi-canal, em série dilute os fármacos de teste de duas vezes por movendo 125 mL de B11-G11-G10 em B10. Misturar por pipetagem de 5 vezes em cada passo. </li><li> Continuar a mover-se 125 uL da coluna a coluna (direita para a esquerda) do outro lado da placa, e depois de parar a coluna 4. </li><li> Depois de se misturar coluna 4, descartar 125 mL num recipiente de desperdícios. As colunas 2 e 3 não deve conter quaisquer drogas para permitir o uso como um fundo (em branco) e controlo de crescimento positivo. NOTA: Como alternativa, siga modelo na Figura 1B para bibliotecas de teste de drogas. Cada placa de 96 poços pode acomodar 58 drogas numa concentração única. Preparar esta placa de drogas utilizando o dobro da concentração final desejada, uma vez que será diluída pela metade na etapa 4.2.4. </li></ol></li><li> O tratamento medicamentoso: <ol><li> Recuperar a placa de 96 poços contendo o Mtb infectados macrófagos (agregados de Mtb / macrófagos). </li><li> Cuidadosamente decantar todos os poços relevantes (B2 através G11) com uma pipeta de multi-canal.Executar esta de um modo em dois passos: primeiro remover 150 mL sem inclinação da placa, e em seguida, retirar o suporte restantes (~ 50 ul) através da inclinação da placa e inserindo a ponta da pipeta para o bordo inferior do poço. Como Mtb / macrófagos agregados são aderentes ao fundo do poço, nenhum material deve ser perdida. </li><li> Suavemente adicionar 100 de meio 7H9-C a todos os poços relevantes (B2 através G11) da placa contendo os agregados / macrófagos de M. tuberculosis. </li><li> Usando uma pipeta de multi-canal, transferir 100 ul do fármaco que contém placa de 96 poços (passo 4.1) aos poços correspondentes da placa de infecção. </li><li> Lugar em saco selado e incubar durante 3 dias a 37 ° C. </li></ol></li></ol> 5. A quantificação da droga Eficácia Utilizando o Ensaio de Microtitulação Resazurina <ol><li> Prepare resazurina solução mãe a uma concentração final de 0,8 mg / ml em H 2 O. Filtra-se através de 0,22 um de tamanho dos poros da membrana de PVDF para a esterilização.</li><li> Preparar solução de trabalho de resazurina por mistura de solução de resazurina, H2O e Tween-80 (solução a 20% em H2O) em uma proporção de 2: 1: 1. As concentrações finais são de 0,4 mg / mL de resazurina e 5% de Tween-80. </li><li> Utilizando um leitor de placa, a instalação de um programa para ler a fluorescência a 530 nm de excitação e 590 nm de emissão a cada 30 min durante 24 h a 37 ° C. leitor de placas pré-quente a 37 ° C. </li><li> Adiciona-se 20 uL de solução de trabalho de resazurina a todos os poços relevantes (B2 a G11) da placa de drogas tratada (passo 4.11) usando uma pipeta de multi-canal. </li><li> Coloque placa no leitor de placa e iniciar o programa criado no passo 5.3. NOTA: Para facilitar o processamento de grandes volumes de placas de ensaio, que é suficiente para desenvolver o ensaio em uma incubadora a 37 ° C e realizar único fluorescência lê a cada 24 h. Isto também se aplica a usuários que não têm acesso a um leitor de placas com capacidades cinéticas e incubação. </li></ol>

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The authors have nothing to disclose.

Aqui, nós descrevemos em detalhe um modelo de infecção por Mtb alternativa adequada para testes de eficácia da droga. Este modelo leva em conta dois fatores-chave que devem ser dadas mais consideração durante o processo de início de desenvolvimento TB: a presença de barreiras fisiologicamente relevantes para a penetração da droga e as alterações metabólicas de Mtb durante a infecção. Embora tenhamos mostrado previamente os benefícios do nosso modelo de infecção e propôs a possibilida...

<html> A tuberculose (TB) continua a ser uma séria ameaça à saúde global, apesar da disponibilidade de regimes de quimioterapia anti-tuberculose há mais de 40 anos 1. Isto é devido em parte à exigência de períodos de tratamento longo de mais de seis meses usando várias combinações de medicamentos, o que leva a paciente a não conformidade 2. O surgimento de tuberculose resistente a medicamentos nos últimos anos tem agravado os problemas em um campo onde o desenvolvimento bem sucedido de drogas clinicamente aprovados é praticamente inexistente 3. Na verdade, apesar do desenvolvimento exaustiva de drogas anti-TB, apenas uma única droga foi aprovada pela FDA para uso clínico nos últimos 40 anos 4. Assim, as novas gerações de medicamentos anti-tuberculose são urgentemente necessárias para resolver este problema. Um problema fundamental na descoberta TB é a falta de sucesso da transferência a partir de compostos com actividade in vitro para a eficácia no ambiente clínico= &quot;xref&quot;&gt; 5, 6, 7. Inicialmente, as abordagens baseadas alvo foram utilizadas para pesquisar drogas anti-Mtb 5, que não se traduziu em células bacterianas inteiras. Mesmo quando são utilizadas células de M. tuberculosis, que é frequentemente realizada usando caldo cultivado culturas, que não prevêem com precisão a eficácia do fármaco in vivo, 8, 9. Estes problemas têm sido reconhecidas e ensaios de rastreio de drogas contra macrófagos contendo M. tuberculosis ou M. tuberculosis latente foram estabelecidas com êxito 8, 10, 11, 12. No entanto, mesmo esses ensaios mais avançados, não dão conta suficiente para as barreiras de penetração que as drogas encontram nas lesões pulmonares não vascularizados, e na focos de necrose no local da infecção. De fato, Até mesmo para a primeira linha TB rifampicina droga, a dose sub-óptima tem sido questionada devido à inadequada no tecido vivo e líquido cefalorraquidiano (LCR) penetração 13, 14, 15, bem como diminuição da eficácia contra intracelular Mtb 8, 9. Como tal, os novos modelos e ensaios que tenham em conta esses parâmetros durante o processo de desenvolvimento de liderança no início, sem dúvida, melhorar a TB esforços de descoberta de drogas. Para atender a essa necessidade, que recentemente estabeleceu um 2 BSL-modelo de infecção barato, rápido e compatível alternativa para Mtb droga testes de eficácia 16. Este modelo de infecção produzida densamente Mtb dentro de grandes estruturas agregadas de macrófagos, que recapitulou as barreiras de penetração celular fisiologicamente relevantes e gerou macrófagos passadas <em&gt; Mtb com um estado fisiológico alterado assemelhando-se latente Mtb. Mtb derivado deste modelo de infecção foi combinado com o ensaio de microplaca com resazurina (REMA) para avaliar a eficácia da droga, o que produziu resultados consistentes com outros modelos de infecção intracelular e bem correlacionado com a capacidade reportada das drogas comuns TB para atingir concentrações elevadas de CSF em relação às concentrações séricas 16. Aqui nós descrevemos em detalhe a geração de Mtb / estruturas de agregação de macrófagos para produzir e macrófagos passadas Mtb apropriado para o teste de sensibilidade às drogas usando REMA. Em particular, mostra-se como este sistema de infecção poderia ser adaptado a um formato de 96 poços para compatibilidade com throughput screening de candidatos de drogas anti-TB.

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

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

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

Para confirmar a robustez de adaptar este modelo de infecção de 96 poços formato de placa, que aqui examinada a susceptibilidade fármaco de M. tuberculosis, derivado da 96-bem adaptado modelo de infecção à rifampicina (RIF) e moxifloxacina (moxi) de acordo com o modelo dado na figura 1A. Nós demonstramos que a geração do M. tuberculosis / macrófagos estruturas agregadas chave para este ensaio podem ser produzidos de forma fiável num formato ...

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

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

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

Um ensaio Compatível de alta capacidade para avaliar droga Eficácia contra macrófagos passadas<em&gt; Mycobacterium tuberculosis</em

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

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

Research

JoVE Journal

Immunology and Infection

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

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

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

Growth of Mycobacterium tuberculosis Biofilms

Mycobacterium tuberculosis, the etiologic agent of human tuberculosis, has an extraordinary ability to survive against environmental stresses including antibiotics. Although stress tolerance of M. tuberculosis is one of the likely contributors to the 6-month long chemotherapy of tuberculosis 1, the molecular mechanisms underlying this characteristic phenotype of the pathogen remain unclear. Many microbial species have evolved to survive in stressful environments by self-assembling in highly organized, surface attached, and matrix encapsulated structures called biofilms 2-4. Growth in communities appears to be a preferred survival strategy of microbes, and is achieved through genetic components that regulate surface attachment, intercellular communications, and synthesis of extracellular polymeric substances (EPS) 5,6. The tolerance to environmental stress is likely facilitated by EPS, and perhaps by the physiological adaptation of individual bacilli to heterogeneous microenvironments within the complex architecture of biofilms 7.
In a series of recent papers we established that M. tuberculosis and Mycobacterium smegmatis have a strong propensity to grow in organized multicellular structures, called biofilms, which can tolerate more than 50 times the minimal inhibitory concentrations of the anti-tuberculosis drugs isoniazid and rifampicin 8-10. M. tuberculosis, however, intriguingly requires specific conditions to form mature biofilms, in particular 9:1 ratio of headspace: media as well as limited exchange of air with the atmosphere 9. Requirements of specialized environmental conditions could possibly be linked to the fact that M. tuberculosis is an obligate human pathogen and thus has adapted to tissue environments. In this publication we demonstrate methods for culturing M. tuberculosis biofilms in a bottle and a 12-well plate format, which is convenient for bacteriological as well as genetic studies. We have described the protocol for an attenuated strain of M. tuberculosis, mc27000, with deletion in the two loci, panCD and RD1, that are critical for in vivo growth of the pathogen 9. This strain can be safely used in a BSL-2 containment for understanding the basic biology of the tuberculosis pathogen thus avoiding the requirement of an expensive BSL-3 facility. The method can be extended, with appropriate modification in media, to grow biofilm of other culturable mycobacterial species.
Overall, a uniform protocol of culturing mycobacterial biofilms will help the investigators interested in studying the basic resilient characteristics of mycobacteria. In addition, a clear and concise method of growing mycobacterial biofilms will also help the clinical and pharmaceutical investigators to test the efficacy of a potential drug.

Growth of Mycobacterium tuberculosis Biofilms

Mycobacterium tuberculosis forms drug tolerant biofilms when cultured in certain conditions. Here we describe methods for culturing M. tuberculosis biofilms and determining the frequency of drug tolerant persisters. These protocols will be useful for further studies into the mechanisms of drug tolerance in M. tuberculosis.

Crescimento de Mycobacterium tuberculosis Biofilmes

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

Imunologia e Infecção

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

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

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

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

Um modelo experimental para estudar a Tuberculose-Malária Coinfection sobre Transmissão Natural de Mycobacterium tuberculosis E Plasmodium berghei</i

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

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

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

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

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

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 ...

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

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

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

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

Criação e Otimização de um Setup de Alto Rendimento para o Estudo<em&gt; Staphylococcus epidermidis</em&gt; E<em&gt; Mycobacterium marinum</em&gt; Infecção como um modelo para a Descoberta de Drogas

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

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

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

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

De alto rendimento quantitativo em tempo real de RT-PCR Ensaio para Determinar Perfis de Expressão do tipo I e III do interferão subtipos

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

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

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

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

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

Um Modelo Pneumonia robusta em imunocompetentes Roedores para avaliar antibacteriano Eficácia contra<i&gt; S. pneumoniae</i&gt;,<i&gt; H. influenzae</i&gt;,<i&gt; K. pneumoniae</i&gt;,<em&gt; P. aeruginosa</em&gt; ou<em&gt; A. baumannii</em

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

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

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

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

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

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 ...

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

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

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

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

A<em&gt; In Vitro</em&gt; Caseum Ensaio de Ligação ao que prevê a penetração da droga na tuberculose Lesões

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

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

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

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

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

Mycobacterium tuberculosis 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 ...

Modeling Tuberculosis in Mycobacterium marinum Infected Adult Zebrafish

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

Modeling Tuberculosis in Mycobacterium marinum Infected Adult Zebrafish

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

Modelagem de tuberculose no Zebrafish adulto infectado de Mycobacterium marinum

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